Effects of electroacupuncture on the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of rats with vascular dementia

This study investigated the mechanism underlying electroacupuncture therapy for vascular dementia through electroacupuncture at the acupoints of Baihui （DU20）, Dazhui （DU14）, and bilateral Shenshu （BL23） in a rat model of vascular dementia produced by bilateral middle cerebral artery occlusion. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in vascular dementia rats was significantly increased after electroacupuncture, compared with the model group that was not treated with acupuncture. The average escape latency was also shortened after electroacupuncture, and escape strategies in the spatial probe test improved from edge and random searches, to linear and trending swim pathways. The experimental findings indicate that electroacupuncture improves learning and memory ability by up-regulating expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of vascular dementia rats.

AMPK-associated signaling to bridge the gap between fuel metabolism and hepatocyte viability

The adenosine monophosphate-activated protein kinase (AMPK) and p70 ribosomal S6 kinase-1 pathway may serve as a key signaling flow that regulates energy metabolism; thus, this pathway becomes an attractive target for the treatment of liver diseases that result from metabolic derangements. In addition, AMPK emerges as a kinase that controls the redox-state and mitochondrial function, whose activity may be modulated by antioxidants. A close link exists between fuel metabolism and mitochondrial biogenesis. The relationship between fuel metabolism and cell survival strongly implies the existence of a shared signaling network, by which hepatocytes respond to challenges of external stimuli. The AMPK pathway may belong to this network. A series of drugs and therapeutic candidates enable hepatocytes to protect mitochondria from radical stress and increase cell viability, which may be associated with the activation of AMPK, liver kinase B1, and other molecules or components. Consequently, the components downstream of AMPK may contribute to stabilizing mitochondrial membrane potential for hepatocyte survival. In this review, we discuss the role of the AMPK pathway in hepatic energy metabolism and hepatocyte viability. This information may help identify ways to prevent and/or treat hepatic diseases caused by the metabolic syndrome. Moreover, clinical drugs and experimental therapeutic candidates that directly or indirectly modulate the AMPK pathway in distinct manners are discussed here with particular emphasis on their effects on fuel metabolism and mitochondrial function.

P70S6 Kinase Phosphorylation： A New Site to Assess Pharmacodynamy of Sirolimus

Background：The phosphorylation ofp70S6 kinase （p70S6K） represents an important target for sensitive detection on pharmacodynamic effects of sirolimus,but the methods of assessing p70S6K phosphorylation are still unclear.The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target ofrapamycin （mTOR） pathway in peripheral blood mononuclear cells （PBMCs） of liver transplant patients through different methods.Methods：Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study.Patients were divided into three groups,patient treated with sirolimus （n =22）,patient treated with tacrolimus （n =30）,patient treated with cyclosporine （n =23）.The p70S6K phosphorylation of PBMCs in patients and healthy control （HC,n =12） were analyzed by phospho-flow cytometry and Western blotting.A correlation analysis of data from phospho-flow cytometry and Western blotting was performed.Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.Results：Intra-assay variability ofp70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%.The p70S6K phosphorylation in patients receiving a sirolimus （19.5 ± 7.7） was significantly lower than in HC （50.1 ± 11.3,P 〈 0.001）,tacrolimus （37.7 ± 15.7,P 〈 0.001） or cyclosporine treated patients （41.7 ± 11.7,P 〈 0.001）.The p70S6K phosphorylation in HC （50.1± 11.3） was significantly higher than in tacrolimus （37.7 ± 15.7,P 〈 0.01） or cyclosporine-treated patients （41.7 ± 11.7,P 〈 0.01）.There was correlation between data from phospho-flow cytometry and data from Westem blotting （r =0.88,P 〈 0.001）.Conclusions：The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Westem blotting.Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

Hepatitis C virus infection and insulin resistance

Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)and is the primary cause for liver transplantation in the western world.Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of typeⅡdiabetes.Insulin resistance plays an important role in the development of various complications associated with HCV infection.Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis,steatosis,HCC and resistance to anti-viral treatment.Thus,HCV associated insulin resistance is a therapeutic target at any stage of HCV infection.HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway.Various mechanisms have been proposed in regard to HCV mediated insulin resistance,involving up regulation of inflammatory cytokines,like tumor necrosis factor-α,phosphorylation of insulin-receptor substrate-1,Akt,up-regulation of gluconeogenic genes like glucose 6 phosphatase,phosphoenolpyruvate carboxykinase 2,and accumulation of lipid droplets.In this review,we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.

Effect of Metformin-Induced Stimulation on the Expression of Insulin Receptor Substrate 1 through Negative Regulation of P70S6k

Objective:The aim is to study the effects of metformin on the expression of 70 kDa ribosomal protein S6 kinase(P70S6k),insulin receptor substrate 1(IRS-1),and IRS-1Ser307 phosphorylation in human luteinized granulosa cells.Methods:Granulosa cells in the experimental group were cultured in M199 medium containing 0.1 mmol/L metformin for 24 h and those in control group were cultured in M199 medium.The expression levels of P70S6k and IRS-1 mRNA were detected by reverse-transcriptiom polymerase chain reaction(RT-PCR)and real-time PCR.P70S6k,IRS-1,p-ser307-IRS-1,and p-thr389-P70S6k protein expression levels were detected by immunofluorescence and western blotting.Results:P70S6k mRNA level was higher and IRS-1 was significantly lower in the experimental group than those in the control group.IRS-1 and p-ser307-IRS-1 were expressed in cell plasma,and P70S6k and p-thr389-P70S6k were expressed in cell nucleus.The results of Western blot analysis indicated that the expression levels of P70S6k,p-thr389-P70S6k,IRS-1,and p-ser307-IRS-1 proteins had significant difference between the experimental group and the control group.Compared to the control group,the relative intensity illustrated that the expression levels of P70S6K and p-thr389-P70S6k significantly increased in the experimental group;however,those of IRS-1 and p-ser307-IRS-1 proteins significantly decreased.Conclusion:Metformin can inhibit the P70S6k mRNA and protein expression levels in the granulosa cells and improve insulin sensitivity by regulating IRS-1 expression through Akt/P70S6k/IRS-1-dependent pathway.

Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator

The mammalian target of rapamycin （mTOR） pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I （10 pg）, induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase （p70 S6K） and eukaryotic initiation factor 4E-binding protein 1 （4E-BP1）, in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4E- BP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses （spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity）. Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain.

Angiotensin IV upregulates the activity of protein phosphatase 1α in Neura-2A cells

The peptide angiotensin IV(Ang IV)is a derivative of angiotensin II.While insulin regulated amino peptidase(IRAP)has been proposed as a potential receptor for Ang IV,the signalling pathways of Ang IV through IRAP remain elusive.We applied high-resolution mass spectrometry to perform a systemic quantitative phosphoproteome of Neura-2A(N2A)cells treated with and without Ang IV us-ing sta ble-isotope labeling by amino acids in cell culture(SILAC),and identifi ed a reduction in the phosphorylation of a major Ser/Thr protein phosphorylase 1(PP1)upon Ang IV treatment.In addition,spinophilin(spn),a PP1 reg-ulatory protein that plays important functions in the neural system,was expressed at higher levels.Immunoblotting revealed decreased phosphorylation of p70S6 kinase(p70S6K)and the major cell cycle modulator retinoblas-toma protein(pRB).These changes are consistent with an observed decrease in cell proliferation.Taken together,our study suggests that Ang IV functions via regulating the activity of PP1.

原发性开角型青光眼房水差异表达蛋白分析

目的分析原发性开角型青光眼(POAG)患者房水的蛋白质表达变化,探寻与疾病发生相关的生物学标志及潜在治疗靶点.方法采用回顾性病例-对照研究设计.纳入2016年10月至2017年12月由天津医科大学眼科医院收治的行白内障手术的年龄相关性白内障患者10例10眼作为年龄相关性白内障组,行青光眼联合白内障手术的POAG合并白内障患者10例10眼作为POAG合并白内障组.术中借助手术通道用1号针头接1ml针筒进入前房中部吸取约100μl房水.通过非标记定量蛋白质组学质谱分析技术分析房水中提取的蛋白,采用Maxquant significancesA的方法进行差异显著性检验,以P<0.05、差异倍数>2的标准筛选得到2个组患者的差异蛋白,并将生物大数据通过GO功能、KEGG显著性富集分析对差异蛋白的功能及调控的信号通路加以注解.结果本次蛋白组学分析共检测到97个差异蛋白,其中包括48个上调蛋白和49个下调蛋白.GO分析显著差异蛋白功能涉及诸多方面,其中与炎症反应有关的有脂多糖结合蛋白(LBP)、CD163、C-反应蛋白(CRP)和膜联蛋白A1(ANXA1);与氧化还原有关的蛋白有谷胱甘肽S转移酶P(GSTP1)和硫氧还原蛋白(TXN);与细胞黏附运动有关:软骨寡聚基质蛋白(COMP)、桥粒胶蛋白(DSC2)和层连蛋白(LAMB2);与纤维化有关的有原胶原蛋白(PLOD1)和固生蛋白(TNC);与神经生长有关:颤蛋白(RELN)、脑信号蛋白(SEMA3F)和轴突导向因子(SEMA4B);与代谢相关的蛋白有丙酮酸激酶(PKM)和羧肽酶N亚型2(CPN2)等.KEGG分析表明NrF2/ERK信号通路、TGF-β/NF-κB信号通路表达在2个组细胞间存在差异.结论POAG患者房水中GSTP1、TXN表达显著降低,可能通过调控NrF2/ERK1/2及TGF-β/NF-κB信号通路,调节细胞黏附性和活性以及细胞外基质表达来参与疾病的发展.GSTP1、TXN可能成为潜在生物学标志及治疗靶点.

桂皮醛对糖尿病小鼠血糖水平的影响及机制

目的：观察桂皮醛（cinnamaldehyde,CA）降血糖作用,并探讨其作用机制。方法：6~8周龄雄性db/db小鼠24只,按血糖随机分为模型组,二甲双胍组（0.2 g·kg^-1）,桂皮醛低剂量组（0.025 g·kg^-1）和桂皮醛高剂量组（0.05 g·kg^-1）,每组6只,另设同周龄C57BL/6J小鼠6只为正常组。治疗4周后,检测各组小鼠空腹血糖（fasting blood glucose,FBG）,总胆固醇（total cholesterd,TC）,甘油三酯（total triglyceride,TG）,游离脂肪酸（free fatty acids,FFA）,天门冬氨酸氨基转移酶（aspartate aminotransferase,AST）,血清胰岛素（fasting insulin,Fins）水平,计算胰岛素抵抗指数（homa insulin-resistance,HOMA-IR）;取肝脏组织检测肝糖原含量以及高碘酸-希夫（periodic acid-Schiff stain,PAS）染色;采用实时荧光定量聚合酶链式反应（Real-time PCR）,蛋白免疫印迹法（Western blot）检测肝脏相关mRNA和蛋白表达。结果：治疗4周后,与模型组比较,CA组小鼠体质量,FBG,Fins,HOMA-IR,血脂明显降低（P〈0.05,P〈0.01）,肝脏糖原含量显著升高;小鼠肝脏葡萄糖-6-磷酸酶（glucose-6-phosphate,G-6-P）,磷酸烯醇式丙酮酸羧激酶（phosphoenolpyruvate carboxykinase,PEPCK）mRNA表达显著降低（P〈0.01）,p-蛋白激酶B（protein kinase B,Akt）,p-糖原合成酶激酶-3β（GSK-3β）蛋白表达显著升高（P〈0.01）。结论：CA具有降低血糖作用,相关机制可能是通过上调肝脏胰岛素信号通路Akt,GSK-3β磷酸化水平,抑制G-6-P,PEPCK mRNA表达实现。