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.

Expression of protein phosphatase 4 in different tissues under hypoxia

Relevant research data shows that there is a certain degree of energy metabolism imbalance in highland residents. Protein phosphatase 4 (PP4) has been found as a new factor in the regulation of sugar and lipid metabolism. Here, we investigate the differential expression of PP4 at a simulated altitude of 4,500 m in the heart, lung, and brain tissues of rats. A hypoxic plateau rat model was established using an animal decompression chamber. A blood routine test was performed by an animal blood cell analyzer on rats cultured for different hypoxia periods at 4,500 m above sea level. Quantitative polymerase chain reaction (qPCR) and western blot were used to detect the changes of protein phosphatase 4 catalytic subunit (PP4C) gene and protein in heart, lung, and brain tissues. The PP4C gene with the highest expression level found in rats slowly entering the high altitude area (20 m-2200 m-7 d-4500 m-3 d) was about twice as high as the low elevation group (20 m above sea level). The simulated high-altitude hypoxia induced an increase of PP4C expression level in all tissues, and the expression in the lung tissue was twice as expressed as heart and brain tissue at high altitude (P < 0.05). These results suggest that the PP4 phosphatase complex is ubiquitously expressed in rat tissues and likely involved in adaptation to or disease associated with high-altitude hypoxia.

Protein phosphatase 4 catalytic subunit (PP4C) increases hepatitis B virus X protein levels and promotes its biological functions / 中华微生物学和免疫学杂志

Objective:To investigate the role of protein phosphatase 4 catalytic subunit (PP4C) in regulating hepatitis B virus X protein (HBx) levels and its effects on the biological functions of HBx, thus to provide a potential therapeutic targets for hepatitis B virus (HBV)-related hepatocellular carcinoma.Methods:In vivo and in vitro interactions between HBx and PP4C were analyzed by co-immunoprecipitation (Co-IP) and GST pull-down assay. Recombinant plasmids of PP4C and HBx were co-transfected with Lipofectamine 3000 reagents into hepatoma cells to detect the protein levels of HBx by Western blot. The half-life of HBx in the transfected cells treated with cycloheximide (CHX) were detected. The phosphorylation assay was used to evaluate the effects of PP4C on HBx phosphorylation. CCK8 assay, wound healing assay and Matrigel invasion chamber assay were used to analyze the effects of PP4C on the biological functions of HBx. Results:PP4C interacted with HBx in vivo and in vitro. PP4C overexpression significantly increased the protein level and stability of HBx and the phosphorylation assay confirmed that PP4C overexpression decreased the serine phosphorylation of HBx in hepatoma cells. PP4C overexpression enhanced the migration and invasion of hepatoma cells, but had no significant effects on the proliferation. Conclusions:The interactions between HBx and PP4C promoted the stability of HBx and ultimately enhanced the migration and invasion of hepatoma cells, and the mechanisms might be related to the decrease of HBx serine phosphorylation by PP4C. This study provided a theoretical basis for further investigation of the pathogenic mechanisms of HBx, and targeting PP4C and HBx interaction might provide insights for developing novel treatment for HBV-related hepatocellular carcinoma.

A case of mental retardation autosomal dominant 35 with neonatal onset / 中华围产医学杂志

This article reported a male patient with neonatal onset mental retardation autosomal dominant 35 (MRD35). The boy presented with repeated convulsions, hypotonia, enlarged head circumference, congenital muscular torticollis and feeding difficulties in the neonatal period. Dynamic electroencephalogram showed paroxysmal epileptic discharges in the left central-temporal region. High-throughput whole-exome sequencing revealed a heterozygous mutation of c.139G>A (p.Glu47Lys) in the PPP2R5D gene, which was a de novo mutation not inherited from his parents. The child had significant developmental delay at the age of one year. MRD35 lacks typical clinical manifestations and requires whole-exome sequencing for definitive diagnosis. Currently, there is no specific treatment for MRD35 and symptomatic treatments, including rehabilitation training, language training and seizure control, are mostly adopted.

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.

Expressions and effects of serum miR-23a-3p and miR-27a-3p in mice with ulcerative colitis / 上海交通大学学报（医学版）

Objective: To explore the expression levels of miR-23a-3p and miR-27a-3p in the sera of mice with ulcerative colitis (UC) and their potential action mechanisms. Methods: Twenty C57BL/6 mice were randomly divided into the control group and the model group with 10 mice in each group. The model group mice were induced orally by water with 5% dextran sulfate sodium (DSS) for 7 d. During the induction period, the general condition, fecal morphology and occult blood status of the mice were observed. After 7 d, the whole blood and colon tissues of mice were collected, and the colon lengths and wet weights were measured. The expressions of miR-23a-3p and miR-27a-3p in the sera were detected by qRT-PCR. The expressions of peroxisome proliferator-activated receptor γ, coactivator-1α (PPARGC1A), PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), B-cell lymphoma-2 (BCL-2), BCL-2 associated X protein (BAX), cytochrome c (CYT-C) and cleaved cysteine-containing aspartate-specific protease (cleaved-caspase-3) were detected by Western blotting. Results: After DSS induction, the model group mice showed mental depression, weight loss, diarrhea, and bloody stool, whose colon lengths were shortened and colon wet weights decreased. The UC model was constructed successfully. In the model group, the expressions of miR-23a-3p and miR-27a-3p in the sera decreased significantly (P<0.05), and the expressions of PPARGC1A, PHLPP2, BAX, CYT-C and cleavedcaspase- 3 in the colon tissues increased significantly (P<0.05), but BCL-2 decreased (P<0.05). Conclusion: The expressions of miR-23a-3p and miR- 27a-3p are low in the sera of UC mice, which may be involved in the occurrence and development of UC by up regulating the expressions of PPARGC1A and PHLPP2 in the colons and triggering mitochondrial pathway to induce apoptosis.

Bone marrow mesenchymal stem cell derived exosomes miR-21-5p promotes proliferation, migration and invasion of prostate cancer PC-3 cell by downregulating PHLPP2 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of exosome originated from bone marrow mesenchymal stem cell (BMSCs) on proliferation, migration and invasion of prostate cancer PC-3 cell and its mechanism. Methods: qPCR was used to detect the expression level of miR-21-5p in prostate cancer cell lines. The morphology of exosomes isolated from BMSCs was observed with an electron microscope. Western blotting was used to detect the expressions of exosome surface markers and the epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin and Vimentin). Dual luciferase reporter gene experiment was used to detect the targeted regulation relationship between miR-21-5p and PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2). PC-3 cells were co-cultured with 10 μl BMSCs exosomes suspension (Exo group), transfected with sh-PHLPP2 or antagomiR, then CCK-8 and Transwell experiments were used to detect changesinproliferation,migrationandinvasionofPC-3cell.Results: miR-21-5p was highly expressed in prostate cancer PC-3 cell line. The exosomes in the supernatant of BMSCs culture fluid were successfully isolated, and the typical vesicle-like structures of exosomes were observed under transmission electron microscope. Exosomes expressed specific proteins such as CD9, CD63 and CD81. In the Exo group, the proliferation, invasion, migration, as well as the expressions of N-cadherin, Vimentin and miR-21-5p in PC-3 cells were significantly higher than those in the control group (all P<0.05). PHLPP2 is a target gene of miR-21-5p. Compared with the control group, the expression of PHLPP2 in PC-3 cells of Exo group and sh-PHLPP2 group was significantly reduced (0.66±0.09, 0.42±0.05 vs 1.09±0.08, all P<0.01); cell viability, invasion and migration were significantly improved (all P<0.01); and E-cadherin expression level was significantly reduced while N-cadherin and Vimentin expressions were significantly increased (both P<0.05). Conclusion: miR-21-5p is highly expressed in prostate cancer PC-3 cell line. BMSC exosome miR-21-5p can increase the proliferation, migration and invasion ability of PC-3 cells through targeted down-regulation of PHLPP2.

Molecular Characterization of A Protein Phosphatase 2C Gene of Subgroup A in Dendrobium officinale / 中国药学杂志

OBJECTIVE: To characterize a protein phosphatase (PP) encoding gene DoPP2C2 in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression profiling. METHODSE: RACE and RT-PCR were used to isolate the full length cDNA of DoPP2C2. The physiochemical properties, conserved domains and subcellular localization of the deduced DoPP2C2 protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 7.0 and MEGA 6.0 softwares, respectively. Quantitative PCR was used for gene expression analysis. RESULTS: The full length cDNA of DoPP2C2 (GenBank accession KT957553) was 1 624 bp in length, and encoded a 387-aa protein with a molecular weight of 42.76×103 and an isoelectric point of 7.03. The deduced DoPP2C2 protein sequence had two PP2C domains (91-152, 216-376), which are all conserved among the PP2Cs. The protein without a signal peptide or a transmembrane region, was predicted to locate in nucleus with hydropathicity. DoPP2C2 had high identities (47.8%-73.4%) with various PP2C proteins from several plants. DoPP2C2 protein belonged to the subgroup A of Arabidopsis and rice PP2C evolutionary tree, and was closely related to OsPP2C30; DoPP2C2 was differentially expressed in the three included organs. The transcripts were less in the roots and stems, being 0.76 and 0.59 folds, respectively, of that in the leaves. CONCLUSION: The molecular characteristics of a subgroup protein phosphatase encoding gene DoPP2C2 of the full length cDNA in D. officinale was obtained. The results will be useful for further functional determination of the gene involving in the growth and development of D. officinale.

Down-regulation of Protein Phosphatase 2A Catalytic Subunit Involved in Mitochondria Fission/fusion Dynamics Imbalance and Functional Impairment Induced by Human tau / 中国医学科学院学报

To explore whether the downregulation of protein phosphatase 2A catalytic subunit(PP2Ac)involved in the pathogenesis of mitochondria fission/fusion dynamics and functional imbalance induced by human tau accumulation. After cotransfection with mito-dsRed plasmids and pIRES-eGFP-tau40 plasmids 48 hours,the rat primary hippocampal neurons were observed with a laser scanning confocal microscope for their changes in shape and distribution of mitochondria.The expressions of mitochondria fission/fusion protein and PP2Ac and PP2Ab were detected by Western blotting.Furthermore,the shape and distribution of mitochondria of rat primary hippocampal neuron and wild type 293wt cells were assayed 48 hours after co-transfection with siPP2Ac-EGFP plasmids and mito-DsRed plasmids,and the fission/fusion dynamics of 293wt cells was captured with live cell time-lapse imaging after co-transfection with siPP2Ac plasmids and mito-Dendra2 plasmids.After transfection with siPP2Ac plasmids,the relative level of mitochondria fission/fusion protein of 293wt cells was assayed by Western blotting,and mitochondria membrane potential was detected by JC-1 staining,and the cellular viability was measured by CCK8 assay.Finally,the shape and distribution and membrane potential of mitochondria of HEK293 cells with stable transfection of htau40(293htau)were detected after co-transfection with PP2Ac and mito-dsRed plasmids. Human tau40 expression decreased distribution of mitochondria and significantly lowered PP2Ac level in primary hippocampal neuron(=4.814, =0.0086).Down-regulation of PP2Ac caused mitochondria elongation and perinuclear accumulation in primary hippocampal neuron and 293wt cells;in addition,down-regulation of PP2Ac in 293wt cells significantly increased mitochondria fusion rate(=2.857, =0.0074)and the levels of mitochondria fusion protein mitofusin(MFN)1(=6.768, =0.0025),MFN2(=3.121, =0.0035),and optic atrophy 1(=3.775, =0.0199);however,the levels of dynamin-like protein-1 and Fis1 remained unchanged.The down-regulation of PP2Ac in 293wt cells led to the significant decrease in mitochondria membrane potential(=2.300, =0.0270)and cell viability(=6.249, <0.0001).Finally,up-regulation of PP2Ac attenuated the abnormalities in the shape,distribution and function of mitochondria in the 293htau cells. Down-regulation of PP2Ac is involved in the abnormal shape and distribution of mitochondria and its dysfunction induced by human tau40 in rat primary hippocampal neurons and HEK293 cells.

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.

Compensatory role of wild-type p53-induced phosphatase in trophoblastic apoptosis in response to hypoxia / 中华围产医学杂志

Objective@#To investigate the mechanism of wild-type p53-induced phosphatase (Wip1) in regulating p53-dependent apoptosis of trophoblasts for further understanding the etiology of preeclampsia (PE).@*Methods@#Placenta tissues were collected from normal (n=15) and PE (n=13) gravidas who underwent caesarean section in the First Affiliated Hospital of Chongqing Medical University from June 2017 to December 2018. Chorionic villus and decidua tissues were collected from another 10 women who aborted in early pregnancy. Two in vitro trophoblastic hypoxia cultures were established by subjecting human chorionic trophoblast cells (HTR8/SVneo) to either hypoxia intervention in incubator (HII) or simulated ischemic buffer (SIB). Wip1 expressions at the transcriptional and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. The localization of Wip1 in placental tissues and HTR8/SVneo cells was determined by immunohistochemistry and immunofluorescence. Cell apoptosis was assessed by flow cytometry after viral infection and hypoxia. And the changes of pathway-related molecules including p53, phospho-p53 (p-p53), mouse double minute 2 homolog (Mdm2) and cleaved caspase3 (cl-cas3) were measured by Western blotting. The impact of Wip1 on Mdm2-p53 interaction was examined by co-immunoprecipitation. NVP-CGM097, an Mdm2-p53 specific inhibitor, was administered in PE cell models to verify the regulation of Wip1 on trophoblastic apoptosis through Mdm2-p53 pathway. Independent student's t-test, Welch's t-test and one-way analysis of variance were used as statistical methods.@*Results@#(1) Wip1 expression, which was mainly in trophoblast cells, was significantly elevated in human PE placentas (mRNA: 1.711±0.141 vs 0.860±0.126, t=4.496; protein: 0.449±0.027 vs 0.192±0.019, t=7.902) and in both in vitro trophoblastic PE models (protein in HII: 1.376±0.086 vs 0.977±0.114, t=2.792; SIB: 1.243±0.057 vs 0.381±0.045, t=11.910) compared with the corresponding control groups (all P<0.05). (2) Compared with corresponding control groups, overexpression of Wip1 suppressed the hypoxia-induced upregulation of p53 (HII: 0.185±0.024 vs 0.572±0.072; SIB: 0.400±0.067 vs 0.803±0.064), cl-cas3 (HII: 0.243±0.034 vs 0.529±0.072; SIB: 0.179±0.011 vs 0.368±0.025) and p-p53/p53 protein expression (HII: 1.326±0.129 vs 2.100±0.187; SIB: 0.473±0.028 vs 0.925±0.036) and also reduced the apoptosis rate [HII: (8.925±1.092)% vs (17.610±1.980)%; SIB: (13.910±1.886)% vs (24.650±1.622)%], which in turn promoted Mdm2-p53 binding (all P<0.05). However, knockdown of Wip1 gene expression in HTR8/SVneo cells brought about opposite effects (all P<0.05). (3) Neither overexpression nor knockdown of Wip1 influenced p53 or cl-cas3 expression when Mdm2-p53 interaction was blocked by NVP-CGM097.@*Conclusions@#Mdm2-p53 interaction promoted by Wip1 upregulation could compensate for the trophoblastic p53 accumulation in response to hypoxia, while exogenous upregulation of Wip1 in trophoblasts may reverse hypoxia-induced apoptosis. Therefore, this might provide a new therapeutic target for PE.

Compensatory role of wild-type p53-induced phosphatase in trophoblastic apoptosis in response to hypoxia / 中华围产医学杂志

Objective To investigate the mechanism of wild-type p53-induced phosphatase (Wip1) in regulating p53-dependent apoptosis of trophoblasts for further understanding the etiology of preeclampsia (PE). Methods Placenta tissues were collected from normal (n=15) and PE (n=13) gravidas who underwent caesarean section in the First Affiliated Hospital of Chongqing Medical University from June 2017 to December 2018. Chorionic villus and decidua tissues were collected from another 10 women who aborted in early pregnancy. Two in vitro trophoblastic hypoxia cultures were established by subjecting human chorionic trophoblast cells (HTR8/SVneo) to either hypoxia intervention in incubator (HII) or simulated ischemic buffer (SIB). Wip1 expressions at the transcriptional and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. The localization of Wip1 in placental tissues and HTR8/SVneo cells was determined by immunohistochemistry and immunofluorescence. Cell apoptosis was assessed by flow cytometry after viral infection and hypoxia. And the changes of pathway-related molecules including p53, phospho-p53 (p-p53), mouse double minute 2 homolog (Mdm2) and cleaved caspase3 (cl-cas3) were measured by Western blotting. The impact of Wip1 on Mdm2-p53 interaction was examined by co-immunoprecipitation. NVP-CGM097, an Mdm2-p53 specific inhibitor, was administered in PE cell models to verify the regulation of Wip1 on trophoblastic apoptosis through Mdm2-p53 pathway. Independent student's t-test, Welch's t-test and one-way analysis of variance were used as statistical methods. Results (1) Wip1 expression, which was mainly in trophoblast cells, was significantly elevated in human PE placentas (mRNA: 1.711±0.141 vs 0.860±0.126, t=4.496; protein: 0.449±0.027 vs 0.192±0.019, t=7.902) and in both in vitro trophoblastic PE models (protein in HII: 1.376±0.086 vs 0.977±0.114, t=2.792; SIB: 1.243±0.057 vs 0.381±0.045, t=11.910) compared with the corresponding control groups (all P<0.05). (2) Compared with corresponding control groups, overexpression of Wip1 suppressed the hypoxia-induced upregulation of p53 (HII: 0.185±0.024 vs 0.572±0.072; SIB: 0.400±0.067 vs 0.803±0.064), cl-cas3 (HII: 0.243±0.034 vs 0.529±0.072; SIB:0.179±0.011 vs 0.368±0.025) and p-p53/p53 protein expression (HII: 1.326±0.129 vs 2.100±0.187; SIB:0.473±0.028 vs 0.925±0.036) and also reduced the apoptosis rate [HII: (8.925±1.092)% vs (17.610±1.980)%;SIB: (13.910±1.886)% vs (24.650±1.622)%], which in turn promoted Mdm2-p53 binding (all P<0.05). However, knockdown of Wip1 gene expression in HTR8/SVneo cells brought about opposite effects (all P<0.05). (3) Neither overexpression nor knockdown of Wip1 influenced p53 or cl-cas3 expression when Mdm2-p53 interaction was blocked by NVP-CGM097. Conclusions Mdm2-p53 interaction promoted by Wip1 upregulation could compensate for the trophoblastic p53 accumulation in response to hypoxia, while exogenous upregulation of Wip1 in trophoblasts may reverse hypoxia-induced apoptosis. Therefore, this might provide a new therapeutic target for PE.

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.

Mechanism of action of protein phosphatase 2A in the promotion and inhibition of hepatocellular carcinoma / 临床肝胆病杂志

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase involved in many signaling pathways associated with cell growth and proliferation, and it also regulates many cellular processes. With an in-depth exploration of PP2A in the process of cell activity, especially malignant tumors, the association between PP2A and hepatocellular carcinoma (HCC) has attracted more and more attention in recent years; however, there is still a controversy over whether PP2A can promote or inhibit HCC. This article reviews the research advances in the mechanism of action of PP2A as a tumor factor in the regulation of HCC and target therapy.

Effect of receptor tyrosine phosphatase D on the characteristics of breast cancer stem cells / 临床与实验病理学杂志

Purpose To investigate the effect of protein tyrosine phosphatase receptor type D (PTPRD) on the characteristics of breast cancer stem cells. Method PTPRD expression in breast cancer cell line MDA-MB231 was down-regulated by small interference RNAs (siRNAs). Self-renewal ability of breast cancer stem cells (BCSCs) was detected by mammosphere formation assay. The holocolony forming ability was detected by colony formation assay. The proportion of CD44+/CD24- BCSCs was detected by flow cytometry. The ability of tumorigenesis of breast cancer cells in mice was sdudied with mouse tumorigenesis test. Separation of CD44+/CD24- stem cell population and non-stem cell population was isolated by immunomagnetic beads. Expression of PTPRD in stem cell and non-stem cell population was detected by Western blot and immunofluorescent. Results Down-regulation of PTPRD promoted the expression of stem cell markers ALDH1 and OCT-4. The expression of PTPRD in breast cancer stem cells was lower than than in non-stem cells (P<0.05). After PTPRD was down-regulated, the number of mammosphere (147±3.51) was significantly higher than that of the control group (106±12.5) (P<0.05), the proportion of holoclone [(35.9±3.4) ％] was significantly higher than that of the control group [(11.2±5.3) ％] (P<0.05), the proportion of CD44+/CD24- cells[(2.88±1.2) ％]was significantly higher than that of the control group [(0.6±0.4) ％], the in vivo tumorigenicity was significantly enhanced in nude mice (P<0.05). Conclusion The expression of PTPRD is lower in BCSCs. PTPRD may inhibit the self-renewal ability of breast cancer stem cells.

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.

Functional identification of protein phosphatase 1-binding consensus residues in NBCe1-B

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, ⁹²²FMDRLK⁹²⁷ , in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922–927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO₃⁻ transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO₃⁻ secretion in several types of epithelia.