Common pathogenesis of type 2 diabetes and Alzheimers disease: a potential mechanism

AIM: Some epidemiologic evidences suggest an association between type 2 diabetes mellitus (T2DM) and Alzheimer disease (AD). The present study was designed to explore the common pathogenesis in these two diseases by comparing the pathological changes in pancreas and hippocampus, the activity of glucogen synthase kinase 3β (GSK-3β) and protein phosphatase 2A (PP2A) in T2DM rats with those in AD rats. METHODS: The SD rats were fed with high glucose, high fat and high protein for 8 weeks, and then injected with streptozocin (STZ) to establish the T2DM models. The model of AD was made by injection of STZ in hippocampus. The levels of hyperphosphorylated tau protein and β-amyloid protein (Aβ) in hippocampus and pancreas were analyzed by Western blotting and immunochemistry. The activities of GSK-3β and PP2A were determined by using γ-32P-ATP and the specific peptide substrate. RESULTS: In hippocampus of T2DM rats, the tau protein was hyperphosphorylated, the Aβ level was raised, the activity of PP2A was increased and the activity of GSK-3β was decreased, which were also observed in the hippocampus of AD rats. In the AD rats, the Aβ level in pancreas was found to rise, the activity of PP2A decreased and the activity of GSK-3β increased, which were similar to the changes in pancreas of T2DM rats. However, tau protein was not detected in the pancreas of both groups. CONCLUSION: These findings suggest that there are some common pathological changes between T2DM and AD. The hyperphosohorylation of tau protein in hippocampus, the raised Aβ level in hippocampus and pancreas, as well as the corresponding activities of PP2A and GSK-3β may contribute to the pathogenesis in the development of T2DM and AD.     

Effect of tenuigenin on tau protein phosphorylation at Ser396 site in neurons of AD rats induced by Aβ1-40

AIM:To investigate the effect of tenuigenin(TEN) on hyperphosphorylation of tau protein in neurons of amyloid β-peptide_1-40(Aβ_1-40) -induced Alzheimer disease(AD) rats. METHODS:Aβ_1-40 was injected into hippocampus CA1 region of the rats to establish AD model. TEN at different doses(18.5 mg/kg, 37.0 mg/kg and 74.0 mg/kg) was intragastrically administered. The protein expression of protein kinase A(PKA),protein phosphatase 2A(PP2A), total tau and p-tau(Ser³⁹⁶) in the neurons was observed by the method of immunohistochemistry. The protein content of total tau and p-tau(Ser³⁹⁶), and the expression level of PKA and PP-2A were detected by Western blotting analysis. RESULTS:In Aβ_1-40 group, the level of total tau, the phosphorylation of tau protein and the expression of PKA were significantly increased compared with those in sham operation group. Meanwhile, the expression of PP2A in Aβ_1-40 group was lower than that in sham operation group. In TEN treatment group, the level of total tau, the phosphorylation of tau protein and the expression of PKA were markedly decreased, and the expression of PP2A was increased as compared with Aβ_1-40 group. CONCLUSION:TEN may protect the neurons from the toxic effect of Aβ_1-40 and reduce the hyperphosphorylation of tau(Ser³⁹⁶) in the neurons of AD rats by activating the expression of PP2A and inhibiting the expression of PKA.     

Adiponectin attenuates H2O2-induced SH-SY5Y cell injury and tau hyperphosphorylation via activating PP2A

AIM: To study the effects of adiponectin on H₂O₂-induced cell injury and tau hyperphosphorylation in human neuroblastoma SH-SY5Y cells. METHODS: Cell viability was determined by MTT assay. H₂O₂-induced cell injury and morphological changes in the SH-SY5Y cells with or without adiponectin treatment were observed. The level of tau phosphorylation as well as the activities of protein phosphatase 2A(PP2A) and of glycogen synthase kinase-3β(GSK-3β) were examined by Western blotting. RESULTS: Adiponectin significantly attenuated H₂O₂-induced cell injury(P<0.01). Adiponectin upregulated the activity of PP2A and decreased phosphorylation levels of tau under the stimulation with H₂O₂ (P<0.01). Okadaic acid, a specific inhibitor of PP2A, blocked the protective effects of adiponectin(P<0.01). Adiponectin increased the phosphorylation level of GSK-3β at Ser9 site under H₂O₂ stimulation(P<0.01). CONCLUSION: Adiponectin protects SH-SY5Y cells against H₂O₂-induced cell injury and decreases tau hyperphosphorylation by activating PP2A and inactivating GSK-3β.     

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.     

Transfection with GSK-3β in vivo induces tau hyperphosphorylation at PHF-1 sites

AIM: To probe into tau hyperphosphorylation at PHF-1 sites induced by glycogen synthase kinase-3β (GSK-3β) in vivo. METHODS: Twenty-one rats were randomly allocated to three groups as follows: GSK-3β transfection group, vector group and control group; 0.1 μg/3 μL GSK-3β-HA plasmid or vector was injected bilaterally into cerebrum of the rats respectively, rats without injection were controls. Western blotting and immunohistochemical staining of cortex were carried out to detect the expression of GSK-3β-HA plasmid and tau phosphorylation using phosphorylation-dependent tau antibody PHF-1. RESULTS: After transfection with GSK-3β-HA for 48 h, GSK-3β-HA was expressed in GSK-3β transfection group; and hyperphosphorylated tau at PHF-1 sites accumulated in neurons in the transfected areas. The hyperphosphorylated tau colocalized largely with GSK-3β expressed by the transfected GSK-3β plasmid. CONCLUSIONS: Transfection with GSK-3β in vivo can induce tau hyperphosphorylation involving the pathogenesis of neurodegenerative disorders. These data further prove that GSK-3β is a key kinase to induce tau hyperphosphorylation and may be a therapeutic target for tauopathy-related neurodegenerative diseases.     

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.     

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.     

Folic acid reduces monocyte chemoattractant protein-1 expession in aorta of rats with hyperhomocysteinemia

AIM: To investigate the effects of folic acid on the expression of monocyte chemoattractant protein-1 (MCP-1) in aorta of rats with hyperhomocysteinemia induced by ingestion of execess methionine (Met). METHODS: Thirty male SD rats ［(200±20） g］ were divided into 3 groups (n=10 for each group): control group (fed with normal diet), high Met group (fed with normal diet enriched by 1.7% Met) and Met plus folate group (fed with normal diet plus 1.7% Met and 0.006% folate). The animals were fed with different regiments for 45 days. Levels of total plasma homocysteine (Hcy) were detected. The expression of MCP-1 protein in aorta was detected by immunohistochemistry and Western blotting. RESULTS: The high-methionine diet resulted in a significant increase in plasma Hcy levels (P<0.01). Serum Hcy levels were significantly lower in rats fed with high-methionine plus folate-rich diet than that in rats fed with high-methionine diet (P<0.01). The expression of MCP-1 were higher in aorta of rats fed with high-methionine diet than those in control rats (46.41±4.23 vs 15.73±2.74, P<0.05). The expression of MCP-1 was significantly reduced in aorta of rats fed with high-methionine plus folate-rich diet compared with rats fed with high-methionine diet (23.12±4.40 vs 46.41±4.23, P<0.05). CONCLUSION: High methionine diet for 45 days is sufficient to induce hyperhomocysteinemia. Folate supplementation to the rats fed with the high-methenine diet prevents elevation of Hcy levels in blood, and reduces the expression of MCP-1 in aorta of rats with hyperhomocysteinemia.     

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.     

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.     

PP2A activation in mesenteric arteries of angiotensin Ⅱ-induced hypertensive rats

AIM: To investigate the mechanism of protein phosphatase 2A (PP2A) activation in mesenteric arteries of angiotensinⅡ(AngⅡ)-induced hypertensive rats. METHODS: Adult male Sprague-Dawley (SD) rats were subjected to AngⅡinfusion (500 ng·kg^-1·min^-1) using osmotic minipump up to 14 d to established the hypertension model. The rats (n=40) were randomly divided into 4 groups:control group (n=10), AngⅡgroup (n=10), candesartan (CAN; AngⅡtype 1 receptor blocker)+AngⅡgroup (n=10) and CAN group (n=10). The rats in CAN+AngⅡgroup and CAN group were administered with candesartan ester at the dose of 10 mg·kg^-1·d^-1 by gavage on the first day after implantation of osmotic minipump. The rats were sacrificed on the 15th day after minipump implantation. Serum and mesenteric arteries were collected. Systolic blood pressure was measured by tail-cuff method. The serum levels of AngⅡ were measured by ELISA. The protein levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser1177), PP2A catalytic subunit (PP2Ac), phosphorylated PP2Ac (Tyr307) and PP2A inhibitor 2 (I₂^PP2A) in the mesenteric arteries were determined by Western blot. The activity of PP2A in the arteries was detected using PP2A activity assay kit. RESULTS: Compared with control group, the systolic blood pressure in AngⅡgroup was significantly increased(P<0.05), while those in CAN+AngⅡgroup and CAN group were significantly decreased (P<0.05). The serum levels of AngⅡ in AngⅡ group and CAN+AngⅡ group were significantly higher than that in control group (P<0.05). Compared with control group, the phosphorylation levels of eNOS Ser1177 were decreased in AngⅡgroup (P<0.05), but the activity of PP2A was significantly increased (P<0.05), and Pearson correlation analyses showed a negative correlation between PP2A activity and eNOS S1177 phosphorylation (r=-0.842, P<0.05). Compared with AngⅡgroup, the phosphorylation levels of eNOS Ser1177 in CAN+AngⅡgroup were significantly increased (P<0.05), but the activity of PP2A was reduced (P<0.05). Compared with control group, the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries were decreased in AngⅡgroup (P<0.05), but increased in CAN+AngⅡgroup (P<0.05). No significant difference in all above-mentioned measures between control group and CAN group, nor in the levels of total eNOS and PP2Ac protein expression among all the groups was observed. CONCLUSION: AngⅡmay reduce the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries of AngⅡ-induced hypertensive rats through AngⅡ/AngⅡ type 1 receptor-mediated signaling pathway, resulting in the activation of PP2A, then leading to down-regulation of eNOS S1177 phosphorylation, which ultimately mediates the occurrence of vascular endothelial dysfunction.     

Role of glycogen synthase kinase-3β signal molecule in myocardial hypertropy

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine protein kinase, which takes part not only in glycogen metabolism, but also in cell proliferation, differentiation and apoptosis. GSK-3β is inhibited by growth factors and hypertrophic stimuli through phosphorylation of its N-terminal end serine (Ser9) residue. It is also activated by phosphorylation of its tyrosine (Tyr216) residue. GSK-3β is profoundly inactivated in mice with hypertrophic cardiomyopathy (HCM) and plays a secondary role in myosin heavy chain mutation. However, the role of GSK-3β in HCM was controversial. Recent studies have demonstrated that the activation of GSK-3β inhibits the myocardial hypertrophy, and is regulated by Wnt/Frizzld and PI3-K/Akt signaling pathways. This article introduces the molecular role of glycogen synthase kinase-3β signaling in myocardial hypertrophy and the different pathways on the activity of glycogen synthase kinase-3β (GSK-3β)     

Glycogen synthase kinase-3 beta overexpression enhances tau protein phosphorylation and attenuates tubulin acetylation in SH-SY5Y cells

AIM: To construct GSK3β-overexpressed SH-SY5Y cells and to observe the effects of GSK3β-overexpression on tau protein phosphorylation and tubulin acetylation in SH-SY5Y engineered cells. METHODS: The cDNA of GSK3β construct was subcloned into mammalian expression vector pBudCE4.1. The integrity of the GSK3β construct was confirmed by sequence analysis. GSK3β was transiently transfected into SH-SY5Y cells using Lipofectamine2000. Western blotting was used to measured protein levels of GSK3β and phosphorylating GSK3β, as well as, the total tau and phosphorylated tau protein and acetylated tubulin. RESULTS: 36 h after transfection, the levels of GSK3β and phosphorylating GSK3β in SH-SY5Y cells were significantly increased compared with non-transfection group and vector group. After 48 h, the levels of phosphorylated tau protein (Ser199/202, Thr231 and Thr205 residues) but not total tau protein were markedly increased in GSK3β-overexpressed SH-SY5Y cells. In addition, the level of acetylated tubulin was lower than that in non- transfection group and vector group. CONCLUSION: The over-expression of GSK3β in SH-SY5Y cells results in robust increases in tau protein phosphorylation at Ser199/202, Thr231 and Thr205 residues, and decreases in tubulin acetylation.     

Protection of melatonin on isoproterenol-induced hyperphosphorylation of tau and spatial memory impairment in rats

AIM: To study the in vivo effect of melatonin (MEL) on Alzheimer-like tau hyperphosphorylation and spatial memory retention impairment induced by isoproterenol (IP). METHODS: Simultaneous injection of 2 μL IP(10 mmol/L) or IP plus MEL(0.1 μg,1μg)into rat hippocampus bilaterally with stereotexin; The phosphorylation of tau was analyzed by immunohistochemistry and Western blot and the memory retention was tested by Morris Water Maze. RESULTS：Injection of IP induced hyperphosphorylation of tau at Tau-1 and PHF-1 epitopes and increased latency of rats to find the hidden platform in the Morris water maze. MEL significantly restored the IP-induced impairment in spatial memory（P＜0.05）and tau hyperphosphorylation determined by both immunocytochemistry and Western blot. CONCLUSION: MEL inhibits partially the IP-induced pathological processes in tau hyperphosphorylation and spatial memory impairment in rat brain.     

AngⅡ/AT1R pathway leads to down-regulation of endothelial nitric oxide synthase phosphorylation

AIM:To investigate the mechanism of angiotensinⅡ (AngⅡ)/angiotensinⅡ type 1 receptor (AT₁R) pathway activating protein phosphatase 2A (PP2A) which leads to down-regulation endothelial nitric oxide synthase (eNOS) phosphorylation level in mesenteric arteries of rats. METHODS:The mesenteric arteries of adult male SD rats (weighing 160~180 g; n=90) were isolated under aseptic conditions. Firstly, to determine the effect of angiotensinⅡ down-regulated eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into normal control (control) group and AngⅡ group. The mesenteric arteries in AngⅡ group were incubated with AngⅡ at 1×10^-7 mol/L, 1×10^-6 mol/L and 1×10^-5 mol/L for 6 h, 12 h and 24 h, respectively. Secondly, to investigate the molecular mechanism by which angiotensinⅡ activated PP2A leading to down-regulation eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into control group, AngⅡ group and candesartan (CAN; a specific AT₁R blocker)+AngⅡ group. The mesenteric arteries were pretreated with 1×10^-5 mol/L CAN for 1 h, then incubated with 1×10^-7 mol/L AngⅡ for 12 h in CAN+AngⅡ group. The protein levels of eNOS, p-eNOS (Ser1177), PP2Ac, p-PP2Ac (Tyr307) and protein phosphatase 2A inhibitor 2 (I₂^PP2A) in the arteries were determined by Western blot. The activity of PP2A in the arteries was detected by PP2A activity kit. RESULTS:Compared with the control group, the protein level of p-eNOS (Ser1177) in the mesenteric arteries was decreased after incubated with AngⅡ for 6 h, 12 h and 24 h (P<0.05). The decreasing tendency of p-eNOS (Ser1177) showed concentration-dependently, especially in 12 h and 24 h groups. The expression of eNOS protein showed no significant difference in each group. Compared with the control group, the mesenteric arteries of the rats were incubated with AngⅡ at 1×10^-7 mol/L for 12 h in vitro, the protein levels of p-eNOS (Ser1177) were down-regulated (P<0.05); pretreatment with CAN significantly increased the protein level of p-eNOS (Ser1177) (P<0.05); the protein levels of eNOS showed no significant difference in each group. Compared with the control group, the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A were decreased after the mesenteric arteries were treated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A (P<0.05), however the expression of PP2Ac protein showed no significant difference in each group. Compared with the control group, the activity of PP2A was increased in the mesenteric arteries incubated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesarten pretreatment inhibited the activity of PP2A significantly (P<0.05). CONCLUSION:AngⅡ increases PP2A activity via AT₁R pathway, thus leading to down-regulation eNOS (Ser1177) phosphorylation level in mesenteric arteries. The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A.     

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.     

Effects of tyrosine phosphatase inhibitor on tau phosphorylation in vivo

AIM: To explore the effect of receptor tyrosine kinase system mediated by phosphotyrosine phosphatase (PTP) on tau phosphorylation in rat hippocampus. METHODS: Pervanadate (PVN), inhibitor of PTP or inhibitor of glycogen synthase kinase-3 (GSK-3), LiCl were injected into rat hippocampus by stereotaxy technique. The level of tau phosphorylation was detected by Western blot and immunohistochemistry after 24 h of injection. RESULTS: PVN significantly inhibited tau phosphorylation at PHF-1 epitope and the inhibition of tau phosphorylation by PVN was stronger than that of LiCl (P<0.01),and tau-1 epitope non-phosphorylated tau increased significantly in LiCl+PVN group than in control group (P<0.05). The level of total tau determined by R111d was significantly lower in PVN and LiCl+PVN treated groups (P<0.05) than that in LiCl and control groups. CONCLUSION: Tyrosine phosphatase inhibitor inhibited tau phosphorylation of hippocampus in rats. The underlie mechanism might be at least partially through the inhibition of GSK-3.     

Effect of Kang Xianling decoction on TGF-β1-Smad pathway in a unilateral ureteral obstruction rat model

AIM: To study the effect of Kang Xianling decoction,comprised of dahuang,danshen,taoren,niuxi and danggui,on TGF-β₁-Smad pathway in unilateral ureteral obstruction rat model.METHODS: Eighteen male SD rats were divided into 3 groups,sham group,model group and model group treated with Kang Xianling decoction randomly.Renal interstitial fibrosis model was established in rats by unilateral ureteral obstruction (UUO).After treatment for additional 14 d,parameters of hydroxyproline in obstructed kidney from 3 groups were analyzed.Rats were sacrificed and the pathological statuses of their kidneys were checked by HE staining and electron microscopy.Transforming growth factor-β₁ (TGF-β₁) mRNA in kidney tissue was determined by RT-PCR.TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),phosphorylated Smad2 and Smad2 protein were determined by Western blotting.RESULTS: Parameters of hydroxyproline in animals of model group were significantly increased than those in sham operation group (P<0.05).The mRNA expression of TGF-β₁ and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2 in kidney tissue of animals in model group were significantly up-regulated.After intervention with Kang Xianling decoction,the above-mentioned up-regulated parameters,except TGF-β₁,were all significantly inhibited.Compared to model group,the pathological changes in renal tissues in treatment group were remarkable improved.CONCLUSION: Kang Xianling decoction inhibits the TGF-β₁-Smad pathway and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2,so as to decrease the level of collagen in obstructed kidney and to alleviate the renal interstitial fibrosis in UUO rats.     

Phosphodiesterase 4 inhibitor rolipram prevents depression- and anxiety-like behaviors in rats exposed to chronic restraint stress

AIM: To discuss the impact of phosphodiesterase 4 (PDE4) inhibitor rolipram on chronic restraint stress-induced depression- and anxiety-like behaviors in rats. METHODS: (1)Forty SD rats were randomly divided into 4 weight-matched groups: unstressed animals injected with vehicle of lithium chloride (LiCl) and rolipram, restraint-stressed animals injected daily with vehicle prior to stress, restraint stress plus 100 mg/kg LiCl group and restraint stress plus 1 mg/kg rolipram group. The open field test was conducted 24 h before the first stress and drug administration,and then the rats received drugs daily 1 h prior to restraint stress (6 h/d) for 25 d. Daily body weight recording, forced swimming test, elevated plus-maze and open field test were conducted to determine the changes of depression- and anxiety-like behaviors. The expression of phosphorylated cAMP response element-binding protein (p-CREB), brain-derived Reurotrophic factor (BDNF), p-Ser21-glycogen synthase kinase (GSK) 3α, p-Ser9-GSK3β, p-Tyr279-GSK3α, p-Tyr216-GSK3β, total GSK3α and total GSK3β was measured by Western blotting. (2)Thirty SD rats were randomly divided into 6 groups and the cannula was surgically placed above the CA1 region in the hippocampus. Seven days after the surgery, the restraint stress was conducted for 21 d after microinjection of protein kinase A (PKA) antagonist H89 and intraperitoneal injection of LiCl and rolipram everyday. The expression of PDE4D, PKA, p-CREB and p-Ser9-GSK3β was measured by Western blotting. RESULTS: (1)No difference of the locomotor activity among all groups before stress was observed. After repeated stress, the body weight,and the crossing, rearing and grooming in open field test were lower than those in control group, and LiCl and rolipram reversed these effects significantly. In addition, in comparison with control group, the immobility in forced swimming test was increased, the climbing in forced swimmming test and the open-arm exploration in elevated plus-maze were decreased and the expression of p-CREB, BDNF, p-Ser21-GSK3α and p-Ser9-GSK3β was down-regulated. Stress induced depression- and anxiety-like behaviors, and rolipram reversed these changes. The LiCl showed similar effects as rolipram except for the expression of p-CREB and BDNF. No significant difference of the expression of p-Tyr279-GSK3α, p-Tyr216-GSK3β, total GSK3α and total GSK3β among all groups was found. (2)The expression of PDE4D was increased, the expression of PKA, p-CREB and p-Ser9-GSK3β was decreased in the hippocampus induce by restraint stress. However, the effect of rolipram on the expression of PKA, p-CREB and p-Ser9-GSK3β was blocked by PKA inhibitor H89. CONCLUSION: Rolipram significantly reduces the depression- and anxiety-like behaviors, possibly through CREB/BDNF signaling and inhibitory serine-phosphorylation of GSK3-mediated signaling. Importantly, the CREB/BDNF signaling also plays a key role in the down-regulation of serine-phosphorylation of GSK3.