Studies On The Molrcular Mechanism Of Mitophagy Dysfunction In Regulatory T Cell In MG Patients

Objective:To explore the molecular mechanism of abnormal mitochondrial autophagy in regulatory T cells of MG patients,and to provide theoretical basis for exploring new MG-specific treatment approaches.Methods:(1)Selection and treatment of research subjects: Fifteen confirmed MG patients were selected,30 m L of peripheral blood was extracted and Treg cells were sorted out.Treg cells from MG patients were divided into Contol group without any treatment,CCCP treatment group,Cs A treatment group,CCCP+MHY1485 treatment group,and Cs A+Rapamycin treatment group,and cultured in vitro for 4 hours.(2)Cells in each group were stained with mitochondrial probe,and the damage of mitochondrial quality in each group was detected by flow cytometry.(3)Cells in each group were stained with ROS probes,and ROS levels in each group were detected by flow cytometry.(4)Cells in each group were labeled with AV+PI dye,and apoptosis was detected by flow cytometry.(5)Total protein was extracted from Contol group,CCCP group,Cs A group,CCCP+MHY1485 group,and Cs A+Rapamycin group.Phosphorylation levels of AKT and p70S6 K in each group were detected by WES system.(6)Statistical Methods: SPSS25.0 statistical software was used for statistical analysis.Mean ± standard deviation(x±s)was used to represent measurement data following normal distribution.One-way ANOVA was used to compare the mean of multiple groups,and LSD method was used for pair comparison..Results:(1)The quality of mitochondria was(24.91±1.67)%,(15.44±1.54)%,(35.67±1.01)%,(28.30±3.42)%,(9.85±1.02)% in the Contol group,CCCP group,Cs A group,CCCP+MHY1485 group and Cs A+Rapamycin group respectively.Compared with the control group,the proportion of mitochondrial damaged cells in CCCP group decreased,while the proportion of mitochondrial damaged cells in Cs A group was significantly increased(P<0.05).Compared with CCCP group,the proportion of mitochondrial damaged cells in CCCP+MHY1485 group was increased(P<0.05).Compared with the Cs A group,the proportion of mitochondrial damaged cells in the Cs A+Rapamycin group was decreased(P<0.05).(2)The ROS of Tregs: The average ROS level in control group was(2.36±0.36)×104,and the proportion of normal ROS cells was(88.22±0.21)%.The average ROS level of Treg cells in CCCP group was(1.58±0.27)×104,and the proportion of normal ROS cells was(91.19±0.61)%.The average ROS level in Cs A group was(6.98±0.34)×104,and the proportion of normal ROS cells was(40.58±1.83)%.The average ROS level in CCCP+MHY1485 group was(3.03±0.52)×104,and the proportion of normal ROS cells was(85.04±0.95)%.The average ROS level in Cs A+Rapamycin group was(4.27±0.44)×104,and the proportion of normal ROS cells was(80.91±1.13)%.Compared with control group,the ROS level in CCCP group was decreased,and that in Cs A group was increased(P<0.05).Compared with CCCP group,ROS level in CCCP+MHY1485 group was increased(P<0.05).Compared with the Cs A group,the ROS level in the Cs A+Rapamycin group decreased(P<0.05).(3)The apoptosis level of Tregs: Compared with the Control group(9.86±0.21)%,the proportion of apoptotic cells in the CCCP group(4.98±0.49)% was decreased(P<0.05),the proportion of apoptotic cells in the Cs A group(16.64±0.77)%was increased(P<0.05).Compared with the CCCP group,the CCCP+MHY1485 group(9.63±0.77)% increased,and Compared with the Cs A group,the Cs A+Rapamycin group decreased(8.94±0.54)%(both P<0.05).(5)The protein phosphorylation levels of Akt and P70S6 K in each group:Compared with the control group,the protein phosphorylation levels of AKT and P70S6 K in CCCP group were decreased,but those in Cs A group were increased(P<0.05).Compared with CCCP group,the protein phosphorylation levels of AKT and P70S6 K in CCCP+MHY1485 group were increased(P<0.05).Compared with the Cs A group,the protein phosphorylation levels of Akt and p70S6 K in Cs A+Rapamycin group were decreased(P<0.05).Conclusion:Phosphorylation levels of AKT and p70s6 k in Treg cells of MG patients were increased.Different mitochondrial autophagy states of Treg cells in MG patients were regulated in vitro,and the phosphorylation levels of AKT and p70s6 k were different.When the phosphorylation levels of AKT and P70s6 k were regulated in vitro,the quality of mitochondria,reactive oxygen species and apoptosis status of Treg cells in MG patients were different.Abnormal mitochondrial autophagy in Treg cells of MG patients may be related to abnormal activation of mTOR pathway caused by phosphorylation of AKT and p70s6 k.