Research On The Function And Mechanism Of RRS1 In MPP~+-Induced Apoptosis Of SH-SY5Y Cells Model Of Parkinson’s Disease

Objective:As the second largest neurodegenerative disease,Parkinson disease(PD)has a complex pathogenesis,and the process of dopaminergic neuron apoptosis is difficult to reverse once the patient is diagnosed,so there still has no cure medicine for PD.Regulator of ribosome synthesis 1(RRS1)is a conservative regulator that not only participates in the synthesis of 5S RNP and the assembly of 60S subunits,but also plays an important role in the regulation of cell cycle and the apoptosis of cells.In recent years,RRS1 has been found to had an abnormal expression in Huntington’s chorea and tumors.In the early study of this paper,we found that the expression of RRS1 decreased in vitro PD cell model,suggesting that RRS1 might be related to the apoptosis of SH-SY5Y cells induced by MPP+.Therefore,This paper mainly discussed the effect of RRS1 on MPP+-induced apoptosis of SH-SY5Y cells by lentivirus overexpression and knockdown technology,and then analyzed the possible correlation and mechanism between RRS1 and dopaminergic neuron apoptosis so as to provide a new target and basis for the early diagnosis and treatment of PD.Methods:SH-SY5Y cells were cultured in the medium containing 0,100,200 and 400μM MPP~+,and each index was detected.1.Detection of cell proliferation,apoptosis and related gene expression in MPP~+injury model:CCK-8 method was used to measure the proliferation activity of cells in each group at 24,48and 72 hours,JC-1 method was used to detect the effect of MPP~+concentration on the mitochondrial membrane potential of SH-SY5Y cells,ELISA method was used to measure the change of ATP level in cells,DAPI staining was used to detect the pyknosis of nuclei,Annexin V-APC/PI double staining was used to measure the apoptosis of cells in each group and Western blot(WB)was used to detect the protein expression of P53,Bcl-2,Bax and RRS1.2.After RRS1 intervention,cell proliferation,apoptosis and the changes of related gene expression were detected to verify the correlation and mechanism between RRS1 and apoptosis:the lentivirus-RRS1 expression vector(Lv-RRS1),lentivirus RNA interference vector of RRS1(shRNA-RRS1)and the corresponding two negative control lentivirus vectors(LV-control,shRNA-control)were constructed respectively.The SH-SY5Y cells were transfected with lentivirus for 48 h,and the transfection efficiency was detected by qPCR and WB,the transfected cells were treated with 400μM MPP~+for 48 hours,and then,CCK-8method was used to detect the proliferation of cells in each group,DAPI staining was used to measure the pyknosis of nuclei,Annexin V-APC/PI double staining was used to detect the apoptosis of cells in each group and Western blot(WB)was used to measure the protein expression of P53,Bcl-2,Bax and RRS1.Results:Detection of cell proliferation,apoptosis and related gene expression in MPP~+injury model:(1)The proliferation activity of SH-SY5Y cells was decreased significantly with the increase of MPP~+concentration(P<0.01);(2)Compared with the 0μM MPP~+group,the mitochondrial membrane potential and ATP level of 100,200 and 400μM MPP~+group was decreased significantly(P<0.05),suggesting that the mitochondrial function was damaged;(3)The number of nuclear pyknosis were elevated significantly with the increase of MPP~+concentration(P<0.01),and the proportion of apoptotic cells was increased significantly with the increase of MPP~+concentration(P<0.05);(4)With the increase of MPP~+concentration,the protein expression of RRS1 was decreased(P<0.05),the protein expression of P53 was increased(P<0.05),the protein expression of apoptosis related factor Bax was increased(P<0.05),while the protein expression of Bcl-2was decreased(P<0.05),and the apoptosis pathway was activated.2.After lentivirus transfection,compared with the control group,the expression of m RNA and protein of RRS1 gene in the overexpression group were increased significantly(P<0.01),while the expression of mRNA and protein of RRS1 gene in the interference group were decreased significantly(P<0.01).3.After the intervention of the expression of RRS1 gene,the stable transfected cells were treated with 400μM MPP~+for 48 hours,and the proliferation,apoptosis and related gene expression were detected:(1)Compared with Lv-control group,Lv-RRS1 group had a significant increase in cell proliferation activity(P<0.05),and significant reductions in the number of nuclear pyknosis and the rate of apoptosis(P<0.01);(2)Compared with Lv-control group,Lv-RRS1 group had a significant increase in the protein expression of RRS1(P<0.01),significant reductions in the protein expression of P53 and Bax(P<0.01),and a significant increase in the protein expression of Bcl-2(P<0.01),overexpression of RRS1 inhibits cell apoptosis by regulating the protein expression of P53,Bax and Bcl-2.(3)Compared with shRNA-control group,shRNA-RRS1 group had a significant reduction in cell proliferation activity(P<0.05),and significant increases in the number of nuclear pyknosis and the rate of apoptosis(P<0.05);(4)Compared with shRNA-control group,shRNA-RRS1 group had a significant reduction in the protein expression of RRS1(P<0.01),significant increases in the protein expression of P53 and Bax(P<0.05),and a significant reduction in the protein expression of Bcl-2(P<0.01),knockdown of RRS1 aggravates cell apoptosis by regulating the protein expression of P53,Bax and Bcl-2.Conclusion:RRS1 involves in MPP~+-induced apoptosis by regulating the protein expression of P53,Bax and Bcl-2.