Mechanisms Of Platelet-Derived Growth Factor-BB In Restoring HIV Tat-Cocaine-Mediated Impairment Of Neuronal Differentiation

BackgroundAIDS acquired immunodeficiency syndrome(immunodeficiency syndrome acquired,AIDS).This is a chronic fatal infectious disease caused by the human immunodeficiency virus(immunodeficiency virus human,HIV)?China found the first case of AIDS infection in 1985.TO the end of OCT,2015,the national report on the survival of HIV infected persons and patients with a total of cases 575,000,the death was about 177,000.the average monthly increase new report about 3800 cases of HIV.AIDS has become a serious social and public health problem in our country.HIV infection in patients with early stage will have a lesser degree of cognitive impairment,progressive development of dementia,that is,HIV encephalopathy.The incidence of HIV encephalopathy in patients with AIDS was 7%-14%,and 1/3 was AIDS in the patients with dementia when death.Patients of HIV infected are most drugs abuse,and drug abuse will lead to multiple organ damage,especially in the brain dysfunction?Provided by the Chinese Ministry of public security of the 2015 report on drug control showed that the number of country registered drug addicts is about 295 million,and Cocker was one of most widely used one of the drug,cocaine has various toxicological and pharmacological properties,long-term abuse can cause neural biology,pathology and behavioral change.In recent years,studies have shown that cocaine is highly toxic to the central nervous system,induce damage change of the neural structure and function,it can stimulate the cerebral cortex,vision,hearing,touch and other hallucinations,self injurious behavior.Therefore,HIV infected patients taking cocaine will increase the occurrence and development of HIV encephalopathy.However,the present study still can not explain the specific mechanism of neurotoxicity of these patients,and the lack of effective treatment and intervention measures for their toxic effects.This study to HIV and cocaine induced neural stem cell differentiation,in order to investigation the mechanism of neurotoxicity and provides new theoretical basis.ObjectiveTo explore the mechanism of HIV-tat and cocaine on neural stem cell differentiation effect and PDGF-BB reversed the original differentiation trend.Methods Selected pregnant 18 day fetal rat,isolated neural stem cells from the fetal rat hippocampus,neural stem cells(NSCs).Then cultured w and passaged in no serum.The isolated neural stem cells were identified by nestin by immunofluorescence.Then the purified neural stem cell spheres suspended,and cultured.The neural stem cells were treated with PDGF-BB(100ng/ml)alone,and HIV-Tat(100ng/ml),cocaine(10 M)and PDGF-BB CO treatment,study in three time point 1 days,3 days,5 days with the method of MTT,Western blot,PCR,immunofluorescence detection,to detect the neuronal viability and neuron and astrocyte's cell marker(beta-?-tubulin and GFAP),TRPC pathway and protein expression of ERK,AKT,CREB,then used specific inhibitors of skf96365,U0126,LY294002,H89 to inhibite TRPC?ERK?AKT?CREB,and Fluo4-AM probe detected the calcium ion of intracellular,observed the effects of HIV-tat and cocaine on the differentiation of neural stem cells,and the repairing effect of PDGF-BB in the differentiation of neural stem cells.Results1 PDGF-BB on neural stem cell differentiationAfter neural stem cells were treated with PDGF-BB treatment(100ng/ml),respectively in different time points 1 day,3 days,5 days for detection of cell viability,neuron sign and glial cell markers found neuronal markers increased gradually and glial cell marker content gradually decreased,but also the vitality of cells without obvious change.2 the effects of HIV-tat and cocaine on the differentiation of neural stem cellsAfter neural stem cells were treated with TAT(100ng/ml)and cocaine(10 m),respectively in different time points of 1 day,3 days,5 days to detect neuronal sign and glial cell markers found neuronal markers decreased gradually and glial cell markers increased gradually.3.Effect of PDGF-BB on the neural stem differentiation induced by HIV-tat and cocaineAfter neural stem cells were treated with TAT(100ng/ml),cocaine(10 m)and PDGF-BB treatment(100ng/ml),respectively in different time points of 1 day,3 days,5 days to detect neuronal sign and glial cell markers found PDGF-BB reversed the original differentiation trend,found neuronal markers increased gradually and glial cell markers decreased gradually.4.TRPC signaling pathway is involved in the differentiation of neural stem cells induced by PDGF-BBPDGF-BB on TRPC signaling pathway ERK/CREB was activation.TRPC,ERK,CREB specific inhibitor skf96365,U0126,H89 down regulated PDGF-BB induced neural stem cell differentiation.5.AKT signaling pathway is involved in the differentiation of neural stem cells induced by PDGF-BBPDGF-BB on AKT signaling pathway has different levels of activation.TRPC specific inhibitor skf96365 and siRNA silencing TRPC method could not down regulate the expression of AKT signaling pathway,indicating that AKT pathway and TRPC pathway are two independent pathways involved in PDGF-BB induced neural stem cell differentiation.Conclusions1.HIV-tat and cocaine induced neural stem cells to differentiate into glial cells,which led to the disorder of neural function.2.PDGF-BB can induce the differentiation of neural stem cells into neurons,which can repair the function of neural stem cells.3.PDGF-BB could reverse the neural stem differentiation induced by HIV-tat and cocaine,and repair the damage of HIV-tat and cocaine to nerve function.4.TRPC signaling pathway and AKT signaling pathway are involved in the differentiation of neural stem cells induced by PDGF-BB.