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.     

Effect of chronic intermittent hypoxia on AMPK pathway in young rats

AIM: To investigate the effect of chronic intermittent hypoxia on AMP-activated protein kinase(AMPK) pathway in the brain of young rats. METHODS: Part one: SD mice(3~4 weeks old) were randomly divided into 4 groups(n=8): simulated air control group for 2 weeks(2AC), chronic intermittent hypoxia group for 2 weeks(2IH), simulated air control group for 4 weeks(4AC) and chronic intermittent hypoxia group for 4 weeks(4IH). Part two: SD mice(3~4 weeks old) were randomly divided into 2 groups(n=8): chronic intermittent hypoxia group for 4 weeks(4IH) and chronic intermittent hypoxia group treated with AMPK inhibitor for 4 weeks(4IHI). After modeling, the eight-arm maze test was performed. TUNEL method was used to detect the neuronal apoptosis in the hippocampal and prefrontal cortical tissues. The mRNA expression of adenosine A2a receptor was examined by RT-qPCR, and the protein levels of phosphorylated AMPK(p-AMPK) and mammalian target of rapamycin(p-mTOR) were determined by Western blot. RESULTS: Compared with control group, the numbers of reference memory error(RME), working memory error(WME) and total error(TE) in 2IH group and 4IH group significantly increased(P<0.01). Compared with 2IH group, the numbers of errors in 4IH group also increased significantly(P<0.01). Compared with 4IH group, the values in 4IHI group significantly decreased. Compared with control group, the neuronal apoptosis of hippocampus and prefrontal cortex in 2IH group and 4IH group increased, and that in 4IH group was more evident(P<0.05). In 4IHI group, the neuronal apoptosis decreased. The mRNA expression of adenosine A2a receptor in the hippocampal and cortical tissues in 2IH group and 4IH group was higher than that in control group. The protein level of p-AMPK was higher, and p-mTOR was lower in 2IH group and 4IH group, and those in 4IH group were more evident(P<0.05). Compared with 4IH group, the protein level of p-AMPK was lower, and p-mTOR was higher in 4IHI group. CONCLUSION: Chronic intermittent hypoxia induces neuronal apoptosis, resulting in impairment of learning and memory in a time-dependent manner by upregulating adenosine A2a receptor, activating AMPK activity, and inhibiting mTOR phosphorylation in rats.     

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.     

miRNA-126 inhibits proliferation,migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

AIM: To investigate the effects of microRNA(miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control(NC) group and control group(P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir(P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group(P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway.     

Proliferative effect of PDGF and anti-proliferative activity of AMPK on vascular smooth muscle cells

AIM: To investigate the proliferative effect of platelet-derived growth factor (PDGF) and anti-proliferative activity of AMP-activated protein kinase (AMPK) on vascular smooth muscle cells (VSMCs). METHODS: The proliferation of VSMCs cultured with PDGF and activation of AMPK were observed. VSMCs were divided in 4 groups: control group; PDGF group; 5-aminoimidazole-4 -carboxamide-1-β-D-riboside (AICAR) group and AICAR+PDGF group. The time course of AMPK activation was determined. The protein level of mTOR was also measured. RESULTS: Compared with control group, the proliferative rate in PDGF group was significantly increased. The growth of VSMCs was inhibited in a time-dependent manner and the activity of p-mTOR was significantly decreased in AICAR group. Compared with control group, the expression of p-AMPK in PDGF group was significantly decreased, and that in AICAR group and AICAR+PDGF group was significantly increased. The expression of p-AMPK in AICAR+PDGF group was higher than that in PDGF group. The activity of p-mTOR in PDGF group was significantly higher than that in control group, while that of AICAR group and AICAR+PDGF group was significantly decreased. The expression of p-mTOR in AICAR+PDGF group was lower than that in PDGF group. CONCLUSION: Stimulation of VSMCs with PDGF promotes the cell proliferation, which can be inhibited by AICAR. The proliferation of VSMCs activated by AMPK is probably correlated with the down-regulation of mTOR expression.     

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.     

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.     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.     

Inhibitory effect of ischemic postconditioning on autophagy induced by focal cerebral ischemia reperfusion in rats

AIM: To investigate the effect of ischemic postconditioning (IPC) on autophagy induced by focal cerebral ischemia reperfusion (I/R) in rats. METHODS: Healthy male SD rats were assigned randomly into sham-operation (sham) group, I/R group and IPC group with 10 rats in each group. The rats in sham group were only exposed the right common, internal and external carotid artery surgically. The rats in I/R group were subjected to right middle cerebral artery occlusion (MCAO) by the modified Longa suture method for 2 h followed by 24 h of reperfusion. The rats in IPC group were subjected to MCAO for 2 h followed by reperfusion of the ipsilateral common carotid artery occlusion for 10 s for 5 episodes, and then reperfusion for 24 h. Autophagy was obeserved by transmission electron microscopy (TEM). The protein levels of mammalian target of rapamycin (mTOR), p-mTOR and microtubule associated protein light chain 3 (LC3)-II in brain tissue of the rats were determined by Western blot. Pathological changes of brain tissue were observed by HE staining. RESULTS: The protein levels of mTOR and p-mTOR in IPC group were significantly higher than those in I/R group (P<0.05). The expression of LC3-II in IPC group was significantly lower than that in I/R group (P<0.01). The cerebral infarction area and brain water content in IPC group were significantly lower than those in I/R group (P<0.01). HE staining showed that neurons degeneration and necrosis in IPC group were significantly alleviated compared with I/R group. TEM observation showed that IPC revealed fewer autophagosomes, with much less severe cell damage than that in I/R group. CONCLUSION: IPC reduces brain ischemia reperfusion damage by decreasing autophagy of brain cells, which might be related to the activation of mTOR.     

Effects of dexmedetomidine on behaviors and expression of BDNF and mTOR in hippocampus of depressive rats

AIM: To investigate the effects of dexmedetomidine (DEX) on the behaviors and the expression of brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) in the hippocampus of depressive rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into 5 groups: sham operation group, model group, and DEX (2.5, 5 and 10 μg/kg) groups. The rats were randomly selected in each group (n=12). The rat depression model was established by chronic unpredictable mild stress and ovariectomy. The rats in DEX groups received daily DEX treatment via intraperitoneal injection for 21 d. The forced swimming immobility time (FSIT) and open-field test were used to evaluate the antidepressant effect of DEX. Escape latency and times of crossing the flat were evaluated by Morris water maze. The histological changes of hippocampal neurons were determined by Nissl staining. The mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were detected by RT-qPCR. The protein expression of IL-1β, IL-6, TNF-α and BDNF, and the phosphorylation levels of protein kinase A (PKA), cAMP response element-binding protein (CREB), tropomyosin-related kinase B (TrkB), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and mTOR in hippocampus were evaluated by Western blot. RESULTS: Compared with model group, the FSIT was significantly reduced and the spontaneous activity was markedly increased in DEX groups. The damage of the hippocampal neurons was obviously attenuated, the escape latency was obviously decreased, and times of crossing the flat were markedly increased (P<0.05 or P<0.01). The levels of IL-1β, IL-6 and TNF-α were obviously decreased, and the protein levels of p-PKA, p-CREB, BDNF, p-TrkB and p-PI3K, p-Akt, p-mTOR in hippocampal tissues were obviously increased (P<0.05 or P<0.01). CONCLUSION: Dexmedetomidine improves the behaviors and the spatial learning and memory ability of depressive model rats, which may be related to its anti-inflammatory effects, as well as up-regulating the protein levels of BDNF and p-TrkB, and activating PI3K/Akt/mTOR signaling pathway in the hippocampus.     

Regulation of retinoid X receptor-mediated-autophagy pathway in rat pulmonary ischemia/reperfusion injury

AIM: To investigate the regulation of retinoid X receptor (RXR)-mediated autophagy pathway in rat pulmonary ischemia/reperfusion (I/R) injury. METHODS: The male SPF-grade SD rats were randomly divided into 7 groups (n=10):normal control (C) group, sham (S) group, sham plus 9-cis-retinoic acid (SRA) group, sham plus HX531 (SH) group, I/R group, I/R plus 9-cis-retinoic acid (RA) group and I/R plus HX531 (HX531) group. The model of pulmonary I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion. The animals in C group didn't receive any treatment. Only sternotomy was performed for the rats in S group, the hilum of lung was not clamped, and the rats were mechanically ventilated for 210 min. The clamping of the left hilum of lung for 30 min followed by 180 min of reperfusion was performed in I/R group. In SRA, SH, RA and HX531 groups, 9-cis-re-tinoic acid (5 mg/kg) was intraperitoneally injected at 90 min before establishment of the model. In SH group and HX531 group, HX531 at 5 mg/kg was intraperitoneally injected at 30 min before establishment of the model. Left lung tissues were removed after 180 min of reperfusion for determing the index of quantitative assessment (IQA) of alveolar damage. The pathological changes of the lung were observed by HE staining, and immunofluorescence staining was used to observe the changes of RXRα in various lung tissues. The mRNA expression of autophagy-associated molecules LC3, beclin 1 and mTOR was detected by RT-PCR. The protein levels LC3-Ⅱ, beclin 1 and p-mTOR in each group were determined by Western blot. RESULTS: Compared with C group, the lung IQA, the mRNA expression of LC3 and beclin 1, and the protein levels of LC3 -Ⅱ and beclin 1 in I/R, RA and HX531 groups were increased significantly, the mTOR mRNA and p-mTOR protein levels were decreased (P<0.05), and the morphological structure of the lung was also impaired. Compared with I/R group, the lung IQA and the expression of LC3 and beclin 1 at mRNA and protein levels were significantly decreased, the mRNA expression of RXRα and mTOR, and the protein level of p-mTOR were increased in RA group (P<0.05), and the structural damage of the lung tissue was also significantly reduced. No statistically significant difference was observed between I/R group and HX531 group. Compared with RA group, the lung IQA and the expression of LC3 and beclin 1 at mRNA and protein levels was increased significantly, the mRNA expression of RXRα and mTOR, and the protein level of p-mTOR were decreased in HX531 group (P<0.05), and the morphological damage of the lung tissue was increased. CONCLUSION: The activation of RXR effectively alleviates the pulmonary I/R injury in rats. The protective role of RXR in lung tissue may be related to the inhibition of autophagy pathway.     

Astragalus polysaccharides improve chronic heart failure by promoting myocardial FFA metabolism via AMPK pathway

AIM: To investigate the effect of Astragalus polysaccharides (APS) on chronic heart failure and its mechanism. METHODS: Male SD rats (n=32) were randomly divided into control group, sham group, model group and APS group (8 rats in each group). The left coronary artery ligation in the rats was conducted to establish myocardial infarction heart failure model. After modeling, the rats in APS group were given APS (3 g·kg^-1·d^-1) by intragastric administration for 6 weeks. Left ventricular diastolic diameter (LVD), left ventricular systolic diameter (LVS), left ventricular ejection fraction (LVEF) and fractional shortening (FS) were detected by echocardiography. HE staining was used to observe the pathological changes. The concentrations of free fatty acid (FFA) in the serum and myocardium were observed by the method of acetyl coenzyme A synthetase and acetyl coenzyme A oxidase (ACS-ACOD). The protein levels of total AMP-activated protein kinase (AMPK), phosphorylated AMP-activated protein kinase (p-AMPK), fatty acid translocase (FAT/CD36) and carnitine palmitoyltransferase I (CPT-1) were measured by Western blotting. RESULTS: No significant difference in each index between sham group and control group was observed. Compared with control group, LVEF and FS in model group was significantly decreased, while LVD and LVS was significantly increased (P<0.05). The LVEF and FS in APS group were significantly improved compared with model group (P<0.05), and there was no significant difference between APS group and control group. LVD and LVS in APS group were obviously improved compared with mo-del group (P<0.05), and the difference was significant compared with control group (P<0.05). Compared with control group, focal myocardial necrosis increased, and residual myocardial cells reduced in model group, while those was much better in APS group as compared with model group (P<0.05). The FFA concentrations in the serum and myocardium in model group increased significantly compared with control group (P<0.05), while those decreased significantly in APS group as compared with model group (P<0.05). The protein levels of p-AMPK, CPT-1, and cell membrane FAT/CD36 in model group decreased significantly compared with control group (P<0.05), and those in APS group increased obviously compared with control group (P<0.05). CONCLUSION: APS improves chronic heart failure by activating the AMPK pathway and promoting myocardial ingestion and utiliation of FFA.     

NVP-BEZ235 induces autophagy of polycystic kidney rat cholangiocytes

AIM: To explore the effect of dual PI3K/Akt/mTOR inhibitor NVP-BEZ235 on autophagy of polycystic kidney (PCK) rat cholangiocytes. METHODS: The protein levels of p-mTOR and p-Akt in the bile duct epithelial cells were examined by immunohistochemistry. The effect of NVP-BEZ235 on the viability of cholangiocytes was detected by WST-1 assay. The levels of PI3K/Akt/mTOR signaling pathway and autophagy-related proteins with NVP-BEZ235 treatment were determined by Western blot. The effects of LC3 and Beclin 1 silencing, and authophagy-specific inhibitor 3-methyladenine (3-MA) on the cell viability were analyzed by WST-1 assay. RESULTS: The protein levels of p-Akt and p-mTOR were highly increased in the bile duct epithelium of the PCK rats. NVP-BEZ235 significantly inhibited the viability of the cholangiocytes in a dose- and time-dependent manner (P<0.05). NVP-BEZ235 significantly reduced the levels of PI3K/Akt/mTOR signaling pathway-related proteins in the PCK rat cholangiocytes. NVP-BEZ235 upregulated the autophagy-specific proteins LC3 II and Beclin 1. The inhibitory effect of NVP-BEZ235 on the cell viability was weakened by treatment with 3-MA and knockdown of LC3 and Beclin 1 (P<0.01).CONCLUSION: The PI3K/Akt/mTOR inhibitor NVP-BEZ235 suppresses the viability of PCK rat cholangiocytes, and the mechanism is closely related with autophagy.     

Shikonin induces apoptosis and autophagy of human cervical cancer HeLa cells by PI3K/Akt/mTOR signaling pathway

AIM:To observe the effects of shikonin on the apoptosis and autophagy of human cervical cancer HeLa cells, and to explore the possible role of PI3K/Akt/mTOR signaling pathway in these processes. METHODS:The HeLa cells were treated with shikonin, and the cell viability was detected by CCK-8 assay. The apoptosis was detected by Annexin V/PI double staining. The autophagosome was observed by transfection with GFP-LC3 into the HeLa cells. After the treatment with shikonin combined with autophagy inhibitor 3-methyladenine (3-MA) or apoptosis inhibitor Z-DEVD-FMK, the protein levels of autophagy-and apoptosis-related molecules microtuble-associated protein 1 light chain 3 (LC3) and cleaved caspase-3 in the HeLa cells were determined by Western blot. The protein levels of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR) were also determined by Western blot. RESULTS:Shikonin significantly inhibited the viability of HeLa cells (P<0.05). Compared with control group, shikonin significantly induced apoptosis of HeLa cells (P<0.05). The results of GFP-LC3 plasmid transfection analysis showed that green dot-like congregate autophagosomes appeared in the cytoplasm of the HeLa cells after shikonin treatment, while the autophagosomes were rarely observed in control group. Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly decreased and cleaved caspase-3 was significantly increased in shikonin+3-MA group (P<0.05). Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly increased and cleaved caspase-3 was significantly decreased in shikonin+Z-DEVD-FMK group (P<0.05). Compared with control group, shikonin significantly decreased the protein levels of p-PI3K, p-Akt and p-mTOR (P<0.05). CONCLUSION:The apoptosis and autophagy of the HeLa cells are induced by shikonin, these two processes are complementary. The mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway.     

Role of retinoic acid X receptor in regulation of autophagy in rat alveolar type Ⅱ epithelial cells induced by hypoxia/reoxygenation

AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation; （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h; （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling, and the rest were treated the same as the H/R group; （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia; （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h, then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure, and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）, beclin 1, LC3, mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK, beclin 1, LC3-Ⅱ, p-mTOR and P62. RESULTS Compared with Con group, the cell viability in H/R, DMSO, 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK, beclin 1 and LC3 was significantly increased, and the protein levels of p-AMPK, beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased, and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R, DMSO and HX531 groups （P<0.05）, and no significant difference among H/R, DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R, and its mechanism may be related to the inhibition of autophagy.     

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.     

miR-34a induces senescence of bone marrow-derived mesenchymal stem cells under high glucose condition by SIRT1

AIM:To detect the effect and potential mechanism of microRNA-34a (miR-34a) on the senescence of bone marrow-derived mesenchymal stem cells (BMSCs) under high glucose condition. METHODS:BMSCs were isolated and cultured from 60~80 g male SD rats. The BMSCs were divided into 5 groups:normal glucose(NG) group, high glucose(HG) group, HG+miR-34a mimic group, HG+miR-34a NC group and HG+miR-34a inhibitor group. In order to confirm whether miR-34a regulated the senescence of BMSCs under high glucose condition by regulating the expression of silent information regulator 1(SIRT1), in addition to the above groups, HG+siRNA-SIRT1 group, HG+siRNA-NT group and HG+miR-34a inhibitor+siRNA-SIRT1 group were added. The expression of miR-34a and SIRT1 mRNA was detected by RT-qPCR. CCK-8 assay and senescence-associated β-galactosidase assay were used to detect cell viability and senescence, respectively. The protein expression of SIRT1, forkhead box O3a (FOXO3a) and P21 in the BMSCs was analyzed by Western blot. RESULTS:The expression of miR-34a in HG group was increased significantly compared with NG group (P<0.01), and long-term exposure of the BMSCs to high glucose lead to decreased cell viability and increased senescence (P<0.05). Compared with HG+miR-34a NC group, the cell viability in HG+miR-34a mimic group was decreased significantly (P<0.01), the senescence of BMSCs was increased significantly (P<0.01), the protein expression of SIRT1 was decreased significantly (P<0.01) and the protein expression of FOXO3a was increased significantly (P<0.01). However, inhibition of miR-34a expression showed the opposite effect to miR-34a mimic. Similar to the HG+miR-34a mimic group, the protein expression of P21 and FOXO3a in HG+siRNA-SIRT1 group were significantly higher than that in HG group (P<0.01). After adding siRNA-SIRT1 into HG+miR-34a inhibitor group, the inhibitory effect of the miR-34a inhibitor on the expression of P21 and FOXO3a in BMSCs were partly weakened (P<0.05). CONCLUSION:miR-34a regulate the senescence of BMSCs under high glucose condition by regulating the expression of SIRT1.     

Carnosine protects against high glucose-induced injury in H9c2 cells

AIM： To explore the protective effect of carnosine (Car) on cardiomyocytes with high glucose (HG)-induced injury. METHODS：Rat H9c2 cardiomyocytes were cultured in vitro and divided into three groups: normal control (NC) group, HG group and Car pretreatment (Car+HG) group. The survival rate of H9c2 cells was measured by MTT assay. Intracellular level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA. The protein expression of caspase-8, caspase-9 and caspase-3 was determined by Western blotting. RESULTS：The survival rate of H9c2 cells decreased with the increases in glucose concentration and time, while pretreatment with 20 mmol/L Car could increase the survival rate significantly (P<0.05). The intracellular level of ROS in HG group was significantly increased compared with NC group (P<0.05), while that in Car+HG group was significantly decreased compared with HG group (P<0.05). The expression of caspase-8, caspase-9 and caspase-3 proteins in HG group was significantly increased compared with NC group (P<0.05). Compared with HG group, the expression of caspase-9 and caspase-3 was significantly decreased in Car+HG group (P<0.05), but the expression of caspase-8 did not obviously change (P>0.05). CONCLUSION：Carnosine can protect H9c2 cells against the injury of oxidative stress and apoptosis induced by high glucose.