ORIGINAL ARTICLE
Xuezhikang Attenuated the Functional and Morphological Impairment of Pancreatic Islets in Diabetic Mice Via the Inhibition of Oxidative Stress Jun Wang, MS,* WeiMin Jiang, MD, PhD,† Yong Zhong, MD, PhD,*‡ Bin Lu, MD, PhD,§ JiaQing Shao, MD, PhD,§ ShiSen Jiang, MD,* and Ping Gu, MD, PhD*§
Abstract: Xuezhikang, purified from red yeast rice, is a traditional Chinese medicine with pleiotropic effects on the cardiovascular system. Oxidative stress plays a crucial role in the dysfunction of pancreas islet in diabetic condition and represents a promising therapeutical target for diabetes mellitus. Therefore, the purpose of this study was to explore the effects and possible mechanisms of xuezhikang on the microenvironment and insulin secretion by pancreatic islets in db/db diabetic mice. Our results showed that xuezhikang decreased the blood glucose level by improving glucose tolerance and insulin secretion in db/db mice. Xuezhikang protected islets from hyperglycemic injury as illustrated by the conserved b-cell content and microenvironment. Furthermore, xuezhikang potently inhibited the expression of key factors in oxidative stress. In addition, administration of xuezhikang caused an upregulated expression of glucose-sensing apparatus. These observations provide evidence that the influence of xuezhikang on oxidative stress may at least partly account for its protective effects on the microenvironment and insulin secretion function of pancreatic islets in diabetes. Key Words: xuezhikang, diabetes, pancreatic islet, oxidative stress, nicotinamide adenine dinucleotide phosphate oxidase (J Cardiovasc Pharmacol 2014;63:282–289)
Received for publication August 15, 2013; accepted November 7, 2013. From the *Department of Cardiology, School of Medicine, Nanjing University, Jinling Hospital/Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu Province, China; †Department of Cardiology, Jiangsu Provincial Hospital of Traditional Chinese Medicine, Nanjing, Jiangsu Province, China; and Departments of ‡Health Care, and §Endocrinology, School of Medicine, Nanjing University, Jinling Hospital/Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu Province, China. Supported by the Natural Science Foundation of China (81000352, 30900697, 81373605, and 81100568), the Natural Science Foundation of Jiangsu Province (BK2011661 and BK20131456), the Postdoctoral Scientific Foundation of China (20100471843), and the Postdoctoral Scientific Foundation of Jiangsu Province (1001027C). The authors report no conflicts of interest. J.W. and W.J. have contributed equally. Reprints: Ping Gu, MD, PhD, Department of Endocrinology, School of Medicine, Nanjing University, Jinling Hospital/Nanjing General Hospital of Nanjing Military Command, 305 Zhongshan East Rd, Nanjing, Jiangsu Province, China 210002 (e-mail: [email protected]); ShiSen Jiang, MD, Department of Cardiology, School of Medicine, Nanjing University, Jinling Hospital/Nanjing General Hospital of Nanjing Military Command, 305 Zhongshan East Rd, Nanjing, Jiangsu Province, China 210002 (email: [email protected]). Copyright © 2013 by Lippincott Williams & Wilkins
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INTRODUCTION Dysfunction of pancreatic islets is a hallmark of diabetes mellitus (DM). Islets maintain a rich capillary network, also termed the microenvironment, which is essential for the optimal secretory function of b cells and thus carbohydrate metabolism. There are several indications that a high glucose concentration induces oxidative stress in pancreatic b cells. For instance, pancreatic islets present the low antioxidant enzymes activity1 and are highly susceptible to cellular injury. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is the major source of reactive oxygen species (ROS) in pancreatic b cells.2 As a multicomponent enzyme, NOX consists of membrane-bound (gp91phox and p22phox) and cytosolic components. The catalytic subunit, gp91phox, plays a critical role in activation of NOX.3 Inhibition of NOX is accepted as one mechanism of reducing oxidative stress. One study demonstrated that silencing NOX attenuated oxidative stress and thus cellular injury in b cells.4 Xuezhikang, extracted from red yeast Chinese rice, has been widely prescribed as a lipid-lowering drug. Several clinical trials have demonstrated the efficacy and safety of xuezhikang in regulating lipid and glucose metabolism, which result into the cardiovascular events declined.5–8 Animal experiments demonstrated that xuezhikang is able to ameliorate insulin resistance in rats.9 However, the exact molecular mechanism remains unknown. Therefore, the present study was designed to evaluate the impact of xuezhikang on pancreatic islets in mice and explore the pathways involved.
MATERIALS AND METHODS Animals This study was reviewed and approved by the Animal Care and Use Committee of Jinling Hospital. Forty 8-weekold male genetically diabetic C57BL/KsJ-db/db mice and their lean nondiabetic C57BL/KsJ-db/m littermates, weighing 34–42 g, were kindly provided by the Research Institute of Nephrology, Jinling Hospital. Mice were housed under standard conditions (room temperature, 20 6 18C; humidity, 60% 6 10%; lights on from 6 AM to 6 PM). Only the db/db mice were randomly assigned to receive xuezhikang (n = 20) or placebo (n = 20). The placebo-treated db/m group served as a nondiabetic control (wild-type control, n = 20). Mice were given either 300 mg/kg/d of xuezhikang (WBL Peking J Cardiovasc Pharmacol Volume 63, Number 3, March 2014
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University Biotech Co, Ltd, Beijing, China) or placebo (0.5% natrium cellulose solution) by gavage for 8 weeks. The administration dosage of xuezhikang was derived from the previous studies10,11 and the body surface area normalization method. Mice were provided with standard chow and water ad libitum.
Metabolism Measurement Body weight, fasting blood glucose (FBG), and fasting serum insulin (FSI) were measured every week. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels were assayed by the enzymatic methods. After fasting for 15 hours, in 10 mice for each group, an intraperitoneal glucose tolerance test was performed with 500 mg/kg glucose injected intraperitoneally. Glucose and insulin levels were determined in blood samples collected at different time points (0, 30, 60, and 120 minutes).
Islet Isolation and Perifusion Animals were anesthetized with sodium phenobarbital before pancreatectomy. Islets were isolated from mice in each group by infusion of collagenase P (Roche, Penzgerg, Germany) followed by purification with a Ficoll step density gradient. Islets were handpicked twice and cultured for 1 hour in RPMI 1640 medium. The in vitro insulin release kinetics were studied with the perifusion system as described previously.12 Fifty sizematched islets were placed in individual columns in a 37ºC water bath. Krebs–Ringer bicarbonate buffer containing 2.8 mM glucose was perfused in each column at 0.5 mL/min for 60 minutes. Islets were stimulated by a high concentration of glucose (16.7 mM). The dripped solution was collected after 20 seconds, 1 minute, and 5 minutes, separately within each phase that started 2, 5 minutes, and 30 minutes after the initiation of stimulation. The solution was assayed to evaluate the amount of insulin secreted by each islet column.
Electron Microscopy Pancreas tissues were cut into small pieces with razor blades. They were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.4) at 48C for 2 hours, washed, fixed, dehydrated, and embedded in Epon 812. Ultrathin sections were stained with 2% uranyl acetate followed by lead citrate, and the stained sections were examined on a JEM-1200EX electron microscope (JEOL, Tokyo, Japan) operating at 80 kV. The relative mitochondrial volume was calculated by counting the mitochondrial area on random micrographs at a 20,000 magnification with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Four micrographs were used per group.
Immunohistochemistry
The pancreas was collected, fixed in 4% paraformaldehyde for 4–6 hours, and embedded in paraffin. The ABC method was employed for the detection of insulin, gp91phox, 8-hydroxy-20-deoxyguanosine (8-OHdG), 4-hydroxynonenal modified protein (4-HNE), and CD31. Specimens were deparaffinized with xylene and rehydrated in an ethanol series. Endogenous peroxidase was blocked with 0.3% H2O2 in methanol, and 10% goat serum was added to block nonspecific
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antigens. After that, the sections were incubated overnight in primary antibodies at 48C. Fourteen hours later, these sections were incubated for 30 minutes at room temperature with the corresponding biotinylated secondary antibodies. Samples were subsequently incubated with horseradish peroxidase–conjugated streptavidin with the LSAB2 kit (Dako). Staining with 3,3’-diaminobenzidine (Sigma, St Louis, MO) was performed before light counterstaining with Mayer hematoxylin (Wako, Tokyo, Japan). Finally, some slides were dehydrated and mounted, whereas others were subjected to Azan staining. The insulin staining density was analyzed semiquantitatively from percentage of stained b cells, with the Image-Pro Plus 5.0.1 image analysis system (Planetron, Tokyo, Japan). The primary antibodies were as follows: guinea pig antihuman insulin antibody (1:2000; Linco, St Charles, MO), goat anti-human gp91phox antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human 8-OHdG antibody (1:100), mouse anti-human 4-HNE antibody (1:50; Japan Institute for the Control of Aging, Shizuoka, Japan), and rat anti-human CD31 antibody (1:200; BD Bioscience, Tokyo, Japan). The secondary antibodies were goat anti-guinea pig immunoglobulin (Ig)G antibody (1:1000; Chemicon, Temecula, CA), rabbit anti-goat IgS antibody (1:200; Dako), goat anti-mouse IgS antibody (1:200; Dako), and goat anti-rat IgG antibody (1:200; Cosmo Bio, Tokyo, Japan).
Morphometric Analysis For immunohistochemistrical analysis, captured digital images from 15 islets per animal, namely 45 islets per group (n = 3), were observed and estimated by Image-Pro Plus 5.0.1. Intensities of the 8-OHdG-, 4-HNE-, and gp91phox-associated immunoreactions was assessed by the semiquantitative ranking as previously described.13 In brief, the average intensity was given a score of 0, 1, 2, or 3 corresponding to the presence of negative, weak, intermediate, or strong staining, respectively. All evaluations were performed in a blinded manner. The insulin-stained domain represented the relative area containing b cells (ie, b cell content), which was indicated by the value of the b-cell mass [b-cell mass (mg) = islet area/whole pancreatic area · pancreas weight (15 duplications per group)]. The coverage was automatically calculated with the Image-Pro Plus 5.0.1 software package.
Western Blot Analysis For gp91phox, glucokinase (GCK), and glucose transporter-2 (GLUT-2) detection and isolated islet aliquots were lysed in lysis buffer [25 mM N-(2-hydroxyethyl) piperazine-N0 -2-ethanesulfonic acid, 50 mM Tris-HCl (pH 7.4), 6% glycerol, 5 mM ethylene diamine tetraacetie acid, 5 mM ethyleneglycol bis(2-aminoethylether)tetraacetic acid, 0.5% Triton X-100, 50 mM NaF, 40 mM glycerophosphate, and 25 mM sodium pyrophosphate with a protease inhibitor mixture]. Equal amounts of protein (50 mg) were loaded onto 10% dodecyl sulfate, sodium salt-Polyacrylamide gel electrophoresis gels, separated by electrophoresis, and transferred onto nitrocellulose membranes. Membranes were blocked by 5% skim milk and incubated with anti-GCK antibody (1:1000; Santa Cruz Biotechnology), anti-GLUT-2 antibody (1:1000; Santa Cruz Biotechnology), and anti-b-actin antibody (1:1000; Santa Cruz Biotechnology) overnight at www.jcvp.org |
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4ºC. After triplicate washes, the membranes were incubated with horseradish peroxidase–conjugated anti-rabbit IgG (1:2500; Santa Cruz Biotechnology) for 1 hour at room temperature. Bands of interest were visualized by enhanced chemiluminescence (Pierce, Rockford, IL). The intensities of the blots were quantified by scanning densitometry and normalized to b actin.
the test was higher than that of the placebo-treated db/db mice (P , 0.05), which suggests that xuezhikang may improve the glucose tolerance in diabetic mice. Experiments involving the islets perifusion in vitro revealed that, in contrast to the db/db control, insulin secretion derived from isolated islets was not noticeably increased in xuezhikang group pretreated with a low-concentration glucose solution (2.8 mM). However, the insulin secretion was moderately increased at 1 minute after perfusion with the highconcentration glucose solution (16.7 mM) (P , 0.05) in the xuezhikang-treated animals compared with placebo-treated animals (Fig. 1C). This result suggests that xuezhikang was capable of enhancing the first-phase insulin secretion, although this secretion was not comparable with that of the db/m group.
Statistical Analysis Statistical analysis was done with SPSS, version 11.0. Data were expressed as mean 6 SD. Means among groups were compared with one-way analysis of variance. Means between 2 groups were compared with least-significant difference test. Data before and after experiment were compared with paired test. A value of P , 0.05 was considered statistically significant.
RESULTS
Morphometric Analysis
To assess the change of b cells’ number, we calculated the insulin staining density. This measurement method reflected the changes in histological immunostaining. Less-intense dark brown staining, which implied a reduced amount of b cells, could be observed in islets from db/db control mice compared with db/m mice (Fig. 2A). Xuezhikang therapy seemed to decline the trend in b-cell reduction (P , 0.05) (Fig. 2B). Similarly, the favorable effect of xuezhikang treatment was presented by the restored b-cell mass (P , 0.05), indicating that this drug could attenuate the b-cell mass loss caused by the diabetic condition (Fig. 2C). Islets contain abundant vascular endothelial cells, a typical feature of endocrine organs, and are essential for endocrine function. As an endothelial cell marker, the CD31 could be used to estimate the endothelial density. The CD31 expression was depleted in db/db diabetic animals. Interestingly, the staining intensity of CD31 was higher in xuezhikang group than in placebo group (Fig. 3A). Thus, xuezhikang may overcome the rarefaction of vasculature endothelium surrounding pancreatic islets in diabetic subjects. On the other hand, b-cell organelles, such as endoplasmic reticulum and Golgi bodies, in mice administered xuezhikang showed an increased resistance to injury. As illustrated by Figure 3B, the mitochondrial swelling was alleviated and the proportion of intact mitochondria was increased in xuezhikang-treated mice versus their placebotreated counterpart.
Metabolism Profiling At baseline, there were no marked differences in the body weight, FBG, FSI, TC, LDL-C, and TG between the xuezhikang and placebo groups. However, all these values were lower in db/m group compared with 2 db/db groups (P , 0.05). In particular, at 8 weeks, db/db mice had a FBG level .15 mmol/L. After treatment with xuezhikang, hyperglycemia development was alleviated, and the FBG and FSI levels at the terminal stage of treatment in db/db mice were markedly lower than that in placebo-treated db/db mice (P , 0.05); however, there was no difference between before and after xuezhikang treatment. Likewise, the TC, LDL-C, and TG levels in the xuezhikang group were markedly reduced compared with the db/db control (P , 0.05). Besides, the 8 weeks of xuezhikang intake blunted weight gain in the db/db mice (P , 0.05) (Table 1).
Glucose Metabolism During the 120-minute glucose tolerance test, the blood glucose level after glucose loading in xuezhikang group was lower than that of placebo group (P , 0.05), which was standardized to that of nondiabetic group (Fig. 1A). Moreover, insulin secretion stimulated by glucose loading in the xuezhikang group was higher than that of db/db control (Fig. 1B). All these results were demonstrated by the differences in the area under the curve (AUC) among the 3 groups. It was noteworthy that the AUCINS0230 in the xuezhikang-treated mice at 30 minutes after
TABLE 1. Baseline and Terminal Metabolic Parameters of Each Group Nondiabetic (n = 20) Baseline BW (g) FBG (mmol/l) FSI (mIU/L) TC (mmol/l) LDL-C (mmol/l) TG (mmol/l)
26.8 5.6 2.5 2.42 1.63 0.56
6 6 6 6 6 6
0.3† 0.7† 0.3† 0.31† 0.23† 0.11†
Placebo (n = 20)
Terminal 32.7 5.8 2.7 2.52 1.71 0.59
6 6 6 6 6 6
0.5* 0.8* 0.4* 0.25* 0.28* 0.15*
Baseline 38.9 17.1 8.8 5.23 3.68 3.45
6 6 6 6 6 6
3.3 4.2 1.5 0.87 0.45 0.56
Xuezhikang (n = 20)
Terminal 47.1 25.9 9.8 6.03 3.98 3.75
6 6 6 6 6 6
4.3 2.2 1.4 0.93 0.53 0.67
Baseline 38.6 18.1 9.2 5.35 3.57 3.35
6 6 6 6 6 6
2.9 4.1 1.4 0.84 0.39 0.48
Terminal 42.3 18.4 7.7 4.13 2.32 1.65
6 6 6 6 6 6
3.8* 3.4* 0.8* 0.57* 0.34* 0.34*
Data are shown as means 6 SD. *P , 0.05 versus the terminal value of placebo group. †P , 0.05 versus the baseline value of placebo group Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group; BW, body weight.
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FIGURE 1. Effects of xuezhikang on blood glucose and insulin secretion in db/db and db/m mice. A, Plasma glucose levels determined at the different time points of the intraperitoneal glucose tolerance test. B, Insulin concentration determined at the different time points of the intraperitoneal glucose tolerance test. C, Insulin secretion kinetics stimulated by low and high glucose concentrations in the in vitro perifusion experiment with pancreatic islet samples isolated from mice. Each bar represents the means 6 SD. Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. *P , 0.05 versus the placebo group.
Protection Against Oxidative Stress To investigate whether xuezhikang could protect against oxidative stress in diabetic, we measured the expression of oxidative stress markers, 8-OHdG, and 4-HNE. The percentage of b cells expressing 8-OHdG and 4-HNE was higher in db/db mice than in db/m mice (P , 0.05) (Figs. 4A,B). More importantly, xuezhikang greatly reduced the expression levels of these 2 markers to the normal level (Figs. 4A,B). Furthermore, we studied the expression levels of gp91phox, which is one of the major components of NOX. Semiquantitative analysis revealed that xuezhikang uptake rectified the expression of gp91phox to nearly the same level as in the wild-type control and to a level below that of the db/db control (P , 0.05) (Figs. 5A,B). Besides, the Western blotting and its densitometric analysis for gp91phox also showed that xuezhikang abolished the elevation of gp91phox expression in placebo group was lowered in xuezhikang group (P , 0.01) (Figs. 5C,D). These results indicate that xuezhikang may inhibit oxidative stress via downregulation of gp91phox. 2013 Lippincott Williams & Wilkins
Overexpression of the Glucose-Sensing Apparatus In the following study, we sought to obtain more detailed insights into the possible targets of xuezhikang for the purpose of screening potential signaling pathways for this drug in glucose metabolism. GLUT-2 and GCK mediate the glucose response pathway in islets. Thus, we measured the protein levels of GLUT-2 and GCK in an attempt to determine a possible impact of xuezhikang on glucose sensing. Western blotting revealed decreased levels of both GLUT-2 and GCK in db/db mice compared with db/m mice (Fig. 6A). Densitometric analysis further validated that the GLUT-2 and GCK expression levels were increased in xuezhikang group as opposed to placebo group (P , 0.01) (Fig. 6B).
DISCUSSION The present study sheds some light on the salutary property of xuezhikang on pancreatic islet function and www.jcvp.org |
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FIGURE 2. Effects of xuezhikang on the b-cell content of pancreatic islets. A, b-cell amounts were evaluated by immunohistochemistry for insulin in pancreatic islet sections. b-cells positive for insulin showed dark brown staining. B, Insulin staining intensity was analyzed semiquantitatively as the percentage of stained b cells of the db/m control. C, b-cell mass was calculated by the following formula: islet b-cell mass (mg) = (the area stained by insulin antibody)/(the area of the whole pancreas) · (pancreas weight). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. Each bar represents the mean 6 SD of the insulin staining intensity or b-cell mass (n = 8). *P , 0.05 versus the placebo group.
morphology in db/db diabetic mice. Xuezhikang has been commonly used as a Chinese traditional medicine for therapy of patients undergoing cardiovascular disorders. Our findings confirmed that xuezhikang corrected both dysglycemia and dyslipidemia in a diabetic animal model. We also documented improved insulin secretion after xuezhikang treatment, which may be one of the reasons for the sustained blood glucose balance. Morphologically, xuezhikang management helped reduce hyperglycemic damage to the islet microenvironment. Our experiments also showed that xuezhikang suppressed oxidative stress by inhibiting the activity of NOX. In addition, the expression levels of 2 proteins of glucose-sensing apparatus were increased by xuezhikang administration. The increasing incidence of diabetes associated with rosuvastatin has raised concerns about the effect of statins on glycemic parameters.14 To date, it remains questionable whether statin treatment has a detrimental or favorable effect on diabetes.15,16 The current study found that xuezhikang, which contains various statins and other ingredients,17 is able to maintain metabolic homeostasis. Data from 2 research groups indicated that xuezhikang treatment could improve insulin resistance in DM patients.18,19 Yang et al20 theorized that xuezhikang enhances insulin secretion in pancreatic islets, which was partially validated by our animal experiments. We found that not only glucose tolerance but also early-stage insulin secretion was involved, which to our knowledge is another novel function for xuezhikang. Hence, these results
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afford a new opportunity to better understand the unique potency of xuezhikang. A sufficient number of b cells is a prerequisite for adequate insulin production. Meanwhile, pancreatic b-cell mass experiences dynamic changes in response to the varied metabolic signature followed by limited regeneration.21 The combination of the amount and mass of b-cell represents the b-cell content, which is crucial for endocrine efficiency. Marchand et al22 found that atorvastatin significantly increased b-cell mass in a rat model of b-cell injury via its antioxidative properties. This result is supported by our results showing that xuezhikang preserved b-cell content in db/db mice. The microendothelium interacts interdependently with b cells in pancreatic islets. The crosstalk connection allows them to communicate fully with each other and establish the endocrine tissue microenvironment, which may be crucial to the physiological process. Apart from providing oxygen and nutrients, the islet endothelium delivers growth factors to endocrine cells.23 Islet endothelium may also induce insulin gene expression and may affect b-cell function and proliferation.24 Pravastatin has been shown to attenuate the decrease in the proliferation and prevent the apoptosis of islet microendothelial cells chronically exposed to hyperglycemia.25 Here, we found that xuezhikang promoted intraislet endothelial survival. Hence, we theorized that xuezhikang’s preservation of the microenvironment may contribute to endocrine function protection in the context of diabetes. Nonetheless, its 2013 Lippincott Williams & Wilkins
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FIGURE 3. Effects of xuezhikang on the pancreatic islet microenvironment. A, The expression of CD31, an endothelial cell marker located in pancreatic islets, was evaluated by immunohistochemistry (3,3’-diaminobenzidine staining, ·200). B, The morphological changes in b-cell ultrastructure was evaluated by electron microscopy (uranyl acetate and lead citrate staining, ·6000). Representative immunostaining and electron microscopy results are shown (n = 5). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. The scale bar represents 100 mm.
effect on b-cell apoptosis needs to be elucidated, which is one limitation of the experimental design of current article. One study has previously described the ultra-structural changes of b cells in db/db mice26 in line with our observations of mitochondria, endoplasmic reticulum, and Golgi bodies. Like in other cell types, mitochondria, one of the main sources of ROS, are also one of the cellular components most susceptible to increased concentrations of ROS.27 Here, we
found that xuezhikang almost completely prevented the mitochondrial swelling and disruption seen in placebo-treated db/db mice. Thus, we inferred that its antioxidant activity would likely mitigate the oxidative stress that accounts for the morphological changes. A high glucose concentration aggravates ROS generation and reinforces oxidative stress during the progression of b-cell dysfunction in type 2 diabetes.28 In contrast, the suppression of oxidative stress by antioxidants
FIGURE 4. Effects of xuezhikang on reactive oxidative species in pancreatic islets. The expression of the oxidative stress markers 8-OHdG (A) and 4-HNE (B) was evaluated by immunohistochemistry (3,3’-diaminobenzidine staining, ·200). Representative immunostaining results are shown (n = 5). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. The scale bar represents 100 mm. 2013 Lippincott Williams & Wilkins
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FIGURE 5. Effects of xuezhikang on gp91phox in pancreatic islets. A, The expression of the NOX subunits gp91phox was evaluated by immunohistochemistry (3,3’-diaminobenzidine staining, ·200). The scale bar represents 100 mm. B, Semiquantitative analysis of the intensity of gp91phox in the immunohistochemistry staining. C, The expression of gp91phox in islets was evaluated by Western blot. b actin was used as an internal control to monitor equal protein loading. D, Densitometric analysis of the staining intensities of gp91phox in the Western blotting. Representative immunostaining results are shown. Similar results were obtained from the other mice of each group (n = 5). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. Each bar represents the mean 6 SD of the gp91phox staining intensity. *P , 0.05 versus the placebo group, **P , 0.01 versus the placebo group.
results in the recovery of insulin biosynthesis and glucose tolerance in db/db mice.29 Oxidative stress has been reported to be important in the disruption of b-cell content, microendothelium, and cellular structures in DM.30,26 For this reason, we attempted to determine the underlying antioxidative bioactivity of xuezhikang. The detection of 8-OHdG and 4-HNE is the predominant approach to the evaluation of oxidative stress in b cells in diabetic conditions.31 This research demonstrated that xuezhikang downregulated the expression of these markers in islets. Although we performed a semiquantitative analysis, our results still indicated that increased oxidative stress is one of the major factors responsible for the reduced endocrine capability of b cells. More importantly, our experiments suggested that xuezhikang ameliorated oxidative stress, in
accordance with analogous results derived from a study in humans,9 and protected against cellular damage. NOX is the most important multicomponent enzyme involved in the generation of ROS in various cell types. The membrane-bound components of NOX, gp91phox, and p22phox are vital for its activation. The upregulation of both subunits has been demonstrated in b cells from rodent models of type 2 diabetes,32 and NOX plays a pivotal role in oxidative stress modulation and insulin secretion. Li et al10 observed that xuezhikang inhibited NOX activity in macrophages. Similarly, we found that xuezhikang effectively curtailed the increased level of gp91phox in db/db mice, suggesting that the drug may control oxidative stress via NOX inhibition. Further investigation is required to determine the gene expression or specific signaling of NOX.
FIGURE 6. Effects of xuezhikang on the glucose-sensing apparatus in pancreatic b-cells. A, The expression of GLUT-2 and GCK in b cells was evaluated by Western blot. b actin was used as an internal control to monitor equal protein loading. B, Densitometric analysis of the staining intensities of GLUT-2 and GCK in the Western blotting. Representative immunostaining results are shown. Similar results were obtained from the other mice of each group (n = 5). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. Each bar represents the mean 6 SD of the gp91phox staining intensity. **P , 0.01 versus the placebo group.
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As the major glucose transporter protein in the principal signaling cascade in b cells, GLUT-2 participates in transmembrane glucose trafficking and initiates glucosestimulated insulin secretion after the uptake of glucose. GCK is the rate-limiting step in glucose metabolism. It confers tight control over the whole process of carbohydrate utilization, oxidation, and insulin secretion.33 Two independent teams found that the inhibition of oxidative stress would impact on the translocation of GLUT-234 or the expression of GCK.35 So far, there is no information in the literature on an association between xuezhikang and glucose sensors in b cells. In the present study, the downregulated GLUT-2 and GCK expression in islets from db/db mice was significantly restored after chronic exposure to xuezhikang. These findings raise the potential for xuezhikang as an alternative for glucose intolerance and could open up intriguing possibilities for identifying a therapeutical target for xuezhikang in glucose-sensing pathway. In conclusion, xuezhikang protects against oxidative stress in pancreatic islets in db/db diabetic mice by inhibiting NOX. Thus, treatment with xuezhikang could counteract the morphological and functional impairments caused by hyperglycemic conditions. These findings provide pharmacological evidence for the clinical use of xuezhikang in the management of diabetes-relevant metabolic disorders. REFERENCES 1. Lenzen S, Drinkgern J, Tiedge M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radic Biol Med. 1996;20:463–466. 2. Oliveira HR, Verlengia R, Carvalho CR, et al. Pancreatic beta-cells express phagocyte-like NADPH oxidase. Diabetes. 2003;52:1457–1463. 3. Lambeth JD, Cheng G, Arnold RS, et al. Novel homologs of gp91phox. Trends Biochem Sci. 2000;25:459–461. 4. Saitoh Y, Hongwei W, Ueno H, et al. Candesartan attenuates fatty acidinduced oxidative stress and NADPH oxidase activity in pancreatic betacells. Diabetes Res Clin Pract. 2010;90:54–59. 5. Zhao SP, Lu ZL, Du BM, et al; China Coronary Secondary Prevention Study. Xuezhikang, an extract of cholestin, reduces cardiovascular events in type 2 diabetes patients with coronary heart disease: subgroup analysis of patients with type 2 diabetes from China coronary secondary prevention study (CCSPS). J Cardiovasc Pharmacol. 2007;49:81–84. 6. Lu Z, Kou W, Du B, et al; Chinese Coronary Secondary Prevention Study Group. Effect of Xuezhikang, an extract from red yeast Chinese rice, on coronary events in a Chinese population with previous myocardial infarction. Am J Cardiol. 2008;101:1689–1693. 7. Li JJ, Lu ZL, Kou WR, et al; Chinese Coronary Secondary Prevention Study Group. Beneficial impact of Xuezhikang on cardiovascular events and mortality in elderly hypertensive patients with previous myocardial infarction from the China Coronary Secondary Prevention Study (CCSPS). J Clin Pharmacol. 2009;49:947–956. 8. Shang Q, Liu Z, Chen K, et al. A systematic review of xuezhikang, an extract from red yeast rice, for coronary heart disease complicated by dyslipidemia. Evid Based Complement Alternat Med. 2012;2012: 636547–636564. 9. Hong XZ, Li LD, Wu LM. Effects of fenofibrate and xuezhikang on high-fat diet-induced non-alcoholic fatty liver disease. Clin Exp Pharmacol Physiol. 2007;34:27–35. 10. Li P, Yang Y, Liu M. Xuezhikang, extract of red yeast rice, inhibited tissue factor and hypercoagulable state through suppressing nicotinamide adenine dinucleotide phosphate oxidase and extracellular signalregulated kinase activation. J Cardiovasc Pharmacol. 2011;58:307–318. 11. Zhu XY, Li P, Yang YB, et al. Xuezhikang, extract of red yeast rice, improved abnormal hemorheology, suppressed caveolin-1 and increased eNOS expression in atherosclerotic rats. PLoS One. 2013;8:e62731.
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12. Gorogawa S, Fujitani Y, Kaneto H, et al. Insulin secretory defects and impaired islet architecture in pancreatic beta-cell-specific STAT3 knockout mice. Biochem Biophys Res Commun. 2004;319:1159–1170. 13. Sakuraba H, Mizukami H, Yagihashi N, et al. Reduced beta-cell mass and expression of oxidative stress-related DNA damage in the islet of Japanese Type II diabetic patients. Diabetologia. 2002;45:85–96. 14. Ridker PM, Danielson E, Fonseca FA, et al; JUPITER Study Group. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med. 2008;359:2195–2207. 15. Freeman D.J, Norrie J, Sattar N, et al. Pravastatin and the development of diabetes mellitus: evidence for a protective treatment effect in the West of Scotland Coronary Prevention Study. Circulation. 2001;103:357–362. 16. Rajpathak SN, Kumbhani DJ, Crandall J, et al. Statin therapy and risk of developing type 2 diabetes: a meta-analysis. Diabetes Care. 2009;32: 1924–1929. 17. Zhang XQ, Zhou FR, JiMing S. HPLC analysis of lovastatin concentration in xuezhikang capsule and other red yeast rice. China J Chin Materia Medica (Chin). 1999;22:222–224. 18. Dai XH, Xue XY, Wang L. The effects of xuezhikang on insulin sensitivity for patients with hypertension complicated with dyslipidemia. Chin J Cardiol (Chin). 2000;20:692–693. 19. Carpentier YA, Portois L, Malaisse WJ. N-3 fatty acids and the metabolic syndrome. Am J Clin Nutr. 2006;83:1499S–1504S. 20. Yang W, Xing X, Lin H. Baseline hypertriglyceridemia, a risk factor for non-insulin dependent diabetes mellitus: a 6-year follow-up study of 432 nondiabetics. Zhonghua Nei Ke Za Zhi. 1995;34:583–586. 21. Atkinson MA, Rhodes CJ. Pancreatic regeneration in type 1 diabetes: dreams on a deserted islet? Diabetologia. 2005;48:2200–2202. 22. Marchand KC, Arany EJ, Hill DJ. Effects of atorvastatin on the regeneration of pancreatic {beta}-cells after streptozotocin treatment in the neonatal rodent. Am J Physiol Endocrinol Metab. 2010;299:E92–E100. 23. Olsson R, Carlsson PO. The pancreatic islet endothelial cell: emerging roles in islet function and disease. Int J Biochem Cell Biol. 2006;38:492–497. 24. Lammert E, Cleaver O, Melton D. Induction of pancreatic differentiation by signals from blood vessels. Science. 2001;294:564–567. 25. Favaro E, Miceli I, Bussolati B, et al. Hyperglycemia induces apoptosis of human pancreatic islet endothelial cells: effects of pravastatin on the Akt survival pathway. Am J Pathol. 2008;173:442–450. 26. Mizuno A, Noma Y, Kuwajima M, et al. Changes in islet capillary angioarchitecture coincide with impaired B-cell function but not with insulin resistance in male Otsuka-Long-Evans-Tokushima fatty rats: dimorphism of the diabetic phenotype at an advanced age. Metabolism. 1999;48:477–483. 27. Jeˇzek P, Hlavata L. Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int J Biochem Cell B. 2005;37: 2478–2503. 28. Evans JL, Goldfine ID, Maddux BA, et al. Are oxidative stress-activated signaling pathways mediators of insulin resistance and beta-cell dysfunction? Diabetes. 2003;52:1–8. 29. Kaneto H, Kajimoto Y, Miyagawa J, et al. Beneficial effects of antioxidants in diabetes—possible protection of pancreatic beta-cells against glucose toxicity. Diabetes. 1999;48:2398–2406. 30. Weiss H, Wester-Rosenloef L, Koch C, et al. The mitochondrial Atp8 mutation induces mitochondrial ROS generation, secretory dysfunction, and b-cell mass adaptation in conplastic B6-mtFVB mice. Endocrinology. 2012;153:4666–4676. 31. Ihara Y, Toyokuni S, Uchida K, et al. Hyperglycemia causes oxidative stress in pancreatic b-cells of GK rats, a model of type 2 diabetes. Diabetes. 1999;48:927–932. 32. Nakayama M, Inoguchi T, Sonta T, et al. Increased expression of NADPH oxidase in islets of animal models of type 2 diabetes and its improvement by an AT1 receptor antagonist. Biochem Bioph Res Commun. 2005;332:927–933. 33. Matschinsky FM, Glaser B, Magnuson MA. Pancreatic beta-cell glucokinase: closing the gap between theoretical concepts and experimental realities. Diabetes. 1998;47:307–315. 34. Rashid K, Das J, Sil PC. Taurine ameliorate alloxan induced oxidative stress and intrinsic apoptotic pathway in the hepatic tissue of diabetic rats. Food Chem Toxicol. 2013;51:317–329. 35. Xiong X, Tao R, Depinho RA, et al. Deletion of hepatic foxO1/3/4 genes in mice significantly impacts on glucose metabolism through downregulation of gluconeogenesis and upregulation of glycolysis. PLoS One. 2013;8:e74340.
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Xuezhikang Attenuated the Functional and Morphological Impairment of Pancreatic Islets in Diabetic Mice Via the Inhibition of Oxidative Stress Jun Wang, MS,* WeiMin Jiang, MD, PhD,† Yong Zhong, MD, PhD,*‡ Bin Lu, MD, PhD,§ JiaQing Shao, MD, PhD,§ ShiSen Jiang, MD,* and Ping Gu, MD, PhD*§
Abstract: Xuezhikang, purified from red yeast rice, is a traditional Chinese medicine with pleiotropic effects on the cardiovascular system. Oxidative stress plays a crucial role in the dysfunction of pancreas islet in diabetic condition and represents a promising therapeutical target for diabetes mellitus. Therefore, the purpose of this study was to explore the effects and possible mechanisms of xuezhikang on the microenvironment and insulin secretion by pancreatic islets in db/db diabetic mice. Our results showed that xuezhikang decreased the blood glucose level by improving glucose tolerance and insulin secretion in db/db mice. Xuezhikang protected islets from hyperglycemic injury as illustrated by the conserved b-cell content and microenvironment. Furthermore, xuezhikang potently inhibited the expression of key factors in oxidative stress. In addition, administration of xuezhikang caused an upregulated expression of glucose-sensing apparatus. These observations provide evidence that the influence of xuezhikang on oxidative stress may at least partly account for its protective effects on the microenvironment and insulin secretion function of pancreatic islets in diabetes. Key Words: xuezhikang, diabetes, pancreatic islet, oxidative stress, nicotinamide adenine dinucleotide phosphate oxidase (J Cardiovasc Pharmacol 2014;63:282–289)
Received for publication August 15, 2013; accepted November 7, 2013. From the *Department of Cardiology, School of Medicine, Nanjing University, Jinling Hospital/Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu Province, China; †Department of Cardiology, Jiangsu Provincial Hospital of Traditional Chinese Medicine, Nanjing, Jiangsu Province, China; and Departments of ‡Health Care, and §Endocrinology, School of Medicine, Nanjing University, Jinling Hospital/Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu Province, China. Supported by the Natural Science Foundation of China (81000352, 30900697, 81373605, and 81100568), the Natural Science Foundation of Jiangsu Province (BK2011661 and BK20131456), the Postdoctoral Scientific Foundation of China (20100471843), and the Postdoctoral Scientific Foundation of Jiangsu Province (1001027C). The authors report no conflicts of interest. J.W. and W.J. have contributed equally. Reprints: Ping Gu, MD, PhD, Department of Endocrinology, School of Medicine, Nanjing University, Jinling Hospital/Nanjing General Hospital of Nanjing Military Command, 305 Zhongshan East Rd, Nanjing, Jiangsu Province, China 210002 (e-mail: [email protected]); ShiSen Jiang, MD, Department of Cardiology, School of Medicine, Nanjing University, Jinling Hospital/Nanjing General Hospital of Nanjing Military Command, 305 Zhongshan East Rd, Nanjing, Jiangsu Province, China 210002 (email: [email protected]). Copyright © 2013 by Lippincott Williams & Wilkins
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INTRODUCTION Dysfunction of pancreatic islets is a hallmark of diabetes mellitus (DM). Islets maintain a rich capillary network, also termed the microenvironment, which is essential for the optimal secretory function of b cells and thus carbohydrate metabolism. There are several indications that a high glucose concentration induces oxidative stress in pancreatic b cells. For instance, pancreatic islets present the low antioxidant enzymes activity1 and are highly susceptible to cellular injury. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is the major source of reactive oxygen species (ROS) in pancreatic b cells.2 As a multicomponent enzyme, NOX consists of membrane-bound (gp91phox and p22phox) and cytosolic components. The catalytic subunit, gp91phox, plays a critical role in activation of NOX.3 Inhibition of NOX is accepted as one mechanism of reducing oxidative stress. One study demonstrated that silencing NOX attenuated oxidative stress and thus cellular injury in b cells.4 Xuezhikang, extracted from red yeast Chinese rice, has been widely prescribed as a lipid-lowering drug. Several clinical trials have demonstrated the efficacy and safety of xuezhikang in regulating lipid and glucose metabolism, which result into the cardiovascular events declined.5–8 Animal experiments demonstrated that xuezhikang is able to ameliorate insulin resistance in rats.9 However, the exact molecular mechanism remains unknown. Therefore, the present study was designed to evaluate the impact of xuezhikang on pancreatic islets in mice and explore the pathways involved.
MATERIALS AND METHODS Animals This study was reviewed and approved by the Animal Care and Use Committee of Jinling Hospital. Forty 8-weekold male genetically diabetic C57BL/KsJ-db/db mice and their lean nondiabetic C57BL/KsJ-db/m littermates, weighing 34–42 g, were kindly provided by the Research Institute of Nephrology, Jinling Hospital. Mice were housed under standard conditions (room temperature, 20 6 18C; humidity, 60% 6 10%; lights on from 6 AM to 6 PM). Only the db/db mice were randomly assigned to receive xuezhikang (n = 20) or placebo (n = 20). The placebo-treated db/m group served as a nondiabetic control (wild-type control, n = 20). Mice were given either 300 mg/kg/d of xuezhikang (WBL Peking J Cardiovasc Pharmacol Volume 63, Number 3, March 2014
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University Biotech Co, Ltd, Beijing, China) or placebo (0.5% natrium cellulose solution) by gavage for 8 weeks. The administration dosage of xuezhikang was derived from the previous studies10,11 and the body surface area normalization method. Mice were provided with standard chow and water ad libitum.
Metabolism Measurement Body weight, fasting blood glucose (FBG), and fasting serum insulin (FSI) were measured every week. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels were assayed by the enzymatic methods. After fasting for 15 hours, in 10 mice for each group, an intraperitoneal glucose tolerance test was performed with 500 mg/kg glucose injected intraperitoneally. Glucose and insulin levels were determined in blood samples collected at different time points (0, 30, 60, and 120 minutes).
Islet Isolation and Perifusion Animals were anesthetized with sodium phenobarbital before pancreatectomy. Islets were isolated from mice in each group by infusion of collagenase P (Roche, Penzgerg, Germany) followed by purification with a Ficoll step density gradient. Islets were handpicked twice and cultured for 1 hour in RPMI 1640 medium. The in vitro insulin release kinetics were studied with the perifusion system as described previously.12 Fifty sizematched islets were placed in individual columns in a 37ºC water bath. Krebs–Ringer bicarbonate buffer containing 2.8 mM glucose was perfused in each column at 0.5 mL/min for 60 minutes. Islets were stimulated by a high concentration of glucose (16.7 mM). The dripped solution was collected after 20 seconds, 1 minute, and 5 minutes, separately within each phase that started 2, 5 minutes, and 30 minutes after the initiation of stimulation. The solution was assayed to evaluate the amount of insulin secreted by each islet column.
Electron Microscopy Pancreas tissues were cut into small pieces with razor blades. They were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.4) at 48C for 2 hours, washed, fixed, dehydrated, and embedded in Epon 812. Ultrathin sections were stained with 2% uranyl acetate followed by lead citrate, and the stained sections were examined on a JEM-1200EX electron microscope (JEOL, Tokyo, Japan) operating at 80 kV. The relative mitochondrial volume was calculated by counting the mitochondrial area on random micrographs at a 20,000 magnification with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Four micrographs were used per group.
Immunohistochemistry
The pancreas was collected, fixed in 4% paraformaldehyde for 4–6 hours, and embedded in paraffin. The ABC method was employed for the detection of insulin, gp91phox, 8-hydroxy-20-deoxyguanosine (8-OHdG), 4-hydroxynonenal modified protein (4-HNE), and CD31. Specimens were deparaffinized with xylene and rehydrated in an ethanol series. Endogenous peroxidase was blocked with 0.3% H2O2 in methanol, and 10% goat serum was added to block nonspecific
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antigens. After that, the sections were incubated overnight in primary antibodies at 48C. Fourteen hours later, these sections were incubated for 30 minutes at room temperature with the corresponding biotinylated secondary antibodies. Samples were subsequently incubated with horseradish peroxidase–conjugated streptavidin with the LSAB2 kit (Dako). Staining with 3,3’-diaminobenzidine (Sigma, St Louis, MO) was performed before light counterstaining with Mayer hematoxylin (Wako, Tokyo, Japan). Finally, some slides were dehydrated and mounted, whereas others were subjected to Azan staining. The insulin staining density was analyzed semiquantitatively from percentage of stained b cells, with the Image-Pro Plus 5.0.1 image analysis system (Planetron, Tokyo, Japan). The primary antibodies were as follows: guinea pig antihuman insulin antibody (1:2000; Linco, St Charles, MO), goat anti-human gp91phox antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human 8-OHdG antibody (1:100), mouse anti-human 4-HNE antibody (1:50; Japan Institute for the Control of Aging, Shizuoka, Japan), and rat anti-human CD31 antibody (1:200; BD Bioscience, Tokyo, Japan). The secondary antibodies were goat anti-guinea pig immunoglobulin (Ig)G antibody (1:1000; Chemicon, Temecula, CA), rabbit anti-goat IgS antibody (1:200; Dako), goat anti-mouse IgS antibody (1:200; Dako), and goat anti-rat IgG antibody (1:200; Cosmo Bio, Tokyo, Japan).
Morphometric Analysis For immunohistochemistrical analysis, captured digital images from 15 islets per animal, namely 45 islets per group (n = 3), were observed and estimated by Image-Pro Plus 5.0.1. Intensities of the 8-OHdG-, 4-HNE-, and gp91phox-associated immunoreactions was assessed by the semiquantitative ranking as previously described.13 In brief, the average intensity was given a score of 0, 1, 2, or 3 corresponding to the presence of negative, weak, intermediate, or strong staining, respectively. All evaluations were performed in a blinded manner. The insulin-stained domain represented the relative area containing b cells (ie, b cell content), which was indicated by the value of the b-cell mass [b-cell mass (mg) = islet area/whole pancreatic area · pancreas weight (15 duplications per group)]. The coverage was automatically calculated with the Image-Pro Plus 5.0.1 software package.
Western Blot Analysis For gp91phox, glucokinase (GCK), and glucose transporter-2 (GLUT-2) detection and isolated islet aliquots were lysed in lysis buffer [25 mM N-(2-hydroxyethyl) piperazine-N0 -2-ethanesulfonic acid, 50 mM Tris-HCl (pH 7.4), 6% glycerol, 5 mM ethylene diamine tetraacetie acid, 5 mM ethyleneglycol bis(2-aminoethylether)tetraacetic acid, 0.5% Triton X-100, 50 mM NaF, 40 mM glycerophosphate, and 25 mM sodium pyrophosphate with a protease inhibitor mixture]. Equal amounts of protein (50 mg) were loaded onto 10% dodecyl sulfate, sodium salt-Polyacrylamide gel electrophoresis gels, separated by electrophoresis, and transferred onto nitrocellulose membranes. Membranes were blocked by 5% skim milk and incubated with anti-GCK antibody (1:1000; Santa Cruz Biotechnology), anti-GLUT-2 antibody (1:1000; Santa Cruz Biotechnology), and anti-b-actin antibody (1:1000; Santa Cruz Biotechnology) overnight at www.jcvp.org |
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4ºC. After triplicate washes, the membranes were incubated with horseradish peroxidase–conjugated anti-rabbit IgG (1:2500; Santa Cruz Biotechnology) for 1 hour at room temperature. Bands of interest were visualized by enhanced chemiluminescence (Pierce, Rockford, IL). The intensities of the blots were quantified by scanning densitometry and normalized to b actin.
the test was higher than that of the placebo-treated db/db mice (P , 0.05), which suggests that xuezhikang may improve the glucose tolerance in diabetic mice. Experiments involving the islets perifusion in vitro revealed that, in contrast to the db/db control, insulin secretion derived from isolated islets was not noticeably increased in xuezhikang group pretreated with a low-concentration glucose solution (2.8 mM). However, the insulin secretion was moderately increased at 1 minute after perfusion with the highconcentration glucose solution (16.7 mM) (P , 0.05) in the xuezhikang-treated animals compared with placebo-treated animals (Fig. 1C). This result suggests that xuezhikang was capable of enhancing the first-phase insulin secretion, although this secretion was not comparable with that of the db/m group.
Statistical Analysis Statistical analysis was done with SPSS, version 11.0. Data were expressed as mean 6 SD. Means among groups were compared with one-way analysis of variance. Means between 2 groups were compared with least-significant difference test. Data before and after experiment were compared with paired test. A value of P , 0.05 was considered statistically significant.
RESULTS
Morphometric Analysis
To assess the change of b cells’ number, we calculated the insulin staining density. This measurement method reflected the changes in histological immunostaining. Less-intense dark brown staining, which implied a reduced amount of b cells, could be observed in islets from db/db control mice compared with db/m mice (Fig. 2A). Xuezhikang therapy seemed to decline the trend in b-cell reduction (P , 0.05) (Fig. 2B). Similarly, the favorable effect of xuezhikang treatment was presented by the restored b-cell mass (P , 0.05), indicating that this drug could attenuate the b-cell mass loss caused by the diabetic condition (Fig. 2C). Islets contain abundant vascular endothelial cells, a typical feature of endocrine organs, and are essential for endocrine function. As an endothelial cell marker, the CD31 could be used to estimate the endothelial density. The CD31 expression was depleted in db/db diabetic animals. Interestingly, the staining intensity of CD31 was higher in xuezhikang group than in placebo group (Fig. 3A). Thus, xuezhikang may overcome the rarefaction of vasculature endothelium surrounding pancreatic islets in diabetic subjects. On the other hand, b-cell organelles, such as endoplasmic reticulum and Golgi bodies, in mice administered xuezhikang showed an increased resistance to injury. As illustrated by Figure 3B, the mitochondrial swelling was alleviated and the proportion of intact mitochondria was increased in xuezhikang-treated mice versus their placebotreated counterpart.
Metabolism Profiling At baseline, there were no marked differences in the body weight, FBG, FSI, TC, LDL-C, and TG between the xuezhikang and placebo groups. However, all these values were lower in db/m group compared with 2 db/db groups (P , 0.05). In particular, at 8 weeks, db/db mice had a FBG level .15 mmol/L. After treatment with xuezhikang, hyperglycemia development was alleviated, and the FBG and FSI levels at the terminal stage of treatment in db/db mice were markedly lower than that in placebo-treated db/db mice (P , 0.05); however, there was no difference between before and after xuezhikang treatment. Likewise, the TC, LDL-C, and TG levels in the xuezhikang group were markedly reduced compared with the db/db control (P , 0.05). Besides, the 8 weeks of xuezhikang intake blunted weight gain in the db/db mice (P , 0.05) (Table 1).
Glucose Metabolism During the 120-minute glucose tolerance test, the blood glucose level after glucose loading in xuezhikang group was lower than that of placebo group (P , 0.05), which was standardized to that of nondiabetic group (Fig. 1A). Moreover, insulin secretion stimulated by glucose loading in the xuezhikang group was higher than that of db/db control (Fig. 1B). All these results were demonstrated by the differences in the area under the curve (AUC) among the 3 groups. It was noteworthy that the AUCINS0230 in the xuezhikang-treated mice at 30 minutes after
TABLE 1. Baseline and Terminal Metabolic Parameters of Each Group Nondiabetic (n = 20) Baseline BW (g) FBG (mmol/l) FSI (mIU/L) TC (mmol/l) LDL-C (mmol/l) TG (mmol/l)
26.8 5.6 2.5 2.42 1.63 0.56
6 6 6 6 6 6
0.3† 0.7† 0.3† 0.31† 0.23† 0.11†
Placebo (n = 20)
Terminal 32.7 5.8 2.7 2.52 1.71 0.59
6 6 6 6 6 6
0.5* 0.8* 0.4* 0.25* 0.28* 0.15*
Baseline 38.9 17.1 8.8 5.23 3.68 3.45
6 6 6 6 6 6
3.3 4.2 1.5 0.87 0.45 0.56
Xuezhikang (n = 20)
Terminal 47.1 25.9 9.8 6.03 3.98 3.75
6 6 6 6 6 6
4.3 2.2 1.4 0.93 0.53 0.67
Baseline 38.6 18.1 9.2 5.35 3.57 3.35
6 6 6 6 6 6
2.9 4.1 1.4 0.84 0.39 0.48
Terminal 42.3 18.4 7.7 4.13 2.32 1.65
6 6 6 6 6 6
3.8* 3.4* 0.8* 0.57* 0.34* 0.34*
Data are shown as means 6 SD. *P , 0.05 versus the terminal value of placebo group. †P , 0.05 versus the baseline value of placebo group Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group; BW, body weight.
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FIGURE 1. Effects of xuezhikang on blood glucose and insulin secretion in db/db and db/m mice. A, Plasma glucose levels determined at the different time points of the intraperitoneal glucose tolerance test. B, Insulin concentration determined at the different time points of the intraperitoneal glucose tolerance test. C, Insulin secretion kinetics stimulated by low and high glucose concentrations in the in vitro perifusion experiment with pancreatic islet samples isolated from mice. Each bar represents the means 6 SD. Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. *P , 0.05 versus the placebo group.
Protection Against Oxidative Stress To investigate whether xuezhikang could protect against oxidative stress in diabetic, we measured the expression of oxidative stress markers, 8-OHdG, and 4-HNE. The percentage of b cells expressing 8-OHdG and 4-HNE was higher in db/db mice than in db/m mice (P , 0.05) (Figs. 4A,B). More importantly, xuezhikang greatly reduced the expression levels of these 2 markers to the normal level (Figs. 4A,B). Furthermore, we studied the expression levels of gp91phox, which is one of the major components of NOX. Semiquantitative analysis revealed that xuezhikang uptake rectified the expression of gp91phox to nearly the same level as in the wild-type control and to a level below that of the db/db control (P , 0.05) (Figs. 5A,B). Besides, the Western blotting and its densitometric analysis for gp91phox also showed that xuezhikang abolished the elevation of gp91phox expression in placebo group was lowered in xuezhikang group (P , 0.01) (Figs. 5C,D). These results indicate that xuezhikang may inhibit oxidative stress via downregulation of gp91phox. 2013 Lippincott Williams & Wilkins
Overexpression of the Glucose-Sensing Apparatus In the following study, we sought to obtain more detailed insights into the possible targets of xuezhikang for the purpose of screening potential signaling pathways for this drug in glucose metabolism. GLUT-2 and GCK mediate the glucose response pathway in islets. Thus, we measured the protein levels of GLUT-2 and GCK in an attempt to determine a possible impact of xuezhikang on glucose sensing. Western blotting revealed decreased levels of both GLUT-2 and GCK in db/db mice compared with db/m mice (Fig. 6A). Densitometric analysis further validated that the GLUT-2 and GCK expression levels were increased in xuezhikang group as opposed to placebo group (P , 0.01) (Fig. 6B).
DISCUSSION The present study sheds some light on the salutary property of xuezhikang on pancreatic islet function and www.jcvp.org |
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FIGURE 2. Effects of xuezhikang on the b-cell content of pancreatic islets. A, b-cell amounts were evaluated by immunohistochemistry for insulin in pancreatic islet sections. b-cells positive for insulin showed dark brown staining. B, Insulin staining intensity was analyzed semiquantitatively as the percentage of stained b cells of the db/m control. C, b-cell mass was calculated by the following formula: islet b-cell mass (mg) = (the area stained by insulin antibody)/(the area of the whole pancreas) · (pancreas weight). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. Each bar represents the mean 6 SD of the insulin staining intensity or b-cell mass (n = 8). *P , 0.05 versus the placebo group.
morphology in db/db diabetic mice. Xuezhikang has been commonly used as a Chinese traditional medicine for therapy of patients undergoing cardiovascular disorders. Our findings confirmed that xuezhikang corrected both dysglycemia and dyslipidemia in a diabetic animal model. We also documented improved insulin secretion after xuezhikang treatment, which may be one of the reasons for the sustained blood glucose balance. Morphologically, xuezhikang management helped reduce hyperglycemic damage to the islet microenvironment. Our experiments also showed that xuezhikang suppressed oxidative stress by inhibiting the activity of NOX. In addition, the expression levels of 2 proteins of glucose-sensing apparatus were increased by xuezhikang administration. The increasing incidence of diabetes associated with rosuvastatin has raised concerns about the effect of statins on glycemic parameters.14 To date, it remains questionable whether statin treatment has a detrimental or favorable effect on diabetes.15,16 The current study found that xuezhikang, which contains various statins and other ingredients,17 is able to maintain metabolic homeostasis. Data from 2 research groups indicated that xuezhikang treatment could improve insulin resistance in DM patients.18,19 Yang et al20 theorized that xuezhikang enhances insulin secretion in pancreatic islets, which was partially validated by our animal experiments. We found that not only glucose tolerance but also early-stage insulin secretion was involved, which to our knowledge is another novel function for xuezhikang. Hence, these results
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afford a new opportunity to better understand the unique potency of xuezhikang. A sufficient number of b cells is a prerequisite for adequate insulin production. Meanwhile, pancreatic b-cell mass experiences dynamic changes in response to the varied metabolic signature followed by limited regeneration.21 The combination of the amount and mass of b-cell represents the b-cell content, which is crucial for endocrine efficiency. Marchand et al22 found that atorvastatin significantly increased b-cell mass in a rat model of b-cell injury via its antioxidative properties. This result is supported by our results showing that xuezhikang preserved b-cell content in db/db mice. The microendothelium interacts interdependently with b cells in pancreatic islets. The crosstalk connection allows them to communicate fully with each other and establish the endocrine tissue microenvironment, which may be crucial to the physiological process. Apart from providing oxygen and nutrients, the islet endothelium delivers growth factors to endocrine cells.23 Islet endothelium may also induce insulin gene expression and may affect b-cell function and proliferation.24 Pravastatin has been shown to attenuate the decrease in the proliferation and prevent the apoptosis of islet microendothelial cells chronically exposed to hyperglycemia.25 Here, we found that xuezhikang promoted intraislet endothelial survival. Hence, we theorized that xuezhikang’s preservation of the microenvironment may contribute to endocrine function protection in the context of diabetes. Nonetheless, its 2013 Lippincott Williams & Wilkins
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FIGURE 3. Effects of xuezhikang on the pancreatic islet microenvironment. A, The expression of CD31, an endothelial cell marker located in pancreatic islets, was evaluated by immunohistochemistry (3,3’-diaminobenzidine staining, ·200). B, The morphological changes in b-cell ultrastructure was evaluated by electron microscopy (uranyl acetate and lead citrate staining, ·6000). Representative immunostaining and electron microscopy results are shown (n = 5). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. The scale bar represents 100 mm.
effect on b-cell apoptosis needs to be elucidated, which is one limitation of the experimental design of current article. One study has previously described the ultra-structural changes of b cells in db/db mice26 in line with our observations of mitochondria, endoplasmic reticulum, and Golgi bodies. Like in other cell types, mitochondria, one of the main sources of ROS, are also one of the cellular components most susceptible to increased concentrations of ROS.27 Here, we
found that xuezhikang almost completely prevented the mitochondrial swelling and disruption seen in placebo-treated db/db mice. Thus, we inferred that its antioxidant activity would likely mitigate the oxidative stress that accounts for the morphological changes. A high glucose concentration aggravates ROS generation and reinforces oxidative stress during the progression of b-cell dysfunction in type 2 diabetes.28 In contrast, the suppression of oxidative stress by antioxidants
FIGURE 4. Effects of xuezhikang on reactive oxidative species in pancreatic islets. The expression of the oxidative stress markers 8-OHdG (A) and 4-HNE (B) was evaluated by immunohistochemistry (3,3’-diaminobenzidine staining, ·200). Representative immunostaining results are shown (n = 5). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. The scale bar represents 100 mm. 2013 Lippincott Williams & Wilkins
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FIGURE 5. Effects of xuezhikang on gp91phox in pancreatic islets. A, The expression of the NOX subunits gp91phox was evaluated by immunohistochemistry (3,3’-diaminobenzidine staining, ·200). The scale bar represents 100 mm. B, Semiquantitative analysis of the intensity of gp91phox in the immunohistochemistry staining. C, The expression of gp91phox in islets was evaluated by Western blot. b actin was used as an internal control to monitor equal protein loading. D, Densitometric analysis of the staining intensities of gp91phox in the Western blotting. Representative immunostaining results are shown. Similar results were obtained from the other mice of each group (n = 5). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. Each bar represents the mean 6 SD of the gp91phox staining intensity. *P , 0.05 versus the placebo group, **P , 0.01 versus the placebo group.
results in the recovery of insulin biosynthesis and glucose tolerance in db/db mice.29 Oxidative stress has been reported to be important in the disruption of b-cell content, microendothelium, and cellular structures in DM.30,26 For this reason, we attempted to determine the underlying antioxidative bioactivity of xuezhikang. The detection of 8-OHdG and 4-HNE is the predominant approach to the evaluation of oxidative stress in b cells in diabetic conditions.31 This research demonstrated that xuezhikang downregulated the expression of these markers in islets. Although we performed a semiquantitative analysis, our results still indicated that increased oxidative stress is one of the major factors responsible for the reduced endocrine capability of b cells. More importantly, our experiments suggested that xuezhikang ameliorated oxidative stress, in
accordance with analogous results derived from a study in humans,9 and protected against cellular damage. NOX is the most important multicomponent enzyme involved in the generation of ROS in various cell types. The membrane-bound components of NOX, gp91phox, and p22phox are vital for its activation. The upregulation of both subunits has been demonstrated in b cells from rodent models of type 2 diabetes,32 and NOX plays a pivotal role in oxidative stress modulation and insulin secretion. Li et al10 observed that xuezhikang inhibited NOX activity in macrophages. Similarly, we found that xuezhikang effectively curtailed the increased level of gp91phox in db/db mice, suggesting that the drug may control oxidative stress via NOX inhibition. Further investigation is required to determine the gene expression or specific signaling of NOX.
FIGURE 6. Effects of xuezhikang on the glucose-sensing apparatus in pancreatic b-cells. A, The expression of GLUT-2 and GCK in b cells was evaluated by Western blot. b actin was used as an internal control to monitor equal protein loading. B, Densitometric analysis of the staining intensities of GLUT-2 and GCK in the Western blotting. Representative immunostaining results are shown. Similar results were obtained from the other mice of each group (n = 5). Nondiabetic, the nondiabetic group; Placebo, the placebo group; Xuezhikang, the xuezhikang group. Each bar represents the mean 6 SD of the gp91phox staining intensity. **P , 0.01 versus the placebo group.
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As the major glucose transporter protein in the principal signaling cascade in b cells, GLUT-2 participates in transmembrane glucose trafficking and initiates glucosestimulated insulin secretion after the uptake of glucose. GCK is the rate-limiting step in glucose metabolism. It confers tight control over the whole process of carbohydrate utilization, oxidation, and insulin secretion.33 Two independent teams found that the inhibition of oxidative stress would impact on the translocation of GLUT-234 or the expression of GCK.35 So far, there is no information in the literature on an association between xuezhikang and glucose sensors in b cells. In the present study, the downregulated GLUT-2 and GCK expression in islets from db/db mice was significantly restored after chronic exposure to xuezhikang. These findings raise the potential for xuezhikang as an alternative for glucose intolerance and could open up intriguing possibilities for identifying a therapeutical target for xuezhikang in glucose-sensing pathway. In conclusion, xuezhikang protects against oxidative stress in pancreatic islets in db/db diabetic mice by inhibiting NOX. Thus, treatment with xuezhikang could counteract the morphological and functional impairments caused by hyperglycemic conditions. These findings provide pharmacological evidence for the clinical use of xuezhikang in the management of diabetes-relevant metabolic disorders. REFERENCES 1. Lenzen S, Drinkgern J, Tiedge M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radic Biol Med. 1996;20:463–466. 2. Oliveira HR, Verlengia R, Carvalho CR, et al. Pancreatic beta-cells express phagocyte-like NADPH oxidase. Diabetes. 2003;52:1457–1463. 3. Lambeth JD, Cheng G, Arnold RS, et al. Novel homologs of gp91phox. Trends Biochem Sci. 2000;25:459–461. 4. Saitoh Y, Hongwei W, Ueno H, et al. Candesartan attenuates fatty acidinduced oxidative stress and NADPH oxidase activity in pancreatic betacells. Diabetes Res Clin Pract. 2010;90:54–59. 5. Zhao SP, Lu ZL, Du BM, et al; China Coronary Secondary Prevention Study. Xuezhikang, an extract of cholestin, reduces cardiovascular events in type 2 diabetes patients with coronary heart disease: subgroup analysis of patients with type 2 diabetes from China coronary secondary prevention study (CCSPS). J Cardiovasc Pharmacol. 2007;49:81–84. 6. Lu Z, Kou W, Du B, et al; Chinese Coronary Secondary Prevention Study Group. Effect of Xuezhikang, an extract from red yeast Chinese rice, on coronary events in a Chinese population with previous myocardial infarction. Am J Cardiol. 2008;101:1689–1693. 7. Li JJ, Lu ZL, Kou WR, et al; Chinese Coronary Secondary Prevention Study Group. Beneficial impact of Xuezhikang on cardiovascular events and mortality in elderly hypertensive patients with previous myocardial infarction from the China Coronary Secondary Prevention Study (CCSPS). J Clin Pharmacol. 2009;49:947–956. 8. Shang Q, Liu Z, Chen K, et al. A systematic review of xuezhikang, an extract from red yeast rice, for coronary heart disease complicated by dyslipidemia. Evid Based Complement Alternat Med. 2012;2012: 636547–636564. 9. Hong XZ, Li LD, Wu LM. Effects of fenofibrate and xuezhikang on high-fat diet-induced non-alcoholic fatty liver disease. Clin Exp Pharmacol Physiol. 2007;34:27–35. 10. Li P, Yang Y, Liu M. Xuezhikang, extract of red yeast rice, inhibited tissue factor and hypercoagulable state through suppressing nicotinamide adenine dinucleotide phosphate oxidase and extracellular signalregulated kinase activation. J Cardiovasc Pharmacol. 2011;58:307–318. 11. Zhu XY, Li P, Yang YB, et al. Xuezhikang, extract of red yeast rice, improved abnormal hemorheology, suppressed caveolin-1 and increased eNOS expression in atherosclerotic rats. PLoS One. 2013;8:e62731.
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