European Journal of Pharmacology 740 (2014) 233–239
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Silybin reduces obliterated retinal capillaries in experimental diabetic retinopathy in rats Hong-Tao Zhang a, Kai Shi b, Attit Baskota a, Fang-Li Zhou a, Ya-Xi Chen a, Hao-Ming Tian a,n a b
Department of Endocrinology and Metabolism, West China Hospital of Sichuan University, Sichuan, Chengdu 610041, PR China Department of Ophthalmology, West China Hospital of Sichuan University, Sichuan, Chengdu 610041, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 28 April 2014 Received in revised form 9 July 2014 Accepted 10 July 2014 Available online 24 July 2014
Silybin has been previously reported to possess anti-inflammatory properties, raising the possibility that it may reduce vascular damage in diabetic retinopathy. Present study was designed to investigate this potential effect of silybin and its underlying mechanisms in experimental diabetic retinopathy. Diabetes was induced with streptozotocin (STZ) plus high-fat diet in Sprague-Dawley rats, and silybin was administrated for 22 weeks after the induction of diabetes. Histochemical and immunofluorescence techniques were used to assess the obliterated retinal capillaries, leukostasis, and level of retinal intercellular adhesion molecule-1 (ICAM-1). Western blot was performed to quantitate the expression of retinal ICAM-1. Results showed that silybin treatment significantly prevented the development of obliterated retinal capillaries in diabetes, compared with vehicle treatment. In addition, leukostasis and level of the retinal ICAM-1 were found to decrease considerably in silybin-treated diabetic groups. In conclusion, these results indicate that silybin reduces obliterated retinal capillaries in experimental diabetes, and the recovered retinal vascular leukostasis and level of ICAM-1 at least partly contributes to the preventive effect of silybin. & 2014 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Silybin (PubChem CID: 31553) Streptozotocin (PubChem CID: 29327) Keywords: Silybin Diabetic retinopathy Obliterated retinal capillary Leukostasis Intercellular adhesion molecule-1
1. Introduction One of the characteristic features of the early diabetic retinopathy is obliterated retinal capillaries, which initially occur in single, isolated capillaries, however, as more and more capillaries become occluded, retinal perfusion decreases, at least locally, leading to retinal ischemia, subsequently increasing production of growth factors, and eventually leading to development of proliferative diabetic retinopathy. Although the exact pathogenesis of obliterated retinal capillaries remains unclear, excess adherent leukocytes in retinal vasculature or leukostasis (Joussen et al., 2004) is considered to play a causal role in the development of obliterated retinal capillaries in diabetes (Miyamoto et al., 1999). The process of leukocyte adhesion to retinal vascular endothelial cells is mediated by intercellular adhesion molecules, such as ICAM-1, a crucial participant in retinal vascular leukocyte adhesion or leukostasis in diabetes (Joussen et al., 2001; Miyamoto et al., 1999). Silybin, a bioactive polyphenolic flavonoid in the seeds of milk thistle (Silybum marianum), has been used as a traditional drug
n
Corresponding author. Tel.: þ 86 028 85422982. E-mail address: [email protected] (H.-M. Tian).
http://dx.doi.org/10.1016/j.ejphar.2014.07.033 0014-2999/& 2014 Elsevier B.V. All rights reserved.
for over 2000 years to treat a range of liver disorders (Flora et al., 1998; Saller et al., 2001). Recent studies also demonstrated that silybin possessed anti-inflammatory properties (Al-Anati et al., 2009; Chittezhath et al., 2008; Giorgi et al., 2012; Gu et al., 2007; Kim et al., 2012; Prabu and Muthumani, 2012; Salamone et al., 2012; Youn et al., 2013). Therefore, silybin might be a suitable protective agent for early diabetic retinopathy. The aim of our study was to investigate the potential protective effect of silybin on retinal vasculature and its underlying mechanisms in experimental diabetes.
2. Materials and methods 2.1. Experimental diabetes All experimental protocols were approved by the Institutional Animal Care and Use Committee of Sichuan University and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. Male SpragueDawley rats (n ¼40, 180 710 g) obtained from the Laboratory Animal Centre of Sichuan University were housed under controlled room temperature (22 72 1C), and humidity (55% 75%) with a 12/12 h light/dark cycle. In order to simulate the pathogenesis of type 2 diabetes, the most common clinical type of diabetes,
234
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
our diabetic rat model was induced with STZ plus high-fat diet. After a 1 week of habituation, animals were randomly divided into high-fat diet (n ¼30) and normal chow (n ¼10) groups. Rats in high-fat diet group were fed on a modified high-fat diet (10% grease, 20% sucrose, 1% bile salt, and 2.5% cholesterol) (Wang et al., 2010), while rats in normal chow group were fed on basic normal rat chow. At the end of week 9, all rats in high-fat diet group were injected with a single dose of 30 mg/kg STZ via abdominal cavity to induce diabetes, while all rats in normal chow group were injected with the drug vehicle citric acid buffer to serve as nondiabetic control. 1 week after STZ injection, fasting blood glucose was measured and only animals with 416.7 mmol/l of blood glucose were included in the diabetic group. Rats in diabetic group were randomly subdivided into vehicle-treated (n ¼10) and silybin-treated (n¼ 20) groups. At the end of week 10, silybin, suspended in 1% carboxymethylcellulose sodium salt, began to be administrated orally at 15 mg/kg/day (n¼ 10) and 30 mg/kg/day (n ¼10), respectively. The total course of treatment was 22 weeks. 4 weeks after STZ injection, high-fat diet was withdrawn, and rats in all groups were fed on the normal rat chow until they were killed.
they were fixed with 4% paraformaldehyde for 24 h, and then washed with PBS for 12 h. Isolated retinas were digested with 3% trypsin at 37 1C for 1.5 h, making an easy isolation of retinal vasculature from retinal tissue. Anti-CD45 antibody was used to confirm the identity of the adherent leukocytes in vessel walls, as CD45 is a marker of leukocytes. After permeated with 0.5% Triton X-100 in PBS (4 1C) for 12 h and blocked with 3% albumin in PBS, the retinal vasculature was incubated in anti-CD45 rabbit polyclonal antibody (1:300) overnight at 4 1C, and then incubated in Alexa Fluor 594-coupled anti-rabbit antibody (1:300) for 2 h at 20 1C after 5 washes with PBS. The adherent leukocytes, labeled with FITC-conjugated concanavalin A and anti-CD45 antibody, in retinal vasculature were observed and imaged under a fluorescence microscope (Olympus X71, Japan). ICAM-1 in retinal vasculature was analyzed with the same procedure. Only adherent leukocytes labeled by both FITC-conjugated concanavalin A and anti-CD45 antibody were counted for quantification. In order to quantify obliterated retinal capillaries, the retinal vasculature was stained with Periodic acid-Schiff (PAS) and hematoxylin. Adherent leukocytes and obliterated retinal capillaries were counted in 4 quadrants of the mid-retina.
2.2. Reagents
2.5. Western blot analysis
Streptozotocin, silybin and trypsin were from Sigma-Aldrich. Anti-ICAM-1 and anti-CD45 rabbit polyclonal antibodies were from Santa Cruz Biotechnology. Fluorescent isothiocyanate (FITC)-labeled Concanavalin A was from Nanocs (New York, NY, USA). Horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG antibody, anti-β-actin mouse monoclonal antibody, and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody were from ZSGB-BiO (Beijing, China). Modified RIPA buffer and SDS sample buffer were from Beyotime (Shanghai, China). All solutions were prepared just before use and protected from light if necessary.
The retinas were homogenized in ice-cold modified RIPA buffer. Lysates were then centrifuged at 12,000 g for 5 min at 4 1C. After transferring the supernatant to a fresh ice-cold tube, protein concentration was determined with a Bio-Rad protein assay kit. Equal concentrations of proteins were mixed with SDS sample buffer and denatured at 95 1C for 5 min. The Equal amounts of protein were loaded in a 6% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) followed by a 10% SDS-PAGE gel. The gels were run for 30 min at 80 V and 50 min at 120 V, respectively. Then the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane at 300 mA for 1.5 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. After blockage, the membranes were incubated overnight with primary antibody for ICAM-1 (1:300) at 4 1C, followed by incubating with horseradish peroxidase-conjugated secondary antibody (1:3000) for 1.5 h at room temperature. Signals were visualized by the molecular imager ChemiDocTMXRS þ detection system (BIO-RAD Laboratories Inc., USA). Protein Bands on the membranes were analyzed by Quantity One software (BIO-RAD Laboratories Inc., USA). To control sample errors, the ratio of band intensity to β-actin was obtained to quantify the relative protein expression level.
2.3. Metabolic characteristics, body weight and white blood cell count measuring Blood samples were collected, after 12 h fast, from tail vein at the end of week 10 and from aortaventralis at the end of week 32, respectively. Fasting serum glucose and lipid profiles were detected by a micro-plate reader (Bio-Rad Laboratories, Inc., USA) with kits (Changchun Huili Biotech Co. Ltd, China). Glycosylated hemoglobin was measured by a DCA Vantage GlycoHemoglobin Analyzer (Siemens AG, Germany). White blood cell count was measured by an automatic blood cell analyzer (Mindray Medical International Ltd, China). 2.4. Immunofluorescence and histochemical staining We labeled leukocytes with FITC-conjugated concanavalin A and anti-CD 45 antibody and isolated retinal vasculature with digesting technique as described previously (Ishida et al., 2003; Kern and Engerman, 1995; Yamashiro et al., 2003), with a slight modification. At the end of week 32, rats were killed and immediately perfused with sterile normal saline (500 ml/kg) with heparin (0.1 mg/ml) in it from a needle inserted into the left ventricle (the descending aorta was clamped) to wash out nonadherent blood cells. One eye was ligatured from retrobulbar vessels and removed for analysis of ICAM-1. The other eye was perfused with 30 ml FITC-conjugated concanavalin A (30 μg/ml in phosphate-buffered saline (PBS), pH 7.4) to label the adherent leukocytes and retinal vascular endothelial cells. Residual concanavalin A was removed by washing with PBS. All perfusions were performed under physiological pressure. After removal of rat eyes,
2.6. Statistical analysis All data were expressed as means 7S.E.M. Analysis of data was accomplished with SPSS 19.0. Statistical comparisons were made using analysis of variance (ANOVA), followed by Dunnett's test wherever appropriate. P value o0.05 was considered significant.
3. Results 3.1. Effect of silybin on metabolic characteristics, body weight, and white blood cell count Diabetic rats were characterized with increases in levels of fasting serum glucose (n ¼ 8, Po 0.05) (Fig. 1A), total cholesterol (n ¼8, P o0.05) (Fig. 1B), and triglyceride (n ¼8, P o0.05) (Fig. 1C), and body weight (n ¼8, Po 0.05) (Fig. 1E) at the end of week 10. However, there were no statistical differences in levels of fasting serum glucose (n ¼8, P 40.05) (Fig. 1A), total cholesterol (n¼ 8,
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
235
Fig. 1. Effect of silybin on metabolic characteristics, body weight, and white blood cell count. Diabetic rats were administrated with silybin at 30 or 15 mg/kg/day for 22 weeks. A: Effect of silybin on fasting serum glucose. B: Effect of silybin on serum total cholesterol. C: Effect of silybin on serum triglyceride. D: Effect of silybin on glycosylated hemoglobin. E: Effect of silybin on body weight. F: Effect of silybin on white blood cell count. Values were presented as mean 7S.E.M. N, non-diabetic control group; D, vehicle-treated diabetic group; S-30 (S-15), 30 (15) mg/kg/day silybin-treated diabetic group.n, P o 0.05 compared with N group.
P 40.05) (Fig. 1B), and triglyceride (n¼ 8, P 40.05) (Fig. 1C), glycosylated hemoglobin (n ¼8, P 40.05) (Fig. 1D), body weight (n ¼8, P4 0.05) (Fig. 1E), and white blood cell count (n ¼8, P 40.05) (Fig. 1F) between vehicle-treated diabetic group and silybin-treated diabetic groups at the end of week 32.
control group. Diabetes of 22 weeks duration significantly increased the numbers of obliterated retinal capillaries (n¼ 8, Po 0.05). Silybin treatment significantly prevented the development of obliterated retinal capillaries, with decreases of 50.02% at 30 mg/kg/day and 26.19% at 15 mg/kg/day (n ¼8, P o0.05), compared with vehicle treatment.
3.2. Effect of silybin on obliterated retinal capillaries 3.3. Effect of silybin on vascular leukostasis in retinal vasculature Obliterated retinal capillaries were quantitated in digest retinal preparation. Fig. 2A shows the original pictures of the effect of silybin on obliterated retinal capillaries in digest retinal preparation with PAS staining. Fig. 2B shows the quantitative analysis of the effect of silybin on obliterated retinal capillaries. The numbers of obliterated retinal capillaries were expressed as mean values in all groups. Data were presented as a percentage of that in normal
Retinal vascular leukostasis was evaluated by counting the adherent leukocytes in retinal vasculature. Fig. 3A shows the representative examples of adherent leukocytes with FITClabeled concanavalin A and anti-CD45 antibody staining in situ in digest retinal preparation. Fig. 3B shows the quantitative analysis of the effect of silybin on leukostasis in retinal vasculature.
236
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
Fig. 2. Effect of silybin on obliterated retinal capillaries. Diabetic rats were administrated with silybin at 30 or 15 mg/kg/day for 22 weeks. A: The original pictures of the effect of silybin on obliterated retinal capillaries in digest retinal preparation stained with PAS ( 400). B: Quantitative analysis of the effect of silybin on obliterated retinal capillaries. Values were presented as mean 7 S.E.M. N, non-diabetic control group; D, vehicle-treated diabetic group; S-30 (S-15), 30 (15) mg/kg/day silybin-treated diabetic group.n, P o0.05 compared with N group; #, P o0.05 compared with D group.
The numbers of adherent leukocytes were expressed as mean values in all groups. Data were presented as a percentage of that in non-diabetic control group. Diabetes resulted in significantly increased retinal vascular leukostasis (n ¼8, P o0.05). In contrast to vehicle treatment, silybin treatment caused a significant decrease, 65.12% at 30 mg/kg/day (n ¼4, P o0.05) and 53.74% at 15 mg/kg/day (n ¼8, Po 0.05), in retinal vascular leukostasis. 3.4. Effect of silybin on ICAM-1 protein expression in retinal vasculature Level of ICAM-1 was assayed with immunofluorescence (in retinal vasculature) and Western blot analysis. Fig. 4A shows the evaluation for the effect of silybin on ICAM-1 protein expression in
digest retinal preparation with immunofluorescence. Fig. 4B shows Western blot analysis of the effect of silybin on ICAM-1 protein expression in retinas. ICAM-1 protein level was expressed as mean values in all groups. We observed that the fluorescence intensity of retinal vasculature was weaker in silybin-treated diabetic groups than in vehicle-treated diabetic group. Furthermore, we confirmed the retinal ICAM-1 expression quantitatively with Western blot analysis. After normalizing to β-actin, level of ICAM-1 protein was presented as a percentage of relative non-diabetic control level. Diabetes induced a significant increase in level of ICAM-1 protein in retinal vasculature (n ¼8, P o0.05). Administration of silybin significantly inhibited ICAM-1 expression. The decreases, 46.64% at 30 mg/kg/day (n ¼8, P o0.05) and 37.54 at 15 mg/kg/day (n¼ 4, Po 0.05), in level of ICAM-1 protein were statistically significant.
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
237
Fig. 3. Effect of silybin on leukostasis in retinal vasculature. Diabetic rats were administrated with silybin at 30 or 15 mg/kg/day for 22 weeks. A: The representative examples of adherent leukocytes with FITC-labeled concanavalin A and anti-CD45 antibody staining in situ in digest retinal preparation (Con A, CD45, and Con A þ CD45, 400; N, D, S-30, and S-15, 200). B: Quantitative analysis of the effect of silybin on vascular leukostasis in retinal vasculature. Values were presented as mean 7S.E.M. Con A, FITC-conjugated concanavalin A; CD45, anti-CD45 antibody; Con A þCD45, Con A merged with CD45; N, non-diabetic control group; D, vehicle-treated diabetic group; S-30 (S-15), 30 (15) mg/kg/day silybin-treated diabetic group.n, Po 0.05 compared with N group; #, P o 0.05 compared with D group.
4. Discussion Previous studies have shown that a large number of flavonoids possess protective effects on diabetes-induced retinal pathological alterations, including vascular hyperpermeability, vascular endothelial cell injury, and excessive retinal proinflammatory cytokines (Chen et al., 2012; Chung et al., 2005; Kumar et al., 2013, 2012; Li et al., 2013; Nakajima et al., 2001; Yang et al., 2009). Present study was designed to explore the potential effect of silybin, also a kind of flavonoid compound, on retinal blood vessels in diabetes that was induced in rats with STZ plus high-fat diet. Obliterated retinal capillaries are a hallmark of early morphological pathology in diabetic retinopathy in human, and also develop in animal models, making obliterated retinal capillaries a potential drug target for diabetic retinopathy. Present study investigated the effect of silybin on retinal capillaries and its underlying mechanisms in diabetes. Our histochemical experiments indicated that silybin significantly reduced diabetes-induced obliterated retinal capillaries in a concentration-dependent manner (Fig. 2). Retinal vascular leukostasis, adherent leukocytes in retinal vasculature, has been accepted as an earlier important pathology,
preceding the formation of obliterated retinal capillaries. Adherent leukocytes are a crucial contributor to retinal vascular damage, including injury, death, or loss in vascular pericytes or endothelial cells (Joussen et al., 2004, 2003) which results in the basement membrane devoid any viable pericytes or endothelial cells and eventually obliterated capillaries (Joussen et al., 2004; Miyamoto et al., 1999). Inhibiting retinal vascular leukostasis prevents the development of obliterated retinal capillaries in diabetes (Joussen et al., 2004). Diabetes-induced retinal vascular leukostasis can be effectively suppressed by lipid regulating or antidiabetic drug (Miyahara et al., 2004; Thakur et al., 2011). Although shown to be an anti-inflammatory agent, silybin has not been reported about its anti-leukostasis property in diabetes. We assessed retinal vascular leukostasis in vehicle or silybin-treated diabetic rats by double labeling technique with FITC-conjugated concanavalin A and anti-CD45 antibody. Our data showed that silybin significantly decreased retinal vascular leukostasis in diabetes (Fig. 3). Studies in diabetic animal models reveal that excess retinal vascular leukostasis is mediated by up-regulated ICAM-1 in vasculature, and inhibiting ICAM-1 expression prevents retinal vascular leukostasis (Joussen et al., 2001; Miyamoto et al., 1999).
238
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
Fig. 4. Effect of silybin on ICAM-1 protein expression in retinal vasculature. Diabetic rats were administrated with silybin at 30 or 15 mg/kg/day for 22 weeks. A: Evaluation for the effect of silybin on ICAM-1 protein expression in digest retinal preparation ( 200) with immunofluorescence. B: Western blot analysis of the effect of silybin on ICAM-1 protein expression in retinas. Values were presented as mean 7 S.E.M. N, non-diabetic control group; D, vehicle-treated diabetic group; S-30 (S-15), 30 (15) mg/kg/ day silybin-treated diabetic group.n, P o 0.05 compared with N group; #, P o0.05 compared with D group.
The concept of ICAM-1 inhibition has become a potential therapeutic strategy for the prevention of diabetic retinopathy (Joussen et al., 2001; Miyamoto et al., 1999). Some flavonoid compounds, such as hesperidin, trans-chalcone, and minocycline, were reported to possess properties to inhibit diabetic-induced (Lamoke et al., 2011) or ischemia-induced (Abcouwer et al., 2013; Shi et al., 2012) ICAM-1 expression in retina. Our immunofluorescence and Western blot analysis also clearly supported that silybin effectively inhibited the up-regulated ICAM-1 protein (Fig. 4) in retinal vasculature in diabetes. In addition, silybin did not significantly alter the levels of blood glucose, blood lipids, and glycosylated hemoglobin, and body weight in our diabetic rat model (Fig. 1). And rats either with increased white blood cell count or focus of infection in skin or viscera were not included in our study.
5. Conclusion In summary, our study has provided evidence that silybin reduces obliterated retinal capillaries in experimental diabetes and the preventive effect is at least partly attributed to the recovered retinal vascular leukostasis and level of ICAM-1.
Acknowledgments The authors are grateful for support from Laboratory of Endocrinology and Metabolism, Laboratory of Ophthalmology, and Center for Molecular and Genetic Medicine, West China Hospital (Grant no. 141040102), Sichuan University.
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
References Abcouwer, S.F., Lin, C.M., Shanmugam, S., Muthusamy, A., Barber, A.J., Antonetti, D. A., 2013. Minocycline prevents retinal inflammation and vascular permeability following ischemia-reperfusion injury. J. Neuroinflamm. 10, 149. Al-Anati, L., Essid, E., Reinehr, R., Petzinger, E., 2009. Silybin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells. Mol. Nutr. Food Res. 53, 460–466. Chen, F., Zhang, H.Q., Zhu, J., Liu, K.Y., Cheng, H., Li, G.L., Xu, S., Lv, W.H., Xie, Z.G., 2012. Puerarin enhances superoxide dismutase activity and inhibits RAGE and VEGF expression in retinas of STZ-induced early diabetic rats. Asian Pac. J. Trop. Med. 5, 891–896. Chittezhath, M., Deep, G., Singh, R.P., Agarwal, C., Agarwal, R., 2008. Silybin inhibits cytokine-induced signaling cascades and down-regulates inducible nitric oxide synthase in human lung carcinoma A549 cells. Mol. Cancer Ther. 7, 1817–1826. Chung, H.K., Choi, S.M., Ahn, B.O., Kwak, H.H., Kim, J.H., Kim, W.B., 2005. Efficacy of troxerutin on streptozotocin-induced rat model in the early stage of diabetic retinopathy. Arzneimittelforschung 55, 573–580. Flora, K., Hahn, M., Rosen, H., Benner, K., 1998. Milk thistle (Silybum marianum) for the therapy of liver disease. Am. J. Gastroenterol. 93, 139–143. Giorgi, V.S., Peracoli, M.T., Peracoli, J.C., Witkin, S.S., Bannwart-Castro, C.F., 2012. Silybin modulates the NF-κb pathway and pro-inflammatory cytokine production by mononuclear cells from preeclamptic women. J. Reprod. Immunol. 95, 67–72. Gu, M., Singh, R.P., Dhanalakshmi, S., Agarwal, C., Agarwal, R., 2007. Silybin inhibits inflammatory and angiogenic attributes in photocarcinogenesis in SKH-1 hairless mice. Cancer Res. 67, 3483–3491. Ishida, S., Usui, T., Yamashiro, K., Kaji, Y., Ahmed, E., Carrasquillo, K.G., Amano, S., Hida, T., Oguchi, Y., Adamis, A.P., 2003. VEGF164 is proinflammatory in the diabetic retina. Invest. Ophthalmol. Vis. Sci. 44, 2155–2162. Joussen, A.M., Murata, T., Tsujikawa, A., Kirchhof, B., Bursell, S.E., Adamis, A.P., 2001. Leukocyte-mediated endothelial cell injury and death in the diabetic retina. Am. J. Pathol. 158, 147–152. Joussen, A.M., Poulaki, V., Mitsiades, N., Cai, W.Y., Suzuma, I., Pak, J., Ju, S.T., Rook, S. L., Esser, P., Mitsiades, C.S., Kirchhof, B., Adamis, A.P., Aiello, L.P., 2003. Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes. FASEB J. 17, 76–78. Joussen, A.M., Poulaki, V., Le, M.L., Koizumi, K., Esser, C., Janicki, H., Schraermeyer, U., Kociok, N., Fauser, S., Kirchhof, B., Kern, T.S., Adamis, A.P., 2004. A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J. 18, 1450–1452. Kern, T.S., Engerman, R.L., 1995. Vascular lesions in diabetes are distributed nonuniformly within the retina. Exp. Eye Res. 60, 545–549. Kim, K.D., Lee, H.J., Lim, S.P., Sikder, A., Lee, S.Y., Lee, C.J., 2012. Silybin regulates gene expression, production and secretion of mucin from cultured airway epithelial cells. Phytother. Res. 26, 1301–1307. Kumar, B., Gupta, S.K., Srinivasan, B.P., Nag, T.C., Srivastava, S., Saxena, R., 2012. Hesperetin ameliorates hyperglycemia induced retinal vasculopathy via antiangiogenic effects in experimental diabetic rats. Vasc. Pharmacol. 57, 201–207.
239
Kumar, B., Gupta, S.K., Srinivasan, B.P., Nag, T.C., Srivastava, S., Saxena, R., Jha, K.A., 2013. Hesperetin rescues retinal oxidative stress, neuroinflammation and apoptosis in diabetic rats. Microvasc. Res. 87, 65–74. Lamoke, F., Labazi, M., Montemari, A., Parisi, G., Varano, M., Bartoli, M., 2011. TransChalcone prevents VEGF expression and retinal neovascularization in the ischemic retina. Exp. Eye Res. 93, 350–354. Li, D., Yang, F., Cheng, H., Liu, C., Sun, M., Wu, K., Ai, M., 2013. Protective effects of total flavonoids from Flos Puerariae on retinal neuronal damage in diabetic mice. Mol. Vis. 19, 1999–2010. Miyahara, S., Kiryu, J., Yamashiro, K., Miyamoto, K., Hirose, F., Tamura, H., Katsuta, H., Nishijima, K., Tsujikawa, A., Honda, Y., 2004. Simvastatin inhibits leukocyte accumulation and vascular permeability in the retinas of rats with streptozotocin-induced diabetes. Am. J. Pathol. 164, 1697–1706. Miyamoto, K., Khosrof, S., Bursell, S.E., Rohan, R., Murata, T., Clermont, A.C., Aiello, L. P., Ogura, Y., Adamis, A.P., 1999. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc. Natl. Acad. Sci. U. S. A. 96, 10836–10841. Nakajima, M., Cooney, M.J., Tu, A.H., Chang, K.Y., Cao, J., Ando, A., An, G.J., Melia, M., de Juan Jr., E., 2001. Normalization of retinal vascular permeability in experimental diabetes with genistein. Invest. Ophthalmol. Vis. Sci. 42, 2110–2114. Prabu, S.M., Muthumani, M., 2012. Silybin ameliorates arsenic induced nephrotoxicity by abrogation of oxidative stress, inflammation and apoptosis in rats. Mol. Biol. Rep. 39, 11201–11216. Salamone, F., Galvano, F., Cappello, F., Mangiameli, A., Barbagallo, I., Li Volti, G., 2012. Silybin modulates lipid homeostasis and inhibits nuclear factor kappa B activation in experimental nonalcoholic steatohepatitis. Transl. Res. 159, 477–486. Saller, R., Meier, R., Brignoli, R., 2001. The use of silymarin in the treatment of liver diseases. Drugs 61, 2035–2063. Shi, X., Liao, S., Mi, H., Guo, C., Qi, D., Li, F., Zhang, C., Yang, Z., 2012. Hesperidin prevents retinal and plasma abnormalities in streptozotocin-induced diabetic rats. Molecules 17, 12868–12881. Thakur, A., Scheinman, R.I., Rao, V.R., Kompella, U.B., 2011. Pazopanib, a multitargeted tyrosine kinase inhibitor, reduces diabetic retinal vascular leukostasis and leakage. Microvasc. Res. 82, 346–350. Wang, Y., Zhang, H.T., Su, X.L., Deng, X.L., Yuan, B.X., Zhang, W., Wang, X.F., Yang, Y. B., 2010. Experimental diabetes mellitus down-regulates large-conductance Ca2 þ -activated K þ channels in cerebral artery smooth muscle and alters functional conductance. Curr. Neurovasc. Res. 7, 75–84. Yamashiro, K., Tsujikawa, A., Ishida, S., Usui, T., Kaji, Y., Honda, Y., Ogura, Y., Adamis, A.P., 2003. Platelets accumulate in the diabetic retinal vasculature following endothelial death and suppress blood-retinal barrier breakdown. Am. J. Pathol. 163, 253–259. Yang, L.P., Sun, H.L., Wu, L.M., Guo, X.J., Dou, H.L., Tso, M.O., Zhao, L., Li, S.M., 2009. Baicalein reduces inflammatory process in a rodent model of diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 50, 2319–2327. Youn, C.K., Park, S.J., Lee, M.Y., Cha, M.J., Kim, O.H., You, H.J., Chang, I.Y., Yoon, S.P., Jeon, Y.J., 2013. Silybin inhibits LPS-induced macrophage activation by blocking p38 MAPK in RAW 264.7 cells. Biomol. Ther. 21, 258–263.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Silybin reduces obliterated retinal capillaries in experimental diabetic retinopathy in rats Hong-Tao Zhang a, Kai Shi b, Attit Baskota a, Fang-Li Zhou a, Ya-Xi Chen a, Hao-Ming Tian a,n a b
Department of Endocrinology and Metabolism, West China Hospital of Sichuan University, Sichuan, Chengdu 610041, PR China Department of Ophthalmology, West China Hospital of Sichuan University, Sichuan, Chengdu 610041, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 28 April 2014 Received in revised form 9 July 2014 Accepted 10 July 2014 Available online 24 July 2014
Silybin has been previously reported to possess anti-inflammatory properties, raising the possibility that it may reduce vascular damage in diabetic retinopathy. Present study was designed to investigate this potential effect of silybin and its underlying mechanisms in experimental diabetic retinopathy. Diabetes was induced with streptozotocin (STZ) plus high-fat diet in Sprague-Dawley rats, and silybin was administrated for 22 weeks after the induction of diabetes. Histochemical and immunofluorescence techniques were used to assess the obliterated retinal capillaries, leukostasis, and level of retinal intercellular adhesion molecule-1 (ICAM-1). Western blot was performed to quantitate the expression of retinal ICAM-1. Results showed that silybin treatment significantly prevented the development of obliterated retinal capillaries in diabetes, compared with vehicle treatment. In addition, leukostasis and level of the retinal ICAM-1 were found to decrease considerably in silybin-treated diabetic groups. In conclusion, these results indicate that silybin reduces obliterated retinal capillaries in experimental diabetes, and the recovered retinal vascular leukostasis and level of ICAM-1 at least partly contributes to the preventive effect of silybin. & 2014 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Silybin (PubChem CID: 31553) Streptozotocin (PubChem CID: 29327) Keywords: Silybin Diabetic retinopathy Obliterated retinal capillary Leukostasis Intercellular adhesion molecule-1
1. Introduction One of the characteristic features of the early diabetic retinopathy is obliterated retinal capillaries, which initially occur in single, isolated capillaries, however, as more and more capillaries become occluded, retinal perfusion decreases, at least locally, leading to retinal ischemia, subsequently increasing production of growth factors, and eventually leading to development of proliferative diabetic retinopathy. Although the exact pathogenesis of obliterated retinal capillaries remains unclear, excess adherent leukocytes in retinal vasculature or leukostasis (Joussen et al., 2004) is considered to play a causal role in the development of obliterated retinal capillaries in diabetes (Miyamoto et al., 1999). The process of leukocyte adhesion to retinal vascular endothelial cells is mediated by intercellular adhesion molecules, such as ICAM-1, a crucial participant in retinal vascular leukocyte adhesion or leukostasis in diabetes (Joussen et al., 2001; Miyamoto et al., 1999). Silybin, a bioactive polyphenolic flavonoid in the seeds of milk thistle (Silybum marianum), has been used as a traditional drug
n
Corresponding author. Tel.: þ 86 028 85422982. E-mail address: [email protected] (H.-M. Tian).
http://dx.doi.org/10.1016/j.ejphar.2014.07.033 0014-2999/& 2014 Elsevier B.V. All rights reserved.
for over 2000 years to treat a range of liver disorders (Flora et al., 1998; Saller et al., 2001). Recent studies also demonstrated that silybin possessed anti-inflammatory properties (Al-Anati et al., 2009; Chittezhath et al., 2008; Giorgi et al., 2012; Gu et al., 2007; Kim et al., 2012; Prabu and Muthumani, 2012; Salamone et al., 2012; Youn et al., 2013). Therefore, silybin might be a suitable protective agent for early diabetic retinopathy. The aim of our study was to investigate the potential protective effect of silybin on retinal vasculature and its underlying mechanisms in experimental diabetes.
2. Materials and methods 2.1. Experimental diabetes All experimental protocols were approved by the Institutional Animal Care and Use Committee of Sichuan University and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. Male SpragueDawley rats (n ¼40, 180 710 g) obtained from the Laboratory Animal Centre of Sichuan University were housed under controlled room temperature (22 72 1C), and humidity (55% 75%) with a 12/12 h light/dark cycle. In order to simulate the pathogenesis of type 2 diabetes, the most common clinical type of diabetes,
234
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
our diabetic rat model was induced with STZ plus high-fat diet. After a 1 week of habituation, animals were randomly divided into high-fat diet (n ¼30) and normal chow (n ¼10) groups. Rats in high-fat diet group were fed on a modified high-fat diet (10% grease, 20% sucrose, 1% bile salt, and 2.5% cholesterol) (Wang et al., 2010), while rats in normal chow group were fed on basic normal rat chow. At the end of week 9, all rats in high-fat diet group were injected with a single dose of 30 mg/kg STZ via abdominal cavity to induce diabetes, while all rats in normal chow group were injected with the drug vehicle citric acid buffer to serve as nondiabetic control. 1 week after STZ injection, fasting blood glucose was measured and only animals with 416.7 mmol/l of blood glucose were included in the diabetic group. Rats in diabetic group were randomly subdivided into vehicle-treated (n ¼10) and silybin-treated (n¼ 20) groups. At the end of week 10, silybin, suspended in 1% carboxymethylcellulose sodium salt, began to be administrated orally at 15 mg/kg/day (n¼ 10) and 30 mg/kg/day (n ¼10), respectively. The total course of treatment was 22 weeks. 4 weeks after STZ injection, high-fat diet was withdrawn, and rats in all groups were fed on the normal rat chow until they were killed.
they were fixed with 4% paraformaldehyde for 24 h, and then washed with PBS for 12 h. Isolated retinas were digested with 3% trypsin at 37 1C for 1.5 h, making an easy isolation of retinal vasculature from retinal tissue. Anti-CD45 antibody was used to confirm the identity of the adherent leukocytes in vessel walls, as CD45 is a marker of leukocytes. After permeated with 0.5% Triton X-100 in PBS (4 1C) for 12 h and blocked with 3% albumin in PBS, the retinal vasculature was incubated in anti-CD45 rabbit polyclonal antibody (1:300) overnight at 4 1C, and then incubated in Alexa Fluor 594-coupled anti-rabbit antibody (1:300) for 2 h at 20 1C after 5 washes with PBS. The adherent leukocytes, labeled with FITC-conjugated concanavalin A and anti-CD45 antibody, in retinal vasculature were observed and imaged under a fluorescence microscope (Olympus X71, Japan). ICAM-1 in retinal vasculature was analyzed with the same procedure. Only adherent leukocytes labeled by both FITC-conjugated concanavalin A and anti-CD45 antibody were counted for quantification. In order to quantify obliterated retinal capillaries, the retinal vasculature was stained with Periodic acid-Schiff (PAS) and hematoxylin. Adherent leukocytes and obliterated retinal capillaries were counted in 4 quadrants of the mid-retina.
2.2. Reagents
2.5. Western blot analysis
Streptozotocin, silybin and trypsin were from Sigma-Aldrich. Anti-ICAM-1 and anti-CD45 rabbit polyclonal antibodies were from Santa Cruz Biotechnology. Fluorescent isothiocyanate (FITC)-labeled Concanavalin A was from Nanocs (New York, NY, USA). Horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG antibody, anti-β-actin mouse monoclonal antibody, and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody were from ZSGB-BiO (Beijing, China). Modified RIPA buffer and SDS sample buffer were from Beyotime (Shanghai, China). All solutions were prepared just before use and protected from light if necessary.
The retinas were homogenized in ice-cold modified RIPA buffer. Lysates were then centrifuged at 12,000 g for 5 min at 4 1C. After transferring the supernatant to a fresh ice-cold tube, protein concentration was determined with a Bio-Rad protein assay kit. Equal concentrations of proteins were mixed with SDS sample buffer and denatured at 95 1C for 5 min. The Equal amounts of protein were loaded in a 6% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) followed by a 10% SDS-PAGE gel. The gels were run for 30 min at 80 V and 50 min at 120 V, respectively. Then the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane at 300 mA for 1.5 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. After blockage, the membranes were incubated overnight with primary antibody for ICAM-1 (1:300) at 4 1C, followed by incubating with horseradish peroxidase-conjugated secondary antibody (1:3000) for 1.5 h at room temperature. Signals were visualized by the molecular imager ChemiDocTMXRS þ detection system (BIO-RAD Laboratories Inc., USA). Protein Bands on the membranes were analyzed by Quantity One software (BIO-RAD Laboratories Inc., USA). To control sample errors, the ratio of band intensity to β-actin was obtained to quantify the relative protein expression level.
2.3. Metabolic characteristics, body weight and white blood cell count measuring Blood samples were collected, after 12 h fast, from tail vein at the end of week 10 and from aortaventralis at the end of week 32, respectively. Fasting serum glucose and lipid profiles were detected by a micro-plate reader (Bio-Rad Laboratories, Inc., USA) with kits (Changchun Huili Biotech Co. Ltd, China). Glycosylated hemoglobin was measured by a DCA Vantage GlycoHemoglobin Analyzer (Siemens AG, Germany). White blood cell count was measured by an automatic blood cell analyzer (Mindray Medical International Ltd, China). 2.4. Immunofluorescence and histochemical staining We labeled leukocytes with FITC-conjugated concanavalin A and anti-CD 45 antibody and isolated retinal vasculature with digesting technique as described previously (Ishida et al., 2003; Kern and Engerman, 1995; Yamashiro et al., 2003), with a slight modification. At the end of week 32, rats were killed and immediately perfused with sterile normal saline (500 ml/kg) with heparin (0.1 mg/ml) in it from a needle inserted into the left ventricle (the descending aorta was clamped) to wash out nonadherent blood cells. One eye was ligatured from retrobulbar vessels and removed for analysis of ICAM-1. The other eye was perfused with 30 ml FITC-conjugated concanavalin A (30 μg/ml in phosphate-buffered saline (PBS), pH 7.4) to label the adherent leukocytes and retinal vascular endothelial cells. Residual concanavalin A was removed by washing with PBS. All perfusions were performed under physiological pressure. After removal of rat eyes,
2.6. Statistical analysis All data were expressed as means 7S.E.M. Analysis of data was accomplished with SPSS 19.0. Statistical comparisons were made using analysis of variance (ANOVA), followed by Dunnett's test wherever appropriate. P value o0.05 was considered significant.
3. Results 3.1. Effect of silybin on metabolic characteristics, body weight, and white blood cell count Diabetic rats were characterized with increases in levels of fasting serum glucose (n ¼ 8, Po 0.05) (Fig. 1A), total cholesterol (n ¼8, P o0.05) (Fig. 1B), and triglyceride (n ¼8, P o0.05) (Fig. 1C), and body weight (n ¼8, Po 0.05) (Fig. 1E) at the end of week 10. However, there were no statistical differences in levels of fasting serum glucose (n ¼8, P 40.05) (Fig. 1A), total cholesterol (n¼ 8,
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
235
Fig. 1. Effect of silybin on metabolic characteristics, body weight, and white blood cell count. Diabetic rats were administrated with silybin at 30 or 15 mg/kg/day for 22 weeks. A: Effect of silybin on fasting serum glucose. B: Effect of silybin on serum total cholesterol. C: Effect of silybin on serum triglyceride. D: Effect of silybin on glycosylated hemoglobin. E: Effect of silybin on body weight. F: Effect of silybin on white blood cell count. Values were presented as mean 7S.E.M. N, non-diabetic control group; D, vehicle-treated diabetic group; S-30 (S-15), 30 (15) mg/kg/day silybin-treated diabetic group.n, P o 0.05 compared with N group.
P 40.05) (Fig. 1B), and triglyceride (n¼ 8, P 40.05) (Fig. 1C), glycosylated hemoglobin (n ¼8, P 40.05) (Fig. 1D), body weight (n ¼8, P4 0.05) (Fig. 1E), and white blood cell count (n ¼8, P 40.05) (Fig. 1F) between vehicle-treated diabetic group and silybin-treated diabetic groups at the end of week 32.
control group. Diabetes of 22 weeks duration significantly increased the numbers of obliterated retinal capillaries (n¼ 8, Po 0.05). Silybin treatment significantly prevented the development of obliterated retinal capillaries, with decreases of 50.02% at 30 mg/kg/day and 26.19% at 15 mg/kg/day (n ¼8, P o0.05), compared with vehicle treatment.
3.2. Effect of silybin on obliterated retinal capillaries 3.3. Effect of silybin on vascular leukostasis in retinal vasculature Obliterated retinal capillaries were quantitated in digest retinal preparation. Fig. 2A shows the original pictures of the effect of silybin on obliterated retinal capillaries in digest retinal preparation with PAS staining. Fig. 2B shows the quantitative analysis of the effect of silybin on obliterated retinal capillaries. The numbers of obliterated retinal capillaries were expressed as mean values in all groups. Data were presented as a percentage of that in normal
Retinal vascular leukostasis was evaluated by counting the adherent leukocytes in retinal vasculature. Fig. 3A shows the representative examples of adherent leukocytes with FITClabeled concanavalin A and anti-CD45 antibody staining in situ in digest retinal preparation. Fig. 3B shows the quantitative analysis of the effect of silybin on leukostasis in retinal vasculature.
236
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
Fig. 2. Effect of silybin on obliterated retinal capillaries. Diabetic rats were administrated with silybin at 30 or 15 mg/kg/day for 22 weeks. A: The original pictures of the effect of silybin on obliterated retinal capillaries in digest retinal preparation stained with PAS ( 400). B: Quantitative analysis of the effect of silybin on obliterated retinal capillaries. Values were presented as mean 7 S.E.M. N, non-diabetic control group; D, vehicle-treated diabetic group; S-30 (S-15), 30 (15) mg/kg/day silybin-treated diabetic group.n, P o0.05 compared with N group; #, P o0.05 compared with D group.
The numbers of adherent leukocytes were expressed as mean values in all groups. Data were presented as a percentage of that in non-diabetic control group. Diabetes resulted in significantly increased retinal vascular leukostasis (n ¼8, P o0.05). In contrast to vehicle treatment, silybin treatment caused a significant decrease, 65.12% at 30 mg/kg/day (n ¼4, P o0.05) and 53.74% at 15 mg/kg/day (n ¼8, Po 0.05), in retinal vascular leukostasis. 3.4. Effect of silybin on ICAM-1 protein expression in retinal vasculature Level of ICAM-1 was assayed with immunofluorescence (in retinal vasculature) and Western blot analysis. Fig. 4A shows the evaluation for the effect of silybin on ICAM-1 protein expression in
digest retinal preparation with immunofluorescence. Fig. 4B shows Western blot analysis of the effect of silybin on ICAM-1 protein expression in retinas. ICAM-1 protein level was expressed as mean values in all groups. We observed that the fluorescence intensity of retinal vasculature was weaker in silybin-treated diabetic groups than in vehicle-treated diabetic group. Furthermore, we confirmed the retinal ICAM-1 expression quantitatively with Western blot analysis. After normalizing to β-actin, level of ICAM-1 protein was presented as a percentage of relative non-diabetic control level. Diabetes induced a significant increase in level of ICAM-1 protein in retinal vasculature (n ¼8, P o0.05). Administration of silybin significantly inhibited ICAM-1 expression. The decreases, 46.64% at 30 mg/kg/day (n ¼8, P o0.05) and 37.54 at 15 mg/kg/day (n¼ 4, Po 0.05), in level of ICAM-1 protein were statistically significant.
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
237
Fig. 3. Effect of silybin on leukostasis in retinal vasculature. Diabetic rats were administrated with silybin at 30 or 15 mg/kg/day for 22 weeks. A: The representative examples of adherent leukocytes with FITC-labeled concanavalin A and anti-CD45 antibody staining in situ in digest retinal preparation (Con A, CD45, and Con A þ CD45, 400; N, D, S-30, and S-15, 200). B: Quantitative analysis of the effect of silybin on vascular leukostasis in retinal vasculature. Values were presented as mean 7S.E.M. Con A, FITC-conjugated concanavalin A; CD45, anti-CD45 antibody; Con A þCD45, Con A merged with CD45; N, non-diabetic control group; D, vehicle-treated diabetic group; S-30 (S-15), 30 (15) mg/kg/day silybin-treated diabetic group.n, Po 0.05 compared with N group; #, P o 0.05 compared with D group.
4. Discussion Previous studies have shown that a large number of flavonoids possess protective effects on diabetes-induced retinal pathological alterations, including vascular hyperpermeability, vascular endothelial cell injury, and excessive retinal proinflammatory cytokines (Chen et al., 2012; Chung et al., 2005; Kumar et al., 2013, 2012; Li et al., 2013; Nakajima et al., 2001; Yang et al., 2009). Present study was designed to explore the potential effect of silybin, also a kind of flavonoid compound, on retinal blood vessels in diabetes that was induced in rats with STZ plus high-fat diet. Obliterated retinal capillaries are a hallmark of early morphological pathology in diabetic retinopathy in human, and also develop in animal models, making obliterated retinal capillaries a potential drug target for diabetic retinopathy. Present study investigated the effect of silybin on retinal capillaries and its underlying mechanisms in diabetes. Our histochemical experiments indicated that silybin significantly reduced diabetes-induced obliterated retinal capillaries in a concentration-dependent manner (Fig. 2). Retinal vascular leukostasis, adherent leukocytes in retinal vasculature, has been accepted as an earlier important pathology,
preceding the formation of obliterated retinal capillaries. Adherent leukocytes are a crucial contributor to retinal vascular damage, including injury, death, or loss in vascular pericytes or endothelial cells (Joussen et al., 2004, 2003) which results in the basement membrane devoid any viable pericytes or endothelial cells and eventually obliterated capillaries (Joussen et al., 2004; Miyamoto et al., 1999). Inhibiting retinal vascular leukostasis prevents the development of obliterated retinal capillaries in diabetes (Joussen et al., 2004). Diabetes-induced retinal vascular leukostasis can be effectively suppressed by lipid regulating or antidiabetic drug (Miyahara et al., 2004; Thakur et al., 2011). Although shown to be an anti-inflammatory agent, silybin has not been reported about its anti-leukostasis property in diabetes. We assessed retinal vascular leukostasis in vehicle or silybin-treated diabetic rats by double labeling technique with FITC-conjugated concanavalin A and anti-CD45 antibody. Our data showed that silybin significantly decreased retinal vascular leukostasis in diabetes (Fig. 3). Studies in diabetic animal models reveal that excess retinal vascular leukostasis is mediated by up-regulated ICAM-1 in vasculature, and inhibiting ICAM-1 expression prevents retinal vascular leukostasis (Joussen et al., 2001; Miyamoto et al., 1999).
238
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
Fig. 4. Effect of silybin on ICAM-1 protein expression in retinal vasculature. Diabetic rats were administrated with silybin at 30 or 15 mg/kg/day for 22 weeks. A: Evaluation for the effect of silybin on ICAM-1 protein expression in digest retinal preparation ( 200) with immunofluorescence. B: Western blot analysis of the effect of silybin on ICAM-1 protein expression in retinas. Values were presented as mean 7 S.E.M. N, non-diabetic control group; D, vehicle-treated diabetic group; S-30 (S-15), 30 (15) mg/kg/ day silybin-treated diabetic group.n, P o 0.05 compared with N group; #, P o0.05 compared with D group.
The concept of ICAM-1 inhibition has become a potential therapeutic strategy for the prevention of diabetic retinopathy (Joussen et al., 2001; Miyamoto et al., 1999). Some flavonoid compounds, such as hesperidin, trans-chalcone, and minocycline, were reported to possess properties to inhibit diabetic-induced (Lamoke et al., 2011) or ischemia-induced (Abcouwer et al., 2013; Shi et al., 2012) ICAM-1 expression in retina. Our immunofluorescence and Western blot analysis also clearly supported that silybin effectively inhibited the up-regulated ICAM-1 protein (Fig. 4) in retinal vasculature in diabetes. In addition, silybin did not significantly alter the levels of blood glucose, blood lipids, and glycosylated hemoglobin, and body weight in our diabetic rat model (Fig. 1). And rats either with increased white blood cell count or focus of infection in skin or viscera were not included in our study.
5. Conclusion In summary, our study has provided evidence that silybin reduces obliterated retinal capillaries in experimental diabetes and the preventive effect is at least partly attributed to the recovered retinal vascular leukostasis and level of ICAM-1.
Acknowledgments The authors are grateful for support from Laboratory of Endocrinology and Metabolism, Laboratory of Ophthalmology, and Center for Molecular and Genetic Medicine, West China Hospital (Grant no. 141040102), Sichuan University.
H.-T. Zhang et al. / European Journal of Pharmacology 740 (2014) 233–239
References Abcouwer, S.F., Lin, C.M., Shanmugam, S., Muthusamy, A., Barber, A.J., Antonetti, D. A., 2013. Minocycline prevents retinal inflammation and vascular permeability following ischemia-reperfusion injury. J. Neuroinflamm. 10, 149. Al-Anati, L., Essid, E., Reinehr, R., Petzinger, E., 2009. Silybin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells. Mol. Nutr. Food Res. 53, 460–466. Chen, F., Zhang, H.Q., Zhu, J., Liu, K.Y., Cheng, H., Li, G.L., Xu, S., Lv, W.H., Xie, Z.G., 2012. Puerarin enhances superoxide dismutase activity and inhibits RAGE and VEGF expression in retinas of STZ-induced early diabetic rats. Asian Pac. J. Trop. Med. 5, 891–896. Chittezhath, M., Deep, G., Singh, R.P., Agarwal, C., Agarwal, R., 2008. Silybin inhibits cytokine-induced signaling cascades and down-regulates inducible nitric oxide synthase in human lung carcinoma A549 cells. Mol. Cancer Ther. 7, 1817–1826. Chung, H.K., Choi, S.M., Ahn, B.O., Kwak, H.H., Kim, J.H., Kim, W.B., 2005. Efficacy of troxerutin on streptozotocin-induced rat model in the early stage of diabetic retinopathy. Arzneimittelforschung 55, 573–580. Flora, K., Hahn, M., Rosen, H., Benner, K., 1998. Milk thistle (Silybum marianum) for the therapy of liver disease. Am. J. Gastroenterol. 93, 139–143. Giorgi, V.S., Peracoli, M.T., Peracoli, J.C., Witkin, S.S., Bannwart-Castro, C.F., 2012. Silybin modulates the NF-κb pathway and pro-inflammatory cytokine production by mononuclear cells from preeclamptic women. J. Reprod. Immunol. 95, 67–72. Gu, M., Singh, R.P., Dhanalakshmi, S., Agarwal, C., Agarwal, R., 2007. Silybin inhibits inflammatory and angiogenic attributes in photocarcinogenesis in SKH-1 hairless mice. Cancer Res. 67, 3483–3491. Ishida, S., Usui, T., Yamashiro, K., Kaji, Y., Ahmed, E., Carrasquillo, K.G., Amano, S., Hida, T., Oguchi, Y., Adamis, A.P., 2003. VEGF164 is proinflammatory in the diabetic retina. Invest. Ophthalmol. Vis. Sci. 44, 2155–2162. Joussen, A.M., Murata, T., Tsujikawa, A., Kirchhof, B., Bursell, S.E., Adamis, A.P., 2001. Leukocyte-mediated endothelial cell injury and death in the diabetic retina. Am. J. Pathol. 158, 147–152. Joussen, A.M., Poulaki, V., Mitsiades, N., Cai, W.Y., Suzuma, I., Pak, J., Ju, S.T., Rook, S. L., Esser, P., Mitsiades, C.S., Kirchhof, B., Adamis, A.P., Aiello, L.P., 2003. Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes. FASEB J. 17, 76–78. Joussen, A.M., Poulaki, V., Le, M.L., Koizumi, K., Esser, C., Janicki, H., Schraermeyer, U., Kociok, N., Fauser, S., Kirchhof, B., Kern, T.S., Adamis, A.P., 2004. A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J. 18, 1450–1452. Kern, T.S., Engerman, R.L., 1995. Vascular lesions in diabetes are distributed nonuniformly within the retina. Exp. Eye Res. 60, 545–549. Kim, K.D., Lee, H.J., Lim, S.P., Sikder, A., Lee, S.Y., Lee, C.J., 2012. Silybin regulates gene expression, production and secretion of mucin from cultured airway epithelial cells. Phytother. Res. 26, 1301–1307. Kumar, B., Gupta, S.K., Srinivasan, B.P., Nag, T.C., Srivastava, S., Saxena, R., 2012. Hesperetin ameliorates hyperglycemia induced retinal vasculopathy via antiangiogenic effects in experimental diabetic rats. Vasc. Pharmacol. 57, 201–207.
239
Kumar, B., Gupta, S.K., Srinivasan, B.P., Nag, T.C., Srivastava, S., Saxena, R., Jha, K.A., 2013. Hesperetin rescues retinal oxidative stress, neuroinflammation and apoptosis in diabetic rats. Microvasc. Res. 87, 65–74. Lamoke, F., Labazi, M., Montemari, A., Parisi, G., Varano, M., Bartoli, M., 2011. TransChalcone prevents VEGF expression and retinal neovascularization in the ischemic retina. Exp. Eye Res. 93, 350–354. Li, D., Yang, F., Cheng, H., Liu, C., Sun, M., Wu, K., Ai, M., 2013. Protective effects of total flavonoids from Flos Puerariae on retinal neuronal damage in diabetic mice. Mol. Vis. 19, 1999–2010. Miyahara, S., Kiryu, J., Yamashiro, K., Miyamoto, K., Hirose, F., Tamura, H., Katsuta, H., Nishijima, K., Tsujikawa, A., Honda, Y., 2004. Simvastatin inhibits leukocyte accumulation and vascular permeability in the retinas of rats with streptozotocin-induced diabetes. Am. J. Pathol. 164, 1697–1706. Miyamoto, K., Khosrof, S., Bursell, S.E., Rohan, R., Murata, T., Clermont, A.C., Aiello, L. P., Ogura, Y., Adamis, A.P., 1999. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc. Natl. Acad. Sci. U. S. A. 96, 10836–10841. Nakajima, M., Cooney, M.J., Tu, A.H., Chang, K.Y., Cao, J., Ando, A., An, G.J., Melia, M., de Juan Jr., E., 2001. Normalization of retinal vascular permeability in experimental diabetes with genistein. Invest. Ophthalmol. Vis. Sci. 42, 2110–2114. Prabu, S.M., Muthumani, M., 2012. Silybin ameliorates arsenic induced nephrotoxicity by abrogation of oxidative stress, inflammation and apoptosis in rats. Mol. Biol. Rep. 39, 11201–11216. Salamone, F., Galvano, F., Cappello, F., Mangiameli, A., Barbagallo, I., Li Volti, G., 2012. Silybin modulates lipid homeostasis and inhibits nuclear factor kappa B activation in experimental nonalcoholic steatohepatitis. Transl. Res. 159, 477–486. Saller, R., Meier, R., Brignoli, R., 2001. The use of silymarin in the treatment of liver diseases. Drugs 61, 2035–2063. Shi, X., Liao, S., Mi, H., Guo, C., Qi, D., Li, F., Zhang, C., Yang, Z., 2012. Hesperidin prevents retinal and plasma abnormalities in streptozotocin-induced diabetic rats. Molecules 17, 12868–12881. Thakur, A., Scheinman, R.I., Rao, V.R., Kompella, U.B., 2011. Pazopanib, a multitargeted tyrosine kinase inhibitor, reduces diabetic retinal vascular leukostasis and leakage. Microvasc. Res. 82, 346–350. Wang, Y., Zhang, H.T., Su, X.L., Deng, X.L., Yuan, B.X., Zhang, W., Wang, X.F., Yang, Y. B., 2010. Experimental diabetes mellitus down-regulates large-conductance Ca2 þ -activated K þ channels in cerebral artery smooth muscle and alters functional conductance. Curr. Neurovasc. Res. 7, 75–84. Yamashiro, K., Tsujikawa, A., Ishida, S., Usui, T., Kaji, Y., Honda, Y., Ogura, Y., Adamis, A.P., 2003. Platelets accumulate in the diabetic retinal vasculature following endothelial death and suppress blood-retinal barrier breakdown. Am. J. Pathol. 163, 253–259. Yang, L.P., Sun, H.L., Wu, L.M., Guo, X.J., Dou, H.L., Tso, M.O., Zhao, L., Li, S.M., 2009. Baicalein reduces inflammatory process in a rodent model of diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 50, 2319–2327. Youn, C.K., Park, S.J., Lee, M.Y., Cha, M.J., Kim, O.H., You, H.J., Chang, I.Y., Yoon, S.P., Jeon, Y.J., 2013. Silybin inhibits LPS-induced macrophage activation by blocking p38 MAPK in RAW 264.7 cells. Biomol. Ther. 21, 258–263.
