The Mechanism Of HIV-Tat Protein And Methamphetamine Synergistically Induced Autophagy In Dopaminergic Neurons Of Tree Shrew And The Intervention Of GsRb1

Despite the clinical implementation of antiretroviral therapy(ART)has significantly prolonged the lifetime of the human immunodeficiency virus(HIV)infected population,HIV-associated neurocognitive disorders(HANDs)remain a critical concern in a considerable number of HIV-positive individuals.According to the world health organization report,about a third of people with HIV develop HIV dementia.The neurotoxic activities of numerous HIV-Tat protein is also implicated in the neuropathogenic process.HIV-Tat(the transactivator of transcription)is a viral protein,leading to alterations in inflammatory molecule production by interacting with cellular gene promoters and cellular receptors to induce apoptosis,autophagy,and cell death,which may be one of the underlying mechanisms of HANDs.It is widely known that drug abuse such as methamphetamine(METH),can greatly increase risk of HIV-associated neurocognitive disorders and AIDS dementia complex,autophagy plays a key role in these pathogeneses.Autophagy,which degrades and recycles macromolecules and organelles in lysosomal and mitochondria,plays an essential role in normal cellular functions.However,extensive autophagy might be involved in the type II programmed cell death or autophagic cell death,which has been linked to non-apoptotic cell death.Our findings and recent studies indicate that HIV-1 Tat protein and methamphetamine can synergistically lead to autophagy of neurons,which causes degeneration,necrosis and apoptosis of cells,but the mechanism has not elucidated.Tree shrew is a promising animal model for several human diseases in Southeast Asia because it is closely related to humans phylogenetically.They are new varieties of experimental animals.Therefore,our study choose tree shrew as animal model to solve the following scientific questions:1.Whether HIV-Tat and METH can synergistic induce autophay in tree shrews;Whether ATG5,ATG7 and p-mTOR proteins are involved in HIV-Tat and METH-induced autophagy;2.Pretreated with Ginsenoside Rbl,we observe whether Rb1 reduce HIV-Tat and METH-induced autophagy.The study was carry out in vivos and vitros.Firstly,the primary dopaminergic neuronal cells were cultured and identificated via TH and Nestin protein of immunofluorescence test for tree shrews,then METH and HIV-Tat treated.Autophagy related proteins Beclin-1 and LC3B were detected by WB to determine the appropriate time and dose of METH and HIV-Tat.METH and HIV-Tat could co-induced autophagy to be verified by TEM and IF.Next,ATG5 and ATG7 protein expression levels were increased which induced by METH and HIV-Tat via WB medold.After ATG5 and ATG7 gene were silencd,the expression of Beclin-1 and LC3B were declined,which suggest that ATG5 and ATG7 could involve in METH and HIV-Tat-induced autophagy.Pretreated with rapamycin,the expression of Beclin-1 and LC3B were increased and p-mTOR was decreased,whereas pretreated with 3-MA,the expression of Beclin-1 and LC3B were declined and p-mTOR were incresed.ROS and GPX protein expression were evevated by Flow cytometry and WB.ROS were increased and GPX protein were declined by METH and HIV-Tat induced,suggesting that METH and HIV-Tat could induce high oxidative stress reaction.Nrf2 pathway was involved in METH and HIV-Tat-induced oxidative stress by detecting the expression of Nrf2 protein via WB and IF.Last,pretreated with GsRb1,it down-regulated the expression of beclin-1 and LC3B,suggesting that GsRb1 could alleviate METH and HIV-Tat-induced autophagy through anti-oxidative stress response.In vitros,different concentrations of METH and HIV-Tat were injected into adult male tree shrews and determined the appropriated concentration by beclin-1 and LC3B expression,and determined that METH could strengthen HIV-Tat-induced autophagy by Bliss independence model to be calculated.HIV-Tat and METH produced oxidative stress in tree shrews’midbrain through increasing the expression of MDA and decreasing the expression of GSH.GsRbl was given in advance,and the expression of beclin-1 and LC3B was decreased in the brain of the tree shrews,while MDA decreased and GSH increased,which suggest that GsRbl could alleviate METH and HIV-Tat-induced autophagy through anti-oxidative stress response in vitro.In conclusion,our study was suggested:1.METH and HIV-Tat induced autophagy in tree shrews’ dopaminergic neurons,and they had synergistic effect.2.ATG5,ATG7 and p-mTOR were involved in the METH and HIV-Tat-induced autophagy of the dopamine neurons in tree shrews.3.GsRb1 alleviate METH and HIV-Tat-induced autophagy through anti-oxidative stress response.This study will be critical for the assessment of therapeutic modalities for patients with HIV with drug abuse.