Anti-apoptosis effect of liraglutide on islet via regulating microRNA-375

AIM: To observe the anti-apoptosis effect of liraglutide on the islet through microRNA-375 (miR-375) for providing additional pharmacodynamic evidence for its clinical application. METHODS: For in vitro study, C57BL/KsJ-db/m mice aged 8 weeks served as normal control group. A total of 40 male genetically diabetic C57BL/KsJ-db/db mice at the same age were randomly divided into diabetic control group （the db/db mice were injected subcutaneously with equivalent amount of saline) and liraglutide group (the db/db mice were injected subcutaneously with liraglutide at dose of 300 μg·kg^-1·d^-1). After 8 weeks of administration, body weight (BW) was measured and blood was collected for detection of fasting blood glucose (FBG), fasting blood insulin (FINS), triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). Before sacrifice, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were conducted. The histopathological features in the islet tissue were examined with HE staining. The apoptosis in the islet tissue was detected by TUNEL staining. The protein levels of caspase-3, Bcl-2 and Bax were determined by Western blot. The level of miR-375 in the islet tissue was detected by qPCR. Forin vitro study, the MIN-6 cells were cultured and divided into control group (incubated with equivalent amount of solvent), miR-375 mimic group and miR-375 mimic+ liraglutide group. The cell viability was examined by MTT assay. The protein levels of caspase-3, Bcl-2 and Bax were detected by Western blot. RESULTS: In thein vitro study, compared with control group, the levels of BW, FBG, FINS, TC, TG and LDL-C were decreased significantly in liraglutide group. The islet apoptosis was reduced by the administration of liraglutide. The expression of Bcl-2 was up-regulated significantly, while the protein levels of caspase-3 and Bax were down-regulated significantly in liraglutide group. The level of miR-375 was decreased significantly. In the in vitro study, the cell viability was decreased in miR-375 mimic group and increased in miR-375 mimic+liraglutide group. Moreover, the expression of Bcl-2 was decreased and the protein levels of caspase-3 and Bax were increased with the incubation of miR-375 mimic, while the expression of Bcl-2 was increased and the protein levels of caspase-3 and Bax were decreased with the co-incubation of miR-375 mimic and liraglutide. CONCLUSION: Liraglutide attenuates islet apotosis, and the mechanism may be associated with its effects of reducing the elevated level of miR-375 in islet tissues.     

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.     

Effects of intranasal Escherichia coli on glucolipid metabolism in high-fat diet-induced obese mice

AIM: To study whether the pulmonary infection of Escherichia coli (E. coli) interferes the glucolipid metabolism in high-fat diet-induced obese mice. METHODS: High-fat diet-induced obese mice (n=48) and normal chow-fed control mice (n=48) were intranasally infused with 40 μL fluid containing 4×10⁹ CFUs E. coli. The serum, periepididymal adipose tissue and liver were obtained at 0 d, 1 d, 2 d, 3 d and 4 d after infection. The body mass, periepididymal adipose tissue and liver were weighed, and the levels of fasting blood glucose (FBG), fasting blood insulin (FINS), free fatty acid (FFA) and very-low-density lipoprotein (VLDL) were measured by ELISA. The serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), and hepatic TG contents were detected, and the hepatic steatosis was observed under microscope with oil red O staining. RESULTS: Compared with day 0, the body mass, fat mass and fat index were decreased significantly from day 1 to day 4 after infection (P<0.05). The levels of FBG, FINS and HOMA-IR were apparently raised from day 2 to day 4 after infection (P<0.05). The contents of serum FFA, TG and VLDL were increased markedly from day 1 to day 4 after infection (P<0.05). However, the concentrations of serum TC, LDL-C and HDL-C were decreased obviously from day 1 to day 3 (P<0.05). The liver mass, liver index and TG content were significantly increased from day 1 to day 4 (P<0.05). Consistently, the lipid droplet accumulation in the liver cells was increased obviously at day 2 and day 4 after infection. Compared with control group, except the levels of serum TC, LDL-C and HDL-C in obese group substantially decreased, the other indexes were increased by different degrees during the whole experiment period (P<0.05). CONCLUSION: Pulmonary infection of Escherichia coli exacerbates the disorder of glucose and lipid metabolism in high-fat diet-induced obese mice, which contributes the development of insulin resistance and hepatic steatosis.     

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.     

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.     

Effects of EGCG on GLUT2/G6PD/GS expression and blood glucose level in type 2 diabetic rats

AIM To investigate whether epigallocatechin gallate （EGCG） improves blood glucose in type 2 diabetic rats through glucose transporter 2 （GLUT2）-glucose-6-phosphate dehydrogenase （G6PD）-glycogen synthase （GS） pathway. METHODS Type 2 diabetes mellitus （T2DM） model was established in male Sprague-Dawley （SD） rats by feeding with high-fat diet and injection of streptozotocin （STZ）. The rats were divided into 5 groups （n=10）： control （Con） group， T2DM model （M） group， metformin （Met； 200 mg/kg， ig） group， T2DM+low-dose （50 mg/kg， ig） EGCG （EL） group， and T2DM+high-dose （100 mg/kg， ig） EGCG （EH） group. Diabetic rats were given drugs for 8 weeks. After 8 weeks of administration， the rats were killed， and the blood and liver tissues were collected. The levels of fasting blood glucose （FBG）， fasting serum insulin （FINS） and serum glycosylated hemoglobin were measured by biochemical tests. Liver glycogen were test by periodic acid-Schiff （PAS） staining. The mRNA expression of G6PD in the liver was detected by real-time PCR. The protein levels of GS and GLUT2 were determined by Western blot and immunohistochemistry. RESULTS T2DM rat model was established successfully. Compared with Con group， the levels of FBG， FINS and serum glycosylated hemoglobin in M group were increased significantly （P<0.05）， while the insulin sensitivity index （ISI）， the liver glycogen， the G6PD mRNA expression， and the protein levels of GS and GLUT2 were decreased significantly （P<0.05）. Compared with M group， the levels of FBG and serum glycosylated hemoglobin in Met group and EH group were decreased significantly （P<0.05）， while the ISI， the liver glycogen， the G6PD mRNA expression， and the protein levels of GS and GLUT2 were increased significantly （P<0.05）. CONCLUSION EGCG reduces the blood glucose level in T2DM rats， which may be related to the regulation of GLUT2-G6PD-GS signaling pathway.     

Expression of scavenger receptor B1 (SR-B1) in the liver of high fat and sugar diet-induced diabetic mice

AIM: To investigate serum lipid and the expression of SR-B1 in the livers of diabetic mice. METHODS: Ten normal diet, female C57BL/6J mice, fifteen high fat and sugar diet female C57BL/6J mice, five fed 8 weeks and ten fed 16 weeks were used in the experiment. Serum total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), insulin (INS) and the expression of SR-B1 in the livers were measured. RESULTS: 1. In the high fat and sugar diet mice, serum TC and FBG at 16 weeks were significantly higher than that in normal diet mice (P<0.05). 2. The expression of SR-B1 protein in the liver of high fat and sugar diet mice was the higher than that in normal mice, and the SR-B1 expression in the liver of the mouse fed 16 weeks was also higher than that fed 8 weeks. CONCLUSION: The expression of SR-B1 protein in the liver of type 2 diabetes mice is higher than that in normal mice, perhaps it is related to the decrease in serum HDL-C.     

Insulin and gliclazide therapies improve liver lipid deposition in type 2 diabetic rats

AIM：To investigate the effect of insulin and gliclazide therapies on the liver fat accumulation in type 2 diabetic rats. METHODS：A high-fat diet plus low-dose streptozotocin was implemented to establish a type 2 diabetic rat model, and the rats were randomly divided into diabetes mellitus (DM) group, diabetic rats treated with insulin (INS) group, diabetic rats treated with gliclazide per os （PO） group, and normal control (NC) group. The diabetic rats in INS group and PO group were given insulin and gliclazide for 3 weeks, respectively. The changes of the liver fatty were evaluated with oil red O staining. Fasting plasma adiponectin concentration was measured by ELISA. The expression of adiponectin receptor 1 (AdipoR1) was detected by real-time PCR. The protein levels of AMP-activated protein kinase (AMPK), phosphorylated AMPK on threonine 172 (Thr172p-AMPK), sterol regulatory element-binding protein 1c (SREBP-1c), phosphorylated SREBP-1c on serine 372 (Ser372p-SREBP-1c), acetyl-CoA carboxylase (ACC), phosphorylated ACC on serine79 (Ser79p-ACC) and immunoglobulin-binding protein (BiP) in the liver homogenate were determined by Western blotting. RESULTS：Compared with the normal rats, in DM group, the presence of cytoplasmic lipid deposits was confirmed by oil red O staining. In INS group, these changes were significantly lower than those in DM group. Similar results were obtained in PO group. Insulin therapy significantly increased the plasma concentration of diponectin and liver tissue levels of AdipoR1 compared with DM group. At the same time, these 2 indicators returned to normal levels after gliclazide therapy. Thr172p-AMPK/AMPK, Ser372p-SREBP-1c/SREBP-1c and Ser79p-ACC/ACC expression ratios were significantly reduced in DM group compared with control values. The expression of BiP was increased on the contrary. After insulin therapy, Thr172p-AMPK/AMPK and Ser372p-SREBP-1c/SREBP-1c were significantly increased, and Ser79p-ACC/ACC and BiP returned to the normal levels. After gliclazide treatment, Thr172p-AMPK/AMPK and Ser372p-SREBP-1c/SREBP-1c returned to the normal levels, the expression ratio of Ser79p-ACC/ACC had no significant improvement compared with DM group, and the expression of BiP significantly declined. CONCLUSION：Both the insulin and gliclazide therapies reduce the lipid deposition in the liver of rats with type 2 diabetes by activating AMPK, but the extent and mechanism are not the same. In insulin therapy, AMPK restrains the expression of SREBP-1c directly, increases the phosphorylation of SREBP-1c, and affects SREBP-1c by inhibiting the endoplasmic reticulum stress. Gliclazide treatment, which has no effect on the lipid oxidation, reduces lipid deposition in the liver only through the phosphorylation of SREBP-1c and the suppression of the endoplasmic reticulum stress.     

Protective effect of adiponectin on glucose and lipid metabolism by suppressing MHCⅡ expression

AIM: To investigate whether the protective effect of adiponectin on glucose and lipid metabolism is achieved through down-regulating major histocompatibility complex class Ⅱ (MHCⅡ) in the adipose tissue. METHODS: Adiponectin knockout (KO) mice and C57BL/6(WT) mice were fed with high-fat diet and standard diet for 24 weeks, respectively. The body weight, fasting blood glucose (FBG), fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), hepatic histology, and class Ⅱ trans-activator (CⅡTA), histocompatibility 2 class Ⅱ antigen E beta (H2-Eb1) and cluster of differentiation 74(CD74) mRNA and MHC Ⅱ protein levels in adipose tissue were measured at sacrifice. siRNA targeting MHC Ⅱ and overexpression vector was used in 3T3-L1 cells to explore the effect of adiponectin on the protein level of MHCⅡ. RESULTS: The levels of body weight, FBG, FINS, HOMA-IR, TC, TG, LDL-C, hepatic steatosis, CⅡTA, H2-Eb1 and CD74 mRNA expression, and MHCⅡ protein expression in the KO mice were higher than those in the WT mice that fed with high-fat diet or standard diet. In 3T3-L1 cells, inhibition of adiponectin reversed MHC Ⅱ protein level induced by specific siRNA. The expression of MHC Ⅱ in adipocytes decreased after adiponectin was overexpressed. CONCLUSION: Adiponectin improves glucose and lipid metabolism through suppressing the expression of MHCⅡ in the adipose tissue.     

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.     

Changes of heart function and serum cardiac troponin I in early type 2 diabetic rats

AIM: To observe the changes of heart function and the expression of serum cardiac troponin I(cTnI) in early type 2 diabetic rats, and to explore the role of cTnI in the development of type 2 diabetes and early diabetic cardiomyopathy.METHODS: The type 2 diabetes rat model was established by an injection of streptozotocin after high fat diet(5 weeks). The rats were randomly divided into control group, model group of 2 weeks, and model group of 4 weeks. M-mode echocardiography was performed for echocardiographic measurements. Fasting blood glucose(FBG), total cholesterol(TC), triglyceride(TG), high density lipoprotein-cholesterol(HDL-C), low density lipoprotein- cholesterol(LDL-C), fasting insulin(FINS) and cTnI levels were tested. HE staining was used to observe the pathological changes of myocardial structures. The alteration of cTnI in myocardium was determined by Western blot.RESULTS: Compared with normal group, the levels of TC, TG and LDL-C in type 2 diabetic rats were significantly increased, HDL-C levels were significantly reduced. Cardiac histological analysis revealed that type 2 diabetes induced cardiomyocytes degeneration and necrosis. The expression of cTnI increased significantly in diabetic groups compared to control group, and that in model group of 4 weeks increased far more than that in model group of 2 weeks(P<0.05).CONCLUSION: The increased level of cTnI and the change of the heart function may be associated with the development diabetic cardiomyopathy. These changes are valuable for the early clinical diagnosis of myocardial injury in diabetic cardiomyopathy.     

Yiqi-Yangyin recipe attenuates myocardial ischemia-reperfusion injury in diabetic rats by activation of ERK/Nrf2/HO-1 pathway

AIM: To explore the effect of Yiqi-Yangyin recipe on myocardial ischemia-reperfusion injury (MIRI) in rats with diabetes mellitus (DM) and the possible mechanism. METHODS: The rats were divided into normal group (control group), DM sham operation (DM-S) group, DM+MIRI group, low-, medium-and high-dose Yiqi-Yang-yin recipe (TL, TM and TH) groups (7.5, 15 and 30 g/kg decoction of Yiqi-Yangyin recipe by gavage), and Nrf2 inhibitor (bardoxolone methyl) group (30 mg/kg bardoxolone methyl by intragastric administration). The gavage volume was 1 mL/kg. There were 15 rats in each group, and they were administered continuously for 7 d. The tail vein blood was collec-ted after the last administration to detect the blood sugar and lipid levels in the rats. The serum levels of cardiac troponin I (cTnI), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-10 were measured by ELISA. Echocardiography was used to detect the changes of cardiac function in the rats after blood collection. After cardiac function test, the rats were sacrificed to obtain cardiac tissues, and the volume changes of myocardial infarction were assessed by triphenylte-trazole chloride staining. The histopathological changes of myocardium was observed by HE staining. The cardiomyocyte apoptosis was determined by TUNEL assay. The protein levels of phosphorylated extracellular signal-regulated kinase (p-ERK), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the myocardium were determined by Western blot. The myocardial activity of superoxide dismutase (SOD) was measured by nitro blue tetrazolium method, the content of malondialdehyde (MDA) was tested by thiobarbituric acid method, and the production of reactive oxygen species (ROS) was analyzed by iron ion reduction method. RESULTS: Compared with control group, the levels of fasting blood glucose (FBG), total cholesterol (TC) and triglyceride (TG) in DM-S group and DM+MIRI group were significantly elevated, while the level of high-density lipoprotein cholesterol (HDL-C) was significantly lowered (P<0.05). Compared with DM-S group and DM+MIRI group, the levels of FBG, TC, TG in TL, TM, TH and bardoxolone methyl groups were significantly decreased, while HDL-C level was significantly increased (P<0.05). Compared with control group and DM-S group, heart rate (HR) and left ventricular end-diastolic pressure (LVEDP) were increased in DM+MIRI group, mean arterial pressure (MAP), left ventricular systolic pressure (LVSP) and left ventricular ejection fraction (LVEF) were decreased, serum levels of cTnI, TNF-α, IL-1β and IL-10 were increased, the myocardial infarction volume percentage was increased, the myocardial cell breakage and necrosis were increased, the myocardial cell apoptotic rate was increased, the protein levels of p-ERK1/2, Nrf2 and HO-1 were decreased, MDA and ROS levels were increased, and the activity of SOD was decreased (P<0.05). Compared with DM+MIRI group, HR and LVEDP were decreased in TL, TM, TH and bardoxolone methyl groups, MAP, LVSP and LVEF were increased, the serum levels of cTnI, TNF-α, IL-1β and IL-10 were decreased, the myocardial infarction volume percentage was decreased, myocardial cell breakage and necrosis were decreased, myocardial cell apoptotic rate was decreased, the protein levels of p-ERK1/2, Nrf2 and HO-1 were increased, the MDA and ROS levels were decreased, and the activity of SOD was increased (P<0.05). CONCLUSION: Yiqi-Yangyin recipe protects the myocardial tissue of DM+MIRI rats from injury and reduces the oxidative stress level, which may be achieved by activating ERK/Nrf2/HO-1 pathway.     

Effect of resveratrol on autophagy and renal interstitial fibrosis in kidney of diabetic mice

AIMTo observe the effect of resveratrol (Res) on renal autophagy level and renal interstitial fibrosis in the mice with diabetes mellitus (DM), and to discuss the possible mechanism. METHODSThe wild-type C57BL/6 mice were randomly divided into 3 groups, including normal control (NC) group, DM group and Res group (8 in each group). The diabetic mouse model was established by injection of streptozotocin. After 8 weeks of successful replication of the diabetic model, Res was given to the mice in Res group by continuous gavage for 12 weeks, and then the mice in each group were sacrificed to detect the relevant biochemical parameters. The pathological changes of the kidney tissues were observed by HE staining and Masson staining. The levels of the proteins related to autophagy, epithelial-mesenchymal transition (EMT) and fibrosis were determined by Western blot. The mRNA expression of collagen type IV (Col IV), α-smooth muscle actin (α-SMA) and E-cadherin were detected by real-time PCR. RESULTSCompared with NC group, fasting blood glucose (FBG), kidney index (KI), serum creatinine, 24-hour urinary albumin excretion rate and 24-hour urine total protein were remarkably increased in DM group (P<0.05). The results of HE and Masson staining indicated that renal tissue presented fibrosis in DM group. The protein levels of E-cadherin, beclin-1, microtubule-associated protein 1 light chain 3-II (LC3-II) were reduced in DM group, while the levels of α-SMA, Col IV and Snail1 were increased (P<0.05). After intervention with Res for 12 weeks, all the relevant biochemical parameters and KI were reduced (P<0.05) except FBG (P>0.05), and renal fibrosis lesions were obviously alleviated. Compared with DM group, the protein levels of E-cadherin, beclin-1 and LC3-II were increased in Res group, but the protein expression levels of α-SMA, Col IV, Snail1 were reduced (P<0.05). Compared with DM group, the mRNA level of E-cadherin was increased in Res group , but the mRNA levels of Col IV and α-SMA were reduced (P<0.05). CONCLUSION Resveratrol significantly inhibits EMT and reduces renal interstitial fibrosis in diabetic mice, and its mechanism may be related to the promotion of renal autophagy.     

Distinct effects of different β-adrenoceptor stimulation patterns on car-diac AMP-activated protein kinase activity

AIM: To investigate the cardiac AMP-activated protein kinase (AMPK) activity and the effects of AMPK activator on cardiac structure and function in the mice with different β-adrenoceptor (β-AR) stimulation patterns. METHODS: Male BALB/c mice were subcutaneously injected with AMPK activator (AICAR, 250 mg· kg^-1·d^-1) or saline, and infused with β-AR agonist isoproterenol (ISO, 5 mg·kg^-1·d^-1) for 14 d. The cardiac functions were evaluated by echocardiography or hemodynamic method, and the hearts were harvested after infusion cessation immediately or 3 d later. Phosphorylated AMPK (p-AMPK) was measured by Western blot. RESULTS: Sustained ISO infusion increased p-AMPK level. AICAR did not further increase p-AMPK but attenuated ISO-induced increase in heart weight. Sustained ISO infusion increased cardiac systolic function as indicated by left ventricular fractional shortening (FS) and maximum rate of pressure rise (+dp/dt_max). The cardiac systolic function was not further increased by AICAR. The cardiac diastolic function as indicated by left ventricular end-diastolic pressure (LVEDP) was not different in each group. In contrast, cardiac p-AMPK level was similar between the control mice and the mice with sustained ISO infusion and ceased infusion for 3 d. In this model, AICAR improved the cardiac systolic and diastolic functions, which were impaired by ISO. Moreover, the increased pattern of p-AMPK level was similar with that of heart rate upon ISO stimulation. CONCLUSION: Sustained ISO infusion increases p-AMPK. After ISO infusion cessation for 3 d, p-AMPK is decreased to the basal level. β-AR-induced inotropic effects should be avoided to investigate the cardioprotective role of AMPK activation in the β-AR stimulation models.     

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.     

Dynamic expression of S-adenosyl-L-homocysteine hydrolase under intervention of fimasartan in early stage of type 2 diabetic cardiomyopathy rats

AIM: To detect the changes of S-adenosyl-L-homocysteine hydrolase (SAHH) signaling pathway and related proteins under the intervention of fimasartan (FIM) in rats with diabetes mellitus (DM), and to explore the pathological mechanism of the occurrence and development of diabetic cardiomyopathy (DCM). METHODS: The type 2 diabetic rat model was established by injection of streptozocin after 5-week high-fat diet. The rats were randomly divided into control group, DM group, and DM+FIM group. Fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and fasting insulin (FINS) levels were tested. M-mode echocardiography was performed for determining the heart functions. High-performance liquid chromatography (HPLC) was used to detect homocysteine (Hcy), S-adenosylmethionine (SAM) and S-adenosyl-L-homocysteine (SAH). The alteration of SAHH in myocardium was determined by Western blot. RESULTS: The success rate of type 2 diabetic rat modeling was 84%. Compared with DM group, the body weight of the rats in DM+FIM group increased significantly (P<0.05), while cardiac index, left ventricular index, FBG and LDL-C were significantly reduced (P<0.05). The results of echocardiography showed that ejection fraction increased (P<0.05) in DM+FIM group. HPLC detection showed that the level of Hcy and the ratio of SAM/SAH were significantly reduced (P<0.05). The results of Western blot showed that the expression of SAHH in DM group was increased compared with control group, while that in DM+FIM group declined (P<0.01). CONCLUSION: The expression of SAHH, Hcy and other related factors may be important during the occurrence and development of early DCM, and FIM may play a role in this process as an inhibitor of SAHH.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Effect of microRNA-7 knockdown on ConA-induced acute liver injury in mice

AIM: To study the effect of microRNA-7 (miR-7) knockdown (KD) on concanavalin A (ConA)-induced acute liver injury (ALI) in mice.METHODS: Wild type (WT) mice and miR-7KD mice were received ConA (30 mg/kg) to induced acute liver injury model by intraperitoneal injection, and the morphological changes, liver weight and weight index were measured 48 h later. The pathological changes of the liver tissues were observed by HE staining. The levels of serum alanine aminotransferase (ALT), IL-4 and IFN-γ were detected by ELISA. The proportional changes of CD4⁺ T cells and the relative levels of IL-4 and IFN-γ were analyzed by flow cytometry.RESULTS: The color of the liver tissue became lighter, and the weight and weight index were changed significantly in miR-7KD mice compared with control group (P<0.05). HE staining showed that the inflammatory cell infiltration was increased in the liver of miR-7KD mice. Moreover, the level of serum ALT was significantly increased (P<0.05). The serum level of IFN-γ elevated significantly (P<0.01), while the IL-4 levels decreased significantly (P<0.01) in the serum of miR-7KD mice. Furthermore, the proportion of CD4⁺ T cells and relative IFN-γ cells increased obviously (P<0.01).CONCLUSION: miR-7 knockdown promotes the pathogenesis of the ConA-induced acute liver injury in mice.     

Losartan inhibits LPS-induced GFAP expression via AMPK activation in mouse hippocampus

AIM: To investigate the effects of losartan on lipopolysaccharide (LPS)-induced glial fibrillary acidic protein (GFAP) expression, and to determine whether adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation is involved in the mechanism.METHODS: Adult male KM mice were divided into control group, LPS model group, losartan treatment group, and losartan and Compound C co-treatment group. To establish a model of central nervous system inflammation, the mice received daily intracerebroventricular injection of LPS (24 μg/d) for 2 d. Daily losartan administration (0.5, 1 or 5 mg·kg^-1·d^-1, ip) initiated at 14 d prior to LPS injection. Compound C (10 mg/kg, ip), a selective AMPK inhibitor, started to be injected daily at 2 d prior to LPS injection. The hippocampal tissues in each group were isolated at 3 d after the last LPS injection, and then the protein levels of GFAP, AMPK, p-AMPK, mammalian target of rapamycin (mTOR) and p-mTOR were determined by Western blot.RESULTS: Twice LPS injections significantly increased the expression of GFAP in the hippocampus (P<0.01). Losartan inhibited LPS-induced GFAP expression in a concentration-dependent way, and losartan at 5 mg·kg^-1·d^-1 significantly inhibited GFAP expression and AMPK activation (P<0.05), but it had no obvious effect on mTOR activation. Furthermore, Compound C significantly reversed the effect of losartan treatment on LPS-induced GFAP expression and AMPK phosphorylation (P<0.05).CONCLUSION: Losartan inhibits LPS-induced GFAP expression in the mouse hippocampus, and AMPK activation but not mTOR, is involved in the mechanism.