Mol Cell Biochem DOI 10.1007/s11010-015-2374-6

Rosmarinic acid modulates the antioxidant status and protects pancreatic tissues from glucolipotoxicity mediated oxidative stress in high-fat diet: streptozotocin-induced diabetic rats. Jayanthy Govindaraj • Subramanian Sorimuthu Pillai

Received: 26 November 2014 / Accepted: 23 February 2015  Springer Science+Business Media New York 2015

Abstract Persistent hyperglycemia and elevated levels of free fatty acids (FFA) contribute to oxidative stress, a proximate cause for the onset and progression of diabetes and its complications. The present study was hypothesized to evaluate the anti-diabetic potential of Rosmarinic acid (RA) during high-fat diet (HFD)—streptozotocin (STZ)induced type 2 Diabetes (T2D) in wistar albino rats. Oral administration of RA (100 mg/kg b.w) significantly (p \ 0.05) increased the insulin sensitivity index (ISI0,120), while the levels of blood glucose, HbA1c, advanced glycation end products (AGE), TNF-a, IL-1b, IL 6, NO, p-JNK, P38 MAPK and NF-jB were significantly reduced, with a concomitant elevation in the plasma insulin levels in diabetic rats. Furthermore, RA treatment significantly (p \ 0.05) reduced the levels of triglycerides, FFA and cholesterol in serum, and reduced the levels of lipid peroxides, AOPP’s and protein carbonyls in the plasma and pancreas of diabetic rats. The diminished activities of pancreatic superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) and the decreased levels of plasma ceruloplasmin, vitamin C, vitamin E and reduced glutathione (GSH) in diabetic rats were also significantly (p \ 0.05) recovered upon RA treatment denoting its antioxidant potential which was confirmed by Nrf-2, hemeoxyenase (HO-1) levels. Histological, ultrastructural and immunohistochemical data demonstrate that oral administration of RA protects pancreatic b-cells from oxidative niche in HFD-STZ-induced experimental diabetes. Our findings suggest that the oral treatment with RA alleviates pancreatic b-cell dysfunction J. Govindaraj  S. Sorimuthu Pillai (&) Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, Tamilnadu, India e-mail: [email protected]

and glucolipotoxicity-mediated oxidative stress during HFD-STZ-induced T2DM, perhaps through its antioxidant potential. Keywords Rosmarinic acid  Cytokines  NF-jB  P38 MAPK  p-JNK  Nrf-2

Introduction Chronic oxidative stress mediated by glucolipotoxicity has been implicated to play a cardinal role in the onset and progression of diabetes and its associated complications. A sudden surge in the blood glucose levels along with increased lipid content elicits excessive generation of reactive oxygen species (ROS) [1, 2]. All cells possess a wellconstructed convolute defence system against ROS toxicity. However, the pancreatic b-cells due to the feeble intrinsic antioxidant capacity succumb to the excessive ROS that behaves in a sporadic and destructive fashion [3]. Thus, chronic oxidative stress is reported to be the key mechanism which results in defective insulin gene expression, insulin secretion and increased b-cell apoptosis that eventually leads to unceasing deterioration of pancreatic b-cells [4]. Recently, activation of biochemical pathways including stress-activated signalling pathways of nuclear factor-jB (NF-jB), Jun kinases/stress-activated protein kinases (JNK/SAPK) and p38 mitogen-activated protein (MAP) kinase has been reported during insulin resistance and b-cell dysfunction [5, 6]. Hence, regulating b-cell homeostasis and its protection against oxidative niche are indispensable which calls for an effective antioxidant therapy as an appendage in the management of diabetes and its secondary complications. Antioxidants are the first line of defence against oxidative damage, and are

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crucial for maintaining optimum health and homeostasis. Antioxidant therapy is considered to be a significant pharmacologic prelude for the management of diabetes, as their benefits are not only attributed to its radical quenching but also to their ability to interact with numerous basic cellular activities [7]. Several synthetic oral hypoglycaemic drugs are available, which possess considerable antioxidant capacity and prevent hyperglycemia; compounds with the significant therapeutic potential, which are devoid of any side effects, are currently being explored [8]. Recently, the antioxidant potential of polyphenols was attributed to its ability to activate Nrf-2, an important cellular defence mechanism to cope with oxidative stress. The role of Nrf-2 in protecting the vascular complications in diabetes has been emerging and is reported to regulate lipid metabolism [9]. Hence, evaluating efficacy of drug candidates like polyphenols in context with Nrf-2 functional status will shed insights upon its antioxidant potential and can offer promising avenues in therapeutic management of DM. Rosmarinic acid (RA), a polyphenol, is one such natural antioxidant widely distributed in herbs of the Lamiaceae species such as rosemary, sage, lemon balm and thyme. RA is an ester of 3, 4-dihydroxyphenyllactic acid and caffeic acid [10] (Fig. 1) and possesses a wide array of biological activities such as anti-cancer [11], anti-microbial, anti-allergic [12], anti-inflammatory and neuroprotective properties [13, 14]. The antioxidant efficacy of the phenolic compound is attributed to the presence of four hydroxyl groups, which makes it a potent therapeutic agent against oxidative stress [15]. Although the antioxidant potency of RA is well established, systemic reports are lacking in documenting the protective effect of RA against oxidative stress-mediated pancreatic b-cell dysfunction during experimental T2DM. Recently, we have reported the efficacy of RA in regulating carbohydrate metabolising enzymes in HFD-STZ-induced experimental diabetic rats [16]. The aim of the present study was to evaluate the ameliorative potential of RA against hyperglycemia-induced oxidative stress in HFDSTZ induced diabetic rats. Metformin, an oral anti-hyperglycemic drug with antioxidant potential, was used as a reference drug [17].

Materials and methods Chemicals Rosmarinic acid (RA) was purchased from Sigma Aldrich (St. Louis, USA), Ultra-sensitive ELISA kit for rat insulin was obtained from Linco Research, Inc. St. Charles, MO, Streptozotocin, 2, 2–diphenyl–1–picrylhydrazyl (DPPH); 2, 20–azino–bis (3–ethylbenzothiaz oline-6-sulfonic acid) diammonium salt (ABTS); and all other chemicals used in this study were of analytical grade and were obtained from standard commercial suppliers. DPPH assay The DPPH radical scavenging activity of RA was determined by the method of Brand-Williams et al. (1995) [18] with slight modifications. The methanolic solution of DPPH (60 lM) was mixed with equivalent aliquot of different concentration (3.125–100 lM) of RA in methanol. The samples were treated with the stable DPPH radical in an ethanol solution. The reaction mixture consisted of adding 0.5 mL of sample, 3 mL of absolute ethanol and 0.3 mL of DPPH radical solution 0.5 mM in ethanol. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The mixture of ethanol (3.3 mL) and sample (0.5 mL) serves as blank. The control solution was prepared by mixing ethanol (3.5 mL) and DPPH radical solution (0.3 mL). The changes in colour (from deep violet to light yellow) were read [Absorbance (Abs)] at 517 nm after 100 min of reaction using a UVVIS spectrophotometer (DU 800; Beckman Coulter, Fullerton, CA, USA). ABTS assay ABTS radical scavenging activity of RA was determined according to the method of Re et al., (1999) [19] with slight modifications. In brief, ABTS radical cation (ABTS•?) was produced by mixing ABTS stock solution (7 mM in water) with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature (RT) for 12–16 h before use. Then, ABTS•? solution was diluted with ethanol to an absorbance of 0.7 at 734 nm. To 3.0 ml of diluted ABTS? solution, 30 ll of different concentration (3.125–100 lM) of RA in ethanol was added and after 1 min, and the change in absorbance was measured at 734 nm spectrophotometrically. Superoxide dismutase-like (SOD-like) scavenging activity assay

Fig. 1 Chemical structure of Rosmarinic acid (RA)

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The SOD-like scavenging activity of RA was determined using the method described by Debnath et al. (2011) [20] with

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slight modifications. Briefly, 30 ll of different concentration (3.125–100 lM) of RA was mixed with 200 ll of pyrogallol solution (7.2 mM in water) and 3 ml of 50 mM Tris–HCl buffer, at pH 8.5, containing 10 mM EDTA. After 10 min, 1 ml of 1 N HCl was added to the above mixture to arrest the reactions and the absorbance was measured at 420 nm.

At the end of the experimental period, the animals were fasted overnight with free access to water, anesthetized with ketamine (80 mg/kg b.w. i.p) and euthanized by cervical dislocation. The blood samples were collected with and without anticoagulants for plasma and serum separation, respectively.

Experimental animals

Preparation of pancreatic tissue homogenate

Male albino Wistar rats weighing around 150–170 g were procured from Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai, and were housed under standard husbandry conditions (12-h light and dark cycle, relative humidity 55 %). The rats were fed with balanced diet (Hindustan Lever Ltd, Bangalore, India) and water ad libitum. The rat diet (pellet) composed of 5 % fat, 21 % protein, 55 % nitrogen-free extract, and 4 % fibre (w/w) with adequate mineral, and vitamin was fed to the animals. The experimental design was performed in accordance with the current ethical norms approved by the Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (IAEC NO: 01/01/2013).

Pancreatic tissues from control and experimental groups of rats were excised, rinsed with ice-cold saline and homogenized in Tris–HCl buffer (0.1 M. pH 7.4) using Teflon homogenizer and centrifuged at 12,000 g for 30 min at 4 C. The supernatant was pooled and used for the biochemical estimations. The protein content in the tissue homogenate was estimated by the method of Lowry et al. (1951) [22].

Induction of experimental diabetes The rats were divided into two dietary regimens by feeding either normal or high-fat diet (HFD) for the initial period of 2 weeks [21]. After 2 weeks of dietary manipulation, the groups of rats fed with HFD was injected intraperitoneally with a single low dose of STZ (35 mg/kg b.w) dissolved in 0.1 M cold citrate buffer, pH 4.5. One week after STZ injection, the rats were screened for blood glucose levels. Rats having fasting blood glucose (FBG) [ 250 mg/dl that exhibited random hyperglycaemia and glycosuria were selected for the experimental design. The rats were allowed to continue to feed on their respective diets till the experimental tenure. Experimental design The experimental animals were divided into the four groups; with comprising a minimum of six rats. Group 1 Group 2 Group 3

Group 4

Control rats fed with normal pellet diet HFD ? STZ (i.p. 35 mg/kg b.w. as described above) induced diabetic rats Diabetic rats orally treated with RA (100 mg/kg b.w./rat/day) in aqueous solution (using oral gavage) for 30 days Diabetic rats orally treated with Metformin (200 mg/kg b.w./rat/day) in aqueous solution (using oral gavage) for 30 days

Determination of fasting blood glucose, plasma insulin, HbA1c and ISI0,120 Commercially supplied kits were obtained from Span diagnostic chemicals, Surat, India, and Lincoplex Ltd, St. Charles, MO, for estimating fasting blood glucose and plasma insulin in control and experimental groups of rats, respectively. Furthermore, the level of glycosylated haemoglobin (HbA1c) was also estimated in the control and experimental groups of rats [23], and the insulin sensitivity was also assessed by ISI0-120 analysis [24]. ISI0–120 was determined using plasma glucose and insulin concentrations from 0 min and 120 min OGTT values obtained from control and experimental groups. The ISI0-120 index was calculated using the following formula.  ISI0120 mg l2 =mmol/mIU/min ¼ ½7500mg ðG0  G120 Þ  0:19  m=½120  Gmean  Log ðImean Þ where 7500 mg is oral glucose load in an OGTT, G0 is Fasting plasma glucose concentration (mg/dl), G120 is Plasma glucose concentration in the 120th min of OGTT (mg/dl), m is Body weight (g), 120 is Duration of OGTT min, Imean is Mean plasma insulin concentration during OGTT (mIU/l), Gmean—Mean plasma glucose concentration during OGTT (mmol/l). All the analyses were performed according to proper instructions of the manufacturer. Assay of lipid profile Levels of serum cholesterol, triglycerides and free fatty acids were estimated by the method of Parekh et al. (1970) [25], Foster et al. (1973) [26] and Itaya (1977) [27], respectively.

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Measurement of hyperglycaemia mediated oxidative stress and AGE The levels of non-enzymatic antioxidants such as vitamin C [28], vitamin E [29], GSH [30] and ceruloplasmin [31] were estimated in the plasma of control and experimental groups of rats. The activities of enzymatic antioxidants such as superoxide dismutase (SOD) (Activity of SOD is expressed as units (U) of SOD/mg of protein, where 1 U of SOD is the amount of enzyme that inhibits the auto-oxidation of epinephrine by 50 %) [32], catalase (CAT) (Activity of CAT is expressed as nmol H2O2 decomposed min-1 mg-1 protein) [33], glutathione peroxiase (GPx) (Activity of GPx is expressed as lmol of GSH consumed/min/mg protein) [34] and glutathione-s-transferase (GST) (Activity of GST is expressed as lmol 1-chloro-2,4-dinitrobenzene conjugate formed min-1 mg-1 protein) [35], were determined in the pancreatic tissue homogenate of control and experimental group of rats. Furthermore, the levels of lipid peroxides and protein oxidation products in terms of thiobarbituric acid reactive substances (TBARS), advanced oxidation protein products (AOPP) [36, 37] and protein carbonyls [38] were determined in the plasma and pancreatic tissue homogenate of control and experimental groups of rats. Serum AGE was also measured in control and experimental groups of rats using ELISA kit (Abcam, Cambridge, UK). Assay of TNF-a, IL-1b and IL-6, NF-jB p65 unit and NO Plasma and pancreatic tissues were subjected to spectrophotometric quantification at 450 nm using ELISA kit for analysing the levels of pro-inflammatory cytokines such as TNFa, IL-1b, IL-6, NO and NF-jB p65 (nuclear fractions of pancreatic tissues) in control and experimental groups of rats. Spectrophotometric analysis was performed using standard protocols recommended by ELISA kit suppliers (TNF-a, IL1b, IL-6 from Biosource, and Camarillo, CA and NF-jB p65 from Imgenex, San Diego, CA). The concentration of proinflammatory cytokines in unknown samples was determined using standard plots constructed using standard cytokines. Furthermore, NO levels in plasma and pancreatic tissues of control and experimental groups of rats were also indirectly estimated by determining the nitrite levels using colorimetric method based on the Griess reaction [39]. Histological observation of pancreas A portion of excised pancreatic tissues from control and experimental group of rats were formalin-fixed (10 % neutral buffered formalin), paraffin embedded, and 5-lm sections were used for hematoxylin-eosin (H & E) staining. Paraffin embedded tissue sections were cleared in xylene thrice and utilised

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for staining and analysis. Histological changes in the stained sections of control and experimental groups of rats were viewed under bright field light microscope at 40X magnification. Immunohistochemical analysis Immunohistochemical analysis was performed with standard protocols with slight modifications. Briefly, 5-lm thick paraffin embedded pancreatic tissue sections on poly-Llysine coated glass slides were deparaffinized by placing the slides in an oven at 60 C for 10 min and then rinsed twice in xylene for 5 min each. The slides were then hydrated in graded ethanol series for 10 min each and then finally washed in double distilled water (dd H2O) for 5 min. The sections were then incubated with 1 % hydrogen perodxide (H2O2) in double distilled water for 15 min at 22 C, to quench the endogenous peroxidase activity. Then the sections were rinsed with Tris–HCl containing 150 mM NaCl (pH 7.4) and blocked with blocking buffer (1X Tris buffered saline (TBS), 0.05 % Tween 20, 5 % non-fat dried milk (NFDM)) for 1 h at 22 C. After washing with 1X TBS containing 0.05 % Tween 20, the sections were incubated with anti-insulin primary antibody (1: 3000) (Rabbit polyclonal) overnight at 4 C, followed by incubation with goat anti-rabbit horseradish peroxidise conjugated (HRP) secondary antibody (1:2000) (Banglaore genei, India) for 1 h at room temperature (RT). After washing with 1X TBS containing 0.05 % Tween 20, the immunoreactivity was analysed by staining the sections with 0.01 % DAB and H2O2 for 3 min and the sections were observed (40X) for brown colour formation under bright field in a microscope. Transmission electron microscopy study A portion of pancreas (about 1 mm3) from control and experimental groups of rats were fixed in 3 % glutaraldehyde in sodium phosphate buffer (200 mM, pH 7.4) for 3 h at 48 C. Tissue samples were washed with the same buffer, post-fixed in 1 % osmium tetroxide and sodium phosphate buffer (200 mM, pH 7.4) for 1 h at 48 C. The samples were again washed with the same buffer for 3 h at 48 C, dehydrated with graded series of ethanol and embedded in Araldite. Thin sections were cut with LKBUM4 ultramicrotome using a diamond knife (Diatome, Aldermaston, Berkshire, England), mounted on a copper grid and stained with 2 % uranyl acetate and Reynolds lead citrate [40]. The grids were examined under a Philips EM201C transmission electron microscope (Philips, Eindhoven, Netherland). Protein extraction and Western blotting Total protein, cytoplasm and nuclear extracts of the pancreatic tissues were used for Western blotting. For extracting the

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total protein, 50 mg of each tissue was homogenized in 0.1 M Tris–HCL buffer (pH 7.4) containing protease inhibitor. The protein was quantified by Bradfrod method. SDS-PAGE was performed using equivalent protein extracts (50 lg) from each sample. The resolved proteins were electrophoretically transferred to nitrocellulose membranes. The blots were incubated in 0.1 % TBST containing 5 % BSA for 1 h in room temperature to block nonspecific binding sites. The blot was incubated with respective primary antibodies for p-p54-JNK, t-JNK, p38 MAPK, NF-jB-p65, Nrf-2, HO-1 and b-actin (internal control) overnight at 4 C. All the antibodies were procured from Santa Cruz Biotech, USA. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After extensive washes the membranes were using 3, 30 diaminobenzidine (DAB) chromogen. The band intensity was quantified using Image—J software. Statistical analysis The values are expressed as mean ± standard deviation (S.D.) for 6 rats in each group. Data analysis was performed with SPSS 16 student software. Hypothesis testing method included one-way analysis of variance (ANOVA) followed by post hoc testing performed with least significance difference (LSD). A value of p \ 0.05 was considered to indicate statistical significance.

Results In vitro antioxidant assay The DPPH, ABTS and SOD-like scavenging activity of RA is depicted in Fig. 2a, b, c respectively. DPPH radical scavenging activity was quantified in terms of percentage inhibition of a preformed free radical by antioxidants. There was a significant (p \ 0.05) improvement in the percentage inhibition of the DPPH• radical by RA. ABTS activity was quantified in terms of percentage inhibition of the ABTS? radical cation by antioxidants in each sample. RA showed 84.7 % inhibition at a concentration of 20 lM in DPPH assay and 90.3 % inhibition at a concentration of 40 lM in ABTS assay and 87.6 % inhibition at a concentration of 30 lM in SOD-like activity assay reflecting its radical scavenging capacity. Effect of RA on blood glucose, HbA1c, plasma insulin and AGE Table 1 shows the levels of fasting blood glucose (mg/dl), HbA1c (%Hb), plasma insulin (ng/ml) and AGE (lg/ml) [Methylglyoxal (MG), carboxyethyl-lysine (CEL) and

Fig. 2 Dose dependent effect of RA in scavenging of 2, 2-Diphenyl1-picrylhydrazyl (DPPH) and 2, 2-Azino-bis (3-ethylbenzothiazoline6-sulfonic acid) diammonium salt (ABTS) and SOD radicals

carboxymethyl-lysine (CML)] in control and experimental groups of rats. Conversely, the levels of fasting blood glucose and HbA1c, AGE in diabetic group of rats were significantly (p \ 0.05) increased as compared with control

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group of rats. The level of plasma insulin was significantly decreased (p \ 0.05) in diabetic group of rats showing relative insulin deficiency. However, oral administration of RA as well as metformin to diabetic groups of rats significantly (p \ 0.05) reduced the levels of fasting blood glucose, HbA1c and increased plasma insulin levels when compared with diabetic group of rats. Effect of RA on insulin sensitivity index (ISI0–120) The insulin sensitivity index (ISI0–120) in control and experimental group of rats is depicted in Fig. 3. The insulin sensitivity of the diabetic rats was increased appreciably on treatment with RA denoting its plausible insulin sensitising effects. The (ISI0–120) are expressed in mg l2/mmol/mIU/min.

Effect of RA on enzymic and non-enzymic antioxidants Table 3 depicts the activities of enzymatic antioxidants such as SOD, CAT, GPx and GST in pancreatic tissues of control and experimental groups of rats. The activities were significantly (p \ 0.05) reduced in the pancreatic tissues of diabetic group of rats. Oral administration of RA as well as metformin, significantly (p \ 0.05) attenuated the altered activities of these enzymic antioxidants to near normalcy in pancreatic tissues of diabetic rats. The levels of plasma non-enzymatic antioxidants such as vitamin C, vitamin E, GSH and ceruloplasmin were also shown in Table 3. Diabetic rats showed a significant (p \ 0.05) decrease in these levels when compared to control rats. Conversely, administration of RA as well as metformin to diabetic group of rats significantly (p \ 0.05) increased the levels to near control values.

Effect of RA on serum lipids and FFA levels Table 2 shows the levels of lipids [total cholesterol (mmol/L), triglycerides (mmol/L)] and free fatty acids (lmol/L) in the serum of control and experimental group of rats. The diabetic group showed significantly (p \ 0.05) high lipid profile when compared to the control group. Treatment with RA significantly (p \ 0.05) reduced the levels of lipids and free fatty acids. Effect of RA on LPO, AOPP and PCO Figure 4 shows the levels of lipid peroxides (LPO), advanced oxidation protein products (AOPP) and protein carbonyls (PCO) in plasma and pancreatic tissues of control and experimental groups of rats. A significant (p \ 0.05) increase was noted in the levels of LPO in terms of TBARS, AOPP and PCO in plasma and pancreatic tissues of diabetic group of rats. Treatment with RA and metformin significantly (p \ 0.05) reduced the levels to near normalcy when compared with diabetic groups of rats.

Fig. 3 Effect of RA on the Insulin sensitivity index (ISI0, 120) in the control and experimental group of rats. Values are expressed as Mean ± S.D. for groups of six rats in each group. Statistical significance was determined by one way ANOVA followed by post hoc test LSD. p \ 0.05. *Compared with control; #compared with diabetic rats

Table 1 Effect of RA on the levels of blood glucose, plasma insulin, HbA1c and serum AGE’s in the control and experimental group of rats Groups

Glucose (mg/dl)

Insulin (ng/ml)

HbA1c (%Hb)

Serum AGE’s (lg/ml)

Control

86.92 ± 5.98

0.86 ± 0.036

6.13 ± 0.23

481.20 ± 14.96

Diabetic

293.45 ± 5.21a

0.69 ± 0.052a

12.29 ± 1.14a

938.54 ± 39.41a

b

b

b

755.27 ± 31.72b

b

721.14 ± 25.23b

Diabetic ? RA Diabetic ? Metformin

130.13 ± 6.68

b

113.24 ± 4.76

0.74 ± 0.044

b

0.78 ± 0.051

9.17 ± 0.69 7.24 ± 0.30

The levels of blood glucose, plasma insulin, HbA1c and serum AGE’s in the control and experimental group of rats. Values are given as Mean ± S.D. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Values are statistically significant at p \ 0.05 Statistical significance was compared within the groups as follows a

compared with control

b

compared with diabetic rats

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Mol Cell Biochem Table 2 Effect of RA on the Serum lipid profile levels in the control and experimental groups of rats Groups

TC (mmol/L)

TG (mmol/L)

FFA (lmol/L)

Control

2.37 ± 0.07

0.72 ± 0.09

451.96 ± 14.46

Diabetic

3.87 ± 0.37a

1.45 ± 0.11a

1022.52 ± 47.03a

Diabetic ? RA

2.71 ± 0.19b

0.92 ± 0.05b

858.41 ± 43.77b

Diabetic ? Metformin

2.34 ± 0.10b

0.88 ± 0.04b

793.77 ± 25.40b

The levels of Serum lipid profile (Total cholesterol, Triglycerides, and free fatty acids) in the control and experimental groups of rats. Values are given as Mean ± S.D. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Values are statistically significant at p \ 0.05 Statistical significance was compared within the groups as follows a

compared with control

b

compared with diabetic rats

Fig. 4 Effect of RA on the level of TBARS, AOPP and protein carbonyls in plasma and pancreatic tissues of control and experimental groups of rats. Values are given as Mean ± S.D. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Values are statistically significant at p \ 0.05. Statistical significance was compared within the groups as follows, *compared with control, #compared with diabetic rats

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compared with control

compared with diabetic rats b

a

Statistical significance was compared within the groups as follows

3.66 ± 0.12 Diabetic ? Metformin

SOD one unit of SOD activity is the amount of enzyme required to inhibit auto-oxidation of epinephrine by 50 %. CAT Unit of catalase activity is nmoles of hydrogen peroxide decomposed/ min/mg of protein. GPx Unit of GPx activity is lmol glutathione oxidized/min/mg of protein. GST Unit of GST activity is lmol 1-chloro-2, 4-dinitrobenzene conjugate formed min-1 mg-1 protein

3.78 ± 0.26 Diabetic ? RA

Depicts the activities of pancreatic SOD, CAT, GPx, GST and levels of Plasma Vit C, VIt E GSH and ceruloplasmin in control and experimental groups of rats. Values are given as Mean ± S.D. for groups of six rats in each. One way ANOVA followed by post hoc test LSD. Values are statistically significant at p \ 0.05

9.11 ± 0.31b 28.36 ± 1.21 0.64 ± 0.03 0.88 ± 0.04 4.22 ± 0.23 5.45 ± 0.28 13.01 ± 0.59

9.97 ± 0.65b 25.94 ± 2.17

b b

0.59 ± 0.06 0.81 ± 0.06

b b b

4.19 ± 0.36 5.89 ± 0.47 12.23 ± 0.96

b

4.56 ± 0.41a

b

5.56 ± 0.50a

b

17.94 ± 0.3 5.54 ± 0.17

1.67 ± 0.14a

Control

Diabetic

b

b b b b b

12.32 ± 0.29 34.22 ± 1.22

16.78 ± 2.44a 0.42 ± 0.04a

0.78 ± 0.03 1.54 ± 0.02

0.32 ± 0.03a 1.45 ± 0.14a

5.66 ± 0.08 7.62 ± 0.18

3.12 ± 0.29a

GSH (mg/dl) Vit E (mg/dl) Vit C (mg/dl) GST GPx CAT

Figure 7a–d shows the representative photomicrographs of hematoxylin-eosin-stained pancreatic tissues of control and experimental groups of rats. Figure 6a shows the section of pancreatic tissue of control rats showing normal islets. Figure 7b portrays the section of pancreatic tissues of diabetic group of rats with degenerative changes of islets, characterized by reduction in the number and size of islets, mononulear infiltration, islet atrophy with b-cell destruction. Figure 7c demonstrates the section of pancreatic tissues of diabetic group of rats treated with RA presenting with less marked b-cells degeneration than that of diabetic group of rats. Moreover, several b-cells are well granulated and increased in islets when compared to the pancreatic tissues of diabetic rats. Likewise, the pancreatic tissues of diabetic rats treated with metformin shows the improved number of granulated cells in islets (Fig. 7d) when compared with control group of rats. The ultrastructural changes occurred in pancreatic bcells of control and experimental groups of rats are shown in Fig. 7f–i. Figure 7f represents the electron micrograph of pancreatic b-cell of control group of rats showing the

SOD

Histological and ultrastructural analysis

Groups

Figure 5a, b, c depicts the levels of TNF-a, IL-1b and IL-6 in the plasma of control and experimental group of rats. The levels of TNF-a, IL-1b and IL-6 in diabetic group of rats were significantly (p \ 0.05) increased when compared to control group of rats. However, oral treatment with RA as well as metformin for 30 days to diabetic groups of rats significantly (p \ 0.05) declined the TNF-a levels to near normalcy, while IL-1b and IL-6 levels remain sustained as compared to diabetic rats. We speculate this outcome as a result of enhanced insulin sensitivity in the peripheral tissues during RA treatment which is presumed to elevate circulating cytokine levels [41]. Figure 6a, b, c represents the levels of NF-jB p65 in the nuclear fractions of pancreatic tissues and NO levels in the serum and pancreas of control and experimental groups of rats. The levels of NF-jB p65 unit in pancreatic tissues of diabetic rats were significantly (p \ 0.05) increased when compared with control group of rats. However, oral administration of RA as well as metformin to diabetic group of rats showed a significant (p \ 0.05) decline in the levels of nuclear NF-jB p65 unit. Diabetic rats showed significant increase in the level of nitrite (NO2-) in both serum and pancreatic tissues as compared to normal control rats, whereas the level of nitrite was significantly (p \ 0.05) reduced in diabetic rats treated with RA as well as in groups treated with metformin (Fig. 6b)

Table 3 Effect of RA on the activities of Pancreatic SOD, CAT, GPx, GST and levels of plasma non-enzymatic antioxidants in the control and experimental groups of rats

Effect of RA on TNF-a, IL-1b, IL-6 NF-jB p65 and NO

Ceruloplasmin (mg/dl)

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Fig. 5 Effect of RA on the levels of a TNF-a, b IL-1b, and c IL-6 in plasma of control and experimental groups of rats. Values are expressed as Mean ± S.D. for groups of six rats in each group. Statistical significance was determined by one way ANOVA followed by post hoc test LSD. p \ 0.05. *compared with control; #compared with diabetic rats

normal cellular organelles such as mitochondria, endoplasmic reticulum, Golgi complex, and large number of secretory granules. The granules composed of a central core, usually with moderate homogenous or slightly heterogeneous electron density, and an external singlelayered membrane. The granules had a space between the core and the membrane. The granules were diffusely distributed in the cytoplasm. The electron micrograph of pancreatic b-cells of diabetic group of rats (Fig. 7g) revealed the destruction of b-cell with loss of nuclear envelope and mitochondrial cristae, vacuolization with ballooning appearance of mitochondria as well as dilation

Fig. 6 Effect of RA on the levels of a NF-jB p65 unit b serum NO c pancreatic NO levels in the control and experimental groups of rats. Values are expressed as Mean ± S.D. for groups of six rats in each group. Statistical significance was determined by one-way ANOVA followed by post hoc test LSD. p \ 0.05, *compared with control; # compared with diabetic rats

of the rough endoplasmic reticulum. The electron micrograph (Fig. 7h) apparently shows the pancreatic b-cell protective nature of RA in diabetic group of rats by means of moderate increase in secretory granules, minimal nuclear membrane damage, minimal loss in the cristae with weak swelling of mitochondria and no vacuolarization of cytoplasmic region of b-cell. Likewise, the electron micrograph of b-cell of diabetic group of rats treated with metformin showed similar pattern of b-cell protection (Fig. 7i) as compared with control group of rats.

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Fig. 7 a–d Shows the representative photomicrographs of H&E staining of pancreatic tissues of control and experimental group of rats. Histological photograph of a control, b diabetic pancreas showing mononuclear infiltration, b-cell atrophy and b-cell destruction, c diabetic ? RA treated group and d diabetic ? Metformintreated group at 40X magnification. Localisation of organelles is marked as 1. Glandular acinus, 2. Islet of Langerhans, 3. Interlobular connective tissue septa, 4. Intralobular duct. e Graph shows the histologic scoring b cells of pancreas. Histological staining was assessed and measured objectively by two independent observers and

gave similar results. For quantitation, data expressing the respective stain was quantified by counting the positively stained cells in ten fields/section by three independent observers in blinded fashion, as compared with control and experimental group of rats. Values are given as Mean ± S.D. for groups of six rats in each group. One-way ANOVA followed by post hoc test LSD. *Compared with control, # compared with diabetic rats. f–i Transmission electron micrographs of f control, g diabetic, h diabetic ? RA and i diabetic ? Metformin at 15,000x magnification. Localisation of organelles is marked as 1. Nucleus, 2. Secretory granules, 3. Central vacuole

Effect of RA on Insulin immunohistochemistry

Effect of RA on p54-JNK, p38 MAPK, NF-jBp65, Nrf2 and HO-1 protein expressions by Western blotting analyses

Figure 8a shows the islets with positive insulin immunoreactivity representing the occurrence of normal insulin secreting cells in the pancreas of normal rats. Diabetic rats showed significantly (p \ 0.05) reduced insulin positive cells (Fig. 8b) when compared with normal rats. RA treated diabetic groups showed significant (p \ 0.05) increase in positive insulin immunoreactivity for the presence of insulin with regular brown insulin granules (Fig. 8c, d) when compared to diabetic groups of rats.

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To further assess the signalling molecules associated with oxidative stress-mediated pancreatic dysfunction during HFD-STZ-induced T2DM, protein levels of p54-JNK, p38 MAPK, NF-jBp65, Nrf-2 and HO-1 were analysed by immunoblot analyses. As shown in Fig. 9, there was a marked increase in p-JNK (Fig. 9a), P38 MAPK (Fig. 9b) and NF-jBp65 (Fig. 9c) protein levels in pancreatic tissues

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Fig. 8 a–d Represents the immunohistochemical analysis of insulin in the pancreas of control and experimental group of rats. a Control, b diabetic, c diabetic ? RA and d Diabetic ? Metformin at 40x magnification. e Represents the percentage of the insulin positively stained cells in pancreas. Positive staining was assessed and measured objectively by two independent observers and gave similar results. For quantitation, data expressing the respective stain was quantified by counting the positively stained cells in ten fields/section by three

independent observers in blinded fashion, and the average was used to denote the total no. of positively stained cells, as compared with control and experimental group of rats. Values are given as Mean ± S.D. for groups of six rats in each group. Statistical significance was determined by one-way ANOVA followed by post hoc test LSD. p \ 0.05. *Compared with control, #compared with diabetic rats

of diabetic rats as compared to control group. RA treatment to diabetic rats significantly (p \ 0.05) mitigated p-JNK, P38 MAPK and NF-jBp65 activation. The expressions of nuclear Nrf-2 and HO-1 levels (Fig. 9d) were significantly (p \ 0.05) reduced in diabetic rats as compared to control group of rats. However, HO-1 and nuclear Nrf-2 levels were significantly higher in RA treated rats as compared to diabetic rats which are comparable to metformin treated rats. The schematic representation of the possible mechanism of action of rosmarinic acid in pancreas during HFD-STZ-induced experimental diabetes mellitus in rats is shown in Fig. 10.

reduce the oxidative stress in experimentally induced DM and they exert wide pharmacological properties with minimal side effects and the studies identifying the mechanisms of polyphenol bioactivity has been a subject of considerable interest in the current treatment strategy [45]. The magnitude of the anti-oxidative potential of natural compounds is expressed by various radical scavenging assays. In the present study, RA exhibited potent DPPH, ABTS and SOD-like radical scavenging activity that clearly enlights its possible antioxidant property that would probably be accounted due to the presence of four hydroxyl groups and CH = CH-COOH functional group that ensures greater efficiency compared to other phenolics [46]. An increase in the plasma glucose and decrease in insulin sensitivity index ISI0, 120 along with a partial reduction in insulin levels (due to loss of pancreatic b-cell mass) is observed in the diabetic rats which ascertains the development of hyperglycemia and insulin resistance. In this line, there are previous documented reports which signify that HFD-STZ-induced diabetic rats exhibit pathological features including hyperglycemia, insulin resistance and oxidative stress [47, 48]. Oral administration of RA to the diabetic rats improves ISI0–120 and protects pancreatic beta cell mass thereby increasing insulin levels and reduces hyperglycemia via its ability to improve insulin sensitivity; the effect of which was similar to that observed on metformin administration. Further, an elevation in the levels of FFA, TC and TG observed in the rats exposed to HFD-STZ imposes hyper-triglyceridemia and hypercholesterolemia,

Discussion A critical imbalance in the pro-oxidant/antioxidant niche due to depletion in antioxidant levels and superfluous free radicals due to persistent hyperglycemia is the proximate feature of DM [42]. Diabetic experimental animal models exhibit high oxidative stress due to chronic hyperglycemia, which deplete the activity of ant oxidative defence system resulting in elevated levels of oxygen free radicals [43]. The combined effect of elevation in glucose and FFA in the HFD-STZ-induced diabetic model may be particularly toxic due to the excessive generation of free radicals that interact with the lipid bilayer and produce lipid peroxides, and is a contiguous cause of inevitable deterioration of bcell function [44]. Plant polyphenols has been reported to

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Mol Cell Biochem Fig. 9 Immunoblot analyses of P-JNK, P38 MAPK, NF-jB, Nrf-2 and HO-1 protein expression levels in pancreas tissues of control and experimental groups of rats. L1 Control, L2 Diabetic, L3 Diabetic ? RA treated, L4 Metformin treated. Proteins levels of a P-JNK, b P38 MAPK, c NF-jB d Nrf-2 & HO-1 were determined by Western blotting analyses and b-actin was used as loading control. Bar graph denotes density analysis of protein expressions in control and experimental groups. Values are expressed as Mean ± S.D. for six rats in each group. Statistical significance was determined by one-way ANOVA followed by post hoc test LSD. p \ 0.05. *Compared with control, # compared with diabetic rats

HFD + STZ

Inflammatory Cytokines TNF-α IL1-β, IL-6 Nf-κB

Total cholesterol Triglycerides Free fatty acids

ROS

Lipid Peroxidation AOPP Protein carbonyls Nitic oxide

Enzymic antioxidants

Non-enzymic antioxidants

Nitric oxide

ROS + RNS

Lipid Peroxidation Hyderoperoxides Protein carbonyls

AGE Formation

Nf-κB JNK P38 MAPK

Nrf-2

Glucose autooxidation Glucose amine synthesis

DNA damage Activation of Poly-ADP-ribose synthsae Decrease cellular NAD

Oxidative Phosphorylation Defective Insulin Production & Secretion

Non-enzymic antioxidants Vitamin C Vitamin E Ceruloplasmin

NAD

Reduced Glutathione

Pancreatic – β cell Decreased Plasma Insulin

Glucose Oxidation

Rosmarinic Acid Inhibits Rosmarinic Acid activates

Fig. 10 Schematic overview of the mechanism of action of rosmarinic acid during HFD-STZ-induced experimental diabetes mellitus in rats

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the condition which is reversed upon oral administration of RA. These data indicate the beneficial effects of RA in preventing the diabetic complications which is supported by the previous studies which demonstrates that RA extract regulates glucose metabolism in rats [49]. Persistent elevation in blood glucose results in glycation of haemoglobin (Hb) that leads to the formation of HbA1C [50]. Estimation of HbA1C % has been found to be particularly useful in monitoring the effectiveness of therapy in diabetes [51]. Agents with antioxidant or free radical scavenging power have been shown to impede oxidative reactions allied with glycation [52]. In this regard, RA administration is found to reduce the levels of AGE’s also along with HbA1C in the diabetic rats. AGE’s are modifications of proteins or lipids that become non-enzymatically glycated and oxidised after contact with the aldose sugars [53, 54]. AGE’s have been implicated in both microvascular and macrovascular complications of diabetes and they modify the extracellular matrix, the action of hormones, cytokines and free radicals via engagement of cell surface receptors [55, 56]. Also, the accumulation of AGE upregulates its receptor RAGE which possesses a binding site for NF-jB and IL-6 [57]. This demonstrates the instrumental role of RA which is similar to that of metformin in attenuating diabetes and its related complications by reversing the oxidative stress associated changes. This finding is further supported by report that denotes that plant-derived agents possess anti-glycation activity [58]. Elevated level of lipid peroxides in the plasma of diabetic rats and lipid peroxidation is one of the distinctive features of chronic diabetes and it might be due to inefficient antioxidant system [59]. Increased lipid peroxidation impairs membrane functions by decreasing membrane fluidity and changing the activity of membrane-bound enzymes and receptors. In the present study, the LPO markers measured as TBARS were found to be increased in the plasma and pancreas of the diabetic rats. Parallel to the lipid peroxidation, the oxidative damage to the proteins is also deleterious and may be simultaneous as the oxidation of proteins can lead to a whole variety of amino acid modification [60]. Similar to LPO, the protein oxidation markers are measured as PCO and AOPP in which AOPP is one of the crucial marker implicated in the pathogenesis of diabetes mellitus [61]. Protein carbonyls in diabetic milieu arises due to carbohydrate auto-oxidation may impose loss of cellular functions and alters membrane permeability thereby facilitating glucose transport to extracellular space leading to protein glycosylation [62]. However, the oral administration of RA to the diabetic rats retrieved the levels of lipid peroxides and protein peroxides thereby showcasing its forceful antioxidant and free radical scavenging potential which is one of the prime mechanisms to mitigate consequences of diabetes mellitus.

Endogenous antioxidant enzymes (SOD, CAT, GPx and GST) and diet derived non-enzymic antioxidants (Vit E, Vit C) are responsible for the detoxification of deleterious oxygen radicals [63]. Since, the pancreatic b cells have feeble intrinsic antioxidant capacity, the antioxidant competence of both the enzymic and non-enzymic antioxidants are ruined due to the overwhelmed production of free radicals [64]. In our study, the oral administration of RA restored the levels of both the enzymic antioxidants and the non-enzymic antioxidants including GSH and ceruloplasmin. Here, we believe that the ability of RA to improve enzymic antioxidants is mediated via its ability to activate Nrf-2, while its ability to enhance non-enzymic antioxidants is supposed to be a result of multiple events that were observed in our study. RA reduces oxidative stress by scavenging free radicals, it reduces hyperglycemia thereby reduces glucose-mediated oxidative stress and more importantly RA is a good penetrator of lipid membranes. Hence by this unique ability to penetrate lipid bilayer, it alters membrane fluidity and protects cell membrane against chain braking free radicals, thereby reducing the necessity of non-enzymic antioxidants to scavenge the free radicals and assisting sustained levels of non-enzymic antioxidants (Vit C and E, GSH) during RA treatment in diabetic rats [65]. Chronic oxidative stress due to inexorable generation of ROS during hyperglycaemia activates an array of stresssensitive signalling pathways, including NF-jB [66] whose activation results in the increased expression of numerous gene products that cause cellular damage and play a major role in the aetiology of diabetic complications. Among the various isoforms of NF-jB, p65-p50 is the predominant form in many cell types including pancreatic b-cells. NFjB resides in cytoplasm as a heterodimer of p65/p50 proteins bound to inhibitory jB (IjB) proteins. In diabetic milieu, conditions like cytokines elevation degrades I jB via ubiquitination pathway and allows nuclear translocation of NF-jBp65 subunit to nucleus and induces transcription of genes including TGF-b chemokine ligand2 (CCL2), intercellular adhesion molecule (ICAM) involved in pathogenesis of DM [67]. Hence, agents that could suppress NF-jB activity will serve better against diabetic ailment. In the present study, diabetic rats showed increased nuclear levels of NF-jB p65 unit in the pancreatic tissues of the diabetic rats denoting activation of NF-jB cascade. However, treatment with RA reduced the levels of NF-jB p65 nuclear subunit in diabetic rats, which signifies that RA by alleviating HFD-STZ-mediated hyperglycemic and oxidative stress protects pancreatic b-cells. Cytokines are important mediators in the dysfunction and destruction of pancreatic b-cells [68], the overproduction of which is triggered by superfluous free radicals that are responsible for perpetuated deleterious effects on pancreatic

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islets [69]. Pro-inflammatory cytokines, IL-1b and IL-6 are involved in pathogenesis of DM and its complications. Cytokines IL-1b and IL-6 are transcriptionally activated by NFjB [70]. Report suggests that elevated IL-1b augment ROS generation, while IL-6 aggravates insulin resistance and TNF-a worsens islet cell destruction [64]. Soaring levels of glucose and elevated levels of pro-inflammatory cytokines (IL-1b, IL-6, and TNF-a) potently induce iNOS and produces NO [71], and the surplus generation of NO in cells may restrain mitochondrial metabolism and contribute to elevated lipid peroxidation (LPO), LDL-oxidation, protein modification and inter-nucleosomal DNA cleavage [72]. In our study, we have observed a significant increase in levels of TNF-a, IL-1b, IL-6 and NO in pancreatic tissues of diabetic rats compared to normal rats. In agreement with previous reports [73], our results demonstrate that RA treatment significantly ameliorated the levels of TNF-a, IL-1b, IL-6 and NO near about to normal values in diabetic rats. Thus, RA might exert its effect preventing the production of deleterious cytokines involved in the development of early injury and progression of late complications of diabetes, probably through the inhibition of NF-jB. There is considerable evidence that an increased level of ROS is responsible for hyperglycemia-induced pancreatic b cell dysfunction [74, 75]. Hyperglycemia-induced ROS production triggers several cellular mechanisms including MAPK and NF-jB pathways in pancreatic beta cells. Regarding the intracellular mechanism, a growing body of evidence has suggested that ROS plays an important role in the activation of MAPK cascade in pancreatic b-cell dysfunction [74]. Here, we examined the MAPK signalling molecules to elucidate the effects of RA on intracellular signalling during HFD-STZ-induced diabetes. Consistent with previous reports [76], in our study a marked activation of p38 and p54-JNK was observed in pancreatic tissues of diabetic rats which were reduced upon RA treatment in diabetic rats. The downstream targets of the MAPKs pathway include transcription factors, such as NF-jB, which is activated and has been linked to pancreatic b-cell dysfunction [77]. Activation of Nrf-2 is an adaptative cellular response to ROS-mediated oxidative damage and serves to maintain intracellular redox homeostasis [78, 79]. Nrf-2 is normally sequestered in the cytoplasm with its inhibitor Keap-1. However, various agents like weak electrophiles and plant phytochemicals alters Nrf-2/Keap-1 complex thereby releasing Nrf-2 to nuclear translocation and binds to promoter of antioxidant response element (ARE) genes and induces their transcription [80, 81]. Reports denotes that STZ-induced diabetes in Nrf-2-null mice exhibited oxidative, nitrostative stress with elevated blood glucose levels, highlights the involvement of Nrf-2 in pathophysiology of DM [82]. In our study, we have demonstrated that Nrf-2

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nuclear levels are weaker in diabetic rats; in contrast RAtreated diabetic rats showed elevated nuclear Nrf-2 levels, which are further supported by elevated by elevated HO-1 levels, a transcriptional product of Nrf-2. This denotes that RA exerts antioxidant role by inducing Nrf-2 activation. This finding agrees with the previous study that demonstrated Rosmarinic acid activates Nrf-2 expression [83]. The pancreatic tissue protective nature of RA in control and experimental groups of rats was further demonstrated by histological as well as ultra structural studies, respectively. Significant morphological changes including breakdown of micro-anatomical features, destruction pancreatic b-cells and ultimate loss of nuclear envelope and mitochondrial cristae, were observed in diabetic condition. Oral treatment of RA to diabetic group improved the degenerative changes and improved the structural and functional integrity of pancreatic b-cells. Immunohistochemical staining of pancreatic sections of diabetic rats treated with RA showed protected b-cell mass and the presence of insulin secretory granules substantiates the protective efficacy of RA against experimentally induced T2DM.

Conclusion Rosmarinic acid attenuated DM in HFD-STZ-induced type 2 diabetic rats through restoration of pancreatic b-cell function. The demonstrated results provide further insights into the mechanisms of HFD-STZ-induced T2DM and highlights role of NF-jB, MAPK components in pathogenesis of DM. Furthermore the present study suggests that anti-diabetic action of RA might be related to its activation of Nrf-2 and inhibition of NF-jB, MAPK expression. Hence, RA may be considered as adjuvant therapeutic agent for the treatment of DM. However, further studies are greatly warranted to establish the above mentioned suggestions. Acknowledgments The Research Fellowship of the University Grant Commission (UGC), New Delhi, India, in the form of UGCBSR-RF to the first author Mrs. G. Jayanthy is gratefully acknowledged. Conflict of interest The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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Rosmarinic acid modulates the antioxidant status and protects pancreatic tissues from glucolipotoxicity mediated oxidative stress in high-fat diet: streptozotocin-induced diabetic rats.

Persistent hyperglycemia and elevated levels of free fatty acids (FFA) contribute to oxidative stress, a proximate cause for the onset and progression...
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