Effects of Berberine chloride on the liver of streptozotocin-induced diabetes in albino Wistar rats

Biomedicine & Pharmacotherapy 99 (2018) 227–236

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Effects of Berberine chloride on the liver of streptozotocin-induced diabetes in albino Wistar rats

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Govindasami Chandirasegarana, Chakkaravarthy Elanchezhiyana, , Kavisa Ghoshb a b

Department of Zoology, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India Unit of Toxicology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Diabetes Oxidative stress Inflammation Apoptosis

The goal of the present study is to evaluate the effect of Berberine chloride (BC) on the liver of streptozotocin (STZ) induced diabetic rat. Diabetic rats were treated with BC (50 mg/kg b.w) or glibenclamide (6 mg/kg b.w), daily for 45 days. After BC treatment in diabetic rats, there was a significant (P < 0.05) decline in the levels of hepatic markers, lipid peroxidation markers such as lipid hydroperoxides (LOOH) and thiobarbituric acid reactive substances (TBARS), and pro-inflammatory mediators like tumor necrosis factor-alpha (TNF-α), phosphorylated nuclear factor kappa-B-p65 (phospho-NF-κB p65), cyclooxygenase (COX-2), nitric oxide synthase (iNOS) as well as pro-apoptotic mediators such as Bax and cytochrome c. A significant (P < 0.05) increase in hexokinase, glucose-6-phosphate dehydrogenase, enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and non-enzymatic antioxidants such as glutathione (GSH), vitamin E and vitamin C, as well as anti-apoptotic protein Bcl-2 were observed in the liver of BC treated diabetic rats. Thus, from these findings, it can be concluded that the administration of BC notably recovered the liver from hyperglycemia induced antioxidant imbalance, inflammation and apoptosis as well as rectified the imbalance in carbohydrate metabolizing enzymes.

1. Introduction Diabetes mellitus is an endocrine disease characterized by chronic hyperglycemia linked with irregularities in carbohydrate, lipid and protein metabolism caused by insufficiency of insulin secretion and/or insulin action, and usually accompanied by a variety of microvascular, macrovascular, neurologic and infectious complications [1]. Globally, the diabetic population had raised from 108 million in 1980 to 422 million in 2014 in which the age group of above 18 years old, has raised from 4.7% in 1980 to 8.5% in 2014 [2]. According to IDF 2015, the diabetic population is expected to rise to 642 million people in 2040, and every 6 s a person dies due to diabetes. In India alone, 69.2 million people have suffered from diabetes, and this will increase to 123.5 million in 2040 [2]. Physiologically, glucose level is regulated by the equilibrium between hepatic glucose production (gluconeogenesis and glycogenolysis) and utilization of glucose by peripheral tissues [3]. Increased hepatic glucose production due to lack of insulin secretion/action is the major cause of hyperglycemia in diabetes [3]. Hyperglycemia promotes oxidative stress, inflammation and apoptosis in tissues [4,5]. Glucose autooxidation and protein glycosylation [6] are responsible for the

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production of free radicals in tissues [7] that promote inflammation and apoptosis [8]. Inflammation can also stimulate more oxidative stress in tissues [9]. Berberine is a plant isoquinoline alkaloid that can be found in many herbs like Hydrastis Canadensis (goldenseal), Rhizoma coptidis (Huanglian), Cortex phellodendri (Huangbai), Coptis chinensis (Coptis or golden thread), Berberis vulgaris (barberry), Berberis aquifolium (Oregon grape), and Berberis aristata (tree turmeric) [10]. All these herbs have long been used in Chinese and Ayurvedic medicines, and BC is the major phytocompound found in these herbs [11]. BC has been used for its various pharmacological activities like anti-microbial, anti-diarrheal, anti-protozoal, anti-trachoma activity, and anti-schistosomal activity [10,12,13]. Berberine has also been reported to be a promising candidate for the treatment of cardiac disorders, hyperlipoidemia and chronic inflammation diseases [11]. The classical technique utilized for berberine isolation is either extraction by alcohol in a neutral medium or by the addition of acetic acid. Further, the purification of berberine is obtained commonly by precipitation as berberine chloride or hydrosulphate [14]. Earlier Moghaddam et al. [15] and Chandirasegaran et al. [16] reported that BC has an ameliorating effect towards STZinduced diabetic rats. Therefore, the goal of this study was to evaluate

Corresponding author. E-mail address: [email protected] (C. Elanchezhiyan).

https://doi.org/10.1016/j.biopha.2018.01.007 Received 21 September 2017; Received in revised form 27 December 2017; Accepted 3 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

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2.5.1. Estimation of liver glycogen Liver glycogen was evaluated by the method of Shirwaikar et al. [22].

the effect of BC on hepatic glucose production, oxidative status, inflammation and apoptosis in the liver of STZ-induced diabetes albino Wistar rats.

2.5.2. Assay of hepatic marker enzymes The hepatic marker enzymes like aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in serum were estimated using diagnostic kits (Span Diagnostics Ltd, Surat, Gujarat, India).

2. Materials and methods 2.1. Chemicals Monoclonal antibodies for pNF-κB p65, iNOS, COX-2, Bcl-2, and cleaved caspase-3 and secondary antibodies were purchased from Cell Signalling Technology, Inc. (CST) (Danvers, Massachusetts). Antibodies for Bax and cytochrome c were purchased from BioLegend Inc. (San Diego, CA) and Abcam, respectively. BC and STZ were procured from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade and were purchased from Himedia, India.

2.5.3. Determination of carbohydrate metabolizing enzymes Activities of hepatic hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase and fructose-1, 6-bisphosphatase were measured by the methods of Brandstrup et al. [23], Ellis and Kirkman [24], Koide and Oda [25] and Gancedo and Gancedo [26], respectively.

2.2. Animals

2.5.4. Estimation of lipid peroxidation and antioxidants Levels of TBARS and LOOH in the liver were measured by the method of Ohkawa et al. [27]. The activity of SOD was evaluated by the method of Kakkar et al. [28]. The activity of CAT enzyme was measured by the method given by Sinha [29]. GPx was evaluated by the method given by Rotruck et al. [30]. The activity of GSH was assessed by the method of Ellman [31]. Vitamin C and E were evaluated by Omaye et al. [32] and Baker and Frank [33] respectively.

Healthy male albino Wistar rats (180–190 g b.w) obtained from the Central Animal House, Annamalai University were housed in polycarbonate cages and maintained under constant 12 h light and dark cycle, and room temperature at 25 ± 2 °C. Rats were fed with standard rodent pellet food (Hindustan Lever Ltd, Mumbai, India) and water was provided ad libitum. Before the start of the experiment, animals were acclimatized to laboratory condition for one week. This study was approved by the Animal Ethics Committee of Rajah Muthiah Medical College and Hospital (Reg No 166/1999/CPCSEA, Proposal No. 1085).

2.6. Histopathology The liver tissue was fixed in 10% formalin for 48 h. It was then followed by dehydration by passing through a series of graded alcohol, beginning with 50% alcohol and progressing in graded step to 100% (absolute) alcohol, and was finally embedded in paraffin. Sections of the liver (5–6 μm thick) were developed using semi-automated rotator microtome, stained with Hematoxylin and Eosin dye and observed microscopically.

2.3. Induction of diabetes in male albino Wistar rats Diabetes was induced in rats by administrating a single dose of intraperitoneal injection of STZ (40 mg/kg b.w) in a buffer (0.1 M citrate buffer, pH 4.5) [17,18]. The STZ treated rats were allowed to drink 5% glucose solution for preventing the drug-induced hypoglycemia. After 3 days of STZ injection, blood was collected from experimental animals from the tail vein, and the blood glucose level was measured. STZ treated rats with fasting blood glucose level of above 230 mg/dl were considered diabetic and used for further study [18].

2.7. Western blotting analysis Total protein from the liver tissue was extracted by homogenizing tissue with 1 ml of a buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and 10 μl protease inhibitor cocktail. The homogenate was then centrifuged at 8000 × g for 2 min at 4 °C, the supernatant was collected and stored at −80 °C. The protein concentration of the supernatant was determined by the method of Lowry et al. [34]. SDS-PAGE was performed using equivalent protein extracts (50 μg) from each sample. The resolved proteins obtained were then electrophoretically transferred to poly vinylidene difluoride membranes. The blots were incubated in 1 × PBS containing 5% non-fat dry milk for 2 h to block nonspecific binding sites. The blots were further incubated with 1:200 dilution of primary antibodies overnight at 4 °C. After washing, the blots were with 1:1000 dilution of horseradish peroxidase-conjugated secondary antibody for 45 min at room temperature. After continuous washes with high and low salt buffers, the immunoreactive proteins were visualized using enhanced chemiluminescence detection reagents (Sigma-Aldrich). Densitometry was performed on IISP flat bed scanner and quantitated with Total Lab 1.11 software.

2.4. Experimental design A total of 24 rats were divided into four groups of six animals each (6 normal rats and 18 diabetic rats). The optimal dosage of BC 50 mg/ kg b.w was fixed based on previous experiments by Chandirasegaran et al. [19]. Group 1- Normal control rats Group 2- Diabetic control rats Group 3- Diabetic + BC (50 mg/kg b.w) treated rats Group 4- Diabetic + Glibenclamide (Reference drug) (6 mg/kg b.w) treated rats [20] BC or glibenclamide were dissolved in distilled water and administered to STZ induced diabetic rats orally by intragastric intubation, daily for 45 days. The experimental design was based on the previous work by Ramachandran et al. [21]. 2.5. Biochemical analysis

2.8. Immunohistochemistry On the morning of 46th day, all the experimental rats were sacrificed by cervical decapitation. The blood was collected from experimental animals by cardiac puncturing and serum was separated for analyzing hepatic marker enzymes. The liver was harvested from all experimental rats, washed with ice-cold saline. The harvested liver was used for histopathology and estimation of lipid peroxidation and antioxidant status, as well as the levels of carbohydrate metabolizing enzymes.

Immunohistochemistry was performed by using super-sensitive polymer-HRP detection system kit, from Biogenex, USA. Briefly, the tissue sections (pancreas) of 4–5 μm were mounted on poly-L-lysinecoated glass slides. They were deparaffinized by placing the slides in an oven at 60 °C for 10 min and then rinsed twice in xylene for 10 min each. The slides were then hydrated in series of graded ethanol (60, 80 228

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and 100%) for 10 min each and finally in double-distilled water for 10 min. Endogenous peroxidases were blocked by exposure to 0.5% hydrogen peroxide. Further, the sections were washed with PBS and incubated overnight with primary antibodies (insulin, caspase 3 and iNOS). The sections were washed in PBS and incubated with biotinylated secondary antibody for 30 min. Then they were incubated for 20 min with 3,3′-diaminobenzidine (DAB). Further, a counterstaining with haematoxylin-eosin was performed. The binding of antibodies was evaluated by examining the slides under a high-power light microscope (LABOMED, No: LX 300) and photomicrographs were recorded at a magnification of 100×.

and experimental rats. TBARS and LOOH levels were significantly (p < 0.05) elevated in diabetic control rats when compared to normal control rats, whereas diabetic rats treated with BC or glibenclamide significantly inhibited the elevated level of TBARS and LOOH as compared with diabetic control rats. 3.5. Effect of Berberine chloride on enzymatic antioxidant enzymes The activity of antioxidant enzymes such as SOD, CAT and GPx was significantly declined in diabetes-induced rats when compared with normal control rats. BC or glibenclamide treated diabetic rats showed significant improvement in the activity of SOD, CAT and GPx in the liver tissue compared to diabetic control rats (Table 2).

2.9. Statistical analysis All data obtained were expressed as mean ± S.D. Statistical significance was evaluated by using one-way ANOVA followed by Tukey multiple comparison test (SPSS program; version 15). P value < 0.05 was considered as significant.

3.6. Effect of Berberine chloride on non-enzymatic antioxidants Table 2 shows the level of non-enzymatic antioxidants in the liver tissue of control and experimental rats. The GSH, vitamin C and E levels declined significantly in diabetic control rats when compared to normal rats. These abnormal levels of non-enzymatic antioxidants were significantly improved to near normal levels in BC or glibenclamide treated diabetic rats.

3. Results 3.1. Effect of Berberine chloride on liver glycogen Fig. 1 shows the level of liver glycogen in normal and experimental rats. The level of liver glycogen was reduced in diabetic control rats when compared to control rats. Treatment with BC had significantly improved glycogen levels in the liver of diabetic rats when compared to diabetic control rats.

3.7. Effect of Berberine chloride on histology of liver Fig. 3 shows the photomicrographs of Hematoxylin and Eosin stained liver tissue sections of control and experimental rats. Diabetic control rats exhibited deformed cellular organization, vacuolization, hypertrophic cells, inflammation, accompanied with the widening of intercellular sinusoids and congestion in the central vein. Diabetic rats treated with BC (50 mg/kg b.w) or glibenclamide showed a lower level of abnormal changes compared to diabetic rats.

3.2. Effect of Berberine chloride on hepatic markers Fig. 2 shows the level of hepatic markers such as AST, ALT and ALP in the serum of control and experimental rats. In diabetic control rats, the level of AST, ALT and ALP were significantly (p < 0.05) elevated when compared to normal rats. BC (50 mg/kg b.w) or glibenclamide treated groups had a significantly lower level of the hepatic markers than in diabetic control rats.

3.8. Effect of Berberine chloride on TNF-α, NF-κB p65, phospho-NF-κB p65, COX-2 and iNOS protein expressions Fig. 4 depicts the protein expression of TNF-α, NF-κB p65, phosphoNF-κB p65, COX-2 and iNOS in the liver of control and experimental rats. The diabetic control rats exhibited an increased level of TNF-α, NF-κB p65, phospho-NF-κB p65, COX-2 and iNOS protein levels when compared to normal control rats. The administration of BC (50 mg/kg b.w) or glibenclamide in diabetic rats resulted in a significant decline in the levels of TNF-α, NF-κB p65, phospho-NF-κB p65, COX-2 and iNOS proteins compared to diabetic rats.

3.3. Effect of Berberine chloride on carbohydrate metabolizing enzymes Table 1 depicts the level of carbohydrate metabolizing enzymes in control and experimental rats. The activity of hepatic hexokinase and glucose-6-phosphate dehydrogenase were significantly declined, whereas glucose-6-phosphatase and fructose-1, 6-bisphosphatase activity were significantly elevated in diabetic control rats. The abnormal levels of carbohydrate metabolizing enzymes were found to be significantly improved by BC (50 mg/kg b.w) or glibenclamide treatment than in diabetic control rats.

3.9. Effect of Berberine chloride on Bax, Bcl-2 and cytochrome c protein expression The western blot result of Bax, Bcl-2 and cytochrome c in the liver of control and experimental rats are shown in Fig. 5. The protein expression of Bax and cytochrome c were notably elevated, whereas Bcl-2 declined in diabetic control rats. In BC treated diabetic rats, Bax and

3.4. Effect of Berberine chloride on lipid peroxidation Table 2 represents TBARS and LOOH levels in the liver of control

Fig. 1. Effect of Berberine chloride on liver glycogen level in diabetic rats. All the data are expressed as the mean ± S.D. for 6 rats. The results with different superscripts (a,b,c..) in each experimental groups are significantly different at p < 0.05.

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Fig. 2. Effect of Berberine chloride on the hepatic markers in diabetic rats. All the data are expressed as the mean ± S.D. for 6 rats. The results with different superscripts (a,b,c..) in each experimental groups are significantly different at p < 0.05.

Table 1 Effect of Berberine chloride on the liver carbohydrate metabolizing enzymes in diabetic rats. All the data are expressed as the mean ± S.D. for 6 rats. The results with different superscripts (a,b,c..) in each experimental groups are significantly different at p < 0.05. Groups

Hexokinase (U*/h/ mgprotein)

Normal control Diabetic control D + BC (50 mg/kg) D + GC (6 mg/kg)

0.34 0.12 0.21 0.26

± ± ± ±

0.03a 0.01b 0.02c 0.02d

Glucose-6-phosphate dehydrogenase (U#/mg protein) 4.78 2.67 3.45 3.98

± ± ± ±

0.36a 0.20b 0.26c 0.32d

Glucose-6 phosphatase (Unit@/min/ mg protein) 5.16 7.92 6.05 5.41

± ± ± ±

0.39a 0.61b 0.46c 0.41a

Fructose 1,6-bis phosphatase (Unit$/h/ mg protein) 13.01 25.46 16.64 13.06

± ± ± ±

0.99a 1.95b 1.27c 1.00a

D – Diabetic rat; BC- Berberine chloride; GC- Glibenclamide. Table 2 Effect of Berberine chloride on lipid peroxidation markers, enzymatic and non-enzymatic antioxidants in the liver of diabetic rats. All the data are expressed as the mean ± S.D. for 6 rats. The results with different superscripts (a,b,c.) in each experimental groups are significantly different at p < 0.05. Parameters

Normal control

Diabetic control

D + BC (50 mg/kg b.w)

D + GC (6 mg/kg b.w)

Lipid peroxidation markers TBARS1 LOOH2

0.89 ± 0.07a 74.10 ± 5.64a

3.76 ± 0.29b 121.02 ± 9.26b

1.81 ± 0.14c 86.79 ± 6.61c

1.57 ± 0.12c 77.30 ± 5.92ac

Enzymatic antioxidants SOD3 CAT4 GPx5

9.54 ± 0.73a 81.03 ± 6.17a 11.01 ± 0.84a

4.48 ± 0.34b 52.69 ± 4.03b 4.88 ± 0.37b

7.85 ± 0.60c 68.73 ± 5.23c 9.40 ± 0.72c

8.89 ± 0.68a 74.30 ± 5.69ac 10.30 ± 0.79ac

Non-enzymatic antioxidants GSH6 Vitamin C7 Vitamin E7

13.06 ± 0.99a 0.86 ± 0.07a 5.74 ± 0.44a

8.02 ± 0.61b 0.41 ± 0.03b 3.66 ± 0.28b

11.56 ± 0.88c 0.69 ± 0.05c 4.55 ± 0.35c

12.48 ± 0.95ac 0.80 ± 0.08a 4.92 ± 0.38c

D – Diabetic rat; BC- Berberine chloride; GC- Glibenclamide. 1 TBARS in tissues were expressed as μmoles/g tissue. 2 LOOH in tissues were expressed as x 10−5 mmoles/g tissue. 3 SOD for tissues were expressed as 50% inhibition of nitroblue tetrazolium reduced in 1 min/mg protein. 4 CAT for tissues were expressed as μmoles of H2O2 consumed/minute/mg protein. 5 GPx for tissues were expressed as μg of GSH consumed /minute/mg protein. 6 GSH for tissues were expressed as μg/mg protein. 7 Vitamin E and vitamin C for tissues were expressed as μmole/mg tissue.

in the immunoreactivity of iNOS and cleaved caspase-3 were observed compared to diabetic control rats.

cytochrome c protein expression were significantly declined, and Bcl-2 protein expression was found to be improved when compared diabetic control rats. The protein expression levels observed in BC treated diabetic rats were found to be similar to glibenclamide treated diabetic rats.

4. Discussion In the diabetic condition, high level of glucose (hyperglycemia) is generated, due to deficiency or inactivity of insulin, which can cause increased oxidative stress, accompanied by inflammation and apoptosis [8]. Hence, in our study, we have investigated the efficacy of BC treatment over hyperglycemia-induced complications on the liver of STZ induced diabetic rats. In this study, STZ was used for the induction of diabetes, as STZ selectively destroys β-cells of the pancreas, without affecting other cells by generating excess ROS and carbonium ion (CH3+). This action of

3.10. Effect of Berberine chloride on immunohistochemical staining of iNOS and cleaved caspase-3 in the liver Figs. 6 and 7 illustrate the immunohistochemical photomicrographs of iNOS and cleaved caspase-3 in the liver of control and experimental rats. In the diabetic control rats, iNOS and cleaved caspase-3 immunoreactivity were found to be augmented in the liver than normal control rats. In BC and glibenclamide treated diabetic rat liver, a decline 230

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Fig. 3. a) Effect of Berberine chloride on histology of the liver in diabetic rats: Photomicrographs of histological changes in hematoxylin and eosin (H & E) stained liver sections of control and experimental rats (X 100). A & D - Control and glibenclamide treated rats liver showing clear central vein (CV). All hepatocytes are arranged as trabeculae from the central portal vein, which are separated by sinusoids (S), B - STZ treated rat liver showing deformed cellular organization accompanied with vacuoles (V), hypertrophic cells, cellular damage (CD), inflammation, widening of intercellular sinusoids (S) and congestion in the central vein (CV). C - BC treated STZ rat liver showing near normal hepatocyte arrangement and sinusoids (S) accompanied with mild central vein congestion (CV). b) Histopathology scoring of hepatic tissue in control and experimental animals.

Fig. 4. Western blot analysis of TNF-α, pNF-κB p65, COX-2 and iNOS in the liver of diadetic rats. A. Representative immunoblot analysis. Protein samples (50 μg/lane) resolved on SDS-PAGE was probed with corresponding antibodies. β-Actin was used as loading control. B. Densitometric analysis. The mean protein expression from control lysates for five determinations was designated as 100% in the graph. Mean ± SD of six determinants is represented in graph for each group. ♣ Significantly different from untreated control (p < 0.05). * Significantly different from diabetic (D) animals (p < 0.05).

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Fig. 5. Western blot analysis of Bax, Bcl-2 and cytochrome c protein expression in the liver of diabetic rats. A. Representative immunoblot analysis. Protein samples (50 μg/lane) resolved on SDS-PAGE was probed with corresponding antibodies. β-Actin was used as loading control. B. Densitometric analysis. The mean protein expression from control lysates for five determinations was designated as 100% in the graph. Mean ± SD of six determinants is represented in the graph for each group. ♣ Significantly different from untreated control (p < 0.05). * Significantly different from diabetic (D) animals (p < 0.05).

STZ finally leads to DNA breaks by alkylation of DNA bases and oxidative damages to cellular components and cell death [35]. At a dose of 40 mg/kg b.w of STZ, there is a partial damage to the β-cells of the pancreas, resulting in a decline in insulin secretion [17,36]. Due to this persistent decline in insulin level, the condition leads to the hyperglycemia [37]. The treatment with a naturally available compound that has free radical scavenging and antioxidant activities may protect the pancreatic islets against the STZ induced diabetic effects [7]. In our previous study, we have reported that BC treatment (50 mg/kg b.w) for 45 days in diabetic rats substantially declined the levels of blood glucose, while plasma insulin was significantly improved when compared to diabetic control rats [38]. In the present study, we investigated the modulatory effect of BC, (50 mg/kg b.w), for 45 days, on hepatic oxidative stress markers, inflammation and apoptosis imbalance in STZ induced diabetic rats. Glycogen is the primary intracellular storable type of glucose. The synthesis of glycogen by the liver is impaired in diabetic condition due to the irregular mechanism of glycogen synthase system and insulin synthesis mechanisms [39]. Treatment with BC significantly increased the liver glycogen levels in diabetic animals, which may be the result of increased insulin levels in the BC or glibenclamide treated diabetic rats, thus revealing a recovered abnormal system of glycogen synthesis system in the liver of diabetic rats. The hepatic markers like AST, ALT and ALP were found to be increased significantly in STZ induced diabetic controls, which might be due to increased inflammation and necrosis of the liver cells that was contributed by the increased glucose levels in the blood circulation. Therefore, an increase in the levels of these markers is primarily the indicator of liver damage, as these enzymes leak out from the cytosol of hepatocytes into circulation, indicating the hepatotoxic effect of STZ [40]. The lowered levels of these hepatic markers in the BC (50 mg/kg b.w) treated diabetic rat revealed the hepatoprotective property of BC. Impaired antioxidant system, increased oxidative stress and lipid

peroxide-mediated damages are hallmarks in the pathogenesis of diabetes mellitus [41,42]. Increased endogenous peroxides may initiate uncontrolled lipid peroxidation, thus leading to cellular infiltration and cell damage [43]. Increased lipid peroxidation impairs membrane functioning by decreasing membrane fluidity and induces free radicalinduced membrane lipid peroxidation, causing increased membrane rigidity and decreased cellular deformability, which collectively leads to the impairment of cell's overall functioning [44]. TBARS and LOOH are common lipid peroxidative markers that are found elevated in STZ induced diabetic rat model [45]. In this investigation, diabetic control rats showed increased levels of TBARS and LOOH. These results indicate elevated levels of oxidative stress in the liver of diabetic control rats. Treatment with BC in diabetic rats significantly reduced the levels of TBARS and LOOH in the liver, indicating antioxidant properties of BC. SOD, CAT and GPx are antioxidant enzymes that function as blockers of the free radical process. SOD is capable of reducing the superoxide radical into hydrogen peroxide (H2O2). The enzymatic antioxidant CAT catalyzes the reduction of hydrogen peroxides and protects the tissues against reactive hydroxyl radicals [46]. In addition, when the cell has increased levels of SOD without a proportional increase in peroxidases (GPx), cells face a peroxide overload challenge. Furthermore, peroxide can react with transitional metals and generate the most harmful hydroxyl radicals [47]. In diabetes mellitus, the high glucose level can inactivate antioxidant enzymes like SOD, CAT and GPx by glycating these proteins and thus initiating oxidative stress, which in turn causes lipid peroxidation [48]. Previously Zhou et al. [49] reported that BC could improve the levels of CAT, SOD and GPx activities in diabetic rats. Therefore, we believe that the near normal levels of CAT, SOD and GPx activities in BC treated diabetic rats strongly indicate the efficacy of BC in attenuating the oxidative stress in diabetic rats. Glutathione (GSH) is a tripeptide, intracellular non-enzymic 232

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Fig. 6. a) Effect of Berberine chloride on immunohistochemical staining of iNOS in the liver of diabetic rats (100 X): (A) Normal control rats, (B) Diabetic control rats, (C) BC treated diabetic rats and (D) Glibenclamide treated diabetic rats. b) Intensity of immunoreaction of iNOS in the liver of control and experimental rats: Intensity of the immunoreaction in each group was measured by the image analysing system (VNT, Beijing, China) and recorded with the range from 0 to 10, Mean ± SD. Each value is mean ± SD for six rats in each group. In each bar, means with different superscript letter (a–c). Significance was accepted at p < 0.05 (Tukey multiple comparison test). D: Diabetic control rats; BC: Berberine chloride (50 mg/kg b.w); G: Glibenclamide (6 mg/kg b.w).

animals expressed near normal levels of GSH, vitamin C and E, which displayed BC's potential to restore the non-enzymatic antioxidant reserves in the diabetic animals. The microscopic photomicrograph sections of the liver of STZ induced diabetic rats showed damage to central vein and surrounding portal triad. Administration of BC recovered the histoarchitecture of the liver to near normal. Thus, these findings collectively suggest that the BC treatment protects the liver from STZ-induced oxidative stress, which can be imparted to its antioxidant activity. The liver is the main site of endogenous glucose production, with only a minor contribution from the kidneys through gluconeogenesis and/or through glycogenolysis [59]. Glycolysis and gluconeogenesis are the two primary and complementary events, balancing the glucose load in the body, which is mainly regulated by insulin. Thus, insulin prevents hyperglycemia, in part, by suppressing hepatic gluconeogenesis and glycogenolysis and facilitating hepatic glycogen synthesis [60]. Hexokinase is the first enzyme of glycolysis, which phosphorylates glucose by transferring phosphoryl group from ATP to form glucose-6phosphate. Induction of diabetes in rats by STZ treatment leads to

antioxidant that protects the cellular system from adverse effects of lipid peroxidation. It directly scavenges free radicals and acts as a cosubstrate for peroxide detoxification by glutathione peroxidases [50]. Increased oxidative stress due to a significant increase in aldehydic products of lipid peroxidation could probably decrease GSH availability in the cell [51]. In the present study, treatment with BC resulted in the elevation of the GSH levels, which in turn protects the cell membrane from the oxidative damage by restoring the redox status [52]. Vitamin C plays a vital role in the antioxidant system, which protects all lipids from undergoing oxidation. It also helps in diminishing the number of apoptotic cells [53] and helps to regenerate vitamin E from its oxidized state [54]. On the other hand, Vitamin E is a non-enzymatic antioxidant, which aids in inhibiting chain reactions associated with lipid peroxidation [55]. Vitamin E is also reported to be very effective in the glycemic control and also helps in lowering the level of HbA1c, the glycated haemoglobin used as a marker to measure average blood glucose [56]. Several reports indicate that the reductions in the levels of non-enzymatic antioxidants are associated with STZ induced diabetic rats [57,58]. In our study, BC administration in STZ induced diabetic 233

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Fig. 7. a) Effect of Berberine chloride on immunohistochemical staining of cleaved caspase-3 in the liver of diabetic rats (100 X): (A) Normal control rats, (B) Diabetic control rats, (C) BC treated diabetic rats and (D) Glibenclamide treated diabetic rats. Diabetic control rats compared with normal control rats and BC treated diabetic rats compared with diabetic control rats. b) Intensity of immunoreaction of cleaved caspase-3 in the liver of control and experimental rats: Intensity of the immunoreaction in each group was measured by the image analysing system (VNT, Beijing, China) and recorded with the range from 0 to 10, Mean ± SD. Each value is mean ± SD for six rats in each group. In each bar, means with different superscript letter (a–c). Significance was accepted at p < 0.05 (Tukey multiple comparison test). D: Diabetic control rats; BC: Berberine chloride (50 mg/kg b.w); G: Glibenclamide (6 mg/kg b.w).

cytotoxicity, necrosis and degeneration of β-cells, which contribute to the deficiency in insulin secretion. This reduced insulin secretion results in decreased glycolysis and glucose utilization for energy production, which in turn impairs hexokinase activity [61]. In this study, BC upturned the activities of hexokinase in STZ induced diabetic rats by enhanced insulin secretion, which may have helped to improve glucose utilization in the cells. Glucose-6-phosphate dehydrogenase is the first and also the ratelimiting enzyme of the pentose phosphate pathway, which is involved in the formation of ribose-5-phosphate and also NADPH [62]. Thus, its low activity leads to decreased NADPH and makes cells very sensitive to oxidant damage. The decreased activity of glucose-6-phosphate dehydrogenase slows down the pentose phosphate pathway in diabetic condition [63]. In the present study, treatment of BC improved the activities of glucose-6-phosphate dehydrogenase in STZ-induced diabetic rats that may be credited to the efficiency of BC in increasing the level of insulin secretion from the β-cells of the pancreas. Glucose-6-phosphatase and fructose-1, 6-bisphosphatase are the regulatory enzymes in the gluconeogenic pathway. The actions of these two enzymes may be ascribable to the increased glucose production during diabetes [64]. The activities of hepatic glucose-6-phosphatase and fructose-1,6-bisphosphatase have been reported to be increased

significantly in diabetic rats [65]. The oral treatment of BC significantly declined the activities of glucose-6-phosphatase and fructose-1, 6-bisphosphatase in STZ-induced diabetic rats, which might have helped in suppressing the glucose production from non-carbohydrate substances. Diabetes-induced hyperglycemia enhances inflammation leading to apoptosis of cells [66]. During the diabetic conditions, inflammation mediators such as IL-6 and TNF-α are found to be increased in the blood of diabetic patients [67,68]. A study by Ingaramo et al. [69] demonstrated that diabetic state induces an increase in the levels of TNF-α and its receptor TNF-R1 in the liver. The binding of TNF-α to TNF-R1 can promote the activation of NF-κB [69]. The pro-inflammatory genes are controlled by phosphorylation. The phospho NF-κB p65, the activated form of NF-κB p65, that encode several proteins, contributing to inflammation, cell proliferation, apoptosis, invasion, etc., and are also involved in impaired insulin signaling by inhibiting the Akt pathway [70]. The NF-κB p65 activation leads to triggering of various transcription genes like iNOS, COX-2, etc., in which iNOS is a well-known inflammation mediator involved in NO production and COX-2 is another inflammation mediator responsible for the increase in the production of prostaglandin, which induces inflammation in cells [71]. In this investigation, the activity of TNF-α, phospho NF-κB p65, COX-2 and iNOS protein expression levels were 234

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and support.

discovered to be significantly elevated in the liver of diabetic control rats when compared to the normal control rats. The increased levels of TNF-α, phospo-NF-κB p65, COX-2 and iNOS proteins were significantly reduced in the BC or glibenclamide treated diabetic rats. These effects may be a result of BC's ability to prohibit TNF-α, COX-2 and iNOS expressions, by suppressing the activation of NF-κB p65. The immunohistochemical staining of iNOS visibly confirmed the declined level of iNOS in the liver of STZ induced diabetic rats. Earlier Li Yuan et al. [72], Kuo et al. [73], Jeong et al. [74] and Lee at al. [75] have also reported that berberine and/or its derivatives can efficiently reduce inflammation through several distinct mechanisms, such as by downregulating COX-2, promoting AMP-activated protein kinase (AMPK) activity or inhibiting NF-kB activation in various cellular and animal models of inflammation. Bcl-2 protein family, a target gene of NF-κB, is a critical regulator of the common pathway of apoptosis [76]. Among these proteins, proapoptotic members such as Bax can trigger apoptosis by lodging on the mitochondrial outer membrane, depolarising, which is followed by inducing the release of apoptogenic factors like cytochrome c and apoptosis inducing from the mitochondria into the cytoplasm. These apoptogenic factors then activate the caspase cascades and subsequently lead to apoptosis of the cell [77,78]. In this study, the results exhibit elevated levels of Bax and cytochrome c in the liver of diabetic control rats, which are caused by the imbalance of NF-κB and oxidative stress. Our result also revealed that BC treatment remarkably down-regulated the Bax and cytochrome c levels, and also down-regulated NF-κB activation, which was evident by the reduced level of phospho NF-κB p65 levels in BC treated diabetic rats. Furthermore, anti-apoptotic Bcl-2 protein expression level was found to be improved in the liver of BC treated diabetic rats compared to diabetic control rats. Further, the immunohistochemical staining of cleaved caspase-3 in the liver of BC treated diabetic rats was found to be a very small region, which also clearly displays the anti-apoptotic activity of BC. Previously in ischemia-induced apoptosis in gerbil model reported that berberine regulates impaired levels of Bax, Bcl-2 and cytochrome c [79]. Thus, all the above effects may be credited to the antioxidant, anti-apoptotic and anti-inflammatory properties of BC.

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5. Conclusion The treatment with BC (50 mg/kg b.w) notably inhibits the oxidative stress, inflammatory and apoptotic protein expressions, along with the improved activity of carbohydrate metabolizing enzymes in the liver of diabetic rats. Thus, these findings clearly display that recovered carbohydrate metabolic enzymes prevent hepatic glucose production and also enhanced the utilization of glucose by cells. Our findings show that BC has the potential to protect against hyperglycemia-induced oxidative stress, inflammation and apoptosis in hepatic cells, and shows promise as both curative and prophylactic drug for diabetes mellitus treatment. This study provides further scope to evaluate the long-term effect of BC on other diabetic mammalian models and explore different molecular pathways to evaluate its safety and efficacy, which would help better understand the curative and/or prophylactic use of BC in diabetes mellitus treatment. Conflict of interest All authors declare that there were no conflicts of interest concerning this publication. Acknowledgements The authors wish to acknowledge the University Grants Commission, New Delhi, Project No.41-178/2012/(SR) for funding this work and also extend our thanks to Department of Zoology (UGC – SAP Sponsored), Annamalai University, for providing infrastructure facility 235

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