Effects of curcumin analogue L6H4 on myocardial tissue of type 2 diabetic rats

AIM: To explore the effects of curcumin analogue L6H4 on the myocardial tissue of type 2 diabetic rats and its mechanism. METHODS: Male Sprague-Dawley rats were randomly divided into normal control (NC) group, high-fat (HF) group, high-fat treatment (FT) group, diabetes mellitus (DM) group and diabetes treatment (DT) group.The rats in the latter 4 groups were fed high-fat diet for 4 weeks, then the rats in DM groups and DT groups were intraperitoneally injected with streptozotocin (STZ) to induce type 2 diabetes, while the rats in FT group and DT group were given L6H4. The blood glucose and lipid levels were detected by biochemical method, and serum adiponectin (APN) levels were detected by ELISA. The serum insulin levels were measured by radioimmunoassay and homeostasis model assessment of insulin resistance (HOMA-IR) were calculated. The morphological changes of myocardium were observed by Masson staining and electron microscopy. The protein expression of adiponectin receptor 1 (AdipoR1) and transforming growth factor β1(TGF-β1) in myocardial tissue were determined by immunohistochemistry. The protein expression of adipoR1 was also detected by Western blot for verification. RESULTS: Compared with NC group, the blood glucose, lipids, insulin, HOMA-IR and TGF-β1 were increased in HF and DM group, but they were decreased after treated with L6H4. Compared with NC group, the concentration of serum APN were decreased and the expression of AdipoR1 in the myocardium were weakened in HF group and DM group, and they increased after treated with L6H4. The myocardial fibrosis was obvious in HF group and DM group, the mitochondria in cardiomyocytes expanded, and the cristae disordered, partial disappeared. These lesions were significantly reduced after L6H4 treatment. CONCLUSION: L6H4 exerts a protective effect on the heart in type 2 diabetic rats. The increased concentration of serum APN, the enhanced expression of AdipoR1, and the expression of TGF-β1 inhibited by APN may be involved in the mechanism of protection.     

Changes of adiponectin and adiponectin receptor 1 in diabetic rats of different courses during myocardial ischemic preconditioning

AIM: To investigate the different effects of adiponectin (APN) and adiponectin receptor 1 (Ad-R1) on myocardial ischemia-reperfusion (IR) injury and ischemic preconditioning (IPC) in different course of diabetic rats in vitro. METHODS: The rat models of type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) were successfully established using streptozotocin and high-fat diet plus streptozotocin, respectively. These rats were divided into 2 groups:4 weeks and 8 weeks. The model of isolated cardiac perfusion was established by Langendorff method. Each group was further divided into control (Con) group, IR group and IPC group. The activity of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent was detected. The serum and myocardial levels of APN were determined by ELISA. The expression of Ad-R1 in the myocardial tissues was detected by Western blot. The area of myocardial infarction was detected, and the ultrastructure of ventricular papillary muscle was observed by transmission electron microscopy. RESULTS: Compared with the corresponding IR group, the activity of LDH and CK in the IPC group at 4 weeks was significantly decreased (P<0.01), and the area of myocardial infarction was significantly reduced. However, no significant difference of each index in DM groups at 8 weeks was observed. Serum APN level was decreased in diabetic rats, especially in T2DM rats (P<0.05). The levels of APN and Ad-R1 in myocardium of normal rats had no difference among Con, IR and IPC groups. The level of APN in myocardium of T1DM rats had no difference in all subgroups, while the expression of Ad-R1 in myocardial tissue of IR group was significantly increased as compared with Con group (P<0.01) and IPC group (P<0.01) both at 4 and 8 weeks. In T2DM rats, the levels of APN in myocardium both at 4 and 8 weeks were decreased in IR group compared with Con group (P<0.05). The level of APN in IR group at 4 weeks was significantly decreased compared with IPC group, but had no significant difference at 8 weeks. The expression of Ad-R1 in myocardial tissue of IR group was significantly increased compared with Con group (P<0.05) both at 4 and 8 weeks. The level of Ad-R1 in IR group at 4 weeks was significantly increased compared with IPC group (P<0.05), but had no significant difference at 8 weeks. CONCLUSION: The protective effect of IPC exists in diabetic rats at 4 weeks, whereas it disappears at 8 weeks. APN and Ad-R1 in myocardium were probably involved in the protective effect of IPC on T2DM rats.     

Study on the role of oxidative stress in the kidneys of diabetic rats

AIM: To investigate the changes of oxidative stress in the kidneys and their roles in nephropathy in diabetic rats. METHODS: Diabetic rats were induced by streptozotocin (STZ). 36 rats were divided into three groups randomly: (1) NC group, normal control rats; (2) DM group, diabetic rats received protamine zinc insulin (PZI) 2U-4U/2 d; (3) DT group, diabetic rats received PZI 9-12 U/kg body weigh/day. 12 weeks later, rats were killed, blood glucose, blood cholesterol, serum creatinine, blood urea nitrogen, HbA1c, urinary creatinine, and urinary protein for 24 h were measured. The activities of antioxidant enzymes in renal cortex, including total superoxide dismutase (TSOD), Cu-Zn superoxide dismutase (Cu-Zn SOD), Mn superoxide dismutase (Mn SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and maleic dialdehyde (MDA) were measured by chromatometry. RT-PCR was performed to detect the expression of different antioxidant enzymes mRNA. RESULTS: For all the targets we measured, there was no significant difference between NC and DT groups. Compared with the other two groups, the levels of blood glucose, cholesterol, trigalloyl glycerol, HbA1c in DM group increased significantly. The activities of TSOD, Cu-Zn SOD and CAT decreased significantly. The activity of GSH-Px increased significantly. There was no significant difference among the activities of Mn SOD in all three groups. The level of MDA in DM group was much higher than that in NC or DT group. The relative expression levels of GSH-Px and Cu-Zn SOD mRNA in DM group were higher than those in other two groups, while the relative expression level of CAT decreased. Mn SOD mRNA was expressed without significant difference in all groups. Compared with NC or DT group, urinary protein in DM group increased significantly, while creatinine clearance rate decreased. CONCLUSIONS: Hyperglycemia affected the expression of antioxidant enzymes. Oxidative stress was caused by hyperglycemia in diabetic rats and may be an important factor in the etiology of diabetic nephropathy.     

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 Qili Qiangxin capsule on serum concentration of adiponectin and heart function in patients with coronary heart disease combined with congestive heart failure

AIM：To investigate the effects of Qili Qiangxin capsule on serum adiponectin (APN), serum N-terminal pro-brain natriuretic peptide (NT-proBNP) and heart function in the patients of coronary heart disease combined with congestive heart failure. METHODS：One hundred and twenty patients were randomly divided into treatment group and control group, and both groups were given anti-failure routine therapy. The patients in treatment group were treated with Qili Qiangxin capsule and the patients in control group were treated with placebo. The patients in the 2 groups were given a certain dose of the drugs for 6 months. The New York Heart Association (NYHA) heart function classification, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD),left ventricular ejection fraction (LVEF), and 6-min walking test (6-MWT) were observed before and after treatment. The levels of APN, NT-proBNP were measured by ELISA before and after treatment. RESULTS：With the increase in the class of NYHA heart function, the serum concentrations of APN and NT-proBNP in the heart failure cases increased significantly. After 6-month treatment, the effective rate in experimental group was 91.7% and that in control group was 75.0%. A significant difference was found between the 2 groups (P<0.01). After treatment, LVEDD and LVESD in both groups were decreased significantly, and LVEF in both groups was increased significantly. The serum concentrations of APN and NT-proBNP decreased significantly (P<0.05). 6-MWT result was improved significantly. Compared with control group, more obvious effect was observed in experimental group (P<0.05). CONCLUSION：Treatment with Qili Qiangxin capsule reduces the levels of APN and NT-proBNP in the patients with coronary heart disease combined with congestive heart failure.     

Effects of T-cadherin on hypoxia/reoxygenation-induced apoptosis of cardiomyocytes from neonatal rats

AIM: To investigate the mechanism of cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) by silencing a new adiponectin receptor T-cadherin through adenovirus-mediated RNA interference. METHODS: The primary cardiomyocytes were isolated from neonatal rats and cultured for 72 h. The cardiomyocytes were randomly divided into control group, H/R group, APN+H/R group, Ad-T-cadherin-siRNA+APN+H/R group and Ad-HK (adenovirus negative control)+APN+H/R group. The transfection ability and efficiency were examined. The expression of T-cadherin at mRNA and protein levels was detected by RT-PCR and Western blotting. The apoptotic rate was analyzed by flow cytometry and TUNEL. RESULTS: High purity of neonatal rat cardiomyocytes was obtained by primary culture. After 48 h, over 90% of myocardiocytes were infected at MOI=100. The transfected myocardiocytes showed a low expression level of T-cadherin under normal physiological condition. Compared with APN+H/R group, the cell apoptotic rate significantly increased in Ad-T-cadherin-siRNA+APN+H/R group (P<0.05). Compared with H/R group, the difference was not statistically significant (P>0.05). CONCLUSION: Ad-T-cadherin-siRNA effectively infects myocardial cells in vitro and successfully reduces the expression of T-cadherin in myocardial cells. The inhibitory effect of adiponectin on H/R-induced cardiomyocyte apoptosis is attenuated by decreasing the expression of T-cadherin.     

Effect of blood glucose control on expression of Smad7 and renal fibrosis in diabetic rats

AIM： To verify the hypothesis that treatment with insulin to control the blood glucose (BG) may relieve or slow down the development of diabetic nephropathy (DN) in diabetic rats by increasing the expression of Smad7. METHODS：The diabetic rat model was established by tail-vein injection of streptozotocin. Sixteen rats were divided into 2 groups. Eight of these animals in diabetes mellitus (DM) group had no treatment. The remaining eight of them in insulin treatment (INS) group were injected with insulin. After 13 weeks, the rats in INS group were given individual treatment with insulin to let the blood glucose level keep within 4 to 7 mmol/L. Meanwhile, 8 rats were used for normal control (NC group). After 16 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and to observe the histophathological changes of the kidney and pancreas. In addition, immunohistochemical staining and Western blotting were employed to detect the protein expression of transforming growth factor β1 (TGF-β1), Smad ubiquitin regulatory factor 2 (Smurf2), Smad7, E-cadherin, α-sooth muscle actin (α-SMA), fibronectin (FN) and collagen I. RESULTS：Compared with NC group, the body weight was significantly reduced in DM group, whereas the body weight in INS group increased gradually. Compared with NC group, the levels of 24 h urine protein (24 h UP), BG and triglyceride (TG) were remarkably increased in DM group. Pathological detection on pancreas indicated that the islet was destroyed. The levels of TGF-β1, Smurf2, α-SMA, FN and collagenⅠ in the kidneys were increased in DM group, and the expression of Smad7 and E-cadherin, which were mainly located in renal tubular epithelial cells, was significantly reduced. Compared with DM group, the levels of 24 h UP and BG were significantly reduced in INS group, and the alleviated renal fibrosis was observed under light microscope. In addition, the protein levels of TGF-β1, Smurf2, α-SMA, FN and collagenⅠ in INS group were decreased compared with DM group, and the expression of Smad7 and E-cadherin was increased significantly. CONCLUSION：Target glucose control with insulin treatment restores the protein expression of Smad7 in the kidney of diabetic rats, reduces the accumulation of extracellular matrix and slows down DN progress. The decrease in TGF-β1 and Smurf2 expression, and the attenuation of Smad7 ubiquitination in renal tissues are the crucial parts in this process.     

Effects of adiponectin on expression of connexin 43 in rat myocardium during ischemia-reperfusion

AIM: To observe the effects of adiponectin(APN) on the expression of connexin 43 (Cx43) in rat myocardium during ischemia-induced arrhythmias. METHODS: The SD rats were randomly divided into 4 groups (n=12): sham operation group (SM group), ischemia and reperfusion group (I/R group), I/R+adiponectin(APN1) group: pre-ischemia with 3.5 μg/kg of APN; I/R+APN2 group: post-ischemia with 3.5 μg/kg of APN. The incidence of ventricular arrhythmias and ventricular arrhythmia score (VAS) were determined. The expression of Cx43 in the ischemic myocardium was studied by the techniques of immunohistochemistry and RT-PCR. The levels of malondialdehyde(MDA) and superoxide dismutase(SOD) were measured by the methods of xanthine oxidase and thiobarbituric acid. The expression of endothelial nitric oxide synthase (eNOS) at mRNA and protein levels was determined by RT-PCR and Western blotting,respectively.The morphological changes of the myocardial tissues were observed under electronic microscope. RESULTS: The VAS and concentration of MDA increased obviously and the activity of SOD was decreased in I/R group as compared with SM group (P<0.01). The expression of Cx43 was evidently decreased and the distribution of Cx43 in the myocardium was disturbed. The expression of eNOS at mRNA and protein levels was decreased in I/R group (P<0.05). The ultrastructure of ventricular myocardium was abnormal in I/R group. Compared with I/R group, APN obviously decreased the VAS caused by ischemia and reperfusion (P<0.01) no matter the drug was given before or after ischemia. APN increased the activity of SOD, inhibited the MDA content in serum, and resulted in normal distribution of Cx43 and increased the expression of Cx43 and eNOS. Compared with I/R group, the changes of heart ultrastructure attenuated greatly in APN group, but didn't recover to normal state. CONCLUSION: Adiponectin antagonizes the arrhythmias during myocardial ischemia and reperfusion via inhibiting oxidative stress and regulating Cx43.     

Effects of adiponectin on reducing endoplasmic reticulum stress injury induced by hypoxia-reoxygenation in cultured rat neonatal cardiomyocytes

AIM: To investigate the effects of adiponectin (APN) on hypoxia-reoxygenation (H/R) induced endoplasmic reticulum stress injury in cultured cardiomyocytes. METHODS: Primary cultured cardiomyocytes were obtained from neonatal rats by enzymatic digestion method. The α-actin expression as molecular marker of the cardiomyocytes was observed by immunocytochemistry. The cells cultured for 72 h were used in the experiment and divided into groups randomly: control group, H/R group, APN+H/R (3 mg/L, 10 mg/L, 20 mg/L, 30 mg/L) groups. The morphological changes of the cardiomyocytes were observed under phase contracted microscope. The content of LDH was measured. The cardiomycocyte apoptosis was detected by flow cytometry. The expression levels of GRP78 and caspase-12 were examined by RT-PCR and Western blotting. RESULTS: The apoptotic rate was significantly increased in H/R group as compared to that in control group (68.20%±1.73% vs 0.73%±0.21%, P<0.05). The levels of LDH in H/R group were also significantly increased. Compared to untreated cells, the protein and mRNA levels of GRP78 and caspase-12 increased significantly in H/R cells. The APN preconditioning significantly reversed these changes. The indexes above improved obviously as compared to H/R group (P<0.05) in a dose-dependent manner. CONCLUSION: Hypoxia/reperfusion induces endoplasmic reticulum stress in rat cardiomyocytes. Adiponectin decreases the endoplasmic reticulum stress injury and plays a protective role by extenuation of cadiomyocyte apoptosis.     

Effect of calories restriction on ER stress in the liver of high fat diet rats

AIM: To study the effect of calories restriction on endoplasmic reticulum(ER) chaperone protein 78-kD glucose regulated protein (GRP78) mRNA expression in the liver of high fat diet rats, in order to explore the mechanism of how calories restriction improves insulin resistance. METHODS: Wistar rats (n=24) were randomly divided into 3 groups: normal chow (NC) group, was fed free normal chow (18.94% of calories as fat) for 12 weeks; high fat group (HF) was fed high fat diet (50.55% of calories as fat) for 12 weeks; calories restriction group (CR) was fed high fat diet for 8 weeks at first, then given 50% of diet consumed by the same age NC group. Changes of body weight, height, and food intake were recorded. At the end of experiment, HOMAIR, the rate of visceral fat (including perirenal fat and epididymal fat) vs weight, plasma protein, blood lipid (including total cholesterol and triglyceride), hepatic GRP78 mRNA and hepatic histological changes (including light microscopic studies and electron microscopic studies) were detected. RESULTS: (1) Animals in HF group had an obviously elevation of fasting insulin (27.51±3.51) mU/L vs (15.46±2.25) mU/L, triglyceride (1.35±0.25) mmol/L vs (0.67±0.10) mmol/L, total cholesterol (2.59±0.34) mmol/L vs (1.41±0.28) mmol/L and insulin resistance index HOMAIR (5.85±0.23 vs 2.85±0.60) compared with NC group, and also had obviously lipid accumulations in the liver. (2) After calories restriction, all the abnormal elevated biochemical indicators were decreased to normal levels, the hepatic lipid accumulations were also improved. (3) The changes of liver ultrastructure in HF group showed rough endoplasmic reticulum enlargement, fragmentation, taking off grain, and with glycogen solution. The changes in CR group were nearly the same as those in NC group. (4) High fat diet induced the expression of GRP78 mRNA, calories restriction might reverse it. CONCLUSION: Reasonable food calories restriction is a good method to improve insulin resistance, partly due to improvement of endoplasmic reticulum stress in liver.     

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.     

Correlation of serum omentin-1 level and insulin resistance in rats

AIM：To establish the insulin resistance rat model for evaluating the correlation of omentin-1 level and insulin resistance. METHODS：SPF male Wistar rats (n=30) were randomly divided into normal control group (NC, n=15) and high-fat diet group (HF, n=15). The rats in NC group were fed with basic diet. The insulin resistant model was established by feeding the rats with high-fat diet in HF group. After 10 weeks, 5 rats in each group were assessed by the technique of hyperinsulinaemic-euglycaemic clamp. After the insulin resistant model was successfully established, the body weight and fasting blood glucose were detected. The concentration of fasting serum omentin-1 was analyzed by ELISA. Fasting serum insulin was measured by radioimmunoassay. RESULTS：No difference of fasting blood glucose between the 2 groups was observed. The level of fasting serum insulin in HF group was significantly higher than that in NC group (P<0.05). The level of serum omentin-1 in HF group were significantly decreased compared with NC group (P<0.01). Pearson’s correlation analysis showed that negative correlations between serum omentin-1 and fasting serum insulin (r=-0.654,P<0.01), serum omentin-1 and free fatty acid (r=-0.446, P<0.05) was found. CONCLUSION：In rats, serum omentin-1 level began to decrease at insulin resistance stage. As serum omentin-1 level decreased, the basal insulin level increased, indicating that decreased serum omentin-1 level may be an early factor of IR, diabetes and cardiovascular diseases.     

Effect of curcumin derivative B06 Bob on synthesis of testosterone from type 2 diabetic rats

AIM: To investigate the effect of curcumin derivatives B06(B06) on the synthesis of testosterone from type 2 diabetic rats. METHODS: Male Sprague-Dawley rats were evenly divided into 5 groups randomly: normal control group (C group), high fat group (H group), high fat treatment group (HT group), diabetes mellitus group (D group) and diabetes treatment group (DT group). The rats in the later 4 groups were fed with high fat diet, after 4 weeks of high fat diet feeding, the rats from D group and DT group were injected with low dose of streptozotocin intraperitoneally to induce diabetes mellitus, while the rats in HT group and DT group were gavaged with B06 at the dose of 0.2 mg·kg^-1·d^-1 for 8 weeks. The blood glucose was detected by glucometer, blood insulin was assayed by ELISA and the insulin resistance index was calculated. The morphology of testes were observed by light and transmission electron microscopy. Serum testosterone and estradiol were measured by radioimmunoassay. The protein expression of steroidogenic acute regulatory protein (StAR) was detected by immunohistochemistry. The mRNA expression of StAR, cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17A1 (P450c17), cytochrome P450 aromatizing enzyme (P450arom), 3β-hydroxysteroid dehydrogenase (HSD) and 17β-HSD was detected by RT-PCR. RESULTS: The levels of blood glucose and insulin resistance index were increased in H group and D group, and serum testosterone was decreased, all of which were reversed after the treatment of B06. Testicular seminiferous tubule was distorted, spermatogenic cells were dropped in H group and D group. In addition, leydig cells were found to have swelling mitochondria in H group and D group, endoplasmic reticulum was reduced, and there was karyopyknosis accompany with sparse chromatin, all of which were ameliorated by B06. The protein expression of StAR was decreased in D group. The mRNA expression of StAR and P450scc was decreased in H group and D group, all of which were increased in B06 treatment group. There was no significant difference in the mRNA expression of P450c17, P450arom, 3β-HSD and 17β-HSD. CONCLUSION: B06 may increase serum testosterone and relieve the damage of testes from type 2 diabetic rats. B06 improves metabolic disorder by up-regulating mRNA expression of StAR and P450scc.     

Serum levels of inflammatory factors and adiponectin in type 2 diabetic retinopathy patients

AIM: To investigate the serum levels of inflammatory factors and adiponectin in type 2 diabetic retinopathy (DR) patients.METHODS: One hundred and ten cases of type 2 diabetic patients were divided into 3 groups: no diabetic retinopathy group (DM, n=35), non-proliferative diabetic retinopathy group (NPDR, n=45), and proliferative diabetic retinopathy group (PDR, n=30). Other 40 normal persons served as controls (NC group). The physical examinated was performed for each patient. Serum levels of fasting plasma glucose (FPG), glycated hemoglobin A1c (HbA1c), 2 h postprandial plasma glucose (2hPG), fasting insulin (FINS), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), adiponectin, intercellular adhesion molecule-1(ICAM-1), tumor necrosis factor-α (TNF-α) and high-sensitivity C-reactive protein (hs-CRP) were measured. Insulin resistance index (HOMA-IR) was also calculated.RESULTS: The systolic blood pressure, body-mass index, waist-hip ratio, serum levels of TG, LDL-C, FPG, 2hPG, HbA1c, ICAM-1, TNF-α, hs-CRP and HOMA-IR were significantly higher in DM group, NPDR group and PDR group than those in NC group (P<0.05). The systolic blood pressure, serum levels of ICAM-1, TNF-α, hs-CRP and HOMA-IR were higher in NPDR group and PDR group than those in DM group (P<0.05). The serum concentration of adiponectin was lower in DM group, NPDR group and PDR group than that in NC group (P<0.05), and that was also lower in NPDR group and PDR group than that in DM group (P<0.05). The negative correlations between adiponectin and ICAM-1 (r=-0.735,P<0.01), TNF-α (r=-0.781,P<0.01), hs-CRP (r=-0.768, P<0.01) or HOMA-IR (r=-0.752, P<0.01) were observed. The relationships between HOMA-IR and ICAM-1 (r=0.857,P<0.01), TNF-α (r=-0.906, P<0.01) or hs-CRP (r=-0.888,P<0.01) were positive.CONCLUSION: The results suggest that inflammatory refactors and adiponectin play important roles in the pathophysiology and progression of DR. The protective effects of adiponectin on DR may be related with its anti-inflammatory reactions to improve insulin resistant.     

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.     

Effect of alpha-lipoic acid on expression of miR-21/Smad7 and fibrosis in kidney of diabetic rats

AIM: To investigate the protective effect of alpha-lipoic acid (ALA) on the kidney of the rats with diabetes mellitus (DM), and to discuss the mechanism. METHODS: The DM rats were divided into normal control (NC) group, DM group and ALA group. After treated with ALA for 6 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and the pathological changes of the kidney tissues were observed by HE staining and Masson staining. The protein levels of transforming growth factor-β1 (TGF-1), p-Smad2/3, Smad7, collagen I and collagen Ⅲ were determined by Western blot. In addition, the expression of microRNA-21 (miR-21) was detected by RT-qPCR. RESULTS: Compared with NC group, the kidney weight/body weight, blood glucose (BG), total cholesterol, triglyceride and 24-h urine protein were remarkably increased in DM group (P<0.05). The pathological observation of the kidney tissues showed fibrosis changes in DM group. The level of Smad7 was reduced in DM group, while the levels of TGF-β1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were increased (P<0.05). After treatment with ALA for 6 weeks, all the relevant biochemical parameters were reduced except BG, and the renal fibrosis lesions were obviously alleviated. Compared with DM group, the levels of TGF-1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were reduced in ALA group, while the level of Smad7 was increased (P<0.05).CONCLUSION: ALA may prevent the development of renal fibrosis in rats through restraining the expression of TGF-β1 and miR-21, increasing the levels of Smad7 protein, and reducing the deposition of extra cellular matrix.     

Protective effect of pyrrolidine dithiocarbamate on cardiac muscles in diabetic rats

AIM: To investigate the effect of pyrrolidine dithiocarbamate (PDTC) on reducing blood glucose level and its protective effect on cardiac muscles in diabetic rats.METHODS: Thirty-seven male Wistar rats were randomly divided into normal control (NC) group and the high-fat diet (HFD) group. After 8 weeks of feeding, the rats in high-fat diet group were given a single dose of streptozotocin (STZ, 27 mg/kg) by intraperitoneal injection to induce type 2 diabetes. The diabetic rats were randomly divided into diabetes mellitus (DM) group and PDTC treatment(PDTC) group. The rats in PDTC group were intraperitoneally injected with PDTC (50 mg/kg) once daily. The rats in NC group and DM group were injected with equivalent volume of saline in the same way. After 1-week treatment, the level of blood glucose was measured, and all animals were killed. The concentration of malondialdehyde (MDA) and the activity of superoxide dismutases (SOD) and glutathione peroxidase (GSH-Px) were determined using commercial kits. The ultrastructural changes of the cardiac tissues were observed under transmission electron microscope. The expression of inducible nitric oxide synthase(iNOS) and content of nitrotyrosine was examined by the method of immunohistochemistry.RESULTS: The levels of blood glucose and MDA were significantly higher, while the activity of SOD and GSH-Px was lower in DM group than those in NC group (P<0.01). Treatment with PDTC markedly decreased the blood glucose and MDA content, and increased the activity of SOD and GSH-Px. Severe degeneration, necrosis, mitochondrial damage and inflammatory cell infiltration were found in the cardiac tissues in DM group. Treatment with PDTC markedly attenuated mitochondrial damage. The expression of iNOS and content of nitrotyrosine in cardiac tissues were significantly higher in DM group than those in NC group, and those were reduced after administration of PDTC.CONCLUSION: High glucose induces oxidative stress, increases the expression of iNOS and content of nitrotyrosine, and impairs the structure and function of myocardium. PDTC reduces blood glucose level, decreases the expression of iNOS and content of nitrotyrosine, and delays or attenuates the development of diabetic cardiomyopathy in diabetic rats.     

Effect of JNK expression in dorsal root ganglia on histological change of foot skin in diabetic rats

AIM: To observe the change of skin histology in diabetic rats and to investigate the possible me-chanism of c-Jun N-terminal kinase (JNK) protein in the dorsal root ganglion (DRG) during the process. METHODS: Diabetic animal model was established in the male SD rats by intraperitoneal injection of streptozotocin. Plantar skin specimens of the rats were collected from control group, DM 2-week group (DM2), DM 4-week group (DM4), and DM 8-week group (DM8). Immunohistochemical staining and HE staining were used to observe the change of PGP 9.5 immunoreactive nerve terminals and the structures of the skin tissues. The protein expression of PGP 9.5 in the plantar skin tissues, and JNK and p-JNK protein in the DRG within lumbar 5, 6 (L5, 6), and sacral 1 (S1) spinal cord segments were detected by Western blotting. RESULTS: PGP 9.5 immunoreactive nerve terminals of the plantar skin of the rats mainly distributed in the basal layer of the epidermis and papillary dermis. Compared with control group, PGP 9.5 positive nerve terminals in DM4 group showed reduced density and sparse distribution. PGP 9.5 positive nerve terminals in DM8 group showed significantly reduced distribution, thinner nerve diameter, shorter length and distorted shape. Histological changes of the thinner epidermal tissue, reduced epidermal cell layers, uneven cell distribution and arrangement in DM4 group, and significantly reduced epidermal cell layers, swollen and blurred cells, increasing cell gap, lack of stratified epidermis arrangement for part of epidermis, atropal and degenerated dermal collagen fiber, significantly decreased subcutaneous fat in DM8 group were observed. The results of Western blotting showed that the protein expression of PGP 9.5 in the plantar skin tissue of DM rats was progressively decreased along with the disease, while the protein level of p-JNK in L5, 6-DRG or S1-DRG showed a gradual increasing trend. PGP 9.5 immunoreactive positive nerve terminal density of plantar skin in DM rats had a negative correlation with the protein level of p-JNK in L5, 6-DRG and S1-DRG (P<0.01), but showed a significant positive correlation with the plantar skin thickness (P<0.01). CONCLUSION: The protein level of p-JNK within L5, 6-DRG or S1-DRG in DM rats shows a progressive enhancement. At the same time, there is a significant change in the skin tissue density and structure. The changes of skin tissue and nerve morphology in DM rat may be related to the activation of JNK/SAPK pathway in L5, 6-DRG or S1-DRG cells. Blocking or inhibiting JNK/SAPK pathway may delay the diabetic peripheral neuropathy and reduce the risk of skin lesions.     

Effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats

AIM:To investigate the effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats. METHODS:The male Sprague-Dawley rats were induced as the model of the type 2 diabetic neuropathic pain rats, they were randomly divided into 6 groups (n=27): type 2 diabetic neuropathic pain (DNP) group, type 2 diabetic neuropathic pain and intraperitoneal injection of curcumin (Cur) group, type 2 diabetic neuropathic pain and solvent control (DSC) group, type 2 diabetic neuropathic pain and JNK inhibitor (DJ) group, type 2 diabetic neuropathic pain and JNK inhibitor solvent control (DJS) group, type 2 diabetic neuropathic pain and monocyte chemoattractant protein 1 (MCP-1) agonist (DM) group. Another 27 normal SD rats were selected as control group. Mechanical withdrawal threshod and thermal withdrawal latency were measured at 3rd d, 7th d and 14th d after dosing, then the lumbar segment 4~6 of the spinal cord and L4~6 DRG were removed at the same time. ELISA was used to measure MCP-1 level. The expression of p-JNK was determined by Western blotting. RESULTS:Compared with DNP group, p-JNK was significantly decreased at 7th d and 14th d in Cur group, DJ group and DM group after treatment (P<0.05). Compared with C group, the MCP-1 was significantly declined in other 6 group after streptozotocin injection (P<0.05). Compared with DNP group, MCP-1 were significantly increased at 7th d and 14th d in Cur group and DJ group after treatment (P<0.05), and that in DM group was greatly decreased (P<0.05). CONCLUSION: The expression of p-JNK and MCP-1 was increased in DNP rats with spinal cord and dorsal root ganglion. The mechanism of curcumin reducing the neuropathic pain in type 2 diabetic rats might be through regulating the JNK/MCP-1 pathway.