Comparison of Effect of Hirudo， Notoginseng Radix et Rhizoma， and Their Combinations on Renal Fibrosis in Rats with Chronic Renal Failure / 中国实验方剂学杂志

ObjectiveTo observe the effects of Hirudo， Notoginseng Radix et Rhizoma， and drug pair on renal pathological morphology and protein phosphatase 2A （PP2A）/adenylate activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signal pathway in rats with chronic renal failure （CRF）. MethodThe 55 male SD rats were randomly divided into a normal group （n=11） and a modeling group （n=44）. The normal group was fed conventionally， and the modeling group was given 0.25 g·kg-1·d-1 adenine by gavage for 28 days to replicate the CRF model. After successful modeling， rats were randomly divided into model group， Hirudo group （3 g·kg-1·d-1）， Notoginseng Radix et Rhizoma group （3 g·kg-1·d-1）， and Hirudo + Notoginseng Radix et Rhizoma group （3 g·kg-1·d-1）， with 9 rats in each group. The normal group and model group were given a constant volume of normal saline by intragastric administration for 30 days. At the end of the experiment， the levels of serum creatinine （SCr） and urea nitrogen （BUN） in all groups were measured. The renal pathological morphology changes were observed by hematoxylin-eosin （HE） staining， Masson staining， and electron microscopy. The mRNA expressions of PP2A， AMPK， and mTOR were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of PP2A， AMPK， phosphorylation（p）-AMPK， mTOR， and p-mTOR in renal tissue were detected by Western blot. ResultCompared with the normal group， the renal pathological structure changes were obvious， and the levels of SCr and BUN were significantly increased. The mRNA expression of PP2A， protein expression of PP2A， and p-mTOR/mTOR expression were significantly increased， and the p-AMPK/AMPK was significantly decreased in the model group （P<0.05）. Compared with the model group， the renal pathological morphology changes were significantly improved， and the levels of SCr and BUN were significantly decreased. The mRNA expression of PP2A， protein expression of PP2A， and p-mTOR/mTOR expression in the renal tissue were significantly decreased， and the p-AMPK/AMPK was significantly increased （P<0.05） in all groups after drug intervention. In addition， the effect in the Hirudo+Notoginseng Radix et Rhizoma group was better. The mRNA expression levels of AMPK and mTOR in the renal tissue were not significantly different among the normal group， model group， and other groups. ConclusionThe efficacy of Hirudo and Notoginseng Radix et Rhizoma pairs in improving renal fibrosis in rats with CRF is significantly better than that of the single drug， and its improvement on renal fibrosis in rats with CRF may be related to the regulation of PP2A/AMPK/mTOR signaling pathway.

Effects of Yizhi Tiaoshen acupuncture on learning and memory function and the expression of phosphorylated tau protein in the hippocampus of Alzheimer's disease model rats / 中国针灸

OBJECTIVE@#To observe the effects of Yizhi Tiaoshen (benefiting mental health and regulating the spirit) acupuncture on learning and memory function, and the expression of phosphorylated tubulin-associated unit (tau) protein in the hippocampus of Alzheimer's disease (AD) model rats, and explore the effect mechanism of this therapy on AD.@*METHODS@#A blank group and a sham-operation group were randomly selected from 60 male SD rats, 10 rats in each one. AD models were established in the rest 40 rats by the intraperitoneal injection of D-galactose and okadaic acid in the CA1 region of the bilateral hippocampus. Thirty successfully-replicated model rats were randomly divided into a model group, a western medication group and an acupuncture group, 10 rats in each one. In the acupuncture group, acupuncture was applied to "Baihui" (GV 20), "Sishencong" (EX-HN 1), "Neiguan" (PC 6), "Shenmen" (HT 7), "Xuanzhong" (GB 39) and "Sanyinjiao" (SP 6); and the needles were retained for 10 min. Acupuncture was given once daily. One course of treatment was composed of 6 days, with the interval of 1 day; the completion of treatment included 4 courses. In the western medication group, donepezil hydrochloride solution (0.45 mg/kg) was administrated intragastrically, once daily; it took 7 days to accomplish one course of treatment and a completion of intervention was composed of 4 courses. Morris water maze (MWM) and novel object recognition test (NORT) were used to assess the learning and memory function of the rats. Using HE staining and Nissl staining, the morphological structure of the hippocampus was observed. With Western blot adopted, the protein expression of the tau, phosphorylated tau protein at Ser198 (p-tau Ser198), protein phosphatase 2A (PP2A) and glycogen synthase kinase-3β (GSK-3β) in the hippocampus was detected.@*RESULTS@#There were no statistical differences in all of the indexes between the sham-operation group and the blank group. Compared with the sham-operation group, in the model group, the MWM escape latency was prolonged (P<0.05), the crossing frequency and the quadrant stay time in original platform were shortened (P<0.05), and the NORT discrimination index (DI) was reduced (P<0.05); the hippocampal cell numbers were declined and the cells arranged irregularly, the hippocampal neuronal structure was abnormal and the numbers of Nissl bodies decreased; the protein expression of p-tau Ser198 and GSK-3βwas increased (P<0.05) and that of PP2A decreased (P<0.05). When compared with the model group, in the western medication group and the acupuncture group, the MWM escape latency was shortened (P<0.05), the crossing frequency and the quadrant stay time in original platform were increased (P<0.05), and DI got higher (P<0.05); the hippocampal cell numbers were elevated and the cells arranged regularly, the damage of hippocampal neuronal structure was attenuated and the numbers of Nissl bodies were increased; the protein expression of p-tau Ser198 and GSK-3β was reduced (P<0.05) and that of PP2A was increased (P<0.05). There were no statistically significant differences in the above indexes between the acupuncture group and the western medication group (P>0.05).@*CONCLUSION@#Acupuncture therapy of "benefiting mental health and regulating the spirit" could improve the learning and memory function and alleviate neuronal injure of AD model rats. The effect mechanism of this therapy may be related to the down-regulation of GSK-3β and the up-regulation of PP2A in the hippocampus, and then to inducing the inhibition of tau protein phosphorylation.

Effect of Danzhi Xiaoyaosan on Phosphorylation of Tau Protein in Rats with Alzheimer's Disease Based on PP2A and GSK-3β / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Danzhi Xiaoyaosan on the phosphorylation of tau protein and different sites of glycogen synthase kinase-3β （GSK-3β） and phosphoseryl/suanyl phosphate protein phosphatase 2A （PP2A） in the hippocampus of rats with Alzheimer's disease （AD） and its mechanism. MethodThe rat model of AD was established by injecting okadaic acid into the bilateral hippocampus of 90 male Wistar rats in SPF grades. The rats with successful modeling were selected and randomly divided into model group， aricept group （0.5 mg·kg-1）， and Danzhi Xiaoyaosan high， medium， and low groups （17.55， 8.77， and 4.38 g·kg-1）， and then gavaged for 42 d， once a day. Morris water maze was used to detect the learning and memory ability of rats， Nissl's staining was used to observe the morphological structure of neurons in the hippocampus， and Real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of tau protein， GSK-3β， and PP2A. Western blot was used to determine the protein expression levels of tau protein， GSK-3β， and PP2A. ResultAs compared with the control group， the learning and memory abilities of the rats in the model group were significantly decreased （P<0.01）， and the hippocampal CA3 region cells had abnormal structure， disorderly arrangement， and decreased number. The expression levels of GSK-3β mRNA， GSK-3β， p-GSK-3β-Tyr216， p-PP2A， and p-tau were increased in the model group as compared with the control group （P<0.01）， and those of p-GSK-3β-Ser9 and PP2A decreased significantly （P<0.01）. As compared with the model group， the learning and memory ability of the Aricept group and the Danzhi Xiaoyaosan groups were improved （P<0.05， P<0.01）， and the cell morphology and the number of hippocampal CA3 regions were better. The mRNA expression levels of PP2A and tau in the Aricept group were significantly up-regulated （P<0.05）， the mRNA expression level of GSK-3β was significantly down-regulated （P<0.01）， and the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-PP2A were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A in the high-dose Danzhi Xiaoyaosan group was significantly up-regulated （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of p-PP2A， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of GSK-3β was significantly down-regulated in the medium-dose Danzhi Xiaoyaosan group （P<0.01）， the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A was significantly up-regulated in the low-dose Danzhi Xiaoyaosan group （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of GSK-3β and p-GSK-3β-Tyr216 were down-regulated （P<0.05， P<0.01）， and those of p-GSK-3β-Ser9 and PP2A were significantly up-regulated （P<0.01）. ConclusionDanzhi Xiaoyaosan can improve the learning and memory ability of rats with AD， and its mechanism may be related to the regulation of the activities of GSK-3β and PP2A protein-related sites and the phosphorylation of tau protein.

Mechanism of Huangjingwan in Inhibiting Tau Hyperphosphorylation in Hippocampal Neurons of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid Resulting in Learning and Memory Disorders / 中国实验方剂学杂志

Objective:To investigate the effect of Huangjingwan （HW） on the activities of glycogen synthase kinase-3β （GSK-3β）， protein phosphatase 2A （PP2A） and the mechanism in inhibiting tau protein hyperphosphorylation in the hippocampal neurons of mice with Alzheimer's disease. Method:After subcutaneous injection with 1.0% D-galactose （0.14 g·kg-1·d-1） into the back and neck of mice for 4 weeks， the right ventricle of mice was injected with 2 μL （75 ng） of okadaic acid for one time to make AD model， and the successfully modeled AD mice were selected by Morris water maze. Then， the selected AD mice were randomly divided into AD model group， memantine group （1.3×10-3 g·kg-1·d-1） and HW group （2.5 g·kg-1·d-1）. In addition， the sham model control group and the normal control group were set up. At the same time， 2 μL normal saline was injected into the right ventricle of mouse in the sham model control group for modeling control. Two weeks after modeling， the mice in the two experimental drug groups were given the corresponding dose of the experimental drug by gavage for 4 weeks. In addition， after 2 weeks of AD modeling， mice in control group and AD model group were intragastrically administrated with the same amount of normal saline daily for 4 weeks. The mice in normal control group were only given daily feed. At the end of gavage， all the mice were tested by the open field experiment and jumping platform experiment to evaluate the differences in exploratory activity ability， anxiety level and learning and memory ability. The number of neurons in CA1 and CA3 areas of hippocampus in all the mice was detected by Nissl staining. Quantitative real-time polymerase chain reaction （Real-time PCR） was used to detect mRNA expressions of GSK-3β and PP2A in hippocampus of mice in each group. Protein expressions of GSK-3β， PP2A， phosphorylated tau （p-tau） and total tau protein （t-tau） in hippocampus of mice in each group were detected by Western blot. Result:Compared with the normal control group， mice in AD model group showed an obvious dementia state， which was characterized by a lower spontaneous activity， lower exploration behavior ability， higher anxiety level， less movement and easier to stay and hide， longer learning response time， significantly increased number of learning and memory errors， and decreased numbers of hippocampal neuron in CA1 and CA3 areas， and reduced mRNA and protein expressions of PP2A， mRNA and protein expressions of GSK-3β， p-tau protein and the ratio of p-tau/t-tau were all increased significantly （P<0.01）， while expression of t-tau protein was decreased， with no significant difference. Compared with the AD model group， mice in the HW group showed a higher spontaneous activity， higher exploration ability， lower anxiety level， higher learning and memory performance， and the numbers of hippocampal neuron in CA1 and CA3 areas increased， while mRNA and protein expressions of PP2A increased， and the mRNA and protein expressions of GSK-3β， the expression of p-tau protein and the ratio of p-tau/t-tau were all decreased significantly （P<0.01）， but with no significant difference in the protein expression of t-tau. Conclusion:HW can inhibit tau hyperphosphorylation in hippocampal neurons of AD mice， restore tau protein function， protect hippocampal neurons， and exert an anti-AD effect， which may be related to the regulatory mechanism in the activity balance between GSK-3β and PP2A in hippocampal neurons.

High-glucose-induced apoptosis, ROS production and pro-inflammatory response in cardiomyocytes is attenuated by metformin treatment via PP2A activation

Metformin has been shown to ameliorate diabetic cardiomyopathy. In the present research we investigatedwhether metformin would reduce cardiomyocyte apoptosis that was induced by high-glucose stimulation in vitrovia activation of PP2A. Primary human and rat cardiomyocytes were subject to high-glucose stimulation. Okadaicacid was used to inhibit PP2A activity. Cell viability and apoptosis was assessed using CCK-8 and by flowcytometry, respectively. Release of HMGB1, TNFa or IL-6 was analyzed by ELISA. Oxidative stress wasevaluated by measuring cellular ROS and mitochondrial superoxide level. PP2A activity was evaluated by Serine/Threonine phosphatase assay system or analyzing Y307 phosphorylation level of PP2A catalytic domain (PP2Ac)by Western blot and the association between PP2Ac and a4 by co-immunoprecipitation. Activation of the NF-jBsignaling pathway was assessed by detecting Ser32 phosphorylation level of IjBa as well as nuclear entry of p65protein by Western blot. Activation of the GSK3b/MCL1 signaling pathway was assessed by detecting Ser9phosphorylation level of GSK3b and protein level of MCL1. We found Metformin pre-treatment attenuatedhuman and rat cardiomyocytes apoptosis, HMGB1, TNFa and IL-6 release and ROS production that were inducedby high-glucose stimulation, and these effects of metformin could be blocked by okadaic acid treatment. Metformin reduced the upregulation of PP2Ac pY307 and the PP2Ac-a4 association, which was not affected byokadaic acid treatment. Metformin pre-treatment reduced NF-jB activation in human and rat cardiomyocytesapoptosis that was elicited by high-glucose stimulation, and this effect of metformin could be blocked by okadaicacid treatment. GSK3b/MCL1 is not part of metformin activating PP2A induced myocardial cell death inhibition.In conclusion, metformin reduced apoptosis, ROS production and inflammatory response in primary human andrat cardiomyocytes in vitro in a PP2A dependent manner.

Comparative analysis of a panel of biomarkers related to protein phosphatase 2A between laryngeal squamous cell carcinoma tissues and adjacent normal tissues / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Laryngeal squamous cell carcinoma (LSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC) worldwide. Protein phosphatase 2A (PP2A) dysfunction has been widely reported in a broad range of malignancies due to its distinctive role in miscellaneous cellular processes. However, it is poorly understood whether aberrant alterations of PP2A are involved in the network of oncogenic events in LSCC. Here, we detected a panel of PP2A-associated proteins using western blot in both laryngeal squamous cell carcinoma tissues and paired adjacent normal tissues from patients (Data S1). We found that phospho-PP2A/C (Y307), α4, cancerous inhibitor of protein phosphatase 2A (CIP2A), Akt, ezrin, phospho-ezrin (T567), 14-3-3, and focal adhesion kinase (FAK) showed increased expression levels in carcinoma tissues relative to normal tissues, while phospho-Akt (T308) showed decreased levels. Our study, thus, provides a rationale for targeting PP2A to develop novel therapies and proposes a combination of interrelated biomarkers for the diagnostic evaluation and prognosis prediction in LSCC.

Inhibitory effects of polyphyllins I and VII on human cisplatin-resistant NSCLC via p53 upregulation and CIP2A/AKT/mTOR signaling axis inhibition / 中国天然药物

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a human oncoprotein that is overexpressed in multiple kinds of cancers including non-small cell lung cancer (NSCLC). CIP2A plays an 'oncogenic nexus' to participate in the tumorigenesis and chemoresistance in several cancer types. AKT and mTORC1 overactivation are detected in NSCLC and many other cancers. Previous studies found that the CIP2A/AKT/mTOR pathway controls cell growth, apoptosis, autophagy process. Polyphyllin I (PPI) and polyphyllin VII (PPVII) are natural components extracted from Paris polyphylla that display anti-cancer properties. In the present study, we investigated whether PPI and PPVII can be used in the cisplatin (DDP)-resistant human NSCLC cell line A549/DDP. Results demonstrated that PPI and PPVII treatment significantly suppressed A549/DDP cell proliferation, migration, invasion and EMT, induced apoptosis and autophagy. Further examination of the mechanism revealed that the PPI and PPVII significantly upregulated the p53, induced caspase-dependent apoptosis and suppressed the CIP2A/AKT/mTOR pathway. The activation of autophagy was mediated through PPI and PPVII induced inhibition of mTOR. We propose that PPI and PPVII might be developed as candidate drugs for DDP-resistant NSCLC.

Telmisartan Inhibits Nitric Oxide Production and Vessel Relaxation via Protein Phosphatase 2A-mediated Endothelial NO Synthase-Ser¹¹⁷⁹ Dephosphorylation

BACKGROUND: Apart from its blood pressure-lowering effect by blocking the renin-angiotensin-aldosterone system, telmisartan, an angiotensin II type 1 receptor blocker (ARB), exhibits various ancillary effects including cardiovascular protective effects in vitro. Nonetheless, the protective effects of telmisartan in cerebrocardiovascular diseases are somewhat variable in large-scale clinical trials. Dysregulation of endothelial nitric oxide (NO) synthase (eNOS)-derived NO contributes to the developments of various vascular diseases. Nevertheless, the direct effects of telmisartan on endothelial functions including NO production and vessel relaxation, and its action mechanism have not been fully elucidated. Here, we investigated the mechanism by which telmisartan regulates NO production and vessel relaxation in vitro and in vivo. METHODS: We measured nitrite levels in culture medium and mouse serum, and performed inhibitor studies and western blot analyses using bovine aortic endothelial cells (BAECs) and a hyperglycemic mouse model. To assess vessel reactivity, we performed acetylcholine (ACh)-induced vessel relaxation assay on isolated rat aortas. RESULTS: Telmisartan decreased NO production in normoglycemic and hyperglycemic BAECs, which was accompanied by reduced phosphorylation of eNOS at Ser¹¹⁷⁹ (p-eNOS-Ser¹¹⁷⁹). Telmisartan increased the expression of protein phosphatase 2A catalytic subunit (PP2Ac) and co-treatment with okadaic acid completely restored telmisartan-inhibited NO production and p-eNOS-Ser¹¹⁷⁹ levels. Of the ARBs tested (including losartan and fimasartan), only telmisartan decreased NO production and p-eNOS-Ser¹¹⁷⁹ levels, and enhanced PP2Ac expression. Co-treatment with GW9662 had no effect on telmisartan-induced changes. In line with in vitro observations, telmisartan reduced serum nitrite and p-eNOS-Ser¹¹⁷⁹ levels, and increased PP2Ac expression in high fat diet-fed mice. Furthermore, telmisartan attenuated ACh-induced rat aorta relaxation. CONCLUSION: We demonstrated that telmisartan inhibited NO production and vessel relaxation at least in part by PP2A-mediated eNOS-Ser¹¹⁷⁹ dephosphorylation in a peroxisome proliferator-activated receptor γ-independent manner. These results may provide a mechanism that explains the inconsistent cerebrocardiovascular protective effects of telmisartan.

Influence of Rotenone on Activity of Protein Phosphates 2A in MN9D Cells / 中国康复理论与实践

Objective To study whether and how rotenone reduces the activity of protein phophatase 2A (PP2A). Methods MN9D cells (mouse midbrain dopaminergic cell line) were divided into normal group (normal cultured), con-trol group (dimethyl sulfoxide of same volume with rotenone group was added in medium), rotenone group (50 nmol/L rotenone was added to the culture medium for 24 hours) and rotenone+C2 group (pretreatment of 5 mol/L C2-ceramide for eight hours, and then exposed to 50 nmol/L rotenone for 24 hours). MTT was used to detect cell viability. Total PP2A levels and tyrosine phosphorylation levels were measured with Western blotting. PP2A activity was detected with colorimetric assay.Results Compared with the control group, the cell viability reduced (P<0.01), phosphorylation of tyrosine 307 of PP2A inceased (P<0.01), and activity of PP2A decreased in the rotenone group (P<0.05). And compared with the rote-none group, the cell viability improved (P<0.01), phosphorylation of tyrosine 307 of PP2A deceased (P<0.01), and activity of PP2A increased (P<0.05) in the rotenone+C2 group. Conclusion Rotenone can inhibit activity of PP2A through increasing phosphorylation of tyrosine 307 at the catalytic subunit of PP2A, which might be involved in reducing cell viability, and implicate a new treatment strategy for Parkinson's disease.

AngⅡ/AT1R pathway leads to down-regulation of endothelial nitric oxide synthase phosphorylation / 中国病理生理杂志

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 syn-thase(eNOS)phosphorylation level in mesenteric arteries of rats.METHODS: 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 angiotensinⅡdown-regulated eNOS(Ser1177)phosphorylation level,the mesenteric arteries were randomly divided into normal control(control)group and AngⅡgroup.The mesenteric arteries in AngⅡgroup were incubated with AngⅡat 1×10 -7mol/L,1×10 -6mol/L and 1×10 -5mol/L for 6 h,12 h and 24 h,respectively.Secondly,to investigate the mo-lecular 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)+AngⅡgroup.The mesenteric arteries were pretreated with 1×10 -5mol /L CAN for 1 h,then incubated with 1×10 -7mol/L AngⅡfor 12 h in CAN+AngⅡgroup.The protein levels of eNOS,p-eNOS(Ser1177),PP2Ac,p-PP2Ac(Tyr307)and protein phosphatase 2A inhibitor 2(IPP2A2 )in the arteries were determined by Western blot.The ac-tivity 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 AngⅡfor 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 1×10-7mol/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 IPP2A2 were decreased after the mesenteric arteries were trea-ted with AngⅡat 1×10 -7mol/L for 12 h(P<0.05).Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307)and IPP2A2 (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 AngⅡat 1× 10-7mol/L for 12 h(P<0.05).Candesarten pretreatment inhibited the activity of PP 2A significantly(P<0.05).CON-CLUSION:AngⅡincreases PP2A activity via AT1R pathway,thus leading to down-regulation eNOS(Ser1177)phospho-rylation level in mesenteric arteries.The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac(Tyr307)and IPP2A2.

Phenotype and mechanism of inducible ppp2r1a knockout mouse model / 中华预防医学杂志

Objective@#Investigate the effects of inducible ppp2r1a knockout on main physiological function in adult mice and study the mechanism.@*Methods@#Ppp2r1aflox/flox mice and CAGG-CreER mice were hybridized to obtain 20 CAGG-CreER ppp2r1aflox/flox and 20 mice in homozygous group. Two groups of mice were divided into 4 groups respectively, finally we got 8 groups with 5 mice in each group. Tamoxifen was injected intraperitoneally to acquire inducible ppp2r1a knockout mice. The knockout efficiency of PP2A Aα in vital organs was measured by Western blot. At 0, 2, 4 and 6 days after injection, we measured body weight, histopathological change, peripheral blood cell counts and blood biochemical. Real-time PCR was performed to measure expression of liver glucolipid metabolism genes.@*Results@#After tamoxifen injection for 6 days, the knockout efficiency of PP2A Aα in vital organs was 35%, 12%, 15%, 60%, 69% and 72%, respectively in heart, liver, spleen, lung, kidney and brain. After tamoxifen injection for 6 days, the weight of homozygous mice was lower than that of wild type mice, with values of (17.42±1.76) g and (21.69±1.82) g, respectively (P<0.05). Moreover, the activity level, abdominal and renal fat were significantly decreased in homozygous mice. Homozygous mice survived no more than 7 days. Compared with wild type mice, the organ coefficient of spleen of homozygous mice was decreased at the 6th day, with values of (0.59±0.10)% and (0.36±0.05)% respectively (P<0.05). Obvious spleen atrophy and marked decrease of nucleated cells were showed by performing HE staining. Tunel staining revealed increased apoptosis ratio of splenic lymphocytes in homozygous mice. The levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) of homozygous mice were higher than wild type mice (P<0.05). The values of ALT and AST in homozygous mice were (153.68±62.80) U/L and (193.2±44.28) U/L. The corresponding values in wild type mice were (41.02±12.91) U/L and (69.40±9.55) U/L. The above results indicated that ppp2r1a knockout caused liver damage. Blood sugar level of homozygous mice was lower than in wild type mice (P<0.05), with values of (4.20±1.99) mmol/L and (8.88±0.65) mmol/L respectively. Plasma total cholesterol (TC), high density lipoprotein (HDL) and β-hydroxybutyric acid (β-HB) level of homozygous mice were higher than those of wild type mice (P<0.05). The values of TC, HDL and β-HB in homozygous mice were (3.12±0.39), (1.53±0.38) and (2.49±0.89) mmol/L. The corresponding values in wild type mice were (1.69±0.92), (0.78±0.50) and (0.45±0.30) mmol/L respectively. The above results indicated that ppp2r1a loss interfered glucose and cholesterol metabolism. In addition, we also found that the white blood cell count (WBC) and lymphocyte count (LYM) of homozygous mice were lower than in wild type mice (P<0.05). The values of WBC and LYM in homozygous mice were (1.88±0.89)×109/L and (0.92±0.37)×109/L respectively. The corresponding values in wild type mice were (3.91±0.80)×109/L and (2.74±0.52)×109/L respectively. The mRNA levels of glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) of homozygous were lower than wild type mice (P<0.05). The fold change of G6P and PEPCK in homozygous mice was 0.46±0.11 and 0.72±0.07 respectively. The corresponding fold change in wild type mice was 1.02±0.07 and 1.02±0.06 respectively.@*Conclusion@#Whole body ppp2r1a is essential for the survival of adult mice, due to the important role in maintaining the metabolism of glucose and cholesterol of liver.

Effect of cornel iridoid glycoside on PP2Ac phosphorylation in okadaic acid-induced neurotoxicity cells / 药学学报

Alzheimer's disease (AD) is the most common neurodegenerative disease in the aging population. Abnormal hyperphosphorylation of tau is the main cause of AD. Protein phosphatases 2A (PP2A) can increase the hyperphosphorylation of tau. Cornel iridoid glycoside (CIG) is one of the main components extracted from Cornus of ficinalis. The aim of the present study was to investigate the effects and the underlying mechanisms of CIG on enhancing PP2A activity. SK-N-SH cells were exposed to 20 nmol·L-1 okadaic acid (OA, an inhibitor of PP2A) for 6 h to induce the hyper-phosphorylation of tau, in order to define the effect of CIG on the activity of PP2A and posttranslational modification of PP2A catalytic subunit C (PP2Ac). We found that OA significantly decreased PP2A activity, increased the phosphorylation of PP2Ac, and enhanced tau hyper-phosphorylation. Pre-incubation of CIG significantly attenuated the OA-induced tau hyper-phosphorylation at Ser 199/202 and Ser 396, and recovered the activity of PP2A. CIG inhibited PP2Ac phosphorylation at Tyr 307 and increased Src phosphorylation. In conclusion, the mechanism of CIG inhibition of tau hyper-phosphorylation was activation of PP2A to reduce the level of p-Src for a reduction of PP2Ac phosphorylation at Tyr307.

Expression of CIP2A, bcl-2 and p63 in papillary thyroid cancer and their significances / 肿瘤研究与临床

Objective To investigate the expression and significance of cellular inhibitor of protein phosphatase 2A (CIP2A), bcl-2 and p63 in papillary thyroid cancer (PTC). Methods Using immunohistochemistry to detect the expression of CIP2A, bcl-2 and p63 in 30 cases of nodular goiter (NG), 30 cases of thyroid adenoma (TA) and 57 cases of PTC [including classical PTC (cPTC) 20 cases, papillary microcarcinoma (PMC) 20 cases, follicular thyroid papillary carcinoma (FPTC) 7 cases]. Results In NG group, TA group and PTC group, positive rates of CIP2A were 0, 0 and 94.74 % (54/57), respectively. The differences were statistically significant. In NG group, TA group and PTC group, positive rates of bcl-2 were 16.67 % (5/30), 13.33 % (4/30) and 85.96 % (49/57), respectively. The differences were statistically significant. In each group, positive rates of p63 were 6.67% (2/30), 3.33% (1/30) and 5.26% (3/57), respectively, no significant difference among them. In PTC, expression of CIP2A and bcl-2 were significantly higher than in NG and TA (χ2 = 105.56, P= 0.00; χ2 = 58.95, P= 0.00). Furthermore, the expression of CIP2A and bcl-2 had correlation in PTC (r=0.94, P=0.00). The expression of CIP2A, bcl-2 and p63 had no significantly difference among all the PTC subtype (χ2 values were 2.02, 2.64, 1.85; all P> 0.05). The expression of CIP2A, bcl-2 and p63 was not associated with patients'age, sex, site, lymph node metastasis (all P>0.05). Conclusions High expression of CIP2A and bcl-2 is associated with PTC, and the expression of CIP2A and bcl-2 has correlation in PTC. The expression of p63 has no correlation with PTC.

Ang Ⅱ/AT1R pathway activates PP2A to down-regulate p-eNOS (Ser1177) in human umbilical vein endothelial cells / 中国病理生理杂志

AIM:To investigate whether angiotensin Ⅱ (Ang Ⅱ)/angiotensin Ⅱ type 1 receptor (AT1 R)pathway down-regulates endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation level in human umbilical vein endothelial cells by activating protein phosphatase 2A (PP2A).METHODS:Human umbilical vein endothelial cells were randomly divided into normal control (control) group,Ang Ⅱ group,candesartan (CAN;specific AT1R blocker) group and CAN pretreatment + Ang Ⅱ group.The protein levels of total eNOS,p-eNOS (Ser1177),PP2Ac,IPP2A2 and p-PP2Ac (Tyr307) were determined by Western blot.The content of NO in the cell culture medium was detected by chemical colorimetry.RESULTS:Compared with control group,the level of p-eNOS (Ser1177) and the content of NO decreased (P ＜0.05).Compared with the same concentration of Ang Ⅱ group,CAN pretreatment increased the level of p-eNOS (Ser1177) and the content of NO (P ＜ 0.05),but the protein expression of eNOS showed no significant difference.Compared with control group,the levels of p-PP2Ac (Tyr307) and IPP2A2 decreased (P ＜ 0.05).Compared with the same concentration of Ang Ⅱ group,CAN pretreatment increased the levels of p-PP2Ac (Tyr307) and IPP2A2 (P ＜ 0.05),but the protein expression of PP2Ac showed no significant difference.CONCLUSION:Ang Ⅱ down-regulates the level of p-eNOS (Ser1177),and decreases the production of NO in human umbilical vein endothelial cells via AT1 R pathway.This effect may be related to the reduction of p-PP2Ac (Tyr307) and protein expression of IPP2A2,which results in the enhancement of PP2A2 activity.Pretreatment with AT1 R blocker CAN increases p-PP2Ac (Tyr307) level and IPP2A2 protein expression,thus reducing the PP2A activity,and ultimately restoring eNOS Ser1177 phosphorylation level and eNOS activity.

Ang Ⅱ/AT1R pathway activates PP2A to down-regulate p-eNOS (Ser1177) in human umbilical vein endothelial cells / 中国病理生理杂志

AIM:To investigate whether angiotensin Ⅱ (Ang Ⅱ)/angiotensin Ⅱ type 1 receptor (AT1 R)pathway down-regulates endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation level in human umbilical vein endothelial cells by activating protein phosphatase 2A (PP2A).METHODS:Human umbilical vein endothelial cells were randomly divided into normal control (control) group,Ang Ⅱ group,candesartan (CAN;specific AT1R blocker) group and CAN pretreatment + Ang Ⅱ group.The protein levels of total eNOS,p-eNOS (Ser1177),PP2Ac,IPP2A2 and p-PP2Ac (Tyr307) were determined by Western blot.The content of NO in the cell culture medium was detected by chemical colorimetry.RESULTS:Compared with control group,the level of p-eNOS (Ser1177) and the content of NO decreased (P ＜0.05).Compared with the same concentration of Ang Ⅱ group,CAN pretreatment increased the level of p-eNOS (Ser1177) and the content of NO (P ＜ 0.05),but the protein expression of eNOS showed no significant difference.Compared with control group,the levels of p-PP2Ac (Tyr307) and IPP2A2 decreased (P ＜ 0.05).Compared with the same concentration of Ang Ⅱ group,CAN pretreatment increased the levels of p-PP2Ac (Tyr307) and IPP2A2 (P ＜ 0.05),but the protein expression of PP2Ac showed no significant difference.CONCLUSION:Ang Ⅱ down-regulates the level of p-eNOS (Ser1177),and decreases the production of NO in human umbilical vein endothelial cells via AT1 R pathway.This effect may be related to the reduction of p-PP2Ac (Tyr307) and protein expression of IPP2A2,which results in the enhancement of PP2A2 activity.Pretreatment with AT1 R blocker CAN increases p-PP2Ac (Tyr307) level and IPP2A2 protein expression,thus reducing the PP2A activity,and ultimately restoring eNOS Ser1177 phosphorylation level and eNOS activity.

Role of Microtubule-associated Protein Tau Phosphorylation in Alzheimer's Disease / 华中科技大学学报（医学）（英德文版）

As a major microtubule-associated protein,tau plays an important role in promoting microtubule assembly and stabilizing microtubules.In Alzheimer's disease (AD) and other tauopathies,the abnormally hyperphosphorylated tau proteins are aggregated into paired helical filaments and accumulated in the neurons with the form of neurofibrillary tangles.An imbalanced regulation in protein kinases and protein phosphatases is the direct cause of tau hyperphosphorylation.Among various kinases and phosphatases,glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) are the most implicated.Accumulation of the hyperphosphorylated tau induces synaptic toxicity and cognitive impairments.Here,we review the upstream factors or pathways that can regulate GSK-3β or PP2A activity mainly based on our recent findings.We will also discuss the mechanisms that may underlie tau-induced synaptic toxicity.

Activity regulation mechanisms of protein phosphatase 2A and its role in Alzheimer’s disease / 国际药学研究杂志

Protein phosphatase 2APP2Ais the main serine/threonine protein phosphatase of the brain, and it is involved in many physiological and pathological processes in the cell. Particularly in the pathogenesis of Alzheimer’s diseaseAD, PP2A is closely related to the main pathological features of AD. Deregulation of PP2A enzymes correlates with increased tau phosphorylation, the formation of amyloid precursor protein, and the missing of neurons. PP2A as the target of drug development may bring new hope for the treatment of AD. This review introduces the structure and activity regulation of PP2A, and the role of PP2A in AD.

Effect of CIP2 A over-expression on proliferation and apoptosis of gastric epithelial cells / 中国病理生理杂志

AIM:To investigate the effects of cancerous inhibitor of protein phosphatase 2A (CIP2A) over-ex-pression on the proliferation and apoptosis of gastric epithelial cells .METHODS:The mRNA expression of CIP2A and cy-clin D1 in the tissues of normal gastric mucosa and gastric polyps was detected by RT -qPCR.The GES-1 cells were divided into control group, Ad-emp group and Ad-CIP2A group.The cell proliferation ability was detected by MTT assay and BrdU assay , and the cell apoptosis was analyzed by flow cytometry .The expression of apoptosis-related molecules was determined by Western blot and RT-qPCR.The levels of inflammatory cytokines were measured by ELISA after GES-1 cells were infec-ted with Ad-emp and Ad-CIP2A.Furthermore, the protein levels of p-Rb, E2F1 and cyclin D1 in the GES-1 cells was de-termined by Western blot after transfected with E2F1 siRNA.RESULTS:The expression of CIP2A and cyclin D1 in ade-nomatous gastric polyps tissues was significantly higher than that in normal gastric mucosa tissues , and no significant change of that between hyperplastic gastric polyps tissues and normal gastric mucosa was observed .After transfected with CIP2A, the proliferation ability of GES-1 cells was increased , the cell apoptosis was inhibited , the concentrations of IL-1βand IL-10 was up-regulated and the protein levels of p-Rb, E2F1 and cyclin D1 were increased, while the protein levels of p-Rb, E2F1 and cyclin D1 were significantly decreased after transfected with E2F1 siRNA.CONCLUSION: CIP2A promotes the proliferation and inhibits the apoptosis of GES-1 cells by activating Rb/E2F1.

Neuroprotective effect of astrocyte protein phosphatase 2 A up-regulation on APP/PS1 double transgenic mice / 中国病理生理杂志

AIM:To investigate the protective effects of astrocyte protein phosphatase 2A (PP2A) up-regula-tion on APP/PS1 double transgenic mice.METHODS:An eGFP-wtPP2A lentivirus with glial fiber acidic protein promoter was constructed to specifically increase PP2A expression in the astrocytes.The mice were divided into wild -type mice +vector virus group (Con), APP/PS1 transgenic mice +vector virus group (APP/PS1) and APP/PS1 transgenic mice +eGFP-wtPP2A lentivirus group (PP2A) by lateral ventricular injection of the lentivirus.Four weeks after injection of the vi-rus, the immunofluorescence of brain slices were used to detect the level of β-amyloid protein ( Aβ) .Golgi staining was used to detect the changes of dendritic spine density and morphology.Electron microscopy was applied to detect the thickness of postsynaptic density (PSD).The Morris water maze test was applied to examine the learning and memory abilities of the mice.RESULTS: Up-regulation of PP2A in the astrocytes attenuated Aβlevel increasing in APP/PS1 group.Up-regulation of PP2A in the astrocytes significantly attenuated both decreases in the dendritic spine density and the percentage of mushroom-like dendritic spines in the hippocampal CA3 region of APP/PS1 mice.Up-regulation of PP2A in the astrocytes significantly attenuated the reduced thickness of PSD in APP/PS1 group.Up-regulation of PP2A in the astro-cytes attenuated the escape latency extending in APP/PS1 group .CONCLUSION: Up-regulation of PP2A in the astro-cytes reduces AD-like pathological changes, and attenuates synaptic impairment, synaptic plasticity deficits and cognitive impairment in the APP/PS1 double transgenic mice.

Activity regulation mechanisms of protein phosphatase 2A and its role in Alzheimer′s disease / 国际药学研究杂志

Protein phosphatase 2A(PP2A)is the main serine/threonine protein phosphatase of the brain,and it is involved in many physiological and pathological processes in the cell. Particularly in the pathogenesis of Alzheimer′s disease(AD),PP2A is closely related to the main pathological features of AD. Deregulation of PP2A enzymes correlates with increased tau phosphorylation, the formation of amyloid precursor protein,and the missing of neurons. PP2A as the target of drug development may bring new hope for the treatment of AD. This review introduces the structure and activity regulation of PP2A,and the role of PP2A in AD.