Thiazolidinedione improves Alzheimer-like changes in the hippocampus of type 2 diabetic rats by Wnt pathway

AIM: The abnormal level of insulin and glycemia in type 2 diabetes mellitus(T2DM) are important risk factors of Alzheimer's disease (AD). To explore the mechanism that thiazolidinedione (TZD) decreases the incidence of AD in T2DM, we use TZD on T2DM rats for an intervention and detect the change of Wnt pathway before and after the intervention.METHODS: To establish a T2DM model, the rats were fed with high glucose, high fat and high protein for 8 weeks, and then injected with STZ. TZD was administered intragastrically for 2 and 4 weeks and the rats were divided into TZD2W and TZD4W groups, respectively. Plasma insulin level was measured by RIA method, and the plasma glucose was detected by glucose-oxidase method. Total tau level, the phosphorylation level of tau at individual phosphorylation sites and the level of amyloid β precursor protein(APP), β-catenin, glycogen synthase kinase-3β (GSK-3β) and PPARγ in rat hippocampus were analyzed by Western blotting. The activity of GSK-3β in the hippocampus of rats was determined using γ- -ATP and the specific peptide substrate. The level of APP was also determined by immunochemistry. The insulin resistant (IR) degree was valued by HOMA-IR.RESULTS: Glycemia level in T2DM and TZD2W groups was obviously higher than that in control group. No significant difference of glycemia level between TZD4W and control group was observed. Plasma insulin levels in all groups were evidently higher than that in control group. The IR degree in T2DM and TZD2W groups increased significantly as compared to control group, but no obvious difference between TZD4W and control group was observed. The level of phosphorylated tau protein at site Ser199/202 and Ser422, and APP level in hippocampus of T2DM rats were found to be notably raised as compared to control group, but the level of phosphorylated tau protein at those sites and APP level were decreased gradually. No change of the PPARγ level was found in the hippocampus in T2DM and control group, but a notable increase was observed after TZD intervention. There was a decrease in β-catenin level in hippocampus of T2DM rats, which increased after TZD intervention for 2 and 4 weeks. There was a rise of GSK-3β activity in T2DM rats, which decreased after intervention.CONCLUSION: These findings suggest that TZD may improve the AD-like changes in the hippocampus of T2DM rats by up-regulation of Wnt pathway, which acts before the insulin signal transduction in the contribution of AD-like changes in T2DM rats.     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.     

Intranasal insulin treatment ameliorates Alzheimer disease-like changes in hippocampus in a rat model of type 2 diabetes

AIM： To investigate Alzheimer disease (AD)-like changes and 2 key components of the insulin signaling pathway in the brain of a rat model of type 2 diabetes (T2D) after insulin treatment. METHODS：The rat model of T2D was established by feeding a high-protein, high-glucose and high-fat diet followed by intrasubcutaneous injection of streptozocin. Intranasal insulin treatment (T2D+I-I) and subcutaneous insulin injection (T2D+S-I) were applied to elevate the insulin level in the brain. The insulin levels in plasma and cerebrospinal fluid as well as the concentration of plasma glucose were measured. Total tau level, the phosphorylation level of tau at some phosphorylation sites, and the activation of GSK-3β and Akt in subcutaneous of the rats were also analyzed by Western blotting.RESULTS：AD-like changes, decreased Akt activation and over-activation of GSK-3β in the hippocampus of the T2D rats were observed. Intranasal insulin treatment for 4 weeks normalized the levels of Akt and GSK-3β, as well as reduced the AD-like changes in the hippocampus of the T2D rats, whereas the treatment with insulin by subcutaneous injection for 4 weeks had minimal effects on the levels of GSK-3β and tau phosphorylation in the hippocampus. CONCLUSION： Intranasal insulin treatment, but not subcutaneous insulin treatment, might decrease the risk of AD in T2D rats by reducing AD-like changes and up-regulating the impaired insulin signaling pathway in the hippocampus,indicating the potential use of intranasal insulin delivery for treatment of AD.     

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.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

PI3K/Akt/GSK-3β pathway mediates ABC transporter regulating multidrug resistance in human colon cancer HCT-15 cells

AIM: To investigate whether the PI3K/Akt signaling pathway regulates the expression of ABC transporter through the downstream glycogen synthase kinase-3β (GSK-3β) pathway and participates in the multidurg resistance of colorectal cancer (CRC) HCT-15 cells. METHODS: Colorectal cancer HCT-15 cells were cultured and then treated with GSK-3β inhibitor (HY-19807) and PI3K/Akt pathway inhibitor (HY-13898), respectively. The median inhibitory concentration (IC₅₀) of oxaliplatin for HCT-15 cells in each group was detected by CCK-8 assay, the inhibition rate and resistance index were also calculated. The protein levels of Akt, p-Akt, GSK-3β, p-GSK3β-Ser9 and ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP-2) in the HCT-15 cells were determined by Western blot. The mRNA expression of ABC transporter in the HCT-15 cells was detected by RT-qPCR. The cell cycle distributions were analyzed by flow cytometry assasy. RESULTS: After GSK-3β inhibitor HY-19807 was used in the HCT-15 cells, the median inhibitory concentration of oxaliplatin was significantly increased, the protein levels of p-GSK3β-Ser9, P-gp and MRP-2 were up-regulated compared with control group (P<0.05), the changes of Akt and p-Akt were not obvious compared with control group (P>0.05). The results of RT-qPCR also showed that the mRNA levels of ABCB1 and ABCC2 were increased (P<0.01). Meanwhile, analysis of the cell cycle distribution showed that GSK-3β inhibitor HY-19807 promoted HCT-15 cell transition from G₁ phase to S phase, and cell proliferation was vigorous. After the PI3K/Akt pathway inhibitor HY-13898 was applied to HCT-15 cells, the IC₅₀ of oxaliplatin was decreased compared with control group (P<0.05). Moreover, the protein levels of p-Akt, p-GSK3β-Ser9, P-gp and MRP-2 were down-regulated (P<0.01). RT-qPCR results also showed that the mRNA expression of ABCB1 and ABCC2 was decreased (P<0.01). At the same time, G₁ phase was prolonged, which inhibited cell transition from G₁ phase to S phase, and inhibited cell proliferation. The protein expression of total GSK-3β was consistent in each group. CONCLUSION: The PI3K/Akt signaling pathway is involved in the proliferation and multidrug resistance of colorectal cancer HCT-15 cells by regulating the phosphorylation of GSK-3β and changing the expression of ABC transporter.     

Effect of short-term high-fructose feeding on liver triglyceride content and hepatic insulin sensitivity in mice

AIM: To investigate the effect of short-term high-fructose feeding on liver triglyceride content and hepatic insulin sensitivity in mice. METHODS: Male C57BL/J6 mice were divided into control group and high (HFru) fructose group. After 3-day feeding, intraperitoneal glucose tolerance test (ipGTT) was performed to evaluate whole-body insulin sensitivity. The mice were sacrificed,and the liver samples were collected for measuring the liver triglyceride content and observing the pathological changes of the liver under light microscope with HE staining. The protein levels of lipogenic enzymes in the liver tissues were measured. To evaluate the hepatic insulin sensitivity, the protein levels (expressed as the ratio) of phosphorylated Akt/total Akt (p-Akt/t- Akt) and phosphorylated GSK-3α/β/total GSK-3α/β(p- GSK-3α/β/t- GSK-3α/β) were compared between 2 groups of the mice with or without insulin injection. RESULTS: After 3-day feeding of high-fructose diet, compared with control group, the area under the curve of ipGTT and triglyceride contents in the liver tissues were significantly increased in HFru group. HE staining of the liver in the mice in HFru group showed obvious lipid droplet formation. Compared with control group, the protein expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD-1) was significantly increased in HFru group. After insulin injection, the ratio of p-Akt/t-Akt and p-GSK-3α/β/t-GSK-3α/β was significantly decreased in HFru group as compared with control group. CONCLUSION: A 3-day short-term high-fructose feeding induces liver steatosis, which is related to the increased protein expression of FAS, ACC and SCD-1. Liver steatosis occurs simultaneously with the development of hepatic insulin resistance.     

Changes of canonical Wnt signaling pathway in brains of rats with autism induced by prenatal exposure to valproic acid

AIM：To investigate the roles of the canonical Wnt pathway in autism. METHODS：Using an autistic model induced by prenatal exposure to valproic acid (VPA), we detected the expression of the signaling molecules of the canonical Wnt pathway in the prefrontal cortex (PFC) and hippocampus formation (HF) of autistic rats. The expression levels of glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β, β-catenin and phosphorylated β-catenin were observed by Western blotting. The mRNA expression of GSK-3β, β-catenin, c-Myc and cyclin D1 was assessed by semi-quantitative RT-PCR. RESULTS：The results of Western blotting showed that inactivated GSK-3β (Ser9) phosphorylation was significantly increased, and inhibitory β-catenin (Ser33/37/Thr41) phosphorylation was obviously decreased compared with control group. The results of RT-PCR showed that the mRNA levels of β-catenin, c-Myc and cyclin D1 increased, and GSK-3β was significantly enhanced in VPA-exposed rats compared with the controls. CONCLUSION：Increased activity of canonical Wnt pathway in the PFC and HF of autistic rats may contribute to the susceptibility to autism.     

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.     

Quercetin inhibits inflammasome activation in diabetic rats and attenuates myocardial injury

AIM:To observed the effect of quercetin on NLRP3 inflammasome activation in the rats with diabetic cardiomyopathy (DCM) and its protective effect on the myocardium. METHODS:Male SD rats (n=40) were randomly divided into normal control group (n=10) and model group (n=30). The rats in model group were intraperitoneally injected with streptozotocin at 60 mg/kg to establish the model of diabetes mellitus (DM). Blood glucose was measured weekly. After 4 weeks, the rats with random blood glucose ≥ 16.6 mmol/L were selected as DM animals. The rats with DM were randomly divided into 3 groups:DM group, DM+vehicle group and DM+quercetin group. The rats in DM+quercetin group were intragastric infusion with quercetin at 100 mg/kg per day. The cardiac function was measured at the end of the 16th week. The methods of Masson staining and HE staining were used to observe the morphological changes of the myocardial tissues. Western blot, ELISA and immunohistochemistry were used to observe the changes of NLRP3, ASC, caspase-1, interleukin (IL)-1β and IL-18. TUNEL staining was used to observe myocardial apoptosis. RESULTS:Quercetin significantly inhibited the activation of NLRP3 inflammasome in the myocardium of the DM rats (P<0.05). The levels of IL-1β and IL-18 in DM+quercetin group were significantly decreased, quercetin reduced cardiac tissue apoptosis, and the cardiac function in DM+quercetin group was significantly improved (P<0.05) compared with DM group and DM+vehicle grpup. CONCLUSION:Quercetin significantly inhibits the activation of NLRP3 inflammasome, and reduces the levels of inflammation and myocardial apoptosis, thus protecting the heart function of DCM rats.     

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.     

Long-term insulin treatment improves postischemic cardiac structure and function via increasing serum BNP levels

AIM：To investigate the influence of long-term insulin treatment on postischemic cardiac structural and functional changes, and to further explore the underlying mechanisms. METHODS：Adult male SD rats were randomly divided into 4 groups (8~10 rats per group): sham group, myocardial infarction (MI) + saline (1 mL·kg-1·d-1, hypodermic injection for 4 weeks) group, MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) group and MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) + wortmannin ［a phosphatidylinositol 3-kinase (PI3K) inhibitor; 15 μg·kg-1·d-1, intraperitoneal injection 15 min before each insulin treatment］ group. The rats in the latter 3 groups were subject to ligation of the left anterior descending coronary artery, while those in sham group underwent the same surgical procedures without tying the sutures. The cardiac structural and functional changes were observed by echocardiogram, heart catheterization and microscopy with HE and Masson trichrome staining. Blood glucose was determined by Roche blood glucose meter, and the serum levels of insulin and brain natriuretic peptide (BNP) were detected by ELISA. The protein expression and phosphorylation of PI3K, Akt, glycogen synthase kinase 3β (GSK3β) and p38 mitogen-activated protein kinase (p38 MAPK) in myocardial tissues were detected by Western blotting. The mRNA expression of BNP, β-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) in myocardial tissues was determined by real-time fluorescence quantitative PCR. RESULTS：At the end of the 4th week, MI rats receiving long-term insulin treatment showed decreased ratio of heart length/heart weight, smaller systolic left ventricle cavity, thicker systolic interventricular septum, and increased cardiac ejection fraction, left ventricular development pressure and instantaneous first derivate of left ventricle pressure (P<0.05 vs MI + saline group). Moreover, insulin treatment significantly increased the phosphorylation of PI3K and Akt and the serum level of BNP, and inhibited the phosphorylation of p38 MAPK (P<0.05 vs MI + saline group), but did not change the mRNA expression of BNP in myocardial tissues. The effects of insulin on BNP were not blocked by wortmannin (P>0.05 vs MI + insulin group). CONCLUSION：Insulin improves postischemic cardiac structure and function by increasing serum BNP levels possibly independent of PI3K-Akt signaling pathway.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Scutellaria barbata flavonoids inhibits NFT aggregation and regulatory mechanism in rats induced by composited Aβ

AIM: To investigate the effects of Scutellaria barbata flavonoids (SBF) on neurofibrillary tangle (NFT) aggregation, tau protein phosphorylation and the regulated mechanism of glycogen synthase kinase (GSK) 3β and protein phosphatase (PP) 2A in the rats induced by amyloid β protein 25-35 (Aβ_25-35) in combination with AlCl₃ and recombinant human transforming growth factor (RHTGF)-β1(composited Aβ). METHODS: The male SD rats were used to establish the simulated Alzheimer disease (AD) model by intracerebroventricular injection of composited Aβ. The Morris water maze was applied for screening the successful model rats with learning and memory deficits. The successful model rats were daily and orally administrated with SBF at doses of 35, 70 and 140 mg/kg or positive control drug Ginkgo biloba leaves flavonoids (GLF) at 140 mg/kg for 37 d. The silver nitrate staining was used to determine the cortical NFT. The protein levels of total tau, phosphorylated protein of tau at Ser199 and Ser214 sites, GSK3β and PP2A in hippocampus and cortex were determined by Western blot. The mRNA expression of GSK3β and PP2A in the hippocampus and cortex was detected by RT-PCR. RESULTS: Compared with sham group, the cell number of positive NFT with silver nitrate staining in model rat cerebral cortex was significantly increased. The protein levels of phosphorylated tau protein at Ser199 and Ser214 sites, GSK3β in the hippocampus and cerebral cortex in the model rats dramatically elevated, and PP2A was marked decreased as compared with the sham group rats. Meanwhile, the mRNA expression of GSK-3β significantly increased but PP2A was decreased. However, these above abnormalities were differently attenuated by treating with SBF at different doses or GLF at 140 mg/kg for 37 d. CONCLUSION: SBF suppresses the NFT aggregation by inhibition of the regulatory functions of GSK-3β and PP2A, thus reducing the phosphorylation of tau protein.     

Effects of sulindac on oxidative stress in an autistic model induced by prenatal exposure to valproic acid

AIM: To investigate the effects of sulindac on oxidative stress in autism. METHODS: With an autistic model induced by prenatal exposure to valproic acid (VPA), we detected the expression of the signaling molecules of canonical Wnt pathway in the prefrontal cortex (PFC) and hippocampus (HC) of autistic rats treated with sulindac. The protein expression levels of glycogen synthase kinase 3β (GSK-3β), β-catenin and 4-hydroxynonenal (4-HNE) were observed by Western blotting. The mRNA expression of thioredoxin(Trx)1 and Trx2 was assessed by semi-quantitative RT-PCR.RESULTS: The protein level of GSK-3β and mRNA levels of Trx1 and Trx2 were lower, whereas the protein expression levels of β-catenin and 4-HNE were higher in VPA group than those in control group. In contrast, the protein levels of GSK-3β were significantly higher in the animals treated with both VPA and sulindac than those in VPA group, while the levels of β-catenin and 4-HNE were decreased.CONCLUSION: Sulindac attenuates oxidative stress in the pathogenesis of autism, suggesting the up-regulation of the Wnt/β-catenin signaling pathway disrupts oxidative homeostasis and further facilitates susceptibility to autism.     

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.     

Overexpression of HBXIP in HepG2 cells induces cell migration and regulates expression of β-catenin

AIM: To investigate the effects of hepatitis B virus X-interacting protein(HBXIP) in hepatic cancer cells on the cell migration and expression of β-catenin. METHODS: Transwell assay was used to assess the cell migration. Gelatin zymography was used to observe the activity of matrix metalloproteinase 9 (MMP-9). The expression of MMP-9, glycogen synthase kinase 3β(GSK-3β), p-GSK3β, β-catenin and p-β-catenin in HepG2 cells was determined by Western blotting. RESULTS: HepG2 cells which stably overexpressed HBXIP (HepG2-HBXIP) exhibited higher migration ability than the control cells. The results of the gelatin zymography assay showed that HBXIP overexpression increased the activity of MMP-9 in HepG2 cells. The results of Western blotting indicated that HBXIP increased the expression of MMP-9 and β-catenin, inhibited the phosphorylation of β-catenin and promoted the phosphorylation of GSK-3β (Ser9). CONCLUSION: HBXIP regulates the GSK-3β/β-catenin signaling pathway, resulting in a significant improvement of hepatocellular carcinoma cell migration.     

rAAV-mediated VLDLR gene transfection reduces tau hyperphosphorylation in hippocampus of type 2 diabetic rats

AIM: Abnormal hyperphosphorylation of tau plays a critical role in the pathogenesis of Alzheimers disease(AD), and tau protein was hyperphosphorylated in type 2 diabetes. The present study was designed to explore the phosphorylation level of tau in hippocampus of type 2 diabetes rats which interrupted by very low density lipoprotein receptor(VLDLR)gene transfection. METHODS: Wistar male rats were randomized into 3 groups. The control group(CTL)was fed with normal food. The T2DM group and T2DM mediated VLDLR gene group were on high sugar, high fat and high protein diet for 3 months. The plasma insulin level was measured by RIA method, and the plasma glucose was determined by glucose-oxidase method. Total tau level, the phosphorylation level of tau at individual phosphorylation sites and the level of VLDLR were analyzed by Western blotting. The activity of glycogen synthase kinase 3β, a key component of insulin signal transduction pathway and a known tau kinase, in the hippocampus of rats was determined by using ［γ-32P］-ATP and the specific peptide substrate. RESULTS: No significant difference of total tau level in hippocampus between T2DM group and T2DM mediated VLDLR gene group was observed. Tau protein in T2DM group was found to be more hyperphosphorylated at several AD-related phosphorylation sites(Ser214, Thr217, Ser396, Ser422 and Ser199/202)than that in CTL, while the immunoreaction at tau-1 site is weaker than that in CTL. VLDLR gene therapy reduced hyperphosphorylation sites of Thr217, Ser396, Ser422 and Ser199/202 of tau to almost the control level, but did not change the phosphorylation of Ser214 or Ser422 on tau. The expression of Ser214 was also observed by immunohistochemical assay. The phosphorylated tau modestly increased in hippocampus in T2DM group compared to CTL, but VLDLR gene treatment did not change the phosphorylation level. The phosphorylation of GSK-3β was decreased dramatically in the hippocampus in T2DM rats, and this phosphorylation was significantly increased after VLDLR gene treatment. CONCLUSION: These findings suggest that Raav mediated VLDLR gene treatment partially reverses tau hyperphosphorylation at several sites in T2DM rat hippocampus, which may mediate by inhibition of GSK-3β activity.     

Effect of caveolin-1 expression on high-salt diet-induced endothelial dysfunction in type 1 diabetic rats

AIM: To investigate the underlying mechanisms responsible for endothelial dysfunction of type 1 diabetes mellitus (DM) rats fed with high-salt diet. METHODS: Type 1 DM was induced by intraperitoneal injection of streptozotocin (70 mg/kg). Normal and diabetic rats were fed high-salt food (HS, 8% NaCl) and standard food for 6 weeks, respectively. Isometric tension of the mesenteric arteries were measured. The expression of Akt, endothelial nitric oxide synthase (eNOS) and caveolin-1 (Cav-1) was examined by Western blot. RESULTS: The rats in DM+HS group exhibited more pronounced impairment of vasorelaxation to acetylcholine and insulin compared with either DM group or HS group (P<0.01). Akt and eNOS phosphorylation levels, and nitric oxide (NO) concentration in DM+HS group were significantly lower than those in DM group (P<0.01). The level of Cav-1 in DM+HS group was significantly higher than that in DM group and HS group. CONCLUSION: Impaired endothelial Akt activation, increased Cav-1 expression and resultant decreased eNOS activation contribute to aggravate high-salt diet-induced endothelial dysfunction and hypertension in DM rats.     

Expression changes of Wnt/β-catenin signaling pathway in diabetic ulcer

AIM: To observe the effect of Wnt/β-catenin signaling pathway on diabetic ulcer. METHODS: Diabetic animal model was established in the female Wistar rats by intraperitoneal injection of low-dose streptozotocin following high-fat diet feeding. A circular wound was made on the dorsum of the rats in both control group and diabetic group. The condition of wound healing was recorded and the structures of the wound tissues were observed by HE staining in the 2 groups at 3, 7 and 14 d after wounding. The expression of β-catenin, GSK-3β and Rspo-3 at mRNA and protein levels in the wound tissues was detected by RT-PCR and ELISA. RESULTS: In diabetic group, the wound healing rate was lower (P<0.05), and the inflammatory cells, fibroblast cells and new capillaries in the wound tissues were fewer than those in control group. The expression of β-catenin and Rspo-3 at mRNA and protein levels in the wound tissues in control group was significantly higher than those in diabetic group, and the expression of GSK-3β was exactly the opposite (P<0.05). CONCLUSION: The down-regulation of Wnt/β-catenin probably resultes from the decreased level of Rspo-3, which may be one of the reasons for delaying the diabetic ulcer healing.