Research Of Mitophagy And Metabolism Of Treg Cells In Patients With Myasthenia Gravis

Objective:Myasthenia gravis(MG)is an autoimmune neuro-muscle junction disease whose pathogenesis is associated with multiple antibodies produced by B cells,whereas regulatory T cells(Treg)can inhibit the activation of autoreactive B cells,reduce the production of pathogenic antibodies,and prevent MG from occurring.However,when the mitophagy of Treg cells is reduced,the immunosuppressive function is impaired.This study intends to use CCCP and Cs A to regulate the mitophagy status of Treg in patients with MG in vitro.Observing the changes of mitochondrial quality,ROS level,lactic acid production and apoptosis to investigate the cellular metabolic mechanism of abnormal mitophagy in peripheral blood Treg involved in the pathogenesis of MG.Methods:1.Fifteen MG patients were selected as the experimental group,and 15 healthy adults were selected as the healthy control group(HCs).2.Took 30 ml peripheral blood from brachial vein,human peripheral blood mononuclear cells(PBMC)were isolated by ficoll density gradient centrifugation,Treg were isolated by Human CD4+CD25+Treg isolation kit based on separation column from PBMC.The viability of Treg was identified by trypan blue dye exclusion assay and the purity of Treg was identified by flow cytometry.3.The Treg of healthy adults were considered as HCs and the Treg of MG patients were divided into three groups: MG group,CCCP induced culture for 24 h group(MG-CCCP)and Cs A induced culture for 72 h group(MG-Cs A).4.The cells in each group were collected,labeled with mitochondrial green fluorescent probe,and the proportion of impaired mitochondrial in each group was detected by flow cytometry.5.The cells in each group were collected,and the standard pore and sample well absorbance [D(λ)] value at 570 nm was measured by a enzyme microplate reader,draw a standard curve according to the standard well,and then calculate the lactate level of each group according to the standard curve.6.The DCFH-DA probe was used to label each group of cells,and the proportion of cells with normal ROS and the average ROS levels of each group were detected by flow cytometry.7.The Annexin V+PI staining was used to label each group of cells,flow cytometry to detect the proportion of apoptotic cells.Results:1.The viability and purity of Treg:The cells were sorted by magnetic beads and then identified by trypan blue and flow cytometry.The viability of Treg was(98.48 ±0.69)%;The purity of CD4+CD25+ Treg was(93.36 ± 1.87)% and the purity of CD4+CD25+CD127low/-Treg was(66.35 ± 1.61)%.2.The mitochondrial quality : About the proportion of impaired mitochondrial in Treg,the MG group(20.81 ± 2.59)% was significantly higher than healthy control group(1.09 ± 0.64%),P < 0.05;Compared with MG group,the MG-CCCP group(12.23 ± 2.31)% was decreased,while that in MG-Cs A group(33.82 ± 1.90)%was significantly higher,all P < 0.05.3.The level of lactic acid : The absorbance value [D(λ)]of each group at 570 nm was measured by enzyme microplate reader.The level of lactic acid was calculated according to the standard curve.Compared with the HCs group(21.32 ± 0.87),the MG group(37.85 ± 1.04)was increased(P < 0.05).Compared with the MG group,the MG-CCCP group(26.94 ± 1.25)was decreased,while the MG-Cs A group(43.28 ± 1.13)was increased(all P < 0.05).4.The ROS of Treg: The HCs group’s average ROS level was(1.21 ± 0.27×104)and the proportion of cells with normal ROS was(94.26 ± 2.05)%,compared with the HCs group,the average ROS level(2.68 ± 0.36×104)was increased and the percentage of cells with normal ROS(75.51 ± 3.28)% decreased in the MG group(P < 0.05).Compared with MG group,the average ROS level(3.89 ± 0.30×104)and the proportion of cells with normal ROS(85.15 ± 2.86)% were increased in MG-CCCP group,the mean ROS level(6.78 ± 0.44×104)increased,while the proportion of cells with normal ROS(46.80 ± 2.45)% decreased in the MG-Cs A group(all P < 0.05).5.The apoptosis level of Treg: Compared with the HCs group(2.87 ± 1.06)%,the proportion of apoptotic cells in the MG group(14.39 ± 2.06)% was increased(P <0.05).Compared with the MG group,the MG-CCCP group(8.35 ± 1.61)%decreased,and the MG-Cs A group increased(21.52 ± 2.26)%(both P < 0.05).Conclusions:The mitochondrial quality of Treg in MG patients is impaired,resulting in increased ROS releasing,decreased proportion of cells with normal ROS,increasedlactic acid produced by glycolysis,and increased apoptosis.CCCP can promote mitophagy in Treg of MG patients,partially eliminate some damaged mitochondria,relieve metabolic stress and reduce apoptosis,while Cs A aggravates metabolic disorder and increases apoptosis by inhibiting mitophagy in Treg of MG patients.Thus,our study showed that abnormal mitophagy in Treg cells of MG patients may be involved in the pathogenesis of MG.