|Year : 2019 | Volume
| Issue : 2 | Page : 80-86
Effects of berberine on high-fat/high-sucrose-induced nonalcoholic steatohepatitis in experimental rats
Elrefaei Eman, Elhorani Hemat, Hibishy Hanaa, Qaseem Safwat
Medical Biochemistry Department, Tanta University, Tanta, Egypt
|Date of Submission||01-Jun-2018|
|Date of Acceptance||01-Sep-2019|
|Date of Web Publication||18-May-2020|
BSc of Medicine and Surgery Faculty of Medicine, Tanta University, Elgarbia, 3151
Background Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the world, characterized by hepatic steatosis, inflammation, hepatocyte injury with or without fibrogenesis, which might lead to cirrhosis. Berberine (BBR) is a natural isoquinoline alkaloid with very impressive health benefits.
Aim The aim of the study is to evaluate the protective effect of BBR in experimental NASH induced by high-fat/high-sucrose diet in male albino rats.
Materials and methods Sixty male albino rats were divided randomly into four equal groups: group I (normal control group), group II (BBR-treated control group), group III (NASH group), and group IV (BBR-treated NASH group). Levels of peroxisome proliferator gamma receptor coactivator one alpha (PGC-1α) in hepatic nuclear extract were measured by enzyme-linked immunosorbent assay, while the activity of cytosolic glycerol-3-phosphate dehydrogenase 1 in the liver tissue homogenate, liver enzymes, lipid profile, and plasma ferric reducing/antioxidant power were measured spectrophotometrically.
Results There was a statistically significant decrease of hepatic PGC-1α, plasma ferric reducing/antioxidant power, serum high-density lipoprotein-cholesterol along with a significant increase in the activity of glycerol-3-phosphate dehydrogenase 1, liver enzymes as well as hyperlipidemia in the NASH group were compared with both normal control and BBR-treated control groups. These pathological disturbances were significantly ameliorated by BBR supplementation.
Conclusion The present study provided unequivocal evidence that disturbed hepatic PGC-1α and altered redox status acted as major contributing factors for the pathogenesis of high-fat/high-sucrose-induced NASH in rats. It also shed some light on the potential therapeutic value of BBR in NASH, partly accredited to its hypolipidemic and antioxidant effects, in addition to upregulating the levels of PGC-1α in hepatic nuclear extracts.
Keywords: berberine, lipin-1, oxidative stress
|How to cite this article:|
Eman E, Hemat E, Hanaa H, Safwat Q. Effects of berberine on high-fat/high-sucrose-induced nonalcoholic steatohepatitis in experimental rats. Tanta Med J 2019;47:80-6
|How to cite this URL:|
Eman E, Hemat E, Hanaa H, Safwat Q. Effects of berberine on high-fat/high-sucrose-induced nonalcoholic steatohepatitis in experimental rats. Tanta Med J [serial online] 2019 [cited 2020 Jun 6];47:80-6. Available from: http://www.tdj.eg.net/text.asp?2019/47/2/80/284493
| Introduction|| |
Nonalcoholic steatohepatitis (NASH) represents an advanced stage of fatty liver disease developed in the absence of alcohol abuse . NASH is the most common chronic liver disease in the Western world, it is primarily a disease of the obese and the prevalence of simple steatosis in obese patients reaches 60%, among those, 20–25% will develop NASH . The disease is characterized by fat accumulation in the liver which results from an imbalance among hepatic lipid intake, synthesis, degradation, and secretion . Patients with simple steatosis progress to NASH according to the multihit theory, liver steatosis sensitizes hepatocytes to other hits, leading to hepatocyte damage, inflammation, and fibrosis. These insults may be increased in oxidative stress and lipid peroxidation, mitochondrial dysfunction, and cytokine/adipokine imbalance ,.
The transcriptional coactivator peroxisome proliferator gamma receptor coactivator one alpha (PGC-1α) regulates many metabolic programs, especially those related to oxidative metabolism. First identified as a coactivator of peroxisomal prolifirator activator receptor gamma (PPARγ) in brown fat-mediated thermogenesis, PGC-1α is now known to interact with many nuclear receptors and other transcription factors outside of this family . In the liver, PGC-1α is strongly induced in fasting and turns on the gene programs of gluconeogenesis, heme biosynthesis, and fatty acid oxidation. This occurs through coactivation of hepatic estrogen-related receptor α, nuclear respiratory factor 1, nuclear respiratory factor 2, PPARα, hepatic nuclear factor-4α, and forkhead box class O1 .
The term oxidative stress refers to the outcome of oxidative damage to biologically relevant macromolecules such as nucleic acids, proteins, lipids, and carbohydrates. Oxidative stress occurs when oxidative stress-related molecules, generated in the extracellular environment or within the cell, exceed intracellular antioxidant defenses .
Oxidative stress represents a key feature of NASH , where activated neutrophils, macrophages, and Kupffer cells are the major source of reactive oxygen species during inflammation.
Glycerol-3-phosphate dehydrogenase (GPDH) is an enzyme that catalyzes the reversible redox conversion of dihydroxyacetone phosphate to glycerol-3-phosphate. It serves as a major link between carbohydrate metabolism and lipid metabolism .
Berberine (BBR) is a proto-BBR alkaloid, and its derivatives exhibit a wide spectrum of pharmacological activities. It has been used in traditional Chinese medicine. BBR along with its derivatives or in combination with other pharmaceutically active compounds or in the form of formulations has applications in various therapeutic areas such as cancer, inflammation, diabetes, depression, hypertension, and various infectious areas . So, it was aimed to evaluate the protective effect of BBR in experimental NASH induced by a high-fat/high-sucrose diet in male albino rats.
| Materials and methods|| |
This study was carried out on 60 male albino rats weighing 200±14 g. During the whole period of the study (12 weeks), animals were housed in wire mesh cages and were allowed free access to water and to either a standard rat chew or high-fat/high-sucrose diet and 10% sucrose in their drinking water. They were kept under constant environmental conditions (temperature 23±2°C and 12 h dark light cycle). Care of the animals and the experimental procedures were performed in the Medical Biochemistry Department, Faculty of Medicine, Tanta University, Egypt, in accordance with guidelines of the ethics committee of Faculty of Medicine, Tanta University, Egypt (Approval code 31085/08/16).
BBR (≥95% purity, CAS NO: 633-65-8) and most other chemicals were purchased from Sigma-Aldrich Chemicals (St Louis, Missouri, USA).
Induction of nonalcoholic steatohepatitis
NASH was induced in the third and fourth groups by a combination of feeding high-fat/high-sucrose diet plus 10% sucrose in their drinking water for 6 weeks. The high-fat/high-sucrose diet contained 20% of energy as protein, 35% as carbohydrates (18% sucrose, 10% maltodextrin, and 7% starch) and 45% as lipids according to Lomba et al. . Subsequently, the fourth group was administered oral BBR at a dose of 200 mg/kg/day for another 6 weeks. Every 5 days, ingredients for diets were mixed, formed into dough with water, rolled into pellets, and stored at −20°C until use.
After being acclimatized for 1 week, the studied animals were divided randomly into four equal groups as follows:
- Group I (control group): this group included 15 rats which received a standard diet for 12 weeks.
- Group II (BBR-treated control group): this group included 15 rats which received a standard diet for 6 weeks followed by oral supplementation of BBR (200 mg/kg/day) for another 6 weeks .
- Group III (NASH group): this group included 15 rats. NASH was induced by feeding rats a high-fat/high-sucrose diet in addition to 10% sucrose in their drinking water for 6 weeks .
- Group IV (BBR-treated NASH group): this group included 15 rats. NASH was induced by feeding rats a high-fat/high-sucrose diet in addition to 10% sucrose in their drinking water for 6 weeks followed by oral supplementation of BBR (200 mg/kg/day) for another 6 weeks .
Collection and preparation of samples
At the end of the experiment, all rats were weighed and fasted overnight. Then, they were sacrificed by cervical decapitation. Blood samples were collected immediately; 1 ml of blood was collected on EDTA-coated tubes to obtain plasma, and the other part of blood was collected on plain tubes to obtain serum. All samples were separated by centrifugation at 3000 rpm for 15 min. The abdomen of the rats was dissected, and the livers were excised, washed by ice-cold saline. One part was homogenized in four volumes of ice-cold 50 mmol/l tri(hydroxymethyl)-aminomethane (Tris) buffer (pH 7.5) containing 1 mmol/l EDTA, 1 mM β-mercaptoethanol, and 0.5% Triton X-100, then centrifugated, and the final supernatant fraction was collected as the source of G3PDH enzyme. Another part was used for the preparation of nucleic acid-free nuclear proteins, by using EpiQuiK nuclear extraction kit (catalog number: #OP-0022), (Epigentek Co., Farmingdale, New York, USA).
Assessment of liver enzymes: serum alanine aminotransferase and aspartate aminotransferase activities were measured by commercially available reagent kit obtained from Spectrum Diagnostic Co. (Cairo, Egypt) According to the method of Reitman and Frankel .
Assessment of lipid profile: serum triglycerides , total cholesterol , and high-density lipoprotein-cholesterol (HDL-C)  were estimated using enzymatic colorimetric methods (Spectrum Diagnostic Co.). Low-density lipoprotein-cholesterol (LDL-C) was calculated by Friedewald’s formula .
Assessment of antioxidant capacity of plasma: the ferric reducing/antioxidant power (FRAP) method was used to determine the total antioxidant capacity level of plasma. This depends on the reduction of ferric tripyridyltriazine complex to ferrous tripyridyltriazine by a reductant at low pH according to Benzie and Strain .
Assessment of PGC-1α level in hepatic nuclear extract: PGC-1α level in hepatic nuclear extract was determined by enzyme-linked immunosorbent assay kit purchased from Sun Red Biotechnology Co. (Shanghai, China), according to the instructions of the manufacturer.
Assessment of G3PDH activity: G3PDH activity was measured according to the method of Kozak and Jensen .
All results were expressed as means±SD. Differences between groups were assessed by one-way analysis of variance, followed by a post-hoc multiple comparison test. The association between the parameters was determined using Pearson’s correlation coefficient. A P value less than 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS, version 20.0 statistical software package (SPSS Inc., Chicago, Illinois, USA).
| Results|| |
[Table 1] summarized the comparative statistics of the studied parameters between all groups. Using the multiple comparison test (Tukey’s test), there were statistically significant increases in body weight values in the high-fat/high-sucrose-fed animals (groups III and IV) as compared with both standard diet-fed control groups (groups I and II). At the endpoint of the experiment (12th week), there was a statistically significant difference among the studied groups. There was a statistically significant decrease of hepatic PGC-1α, plasma FRAP, serum HDL-C along with a significant increase in the activity of GPDH1, liver enzymes as well as serum levels of total cholesterol (TC), triacyl glycerol (TAG), and LDL-C in the NASH group compared with both normal control and BBR-treated control groups. In addition, BBR administration resulted in an increase of hepatic PGC-1α, plasma FRAP, and serum HDL-C along with a significant decrease in the activity of GPDH1, liver enzymes as well as serum levels of TC, TAG, and LDL-C in BBR-treated NASH group compared with the NASH group ([Figure 1]).
|Table 1 Comparative statistics of the studied parameters between all groups|
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|Figure 1 Photomicrograph representation of the liver from the normal control group showing normal liver tissue; hepatocytes are normal polygonal cells with a single central nucleus, no steatosis, and no inflammatory infiltrate. Photomicrograph representation of the liver from the berberine-treated control group showing normal liver tissue; hepatocytes are normal polygonal cells with a single central nucleus, no steatosis, and no inflammatory infiltrate. Photomicrograph representation of nonalcoholic steatohepatitis (NASH) showing macrovesicular steatosis, ballooned hepatocytes, and lobular inflammation (H&E, ×200).|
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| Discussion|| |
The present study depicted that treatment of NASH group with BBR resulted in a significant decrease in the levels of liver enzymes (alanine aminotransferase, aspartate aminotransferase) along with amelioration of these histopathological changes (as evidenced by a decreased number of fat vacuoles as well as decreased necrosis and inflammatory foci) compared with the untreated NASH group, claiming a hepatoprotective effect of BBR. Of relevance to this study, Xing et al.  demonstrated that the intragastric administration of BBR has been demonstrated to partially reverse the macrovesicular steatosis and inflammatory cell infiltration of portal areas and within hepatic lobules induced by a high-fat diet. Additionally, Guo et al.  reported that BBR improves aspects of NAFLD through ameliorating hepatic steatosis and suppressing the proinflammatory responses in primary hepatocytes of mice with diet-induced obesity.
Notably, in the current study daily oral supplementation with BBR for 6 weeks after NASH induction had a hypolipidemic action as evidenced by significantly decreased serum triacylglycerols, total cholesterol, and LDL levels as well as increased HDL levels as compared with the untreated NASH group.
This finding is not without precedence, having been previously noted by Zhao et al.  who provided evidence that BBR exerted a therapeutic effect on hepatic steatosis by inhibiting hepatic lipogenesis and comprehensively regulating the lipid metabolism. Likewise, BBR was shown to decrease hepatic steatosis in high-fat diet-fed rats ,,, and in a randomized, placebo-controlled trial in patients with NAFLD , suggesting an essential role of liver in BBR-mediated lipid homeostasis.
Recently, it has been shown that BBR-induced improvement of hyperlipidemia and fatty liver in obese animals is mediated by the direct and indirect activation of AMP-activated protein kinase (AMPK), in peripheral tissues, notably the liver and muscle, and adipose tissue . Once activated, AMPK is capable of suppressing lipogenesis through phosphorylating and inactivating the lipogenic enzyme acetyl-CoA carboxylase. In addition, a decrease in the production of malonyl-CoA due to AMPK inhibition of ACC brings about an increase in fatty acid oxidation via releasing the inhibitory effect of malonyl-CoA on carnitine palmitoyl transferase 1 ,.
Alternatively, these effects could be also attributed to the observation that BBR modulates the gut microbiota through enriching short-chain fatty acid-producing bacteria and reducing the microbial diversity, which inhibits dietary polysaccharide degradation and decreases additional calorie intake in the gut, which may have beneficial effects to the host’s metabolic status .
The current study displayed that PGC-1α protein levels in hepatic nuclear extracts were significantly diminished in the high-fat/high-sucrose-induced NASH group compared with both normal control and BBR-treated control groups, thus proposing a possible role of altered activity of PGC-1α in the pathogenesis of high-fat/high-sucrose-induced NASH.
This finding came in accordance with that of Aharoni-Simon et al. , who reported a significant reduction in PGC-1α protein levels in steatotic livers of a choline-deficient, ethionine-supplemented-fed mice in comparison to control mice. Consequently, they further confirmed this attenuated PGC-1α activity by chromatin immunoprecipitation analysis, which demonstrated decreased interaction of PGC-1α with promoters of its potential target genes such as those regulating mitochondrial biogenesis and the gluconeogenic gene phosphoenolpyruvate carboxykinase in mice fed the choline-deficient, ethionine-supplemented diet.
In this context, Morris et al.  provided evidence that PGC-1α overexpression in primary hepatocytes produced an increase in the markers of mitochondrial content and function (citrate synthase, mitochondrial DNA, and electron transport system complex proteins), and an increase in fatty acid oxidation, which was accompanied by reduced TAG storage and TAG secretion compared with the control. Also, they stated that PGC-1α-overexpressing hepatocytes were protected from excess TAG accumulation following overnight lipid treatment. Moreover, PGC-1α overexpression in hepatocytes lowered the expression of genes critical to VLDL assembly and secretion ‘apolipoprotein B and microsomal triglyceride transfer protein’ .
Furthermore, the present study revealed that oral supplementation of BBR efficiently caused a significant increase of PGC-1α levels in hepatic nuclear extracts of BBR-treated NASH group as compared with the untreated high-fat/high-sucrose-induced NASH.
SIRT1 has emerged as a key regulator of mammalian metabolism, and has been shown to deacetylate and activate PGC-1α . One hypothesis to explain the effect of BBR on hepatic PGC-1α levels might be based on the study by Sun et al., who has defined BBR as an activator of SIRT1 by using liver-specific SIRT1 knockout mice and SIRT1-/-hepatocytes, thus implying an essential role of SIRT1 in mediating BBR’s induction of autophagy flux and reduction of hepatic fat storage.
This notion was further confirmed by earlier studies indicating that BBR protects against high-fat-diet-induced dysfunction in the muscle mitochondria by inducing (SIRT1)-dependent mitochondrial biogenesis .
The data obtained from the present study clearly indicated that the high-fat/high-sucrose-fed rats exhibited a highly significant increase in hepatic cytosolic enzyme GPDH1 activity compared with both the normal control and BBR-treated control rats. This finding is biologically plausible as enhanced cytosolic GPDH1 activity leads to an increase in glycerol-3-phosphate production which serves as the direct precursor for TAG synthesis . Consequently, its abundance and augmented activity can easily contribute to an enhanced TAG accumulation in the liver and epididymal adipose tissue of high-fat-diet-fed rats ,.Furthermore, this study has shown that treatment with BBR resulted in a significant decrease of the hepatic GPDH1 activity; importantly this attenuation was significant in relation to the untreated high-fat/high-sucrose-fed rats. In agreement with our finding, Ragab et al.  have also found that BBR administration returned the augmented GPDH1 activity in hepatic tissues of high-fat/high-sucrose-fed rats to the control levels.
The present study has reported that NASH was associated with altered redox status as judged by a significant reduction of plasma FRAP levels compared with both normal control and BBR-treated control groups.
This finding correlates well with the earlier studies that have shown that HFD disturbed redox status through antioxidant depletion, thus contributing to NAFLD development ,. Nevertheless, the current study displayed that BBR administration to the high-fat/high-sucrose-induced NASH group resulted in augmentation of their plasma antioxidant capacity as shown by elevated plasma FRAP levels as compared with the untreated NASH group, thus providing an experimental evidence that BBR treatment was able to reduce oxidative stress and to limit the development of NASH.
Several lines of in-vitro and in-vivo evidence corroborate this notion. Zhang et al.  have demonstrated that BBR attenuated ethanol-induced oxidative stress in the liver by reducing hepatic lipid peroxidation, glutathione exhaust, and mitochondrial oxidative damage. Meanwhile, Liu et al.  proved that BBR possessed the anti-oxidative effects as shown by the reduced plasma malondialdehyde level and increased superoxide dismutase activity significantly, thus protecting against the oxidative stress-related damage following the high-glucose high-fat load.
| Conclusion|| |
In conclusion, the present study provided unequivocal evidence that disturbed hepatic PGC-1α signaling and the altered redox status acted as major contributing factors for the pathogenesis of high-fat/high-sucrose-induced NASH in experimental rats. It also shed some light on the potential therapeutic value of BBR in NASH; which might be partly accredited to its hypolipidemic and antioxidant effects. Moreover, BBR was found to significantly upregulate the levels of PGC-1α in hepatic nuclear extracts of BBR-treated NASH group as compared with the untreated NASH group. However, further studies are warranted to justify its therapeutic efficacy before nominating it as a potential drug candidate for preventing or treating NASH and related disorders in humans.
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Conflicts of interest
There are no conflicts of interest.
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