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.

Identification and Expression Analysis of PP2C Gene Family Members in Cannabis sativa / 中国实验方剂学杂志

ObjectiveThe type 2C protein phosphatases （PP2C） are involved in numerous plant signal transduction pathways. They mainly participate in plant stress response and regulate second metabolites biosynthesis via negatively regulating MAPK signaling pathway. Herein，we were to identify and analyze PP2C （CsPP2C） gene family from hemp genome，in hope of providing comprehensive insights for studying CsPP2C function during the development of hemp. MethodMolecular Evolutionary Genetics Analysis （MAGA）-X was used to construct phylogenetic tree. Expert Protein Analysis System （ExPASy），WoLF PSORT，Multiple EM for Motif Elicitation （MEME），Batch Conserved Domain Search （Batch-CD-Search），PlantCare，and TBtools were used，respectively，to predict CsPP2C physicochemical properties，subcellular localization，conserved motifs，protein structure，cis-element in promoter and collinearity with Arabidopsis PP2C. Cannabis sativa transcriptome and Real-time polymerase chain reaction（Real-time PCR） were used to analyze and verify gene expressions，respectively. ResultFifty-two CsPP2C with conserved domains were identified from the entire genome of hemp，encoding proteins ranging from 244 to 1 089 aa in length and with molecular weights ranging from 26.76 to 122.53 kDa. Those genes were mainly distributed in the nucleus，cytoplasm and chloroplast. The 47 CsPP2C were divided into 10 subfamilies，and the remaining 5 were not clustered. Seven pairs of homologous genes between hemp and Arabidopsis thaliana were identified according to collinear analysis. The light-responsive elements and abscisic acid elements are most abundant in the prediction. The gene expression heat map showed varied expression pattern of CsPP2C in different tissues. Real-time PCR results of three CsPP2C were consistent with transcriptome data. Moreover，alternative splicing analysis showed that some CsPP2C had alternative-splicing genes during evolution. ConclusionWe predicted and analyzed CsPP2C gene family in genomic scale and showed that CsPP2C are involved in many biological processes，whereby provides foundation for CsPP2C functional study.

Identification and Expression Analysis of PP2C Gene Family Members in Cannabis sativa / 中国实验方剂学杂志

ObjectiveThe type 2C protein phosphatases （PP2C） are involved in numerous plant signal transduction pathways. They mainly participate in plant stress response and regulate second metabolites biosynthesis via negatively regulating MAPK signaling pathway. Herein，we were to identify and analyze PP2C （CsPP2C） gene family from hemp genome，in hope of providing comprehensive insights for studying CsPP2C function during the development of hemp. MethodMolecular Evolutionary Genetics Analysis （MAGA）-X was used to construct phylogenetic tree. Expert Protein Analysis System （ExPASy），WoLF PSORT，Multiple EM for Motif Elicitation （MEME），Batch Conserved Domain Search （Batch-CD-Search），PlantCare，and TBtools were used，respectively，to predict CsPP2C physicochemical properties，subcellular localization，conserved motifs，protein structure，cis-element in promoter and collinearity with Arabidopsis PP2C. Cannabis sativa transcriptome and Real-time polymerase chain reaction（Real-time PCR） were used to analyze and verify gene expressions，respectively. ResultFifty-two CsPP2C with conserved domains were identified from the entire genome of hemp，encoding proteins ranging from 244 to 1 089 aa in length and with molecular weights ranging from 26.76 to 122.53 kDa. Those genes were mainly distributed in the nucleus，cytoplasm and chloroplast. The 47 CsPP2C were divided into 10 subfamilies，and the remaining 5 were not clustered. Seven pairs of homologous genes between hemp and Arabidopsis thaliana were identified according to collinear analysis. The light-responsive elements and abscisic acid elements are most abundant in the prediction. The gene expression heat map showed varied expression pattern of CsPP2C in different tissues. Real-time PCR results of three CsPP2C were consistent with transcriptome data. Moreover，alternative splicing analysis showed that some CsPP2C had alternative-splicing genes during evolution. ConclusionWe predicted and analyzed CsPP2C gene family in genomic scale and showed that CsPP2C are involved in many biological processes，whereby provides foundation for CsPP2C functional study.

Effect of TSG on GSK3β，PKA and PP2A of APP/PS1/Tau Triple-transgenic Mice Dementia Model / 中国实验方剂学杂志

Objective:To observe the effect of tetrahydroxy stilbene glycoside （TSG） on the expression of glycogen synthase kinase 3<italic>β </italic>（GSK3<italic>β</italic>）， cyclic adenosine monophosphate-dependent protein kinase （PKA） and Serine/threonine phosphatase 2A（PP2A） in the brain of amyloid precursor protein/presenilin-1/Tau （APP/PS1/Tau） triple-transgenic mice dementia model. Method:A total of forty-five 8-month-old APP/PS1/Tau transgenic mice were randomly divided into model group， positive control group （Huperzine-A， 0.15 mg·kg^-1）， low， medium and high dose TSG groups （TSG， 0.033，0.1，0.3 g·kg^-1）， with 9 mice in each group， and another nine C5B7L/6J mice of the same age were selected as normal control group. After 60 days of intragastric administration， the general structure of hippocampal neurons was observed by hematoxylin-eosin （HE） staining， immunohistochemical （IHC） was used to detect the expression of PKA protein in the brain of mice in each group， the mRNA expression levels of GSK3<italic>β</italic>， PKA and PP2A were detected by real time quantitative reverse transcription polymerase chain reaction （Real-time PCR）， and protein expression levels of GSK3<italic>β</italic> and PP2A were detected by Western blot. Result:Compared with the normal control group， the apoptosis level of neurons in the model group was significantly increased， the protein and mRNA expression levels of GSK3<italic>β</italic> and PKA were significantly increased （<italic>P</italic><0.05， <italic>P</italic><0.01）， and the protein and mRNA expression levels of PP2A were significantly decreased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the apoptosis level of neurons in each treatment group was significantly down-regulated， the protein and mRNA expression levels of GSK3<italic>β</italic> and PKA were significantly down-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）， and the protein and mRNA expression levels of PP2A were significantly increased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:The mechanism of TSG in the treatment of Alzheimer's disease （AD） may be related to lowering the transcription and expression of GSK3<italic>β</italic> and PKA， increasing the transcription and expression of PP2A.

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.

SRC kinase inhibitor PP2 inhibits invasion and metastasis of lung cancer A549 cells by upregulating connexin43 expression / 南方医科大学学报

OBJECTIVE@#To investigate the effect of SRC kinase inhibitor PP2 on the invasion and metastasis of lung cancer A549 cells and explore its molecular mechanism.@*METHODS@#MTT assay was used to evaluate the inhibitory effect of PP2 on the proliferation of A549 cells. Cell scratch and Transwell assays were performed to assess the invasion and metastatic capacity of A549 cells after treatment with 1, 2, 4, 8, and 16 μmol/L PP2 for 24 h. Western blotting was used to detect the expressions of connexin43 (Cx43) and MMP-2 in the cells after small interfering RNA (siRNA)-mediated silencing or overexpression of Cx43; the changes in the cell invasion and metastasis in response to PP2 treatment after Cx43 silencing or overexpression were investigated.@*RESULTS@#MTT assay showed that treatment with PP2 at 2, 4, 8, 16, and 32 μmol/L significantly inhibited the proliferation of A549 cells in a concentration-dependent manner. Treatments with PP2 at 1, 2, 4, 8, and 16 μmol/L for 24 h also concentration-dependently lowered the invasion and metastatic abilities of the cells ( < 0.05). At 4 and 8 μmol/L, PP2 significantly increased the expression level of Cx43 protein and decreased the expression level of MMP-2 protein. Overexpression of Cx43 significantly enhanced the inhibitory effect of PP2 on the cell invasion and metastasis, and Cx43 silencing significantly attenuated the inhibitory effect of PP2 ( < 0.05).@*CONCLUSIONS@#PP2 treatment can suppress the invasion and metastasis of A549 cells possibly by modulating the expression of Cx43.

Inhibition of invasion and metastasis of human tonguesquamous cell carcinoma Tca8113 cells by Src kinase inhibitor PP2 / 西安交通大学学报(医学版)

Objective: To investigate the inhibitory effects of Src kinase inhibitor PP2 on migration and invasion of Tca8113 cells. Methods: Tca8113 cells were cultured for 24 h with 5 mol/L, 10 mol/L and 20 micron mol/L of Src kinase inhibitor PP2. The effects of PP2 on the invasion and migration of Tca8113 cells were measured with Transwell chamber and scratch method, respectively. Results: After the treatment with PP2 for 24 h, the expression of p-Src in 5, 10, 20 μmol/L of Src kinase inhibitor PP2 treatment groups was significantly lower than that of the non-drug treatment group (all P<0.05). The number of Tca8113 cells in the non-drug treatment group and the 5, 10, and 20 μmol/L of Src kinase inhibitor PP2 treatment groups was (232.76±28.65), (186.53±21.34), (129.18±17.96), and (37.82±12.41), respectively; the number of migratory cells was (259.38±25.27), (193.45±20.18), (143.24±18.04), and (32.94±14.39), respectively, the cell migration rate was (11.51±0.84)%, (8.06±0.51)%, (5.13±0.57)%, and (3.18±0.12)%, respectively; the overall difference was statistically significant (F=73.852, 85.687, 48.157, all P=0.000). It had a negative correlation with PP2 dose. Conclusion: Src kinase inhibitor PP2 can effectively inhibit the invasion and migration of Tca8113 cells in the concentration-dependent manner, and it may have certain clinical value in the treatment of human tongue squamous cell carcinoma.

Banqiao Codonopisis Pilosula improves cognitive dysfunction in AD model rats by PP2A signaling pathway / 中国药理学通报

Aim To investigate the protective effect of Banqiao Codonopsis pilosula (BCP) on cognitive function in rats with Alzheimer's disease(AD) induced by Okadaic acid (OA) and its possible mechanism. Methods SD rats were randomly divided into DMSO group, OA group and BCP low, medium, high treatment group. The rats were gavage administered in groups of one week and two weeks. The water maze training was continued for five days before modeling, and modeling was started 24 hours after the training. The bilateral hippocampus of DMSO group was injected with 10% DMSO 1.5 |xL. OA group and BCP treatment group were injected with OA (0. 392 mmol • L"1) 1.5 (iL. The water maze test was used to observe the spatial learning ability of rats. Western blot was used to observe the activity of PP2A, the phosphoryla-tion of Tau protein and the expression of synaptic protein in hippocampus, and the Nissl's staining to observe the changes of Nissl bodies in hippocampus CA1 and CA3. Results Water maze experiments showed that BCP could improve spatial memory impairment in AD rats. Western blot results showed that BCP in-creased PP2A activity, increased synaptic protein expression, and decreased Tau protein phosphorylation. Nissl's staining suggested an increase in the number of Nissl bodies in BCP treatment group. Conclusions BCP can up-regulate PP2A activity, decrease the phosphorylation level of Tau protein, increase the expression of synaptic proteins, and repair damaged neurons.

Trillium tschonoskii Maxim improves cognitive dysfunction in Alzheimer’s disease induced by okadaic acid in rats and its possible mechanism / 中国药理学通报

Aim To assess the effects of Trillium Tschonoskii Maxim ( TTM) decoction on learning and memory dysfunction in Alzheimer’ s disease ( AD ) model rats which induced by okadaic acid( OA) and its possible mechanism. Methods The SD rats were di-vided into ten groups,namely,d DMSO control group, OA group, TTM high-dose ( 0. 5 g·kg-1·d-1) group,TTM medium-dose ( 1 g·kg-1·d-1) group, TTM lower-dose (2 g·kg-1·d-1) group,and these groups were divided into one week and two weeks of gavage. Treatment groups were gavaged with TTM de-coction twice a day. After 5 days of Morris water maze training,treatment groups and AD model groups were injected with OA (0.392 mmol·L-1,1. 5 μL) in bi-lateral hippocampal of the rats. The DMSO groups were injected with 10% DMSO. The spatial memory reten- tion wereas detected by water maze at 24 h after injec-tion. After the test, we prepared sample for Western blot and Nissl’s staining. The Western blotting test was used to detect the PP2A activity and the phospho-rylation of Tau protein in the hippocampus. Nissl’'s staining was used to observe the changes of the number of Nissl’s bodies in the hippocampal CA1 and CA3 re-gions. Results The Morris water maze test showed that after injection of OA, the latency of TTM groups wereas shorter than that of OA groups. Western blot showed that the high dose TTM could increase the ac-tivity of PP2A and decrease the level of Tau phospho-rylation at PS-Tau396,,PT-Tau404. The Nissl’s stai-ning results showed that the number of Nissl’s bodies in the hippocampal CA1 and CA3 regions of OA groups wereas significantly attenuated compared with that of the number of Nissl's bodies in the hippocampal CA1 and CA3 regions than DMSO groups. The number of Nissl’s bodies in high groups were morewas larger than that of OA group. Conclusion The results show that TTM can improve the learning and memory dysfunction in AD model rats which induced by OA. The mecha- nism wasis probably that TTM can increase PP2A ac-tivity and then down-regulate the level of Tau phospho-rylation and improve neural development.

Expression of PP2CE in Human Hepatocellular Carcinoma Tissues and the Clinical Significance / 华中科技大学学报(医学版)

Objective To investigate expression of PP2CE in human hepatocellular carcinoma(HCC)tissues and the clinical pathological significance,and test its sub-cellular localization in liver cells and HCC cells.Methods A total of 50 samples of HCC tissues and their para-carcinoma tissues were collected in Tongji Hospital(Wuhan).PP2CE mRNA and protein levels were detected in HCC tissues and their para-carcinoma tissues by qRT-PCR and immunohistochemistry method.Immunofluorescent assay was performed to test the sub-cellular localization of PP2CE in the cells.Results PP2CE mRNA and protein expression levels were significantly decreased in HCC tissues as compared with those in para-carcinoma tissues(both P＜0.05).Immunohistochemistry analysis found that PP2CE expression was associated with tumor size,differentiated degree,portal vein invasion and clinical stage(both P＜0.05).Immunofluorescent assay suggested that PP2CE was mainly located in the cytoplasm and perinuclear area.Conclusion PP2CE is reduced in HCC and may be implicated in the development and progression of HCC.

Phosphatase PP2CB inhibits innate immune response triggered by RNA virus VSV or SeV / 中国免疫学杂志

Objective:To investigate the role of phosphatase PP2CB in the innate immunity against RNA virus and the underlying mechanism.Methods:PP2CB expression in macrophages was silenced with the specific siRNA.The mRNA and protein expression level of type Ⅰ interferon was detected by Q-PCR and ELISA respectively.The phosphorylation level of TBK1 and IRF3 was analyzed by Western blot.Results:RNA virus VSV infection led to the expression change of PP2CB.Overexpression of PP2CB dose-dependently inhibited the activation of IFN-β reporter gene.PP2CB silencing by PP2CB siRNA significantly promoted the production of type Ⅰ interferon triggered by RNA virus VSV or SeV,and inhibited the replication of VSV in macrophages.Furthermore,PP2CB bound TBK1 upon RNA virus infection.PP2CB silencing up-regulated the phosphorylation level of TBK1 and IRF3.Conclusion:Upon RNA virus VSV or SeV infection,phosphatase PP2CB binds TBK1 and inhibits its phosphorylation to negatively regulate the activation of the antiviral innate immune signal pathway,which consequently suppresses the production of type Ⅰ interferon triggered by RNA virus VSV or SeV.

Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.

The Effect of Chemoradiotherapy with SRC Tyrosine Kinase Inhibitor, PP2 and Temozolomide on Malignant Glioma Cells In Vitro and In Vivo / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We investigated the effect of chemoradiotherapy with PP2 and temozolomide (TMZ) on malignant glioma cells using clonogenic assays and in vivo brain tumor model. MATERIALS AND METHODS: The effect of PP2 on radiosensitivity of U251 and T98G cells was investigated using clonogenic assays. The expression of E-cadherin, matrix metalloproteinases 2 (MMP2), Ephrin type-A receptor 2 (EphA2), and vascular endothelial growth factor (VEGF) was measured by Western blotting and an accumulation of γH2AX foci 6 hours after radiotherapy was measured after PP2 treatment. The effect of PP2 on migration, invasion, and vasculogenic mimicry formation (VMF) of U251 cells was evaluated. In an orthotopical brain tumor model with U251 cells, PP2 was injected intraperitoneally with or without oral TMZ before, during and after whole brain radiotherapy. Bioluminescence images were taken to visualize in vivo tumors and immunohistochemical staining of VEGF, CD31, EphA2, and hypoxia-inducible factor 1a was performed. RESULTS: PP2 increased radiosensitivity of U251 and T98G cells without decreasing survival of normal human astrocytes. Chemoradiotherapy with PP2 and TMZ resulted in increased accumulation of γH2AX foci. PP2 induced overexpression of E-cadherin and suppression of MMP2, VEGF, and EphA2. PP2 also compromised invasion, migration, and VMF of U251 cells. In brain tumors, chemoradiotherapy with PP2 and TMZ decreased tumor volume best, but not statistically significantly compared with chemoradiotherapy with TMZ. The expression of VEGF and CD31 was suppressed in PP2-treated tumors. CONCLUSION: PP2 enhances radiosensitivity of malignant glioma cells and suppresses invasion and migration of U251 cells. Chemoradiotherapy with PP2 and TMZ resulted in non-significant tumor volume decrease.

Construction and appraisal of recombinant adeno-associated virus vector for expressing shRNA targeting CIP2A mRNA / 西安交通大学学报(医学版)

Objective To construct the recombinant adeno-associated virus vector carrying cancerous inhibitor of PP2A (CIP2A)short hairpin RNA (shRNA)for preparation of high-titer viruses.Methods The small hairpin RNA of CIP2A (CIP2A shRNA)was designed,synthesized and cloned into pDC31 6-EGFP-U6 plasmid which was double digested by Bam HⅠ and Hin dⅢ.The resultant plasmid pDC31 6-EGFP-shRNA was confirmed and served as template to appraise primers.EGFP-CIP2A shRNA sequence was amplified by PCR,double digested with Eco RⅠand Sal Ⅰ and ligated to pSNAV2.0 plasmid digested with the same enzyme pair.pSNAV2.0-EGFP-CIP2A shRNA plasmid DNA was prepared,purified,identified and transfected into BHK-21 cells.BHK-21 cells expressing CIP2A shRNA (BHK-21/CIP2A-shRNA ) were obtained and subsequently infected with VGTC’s proprietary AAV packaging system to package the rAAV2-CIP2A shRNA.After purification,the functional and infectious virus was obtained and the titer of virus was detected.Real-time PCR and Western blot methods were used to detect the expression of CIP2A after infection with HepG2 cells,and the empty viral vector rAAV2-EGFP was used as control. Results A recombinant adeno-associated virus-2 vector carrying CIP2A shRNA was constructed successfully.The presence of the target sequence in the vector was confirmed by double enzyme digestion and sequencing.By transfecting the pSNAV2.0-EGFP-CIP2A shRNA plasmid into BHK-21 cells,BHK-21/CIP2A shRNA cells were infected with helper virus HSV1-rc/ΔUL2 to package the rAAV2-CIP2A shRNA to obtain a functional and infectious virus.The titer of the recombinant virus was 0.25×10 1 2 v.g./mL.The expression of CIP2A mRNA and screening value of 1×10 5 MOI effected HepG2 cells.Conclusion A high-titer recombinant adeno-associated virus-2 vector carrying CIP2A shRNA has been constructed successfully.

Construction of eukaryotic expression vector of shRNA targeting CIP2 A gene and its inhibition effect on BGC-823 cell proliferation / 基础医学与临床

Objective To construct eukaryotic vectors expressing short hairpin RNAs ( shRNAs ) targeting at the CIP2 A gene and to explore its effects on gastric cell line BGC-823 .Methods Four oligonucleotides targeting the CIP2A gene were synthesized and cloned into the eukaryotic expression plasmid pGPU 6.The recombinant plas-mids, pGPU6/GFP/Neo-CIP2A-shRNA-1, 2, 3 and 4, were introduced into BGC-823 cells by lipofectamine-me-diated transfection and the infection rate was observed by fluorescence microscope .The gene silencing efficiency was measured by real-time PCR and Western blot .The effects on proliferation of BGC-823 cells were detected by CCK-8 .Results DNA sequencing and enzyme digestion analysis confirmed the identity of the four recombinant shRNA expression vectors .Immunofluorescsence demonstrated that transfection efficiency was above 70%.Trans-fection of shRNA-1, 2, 3 and 4, significantly knocked down the expression of CIP 2A mRNA and protein at 24, 48 and 72 h after transfection .Compared with the 2 , 3 and 4 , shRNA-1 had the more strong inhibitory effect on the expression of CIP2A mRNA and protein.The CCK-8 assay showed that the anti-proliferation effect on BGC-823 cells was significant ( P<0.05 ) .Conclusions The recombinant vector may effectively inhibit the expression of CIP2A in BGC-823 cells and depress the proliferation of BGC-823 cells.

Cardamonin Suppresses TGF-beta1-Induced Epithelial Mesenchymal Transition via Restoring Protein Phosphatase 2A Expression

Epithelial mesenchymal transition (EMT) is the first step in metastasis and implicated in the phenotype of cancer stem cells. Therefore, understanding and controlling EMT, are essential to the prevention and cure of metastasis. In the present study, we examined, by Western blot, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy, the effects of cardamonin (CDN) on transforming growth factor-beta1 (TGF-beta1)-induced EMT of A549 lung adenocarcinoma cell lines. TGF-beta1 induced expression of N-cadherin and decreased expression of E-cadherin. CDN suppressed N-cadherin expression and restored E-cadherin expression. Further, TGF-beta1 induced migration and invasion of A549 cancer cells, which was suppressed by CDN. TGF-beta1 induced c-Jun N-terminal kinase (JNK) activation during EMT, but CDN blocked it. Protein serine/threonine phosphatase 2A (PP2A) expression in A549 cancer cells was reduced by TGF-beta1 but CDN restored it. The overall data suggested that CDN suppresses TGF-beta1-induced EMT via PP2A restoration, making it a potential new drug candidate that controls metastasis.

The roles and related mechanisms of PP2A in cognitive dysfunction after the chronic cerebral ischemia / 中华行为医学与脑科学杂志

Objective To explore the roles and related mechanisms of Protein Phosphatase 2A(PP2A) in cognitive dysfunction after the chronic cerebral ischemia.Methods 70 male Sprague Dawley rats in clean degree were divided into sham group,chronic cerebral ischemia group (Bilateral carotid arteries occlusion,2VO),and chronic cerebral ischemia group with PP2A activation group(2VO+aPP2A).The rats were injected intraperitoneally with 1.88 μmol/ml sodium selenate(15 μmol · kg-1 · d-1) or equal volume of saline for 4 weeks.After one month,the chronic cerebral ischemia models were reproduced by the occlusion of bilateral common caroid artery.Then the abilities of learning and memory were tested by Morris water maze,electrophysiological indices were recorded to analyze the LTP changes,and destribution of synaptic vesicles was observed by electron microscope.Results Morris water maze test showed that the 2VO group had significantly longer latent time than sham group in searching platform(P＜0.05),and the 2VO+aPP2A group had dramatically shorter latent time (P＜0.01) than that of 2VO group.Then removing platform to test the rats memory,the data showed that 2VO group spent markedly longer time than sham group to reach the location of the former platform (sham group:(14.50±1.98)s ; 2VO group:(17.30±2.11) s) (P＜0.01),and the 2VO+aPP2A group((15.09± 1.45) s) spent dramatically shorter latent time(P＜0.05) than that of 2VO group.The electrophysiological data showed that 2VO group had the noticeably smaller field excitable postsynaptic potential slope (fEPSP) slope ratio between pre and post of the high frequency stimulations (Long-term potential,LTP) than sham group(sham group:1.69±0.27; 2VO group:2.02±0.137) (P＜0.01),and the 2VO+aPP2A group(1.86±0.19) had strikingly higher ratio than that of 2VO group(P＜0.01).The electromicroscope observation showed that presynaptic vesicles density of 2VO was remarkably lower than that of sham group (sham group:(4.51±0.29) /μm2 ; 2VO group:(2.02±0.14) /μm2) (P＜0.01),and presynaptic vesicles density of 2VO+aPP2A group((3.58±0.50) /μm2) was noticeably higher than that of 2VO group(P＜0.01).Conclusion Activating PP2A can prevent the cognitive dysfunction after chronic cerebral ischemia through regulating LTP and synaptic vesicle density.And PP2A is probably a potential target for preventing and treating the cognitive dysfunction after chronic cerebral ischemia.

Prokaryotic expression of Streptococcus pneumoniae phpP gene and evaluation of PP2C type phosphatase activity of the expressed recombinant protein / 中华微生物学和免疫学杂志

Objective To construct a prokaryotic expression system for expressing the phosphatase-encoding gene phpP in StkP/PhpP signaling couple in Streptococcus pneumonia ( S.pneumoniae) strains, and to further understand the phosphatase activity of the recombinant protein rPhpP.Methods The entire phpP gene of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR products were sequenced.A prokaryotic ex-pression system for expressing the phpP gene was constructed by the genetic engineering technique.The ex-pressed protein rPhpP and the solubility of rPhpP were assessed by SDS-PAGE and gel image analyzer.Ni-NTA affinity chromatography was performed to purify rPhpP.The changes of phpP gene transcription after the treat-ment with sublethal dosages of penicillin and cefotaxime were determined by real-time fluorescent quantitative RT-PCR.The functional domain in the sequence of the phpP gene and its type was analyzed by bioinformatic softwares.The activity of rPhpP in hydrolyzing the substrate of PP2C phosphotase was measured with Serine/Threonine Phosphotase Assay Kit.The enzyme kinetic parameters of rPhpP were calculated.Results The se-quence of the cloned phpP gene was identical with that reported in GenBank.The rPhpP in soluble form was ex-pressed in the constructed prokaryotic expression system.An increased expression of phpP gene at mRNA level was induced by sublethal dosage of penicillin or cefotaxime.The domain of PP2Cc type phosphatase was detec-ted in the sequence of phpP gene.The purified rPhpP protein could hydrolyze phosphopeptides [ RRA ( pT) VA], a substrate of PP2C type phosphatase, in a dose-dependent manner with Km and Kcat values of 277.35μmol/L and 0.71 S-1 ,respectively.Conclusion The protein encoded by phpP gene of S.pneumoniae was a PP2C type phosphatase.The expression of phpP gene could be enhanced by sublethal dosage of penicillin or ce-fotaxime.