Role of SIRT1/AMPK pathway in early intervention of Lira alleviating non-alcoholic fatty liver disease caused by high-fat diet in rats

AIM To investigate the effect of early intervention of glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (Lira) on oxidative stress, glucose tolerance, hepatic steatosis and insulin resistance of the rats with high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD), and to explore the role of silent information regulator 1 (SIRT1)/AMP-activated protein kinase (AMPK) signaling pathway in this process. METHODS Twenty-four male SD rats were randomly divided into normal diet (ND) group, HFD group and HFD+Lira group, with 8 rats in each group. After 1 week of adaptive feeding, the rats in HFD+Lira group were subcutaneously injected with Lira (200 μg/kg) per day at a fixed time point, while the rats in the remaining 2 groups were injected with normal saline at the same volume. During the intervention, the body weight, hair, appetite, defecation and activity of the rats were observed to adjust the dosage timely. The body weight, food intake and blood glucose were recorded weekly. Glucose tolerance test was performed at the end of the 16th week. At the end of the 18th week, hyperinsulinemic euglycemic clamp test was conducted after anesthesia. Blood was taken from the carotid artery. The liver and adipose tissues from different parts were taken after death. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other indicators were detected. HE staining was used to observe the pathological changes of the liver tissue. Lipid accumulation in the liver tissues was observed by oil red O staining. Liver fibrosis was observed by Masson staining and Sirius red staining. Fluorescence staining for reactive oxygen species (ROS) was used to observe the oxidative stress in the liver. The expression of GLP-1 receptor in the liver was observed by immunofluorescence staining. The expression and localization of SIRT1 and phosphorylated AMPK at Thr172 [p-AMPK (Thr172)] were observed by immunohistochemical staining. The protein levels of AMPK, p-AMPK (Thr172), SIRT1, phosphorylated sterol regulatory element binding protein-1c at Ser372 [p-SREBP-1c (Ser372)], phosphorylated acetyl coenzyme A carboxylase at Ser79 [p-ACC (Ser79)], carnitine palmitoyltransferase 1A (CPT1A) and fatty acid synthase (FAS) in liver tissues were determined by Western blot. RESULTS The results of HE and oil red O staining of rat liver tissues in HFD group confirmed the structural disorder and serious lipid accumulation, while Masson and Sirius red staining showed severe fibrosis, suggesting the successful establishment of NAFLD rat model. Compared with ND group, the levels of total cholesterol (TC), triglyceride (TG), AST and ALT in serum, and the levels of malondialdehyde (MDA), TC, TG and ROS in liver tissues in HFD group were significantly increased (P<0.01), while the activity of superoxide dismutase (SOD) was decreased (P<0.01). The protein levels of p-AMPK (Thr172), SIRT1, p-SREBP-1c (Ser372), p-ACC (Ser79) and CPT1A in the liver tissues were significantly reduced (P<0.05 or P<0.01), while the expression of FAS was increased （P<0.01）. Compared with HFD group, lipid accumulation and fibrosis in the liver tissues of the rats in HFD+Lira group were significantly attenuated, the serum levels of TC, TG, AST and ALT, and MDA, TC, TG and ROS in liver tissues were markedly reduced (P<0.05 or P<0.01), while SOD activity was increased (P<0.05). The protein levels of p-AMPK (Thr172), SIRT1, p-SREBP-1c (Ser372), p-ACC (Ser79) and CPT1A in the liver tissues were significantly increased (P<0.05 or P<0.01), while the expression of FAS was decreased （P<0.01）. CONCLUSION Lira attenuates insulin resistance, oxidative stress and fibrosis, and improves liver lipid metabolism in the rats with NAFLD induced by HFD, which may be mediated by SIRT1/AMPK signaling pathway.     

mRNA expression of insulin receptor and leptin receptor in liver of nonalcoholic fatty liver disease from diabetic rats

AIM: To observe the pathologic changes of liver in diabetic rats and to investigate the role of mRNA expression of insulin receptor and leptin receptor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). METHODS: Twenty male Sprague-Dawley rats were divided randomly into two groups: normal control group and diabetic group. After fed with high-fat diet for 4 weeks, diabetic rats were injected with streptozotocin at a dosage of 30 mg/kg intraperitoneally to induce NAFLD model of type 2 diabetes mellitus. Then the diabetic animals were fed with high-fat diet continuously for 12 weeks. At the end of the experiment, the rats were sacrificed, the concentrations of blood glucose, serum lipid, ALT and AST were measured biochemically. The levels of serum leptin and serum insulin were detected by enzyme-linked immunosorbent assay (ELISA) and radio immunoassay (RIA), respectively. The pathologic changes of liver were observed under light microscopy (LM) stained with HE, Sudan Ⅲ and Masson trichrome staining, respectively. The ultra-structural changes of liver were observed under transmission electron microscopy (TEM). Additionally, the mRNA expressions of PEPCK, G6Pase, insulin R and leptin R from rat livers were assayed by semi-quantitative RT-PCR. RESULTS: The levels of blood glucose, serum insulin, serum TG, ALT and AST increased significantly (P<0.01), serum TC elevated (P<0.05), and the levels of serum leptin decreased (P<0.01) in diabetic group compared to those in normal control group. Obvious liver fatty degeneration, piecemeal necrosis with accompanying inflammatory infiltration and fibrosis were found under LM. Hepatocytes pyknosis, lots of lipid deposits in cytoplasm of hepatocytes, proliferation of collagen in space of Disse were observed under TEM in diabetic group. The expression of insulin R and leptin R mRNA in liver from diabetic rats increased significantly (P<0.01) while the expression of PEPCK and G6Pase mRNA remained unchanged. CONCLUSION: Insulin resistance plays an important role in the pathogenesis of NAFLD. Low level of serum leptin, up-regulation of mRNA expression of insulin R and leptin R in liver caused by insulin resistance may be involved in this process.     

Effects of liraglutide on adiponectin and insulin resistance in rats with non-alcoholic fatty liver disease

AIM：To investigate the effects of glucagon-like peptide 1 analog, liraglutide, on adiponectin and insulin resistance in the rats with diet-induced non-alcoholic fatty liver disease (NAFLD). METHODS：Male rats were randomly divided into 3 groups:normal diet (ND) group (n=10), high-fat diet (HFD) group (n=10), and HFD with intraperitoneal injection of liraglutide group (n=10, first 12 weeks with HFD, later 4 weeks with liraglutide). All treatments continued for 16 weeks, and then the rats were killed ethically and the blood samples and liver tissues were collected. The levels of alanine aminotransferase (ALT), fasting blood glucose (FBG), total cholesterol (TC) and triglyceride (TG) were detected by a biochemical automatic analyzer. The levels of free fatty acids (FFAs), fasting insulin (FINS) and adiponectin were measured by RIA and ELISA. RESULTS：Compared with HFD group, the body weight, liver index, homeostasis assessment-insulin resistance (HOMA-IR), the serum levels of TG, TC, ALT and FBG, and the liver levels of TG, TC and FFAs in the rats in liraglutide group were apparently lower, the degree of hepatic steatosis and inflammatory activity significantly decreased (P<0.05), and the level of adiponectin in the serum and liver homogenate increased ob-viously (P<0.05). The level of adiponectin in the liver homogenate was negatively correlated with the levels of FFAs in the liver homogenate. CONCLUSION:Liraglutide is beneficial for NAFLD rats to improve insulin resistance and reduce hepatic steatosis by increasing the level of adiponectin in the serum and liver tissues.     

Effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats and its possible mechanism

AIM:To discuss the mechanism of ginsenoside Rb1 against liver lipid deposition by observing the effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats. METHODS:Totally 32 healthy SPF rats were randomly divided into control group, model group, ginsenoside Rb1 group and simvastatin group. The rats in control group was given the basic feed, while the others were given high-fat diet. The rats in ginsenoside Rb1 group and simvastatin group were given corresponding drugs. The rats in control group and model group were intraperitoneal injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by the automatic biochemistry analyzer. The pathological changes of the liver tissues were observed with HE staining. The protein and mRNA expression levels of pyroptosis-related factors NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were detected by Western blot and RT-qPCR. RESULTS:Compared with control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.01), and the HDL-C content was decreased significantly (P<0.05). The steatotic liver cells covered the visual field. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were increased significantly (P<0.01). Ginsenoside Rb1 significantly decreased the serum levels of TC, TG and LDL-C (P<0.05), and significantly increased the content of HDL-C (P<0.01). Ginsenoside Rb1 also significantly decreased the degree of steatosis, and the number and size of lipid droplets. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were decreased significantly (P<0.05 or P<0.01). CONCLUSION:Ginsenoside Rb1 atte-nuates liver injury and inhibits liver lipid deposition in hyperlipidemia rats by reducing the expression of hepatic pyroptosis-related factors.     

Anthocyanins attenuates hepatic fibrosis and inflammation in non-alcoho-lic fatty liver disease mice

AIM:To explore the therapeutic effect of anthocyanins from Fructus Acanthophorae on high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) in mice and the potential mechanism. METHODS:NAFLD mouse model was established by high-fat diet, and interferred with anthocyanins. The liver weight, and serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC) and low-density li-poprotein cholesterol (LDL-C) were measured. The liver tissues were staining with HE, Oil Red O and Masson's trichrome. The protein levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-10 in the liver tissues were determined by Western blot. The liver macrophage, white blood cell and mononuclear cell infiltration was detected by immunohistochemical method. The chemokines CCL7 and MCP-1 were also measured by immunohistochemical method. RESULTS:Anthocyanins significantly inhibited the increases in the liver weight, ALT, AST, TG, TC and LDL-C induced by high-fat diet. Anthocyanins attenuated the liver fibrosis and inflammatory cell infiltration caused by high-fat diet, and reduced the levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-10 and inflammatory chemokines CCL7 and MCP-1 in the liver tissues. CONCLUSION:Anthocyanins significantly alleviate non-alcoholic fatty liver disease caused by high-fat diet though reducing inflammatory factors, inflammatory cell infiltration and inflammatory chemokines.     

Effects of silybin on mitochondria membrane fluidity in rats with non-alcoholic fatty liver disease

AIM: To explore the effects of silybin on mitochondrial membrane fluidity in the rats with non-alcoholic fatty liver disease (NAFLD).METHODS: NAFLD rats were induced by high-fat diet for 6 weeks. Mitochondrial membrane fluidity, serum lipid levels, hepatic enzymes and peroxidant products were assayed. The histopathological changes of the livers were observed. The effects of silybin on the changes of the above parameters in NAFLD rats were also determined. Rosiglitazone was used as a positive control of treatment. RESULTS: Compared with the control animals, the fluidity of mitochondrial membrane was obviously decreased, and the contents of alanine aminotransferase, aspartate transaminase, triglyceride, total cholesterol, malonaldehyde and superoxide dismutase in serum were markedly increased (P<0.01) in the NAFLD rats. Silybin and rosiglitazone evidently regulated mitochondrial membrane fluidity, decreased the levels of serum lipids, improved the liver functions and meliorated histopathological manifestations of the livers (P<0.05 or P<0.01).CONCLUSION: High-fat diet successfully reproduces a NAFLD animal model. Stabilization of mitochondrial membrane and inhibition of oxidative stress may be the main mechanisms for the hepatoprotective effects of silybin in NAFLD.     

Ginsenoside Rg1 improves liver function by regulating fat metabolism in rats with non-alcoholic fatty liver disease

AIM: To investigate whether ginsenoside Rg1 attenuates high-fat diet (HFD)-induced non-alcoho-lic fatty liver disease (NAFLD) by improving β-oxidation. METHODS: SD rats (n=60) were randomly divided into control group (CON), HFD group, low-dose, medium-dose and high-dose ginsenoside Rg1 groups (LDG, MDG and HDG) and positive drug (sodium ursodeoxycholate) treatment group (PDT). High-fat diet was given for 8 weeks to successfully establish an NAFLD model. The animals were treated with the appropriate medications for 4 weeks and 8 weeks after modeling, and sacrificed to collect the liver tissues for observing the pathologic changes with HE staining and for detecting liver functions and lipid levels. The expression of hepatic acyl-CoA synthetase 1 (CoASH1), carnitine acyltransferase I (CATI) and acyl-CoA oxidase 1 (ACOX1) at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS: After 4-week treatment, the fatty infiltration of the liver tissues in PDT group, LDG group and MDG group was not attenuated except HDG group. After 8 weeks of treatment, a small number of fat particles was observed in PDT group and LDG group, while no infiltration of lipid droplet was found in MDG group and HDG group. Compared with HFD group, the levels of AST, ALT, AKP, TC, TG and LDL-C were significantly decreased after 4-week treatment in PDT group, LDG group, MDG group and HDG group (P<0.05), these indexes were further reduced after 8-week treatment. After 4-week treatment, HDL-C was significantly increased in the 4 treatment groups and almost restored to the level of CON group after 8-week treatment. The levels of CoASH1, CACTI and ACOX1 in the liver tissue of the 4 treatment groups were significantly increased after 4-week treatment (P<0.05) and much improved after 8-week treatment, and those in MDG group and HDG group were better than those in PDT group (P<0.05).CONCLUSION: Ginsenoside Rg1 regulates β-oxidation-related enzymes to improve the fat metabolism, thus playing a therapeutic role in liver injury in the rats with NAFLD.     

Effect of betaine on liver injuries in aging db/db mice induced by lipids

AIM: To evaluate the effect of betaine on lipid metabolism disorder in inherited db/db mice with long-term nonalcoholic fatty liver disease (NAFLD).METHODS: Experimental NAFLD models were established by feeding the db/db mice with high-fat diet. Fifty 7-month-old db/db mice were randomly divided into 5 groups: the mice in low, medium and high dose groups were given betaine by intragastric administration at doses of 200 mg/kg, 400 mg/kg and 800 mg/kg for 6 weeks, respectively,while the mice in saline control group and positive drug group were given normal saline and positive control drug,respctively. All the animals were killed, and serum alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) and glucose tolerance were detected. The pathological changes of the liver tissues were also observed.RESULTS: Betaine significantly decreased the levels of ALT, TC and LDL (P<0.05 or P<0.01). The pathological changes of the liver tissues indicated that the content of lipid in the hepatocytes of betaine treatment groups was less than that in saline control group.CONCLUSION: Betaine significantly improves the lipid metabolism and the liver function in the aging db/db mice, and reduces the accumulation of lipid in the hepatocytes.     

Effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE-/- mice

AIM To observe the effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE^-/- mice. METHODS Thirty-two ApoE^-/- mice were randomly divided into model group, high-dose (60 mg/kg) tanshinone ⅡA group, low-dose (30 mg/kg) tanshinone ⅡA group and simvastatin group, and C57BL/6J mice (n=8) were used as normal control group. The mice in normal control group were given the basic feeding, while the others were given high-fat diet. The mice in tanshinone ⅡA groups and simvastatin group were given corresponding drugs. The mice in normal control group and model group were intraperitoneally injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by automatic biochemistry analyzer. The liver tissues were stained with HE and oil red O. The contents of reactive oxygen species (ROS) and glutathione (GSH) in liver tissues of the mice were measured by commercially available kits. The liver glutathione peroxidase 4 (GPX4) and p53 were detected by immunohistochemical method. The protein and mRNA expression levels of ferroptosis-related factors GPX4, xCT/SLC7A11, p53 and ferritin heavy chain 1 (FTH1) were determined by Wes automatic Western blot quantitative analysis system and RT-qPCR. RESULTS Compared with normal control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.05 or P<0.01), and HDL-C did not change significantly. The fat vacuoles were clearly visible in liver tissue. The content of ROS in liver tissue was increased significantly,and GSH was decreased significantly (P<0.01). The mRNA and protein expression levels of p53 were increased significantly, and GPX4, xCT/SLC7A11 and FTH1 were decreased significantly (P<0.05 or P<0.01). Compared with model group, tanshinone ⅡA significantly decreased the serum levels of TC, TG and LDL-C (P<0.05 or P<0.01), and HDL-C did not change significantly. High-dose and low-dose tanshinoneⅡA also significantly decreased the degree of steatosis, and the size of lipid droplets. The content of ROS in liver tissues was decreased significantly, and GSH was increased significantly (P<0.01). The mRNA and protein expression levels of GPX4, xCT/SLC7A11 and FTH1 were increased significantly, and p53 were decreased significantly (P<0.05 or P<0.01). CONCLUSION Tanshinone ⅡA reduces liver lipid deposition and lipid peroxidation damage in ApoE^-/- mice, which may be related to the intervention of ferroptosis-related proteins in the liver cells.     

Hypothalamic expression of orexin A and lipid metabolism disorder in rats with alimentary obesity

AIM: To evaluate the effect of orexin A in rat hypothalamus on lipid metabolism disorder in rats with alimentary obesity induced by high-fat diet.METHODS: The rat model of alimentary obesity was induced by high-fat diet. The levels of insulin, triglyceride (TG) and total cholesterol (TC) in the serum were detected by luminescent immunoassay and enzymic method. The mRNA expression of orexin A in rat hypothalamus was determined by real-time PCR.RESULTS: There were statistically significant differences of weight, body fat content, and Lee's index between high-fat diet group and control group after 8-week feeding of high-fat diet. Compared to control animals, the levels of insulin, TG and TC in the rats with alimentary obesity significantly increased by 50％, 94％ and 43％, respectively (P<0.05). The expression of orexin A in rat hypothalamus significantly decreased by 57％, and had significant negative correlation with Lee's index, insulin, TG and TC. Their correlation coefficients were r=-0.798 (P<0.05), r=-0.868 (P<0.05), r=-0.981(P<0.05) and r=-0.815 (P<0.05), respectively. CONCLUSION: Alimentary obesity and lipid metabolism disorder induced by high-fat diet are correlated with down-regulation of orexin A expression in rat hypothalamus.     

Effects of pyrrolidine dithiocarbamate on synthesis of hepatic glycogen in diabetic rats

AIM: To determine the effect of pyrrolidine dithiocarbamate on hepatic glycogen synthesis and its mechanism in diabetic rats. METHODS: Male Wistar rats were randomly divided into normal diet group and high-fat diet group. After 8 weeks of feeding, the rats in high-fat diet group were injected intraperitoneally with a single dose of streptozotocin (27 mg/kg) to induce type 2 diabetes. The diabetes rats were randomly divided into 3 groups: diabetes mellitus group (DM), PDTC-treated group (DM+PDTC) and insulin-treated group (DM+INS). The rats in PDTC-treated group were injected with PDTC (50 mg/kg) intraperitoneally daily. At the same time, the rats in normal diet group, DM group and insulin-treated group were injected with equivalent volume of saline in the same way. The rats in insulin-treated group were injected with insulin (1 U/kg) 1 h before killed. After the treatment was taken for 1 week, the levels of blood glucose were measured, then the animals in all groups were killed. The liver glycogen content was detected, and the levels of GSK-3β and Akt phosphorylation in the liver tissues were analyzed by Western blotting. RESULTS: The blood glucose level and liver glycogen content were significantly higher, and the levels of GSK-3β and Akt phosphorylation were lower in DM group than those in normal-diet group (P<0.01). Compared with DM group, the glycogen content, the phosphorylation of Akt and GSK-3β in the liver tissues in DM+PDTC group and DM+INS group increased significantly (P<0.01), and the blood glucose levels decreased (P<0.01). CONCLUSION: PDTC increases the synthesis of liver glycogen and decreases the level of blood glucose by regulating the activity of Akt and GSK-3β in the liver.     

Effects of phytosterol ester on aortic aging and expression of related genes in rats

AIM: To explore the protective effect of phytosterol ester (PSE) on aortic aging in rats. METHODS: The female SD rats (12 months old, n=42) were randomly divided into control group, model group and PSE group. During the experiment, the rats in control group, model group and PSE group were treated with basic feed, high-fat diet (HFD) and HFD with 2% PSE (W/W) for 6 months, respectively. The morphological changes of the aorta were observed by HE staining and Masson staining, and the absolute area of smooth muscle cells and collagen fiber in the vascular wall were measured by image analysis. The levels of advanced glycosylation end products (AGEs), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in the plasma were detected. The expression of silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor γ (PPARγ) at mRNA and protein levels in the vascular tissue was determined by real time PCR and Western blot, respectively. RESULTS: PSE significantly lowered plasma TC and LDL-C, and increased plasma HDL-C level (P<0.05), but had no effect on plasma TG level. PSE significantly attenuated the thickening of intima and media of aging aortic, and decreased the migration of vascular smooth muscle cells (VSMC) and the amount of VSMC and collagen fiber in the aorta (P<0.05). PSE significantly reduced the contents of AGEs and MDA (P<0.05), but had no effect on the activity of SOD and CAT in the plasma. PSE also down-regulated the expression of PPARγ and up-regulated the expression of SIRT1 (P<0.05). CONCLUSION: PSE is able to attenuate the senescence process in the aorta by reducing the production of reactive oxygen species in plasma, and activating SIRT1, or inhibiting the expression of PPARγ in vascular tissues.     

Yunpiheluo decoction alleviates diabetic kidney injury by regulating Sirt1/AMPK signaling pathway and activating autophagy

AIM: To observe the effect on Yunpiheluo decoction (YPHL) on renal injury in type 2 diabetic rats and to explore the mechanism from the perspective of Sirt1-AMPK-autophagy. METHODS: Male Zucker diabetic fatty (ZDF) rats (n=24) were randomly divided into model group, Sirt1 over-expression group and YPHL group, and fed with high-fat and high-sugar diet for 10 weeks. ZL rats were used as normal control and fed with normal diet for 10 weeks. After 10 weeks, urine and blood were collected for renal function detection. The rats were sacrificed and specimen was submitted. In addition, the mRNA expression of Sirt1 was analyzed by real-time PCR. The protein levels of Sirt1, AMPK, p-AMPK, LC3 and P62 in the renal tissues wene determined by Western blot. The renal pathological changes were observed under light microscope with HE and Masson staining. RESULTS: Compared with control group, fasting blood glucose (FBG), urinary protein (UP), urinary albumin (U-ALB) and serum creatinine (SCr) in model group were obviously increased (P<0.01). The mRNA expression of Sirt1 was decreased (P<0.05). The protein levels of SIRT1, AMPK, p-AMPK and LC3-II/-I were decreased (P<0.01), and P62 was increased (P<0.01). Glomerular focal fibrosis, focal renal tubular epithelial cell vacuolation, necrosis, shedding and atrophy, tubular type, and renal interstitial fibrosis were observed. Compared with model group, FBG was obviously decreased in Sirt1 over-expression group (P<0.01), but it showed no significant change in YPHL group (P>0.05). SCr and U-ALB were decreased (P<0.05), Sirt1 mRNA was increased (P<0.05), the protein levels of SIRT1, AMPK, p-AMPK and LC3-II/-I were increased (P<0.01), and P62 was decreased (P<0.01) in Sirt1 over-expression group and YPHL group. HE and Masson staining showed that the renal damage in Sir1 over-expression group and YPHL group was attenuated. CONCLUSION: Yunpiheluo decoction may protect the kidney by increasing the expression level of Sirt1, activating AMPK, and regulating autophagy.     

GLP-1 down-regulates mRNA expression of SOCS-3 and SREBP-1c in rats with nonalcoholic fatty liver disease

AIM: To investigate the effects of glucagon-like peptide-1(GLP-1) on mRNA expression of SOCS-3 and SREBP-1c in the rats with nonalcoholic fatty liver disease. METHODS: Male SD rats were randomly divided into normal control(NC) group, high fat(HF) group and HF+liraglutide(Lira) group. The rats in HF group and HF+Lira group were given high-fat diet for 16 weeks. After 12 weeks of high-fat diet feeding in HF+Lira group, Lira(600 μg·kg^-1·d^-1) was intraperitoneally injected for 4 weeks. At the end of the 16th week, the rats were killed. The pathological changes of the liver were observed under optical microscope. The serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglyceride(TG) and total cholesterol(TC) were detected by automatic biochemical analyzer. TG contents of liver were measured by GPO-PAP method. The fasting insulin(FINS) was determined by ELISA, and insulin resistance index was assessed by homeostasis mode assessment(HOMA-IR). The mRNA expression of SOCS-3 and SREBP-1c in the liver tissues was detected by RT-qPCR. RESULTS: Compared with NC group, HOMA-IR, TG of liver, and the serum levels of ALT, AST, TG, TC and FINS in HF group were obviously increased(P<0.01). Compared with HF group, HOMA-IR, TG of liver, and the serum levels of ALT, AST, TG, TC and FINS in HF+Lira group were all obviously decreased(P<0.05 or P<0.01). The mRNA expression of SOCS-3 and SREBP-1c in HF group was significantly higher than that in NC group(P<0.01). The mRNA expression of SOCSV3 and SREBP-1c in HF+Lira group was significantly decreased as compared with HF group(P<0.05). CONCLUSION: Liraglutide may improve the IR and reduce TG of liver through decreasing the mRNA expression of SOCS-3 and SREBP-1c, so as to play a therapeutic role in nonalcoholic fatty liver disease.     

Effects of oxidative stress on high-fat,high-salt diet and sucrose solution-induced metabolic syndrome in nephropathy rats

AIM: To establish a suitable animal model of nephropathy associated with metabolic syndrome (MS) induced by abnormal diet, and to investigate the effects of oxidative stress on renal damage in MS rats. METHODS: Normal 7-week-old male SD rats were randomly divided into 2 groups.The animals were fed with normal chow (control group, n=10) or high-fat and high-salt diet plus 20% sucrose solution (MS model group, n=10) for 20 weeks. Systolic blood pressure (SBP) was measured monthly. The levels of blood glucose, serum and urinary creatinine (Cr), total cholesterol (TC), triglycerides (TG), fasting insulin (FIns), urinary protein, urinary albumin and urinary sodium were determined. Insulin resistance (HOMA-IR), creatinine clearance (Ccr), urinary protein excretion (UPE), urinary albumin excretion (UAE) and urinary sodium excretion (USE) were calculated. Renal total-antioxidant capacity (T-AOC), inhibiting superoxide anion capacity (ISAC), malondialdehyde (MDA) content, and antioxidant enzyme activity were measured. Renal protein expression of Cu/Zn-SOD, NADPH oxidase subunit p47^phox and p22^phox was detected by Western blotting. In addition, pathological changes of the kidney were observed with PAS and Masson staining,and degree of glomerulosclerosis (GS) and tubulointerstitial injury was evaluated. RESULTS: Compared with control rats, SBP, TC, TG, FIns, USE and UAE were increased in MS rats. Furthermore, the MS rats showed a significant elevation of renal MDA content, p47^phox protein expression and GS score, and reduction of T-AOC, ISAC, SOD activity, and Cu/Zn-SOD protein expression in the kidney. CONCLUSION: SD rats fed with abnormal diet produce a suitable animal model of MS nephropathy that mimics the major features of human MS. Oxidative stress caused by up-regulation of NADPH oxidase expression and down-regulation of SOD expression may be one of the mechanisms leading to MS renal damage.     

Effect of micronised fenofibrate on lipotoxicity and insulin sensitivity in spontaneously hypertensive rats treated with high-fat diet

AIM: To observe the effect of micronised fenofibrate (lipanthyl) on lipotoxicity and insulin sensitivity (IS) in spontaneously hypertensive rats (SHR) with high-fat diet.METHODS: Twenty-seven SHR were randomly divided into three groups: normal chow group (n=9), high-fat diet group (n=9) and micronised fenofibrate treatment group (n=9). Micronised fenofibrate 100 mg·kg-1·d-1 was given orally to SHR, which diet on high-fat diet for three months. Intramuscular lipids were observed and lipids accumulation index (LAI) was calculated. Nonesterified fatty acid, glucose and insulin were determined in all rats.RESULTS: (1) Compared to SHR in normal chow diet group, body weight and the level of serum TG and TC increased significantly and the level of HDL-C decreased significantly in SHR fed with high-fat diet (P<0.05). Micronised fenofibrate significantly decreased systolic blood pressure, body weight, the level of serum TG and TC, increased the level HDL-C (P<0.05). (2) Fasted blood glucose, free fatty acid (FFA), GLU-AUC obviously increased in high-fat diet group compared with normal chow diet group. Insulin sensitivity index (ISI) in high-fat diet group was much lower than that in normal chow diet group (0.0038±0.0007 vs 0.0053±0.0013, P<0.05). No difference was found between fenofibrate-treated group and normal chow diet group. (3) There were more lipid drops in intramuscular cells of SHR treated with high-fat diet than those in fenofibrate-treated group and normal chow diet group (LAI: 6.42±0.59 vs 3.32±0.77 and 1.98±0.97, P<0.05). After covariance analysis, the results above-mentioned also made sense (F=10.46, P<0.05). (4) Inverse association was found between LAI and ISI (r=-0.58, P<0.05). Positive correlations were found between LAI and TG, FFA, body weight.CONCLUSION: In addition to regulating lipid, micronised fenofibrate may reduce BP, body weight, FFA, lipid accumulation in intramuscular cells and improve insulin sensitivity of SHR treated with high-fat diet.     

Expression of perilipin and ADRP in rat liver tissues with abnormal glucose metabolism

AIM: To observe the changes of perilipin and adipose differentiation-related protein (ADRP) du-ring the development of diabetes mellitus and to explore the effect of perilipin and ADRP on abnormal glucose metabolism with non-alcoholic fatty liver disease (NAFLD). METHODS: The rat model of impaired glucose tolerance (IGT) was induced by feeding high-fat diet, and the type 2 diabetes mellitus (T2DM) model was induced by feeding high-fat diet for 4 weeks and intraperitoneally injecting streptozotocin. The morphological change of the liver tissue was observed under optical microscope. The serum contents of perilipin and ADRP were measured by ELISA. The mRNA expression of perilipin and ADRP in the liver tissues was detected by real-time PCR. The protein expression of ADRP in the liver tissues was determined by Western blotting. RESULTS: HE staining showed steatosis in the liver of the rats in IGT group was more serious than that in T2DM group. The biochemical and the pathological processes of rat models were consistent with the clinical feature of related diseases. The serum content of perilipin had no difference among various groups. The mRNA expression of perilipin in IGT group and T2DM group was significantly higher than that in control group. Compared with IGT group, the mRNA expression of perilipin in T2DM group was significantly increased. The serum content of ADRP in T2DM group was significantly lower than that in control group. The mRNA and protein expression of ADRP in model groups was significantly lower than that in control group. Compared with IGT group, the mRNA expression of ADRP in T2DM group was significantly reduced. CONCLUSION: The serum content of ADRP plays a role in the development and progression of T2DM. It is negatively correlated with HOMA-IR. NAFLD occurs during progression of abnormal glucose metabolism induced by feeding high-fat diet. The development of abnormal glucose metabolism with NAFLD is probably related to the increased expression of perilipin and the reduced expression of ADRP.     

Effects of gypenosides on PCSK9 gene expression and blood lipids lowered by simvastatin

AIM: To explore the effect of gypenosides (GPs) on PCSK9 gene expression in hyperlipidemic rat liver and the blood lipids lowered by simvastatin.METHODS: Healthy male SD rats (n=60) were randomized into 5 groups:normal control group, hyperlipidemic model group, simvastatin group, GPs group and GPs combined with simvastatin group (combined group). The rats in all groups were fed high-fat diet except normal control group which were fed with ordinary diet. The rats in control group and hyperlipidemic model group were gavaged with 0.3% CMC-Na every day. The rats in GPs group were gavaged with GPs at 160 mg·kg^-1·d^-1. The rats in simvastatin group were gavaged with simvastatin at 5 mg·kg^-1·d^-1. The rats in combined group were gavaged with GPs and simvastatin. The experiment lasted for 8 weeks. The rats were anesthetized with chloral hydrate, and abdominal arterial blood samples were collected to detect the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). The body weight and the wet weight of the livers were measured, and the liver index was calculated. The pathological changes of the livers were observed under microscope with HE staining. The expression of PCSK9 and low-density lipoprotein receptor (LDLR) at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The model of hyperlipidemia rats was established successfully. Compared with model group, the levels of TC, TG and LDL-C in simvastatin group, GPs group and combined group were obviously decreased (P<0.05), and the HDL-C levels were obviously upregulated (P<0.05). Compared with model group, the liver indexes in simvastatin group, GPs group and combined group were obviously decreased (P<0.05). The pathological changes of the liver tissues showed that hepatic adipose appeared in model group, and that in simvastatin group and GPs group had different degrees of relief, especially in combined group. Compared with model group, the mRNA expression levels of PCSK9 and LDLR in simvastatin group were obviously increased, while the mRNA expression levels of PCSK9 in GPs group and combined group were obviously decreased (P<0.05), and the mRNA expression of LDLR in combined group was obviously increased (P<0.05). Compared with model group, the protein expression of PCSK9 and LDLR in simvastatin group was obviously increased, while the protein expression levels of PCSK9 in GPs group and combined group were obviously reduced, and the LDLR protein levels were obviously increased (P<0.05). CONCLUSION: Gypenosides inhibit the expression of PCSK9 and increase the expression of LDLR in the liver. The combination of gypenosides and simvastatin promotes the lipid-lowering effect of simvastatin and attenuates hepatic steatosis, which may be related to inhibiting the expression of PCSK9 in the liver.     

Effect of glucagon-like peptide 1 on Sesn2/AMPK/mTOR signaling pathway in liver of obese rats induced by high-fat diet

AIM: To explored the effect of glucagon-like peptide 1 receptor agonist liraglutide on Sesn2/AMPK/mTOR signaling pathway in the liver of obese rats.METHODS: Male SD rats were divided into normal chow (NC) group (n=12) and high-fat diet (HF) group (n=33). After 12 weeks, 5 rats of each group were used to assess establishment of obese rat model. The rats in HF group were divided into 4 subgroups, HF group, low dose of liraglutide (LG) group, middle dose of liraglutide (MG) group, and high dose of liraglutide (HG) group, and treated with various doses of liraglutide (0, 50, 100 and 200 μg/kg) via hypodermic injection twice a day for 4 weeks. The body weight and epididymal fat index of the rats at the 16th week were measured. The liver tissue fatty degeneration was observed. The protein levels of Sesn2, AMPK, p-AMPK, mTOR and p-mTOR were determined by Western blot.RESULTS: The body weight of rats in HF group was obviously higher than that in NC group (P<0.01). Compared with NC group, the levels of Sesn2 and p-AMPK/AMPK were significantly decreased in HF group (P<0.01), while the level of p-mTOR/mTOR was not changed. After treatment with liraglutide for 4-week, the body weight of the rats in LG, MG and HG groups was obviously lower than that in HF group (P<0.01), and epididymal fat index of the rats in MG and HG groups was obviously lower than that in HF group (P<0.01). The protein level of Sesn2 in HG group was obviously higher than that in HF group (P<0.01). The level of p-AMPK/AMPK was significantly increased in MG and HG groups (P<0.01). The level of p-mTOR/mTOR was significantly increased decreased in LG, MG and HG groups (P<0.01).CONCLUSION: Glucagon-like peptide 1 receptor agonist liraglutide affects energy metabolism and improves the state of obesity through Sesn2/AMPK/mTOR signaling pathway.