Effects Of TRPM7/PI3K/Akt/mTOR Signaling Pathway On Methamphetamine-induced Hippocampal Neuron Apoptosis

Objectives: To clarify methamphetamine(MA)can induce apoptosis of hippocampal neurons and promote the expression of transient receptor potential M7channel(TRPM7)in nerve cells.To investigate whether TRPM7 regulates phosphoinositol 3-kinase/protein kinase B/ Rapamycin(PI3K/Akt/m TOR)signaling pathway and mediates neuronal apoptosis.New targets of MA abuse prevention and intervention were selected to provide scientific basis for drug abuse disorders and drug withdrawal intervention.Methods:Part1.In vivo experiments: A mouse model of MA subacute poisoning was established,and the expressions of apoptosis-related proteins and TRPM7 were detected1.1A mouse model of MA subacute poisoning was established: 20 male C57BL/6J mice(18-20g)were randomly assigned to control group and MA group;MA group was intraperitoneally injected(2 mg/kg)once at an interval of 12 h for 5consecutive days.The control group was intraperitoneally injected with normal saline of equal volume.Mouse brain tissue was collected by perfusion and sampling.1.2Histopathological observation: Paraffin sections of the whole brain of mice were prepared and stained with hematoxylin-eosin(HE)to observe the morphological changes of the hippocampus of mice.1.3 TRPM7 expression detection: Whole brain paraffin sections of mice were prepared,and TRPM7 expression was detected by immunohistochemistry.1.4 Apoptosis-related protein expression detection: The hippocampal tissues of mice were extracted,and the expressions of apoptosis-related proteins Bax and Bc1-2were detected by Western Blot.Part2.In vitro experiment: A mouse hippocampal neuron cell line model of MA administration was established,and the expressions of apoptosis-related proteins and TRPM7 were detected2.1 Mouse hippocampal neuron cell lines(HT-22 cells)were cultured in vitro and administered with MA to construct an in vitro model of hippocampal neurotoxicity induced by MA.(1)HT-22 cells were treated with gradient concentration of MA for 24 h,and the optimal concentration of MA induced hippocampal neuron injury was screened by CCK-8 kit.After applying the HT-22 cell gradient time with MA(4m M),CCK-8screened the optimal time point for MA induced hippocampal neuron damage in mice.(2)After the HT-22 cells were treated with MA(the optimal concentration and the optimal time),the neuronal marker MAP2 was labeled by immunofluorescence staining,and the morphological changes of the neurons were observed.(3)After the HT-22 cells were treated with MA(optimal concentration and time),the apoptosis level was detected using the in situ end labeling(TUNEL)kit.(4)The expression levels of TRPM7,PI3 K,Akt,m TOR,Caspase-3,Bax and Bc1-2 proteins were detected by WB after the HT-22 cells were treated with MA(optimal concentration and time).2.2 After lentivirus transfection silenced TRPM7 gene in HT-22 cells,MA was administered to HT-22 cells to investigate the role of TRPM7 in MA induced hippocampal neurotoxicity.(1)Establishment of stable cell lines: After purinomycin was treated with gradient concentration,CCK-8 detected cell activity,and the killing curve of purinomycin against HT-22 cells was constructed.After infecting HT-22 cells with lentivirus containing sh RNA(targeting TRPM7),stable transfected cell lines were screened with purinomycin.The silencing effect of TRPM7 was detected by WB method.(2)The apoptosis level of HT-22 cells was detected by TUNEL after the treatment of MA(optimal concentration and time).(3)The expression levels of TRPM7,PI3 K,Akt,m TOR,Caspase-3,Bax and Bc1-2 were detected by WB after the HT-22 cells were treated with MA(optimal concentration and time).Results:Part1.In vivo experiment results1.1 HE staining results indicated that hippocampal edema,nucleolysis and cytoplasmic hyperstaining of mice in MA group were more obvious than those in control group.1.2 Immunohistochemical staining results showed that compared with the control group,TRPM7 expression was increased in the hippocampus of mice in the MA group.1.3 WB results showed that compared with the control group,Bax expression was increased and Bcl-2 expression was decreased in the hippocampus of mice in the MA group.Part2.Results of in vitro experiment2.1 MA induced morphological changes and apoptosis of HT-22 cells,and protein expression changes of TRPM7,PI3 K,Akt,m TOR,Caspase-3,Bax and Bc1-2.(1)The results of CCK-8 showed that the cell activity decreased gradually with the increase of MA concentration and the extension of the treatment time.The optimal concentration of MA induced HT-22 cell damage was 4m M and the optimal treatment time was 24 h.(2)Immunofluorescence results showed that,compared with the control group,the number of cells in the MA group was reduced,synapses were shorter,and cells were swollen.(3)Compared with the control group,TUNEL test results showed that the apoptosis of MA group was significantly increased.(4)WB results showed that compared with the control group,the expressions of TRPM7,m TOR,Caspase-3 and Bax in MA group were increased,while the expressions of PI3 K,Akt and Bc1-2 were decreased.2.2 Silencing TRPM7 alleviates Ma-induced apoptosis of HT-22 cells and the expression changes of TRPM7,PI3 K,m TOR,Bax and Bc1-2 proteins.(1)The expression of TRPM7 could be effectively silenced by killing curve construction,lentivirus transfection and purinomycin screening.(2)WB results showed that the silencing of TRPM7 could reduce the upregulation of m TOR and Bax induced by MA,and the downregulation of PI3 K and Bc1-2 induced by MA.Conclusion:1.Methamphetamine induced apoptosis of hippocampal neuron HT-22 cells.2.TRPM7 can reduce methamphetamine-induced hippocampal neuronal apoptosis by regulating PI3K/m TOR/ Akt signaling pathway.