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.

Involvement of 90-kuD ribosomal S6 kinase in collagen type Ⅰ expression in rat hepatic fibrosis

AIM： To investigate the relationship between 90-kuD ribosomal $6 kinase （pg0RSK） and collagen type I expression during the development of hepatic fibrosis in vivo and in vitro.METHODS： Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type I were determined by co-immunofluoresence and confocal microscopy.Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell （HSC） line. The expression of collagen type I in HSC-T6 cells was assessed by Western blotting and real-time polymerase chainreaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, thencollagen type I promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated withor without platelet-derived growth factor （PDGF）-BB at a final concentration of 20μg/L and the cell growthwas determined by MTS conversion.RESULTS： In fibrotic liver tissues, p90RSK was over-expressed in activated HSCs and had a significantpositive correlation with collagen type I levels.In HSC-T6 cells transfected with RNAi targeted top90RSK, the expression of collagen type I was down-regulated （61.8% in mRNA, P 〈 0.01, 89.1% inprotein, P 〈 0.01）. However, collagen type ] promoteractivity was not increased with over-expression of p90RSK and not decreased with low expression either,compared with controls in the same cell line （P = 0.076）.Furthermore, p90RSK siRNA exerted the inhibitionof HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation.CONCLUSION： p90RSK is over-expressed in activatedHSCs and involved in regulating the abnormalexpression of collagen type I through initiating theproliferation of HSCs.

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.

LRP6 Bidirectionally Regulates Insulin Sensitivity through Insulin Receptor and S6K Signaling in Rats with CG-IUGR

Objective Intrauterine growth restriction followed by postnatal catch-up growth(CG-IUGR)increases the risk of insulin resistance-related diseases.Low-density lipoprotein receptor-related protein 6(LRP6)plays a substantial role in glucose metabolism.However,whether LRP6 is involved in the insulin resistance of CG-IUGR is unclear.This study aimed to explore the role of LRP6 in insulin signaling in response to CG-IUGR.Methods The CG-IUGR rat model was established via a maternal gestational nutritional restriction followed by postnatal litter size reduction.The mRNA and protein expression of the components in the insulin pathway,LRP6/β-catenin and mammalian target of rapamycin(mTOR)/S6 kinase(S6K)signaling,was determined.Liver tissues were immunostained for the expression of LRP6 andβ-catenin.LRP6 was overexpressed or silenced in primary hepatocytes to explore its role in insulin signaling.Results Compared with the control rats,CG-IUGR rats showed higher homeostasis model assessment for insulin resistance(HOMA-IR)index and fasting insulin level,decreased insulin signaling,reduced mTOR/S6K/insulin receptor substrate-1(IRS-1)serine307 activity,and decreased LRP6/β-catenin in the liver tissue.The knockdown of LRP6 in hepatocytes from appropriate-for-gestational-age(AGA)rats led to reductions in insulin receptor(IR)signaling and mTOR/S6K/IRS-1 serine307 activity.In contrast,LRP6 overexpression in hepatocytes of CG-IUGR rats resulted in elevated IR signaling and mTOR/S6K/IRS-1 serine307 activity.Conclusion LRP6 regulated the insulin signaling in the CG-IUGR rats via two distinct pathways,IR and mTOR-S6K signaling.LRP6 may be a potential therapeutic target for insulin resistance in CG-IUGR individuals.

Effect of moxibustion on mTOR-mediated autophagy in rotenone-induced Parkinson's disease model rats

Defects in autophagy-mediated clearance of α-synuclein may be one of the key factors leading to progressive loss of dopaminergic neurons in the substantia nigra. Moxibustion therapy for Parkinson’s disease has been shown to have a positive effect, but the underlying mechanism remains unknown. Based on this, we explored whether moxibustion could protect dopaminergic neurons by promoting autophagy mediated by mammalian target of rapamycin （mTOR）, with subsequent elimination of α-syn. A Parkinson’s disease model was induced in rats by subcutaneous injection of rotenone at the back of their necks, and they received moxibustion at Zusanli （ST36）, Guanyuan （CV4）and Fengfu （GV16）, for 10 minutes at every point, once per day, for 14 consecutive days. Model rats without any treatment were used as a sham control. Compared with the Parkinson’s disease group, the moxibustion group showed significantly greater tyrosine hydroxylase immunoreactivity and expression of light chain 3-II protein in the substantia nigra, and their behavioral score, α-synuclein immunoreactivity,the expression of phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase （p-p70S6K） in the substantia nigra were significantly lower. These results suggest that moxibustion can promote the autophagic clearance of α-syn and improve behavioral performance in Parkinson’s disease model rats. The protective mechanism may be associated with suppression of the mTOR/p70S6K pathway.

Inhibitory effects of rapamycin on the different stages of hepatic fibrosis

AIM: To investigate and compare the inhibitory effects of rapamycin in the different stages of liver fibrosis.

p85S6K sustains synaptic GluA1 to ameliorate cognitive deficits in Alzheimer’s disease

Background Ribosomal protein S6 kinase 1(S6K1)is a serine-threonine kinase that has two main isoforms:p70S6K(70-kDa isoform)and p85S6K(85-kDa isoform).p70S6K,with its upstream mammalian target of rapamycin(mTOR),has been shown to be involved in learning and memory and participate in the pathophysiology of Alzheimer’s dis-ease(AD).However,the function of p85S6K has long been neglected due to its high similarity to p70S6k.The role of p85S6K in learning and memory is still largely unknown.Methods We fractionated the postsynaptic densities to illustrate the differential distribution of p85S6K and p70S6K.Coimmunoprecipitation was performed to unveil interactions between p85S6K and the GluA1 subunit of AMPA receptor.The roles of p85S6K in synaptic targeting of GluA1 and learning and memory were evaluated by specific knockdown or overexpression of p85S6K followed by a broad range of methodologies including immunofluorescence,Western blot,in situ proximity ligation assay,morphological staining and behavioral examination.Further,the expression level of p85S6K was measured in brains from AD patients and AD model mice.Results p85S6K,but not p70S6K,was enriched in the postsynaptic densities.Moreover,knockdown of p85S6K resulted in defective spatial and recognition memory.In addition,p85S6K could interact with the GluA1 subunit of AMPA receptor through synapse-associated protein 97 and A-kinase anchoring protein 79/150.Mechanistic studies demonstrated that p85S6K could directly phosphorylate GluA1 at Ser845 and increase the amount of GluA1 in syn-apses,thus sustaining synaptic function and spine densities.Moreover,p85S6K was found to be specifically decreased in the synaptosomal compartment in the brains of AD patients and AD mice.Overexpression of p85S6K ameliorated the synaptic deficits and cognitive impairment in transgenic AD model mice.Conclusions These results strongly imply a significant role for p85S6K in maintaining synaptic and cognitive function by interacting with GluA1.The findings provide an insight into the rational targeting of p85S6K as a therapeutic potential for AD.

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.

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.

Oral everolimus inhibits intimal proliferation in injured carotid artery in rats

Background Everolimus, a derivative of sirolimus, is a potent immunosuppressant that has important anti-proliferative properties. In the present study, we demonstrated the inhibiting neointimal hyperplasia in injured carotid arteries in rats by using two different doses of everolimus administrated via the oral route for a long time. Methods A rat model of carotid artery injury was established by balloon inflation. Eighty rats were randomly divided into the sham-operated group （n=20）, injury group （n=20）, low dosage of everolimus group （n=20）, and high dosage of everolimus group （n=20）. The low close of everolimus （1.5 mg/kg） was given one day before injuring the carotid artery by balloon, followed by 0.75 mg/kg per day for 28 days via intragastric gavage. High dose everolimus （2.5 mg/kg） was given one day before injuring the carotid artery by balloon, followed by 1 mg/kg per day for 28 days. Expression of eukaryotic translation initiation factor 4E （elF-4E） and phosphorylation of ribosomal proteinS6 kinase 1 （P70S6K） were determined by reverse transcription-polymerase chain reaction and Western blotting analysis. Results In the injured carotid artery, neointimal hyperplasia was normally observed four weeks after injury. Everolimus inhibited neointimal hyperplasia after balloon injury in a dose dependent manner. At the same time, the study demonstrated that everolimus reduced the expression of P-P70S6K, elF-4E, transforming growth factor （TGF）-131 and of proliferating cell nuclear antigen （PCNA）. Conclusions Everolimus significantly inhibited neointimal hyperplasia of the injured carotid artery. The effect depended on dosaqe and was associated with the reduction of phosphorylation of P70S6K and the elF-4E expression level.

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.