Effects Of Astrocytes On The Expression Of Heat Shock Proteins In Paraquat-treated Neurons

ObjectiveTo investigate the effects of astrocytes under normal conditions and paraquat(PQ)induction on the expression of heat shock protein and TDP43 protein in PQ-treated neurons and the survival of neurons.Methods1.In order to establish a co-cultivation system of astrocytes and nerve cells,U87 cells were used as the astrocyte model,and SH-SY5 Y cells were used as the nerve cell model.Trans-well co-culture system was used to co-culture U87 cells and SH-SY5 Y cells.2.In order to establish a paraquat-treated SH-SY5 Y cell model,select the appropriate paraquat-treated concentration,the experiment is divided into a blank control group(without paraquat-treated SH-SY5 Y cells or U87 cells),paraquat-treated group(paraquat-treated SH-SY5 Y cells or U87 cells).The survival rate of U87 cells and SH-SY5 Y cells at the paraquat concentration of 0 ?M,100 ?M,200 ?M,300 ?M,400 ?M,500 ?M was tested by CCK8 method.Immunofluorescence was used to detect the localized expression of TDP43 protein aggregation in SH-SY5 Y cells induced by 100 ?M paraquat.3.In order to study the effect of U87 cells on paraquat-treated SH-SY5 Y cells under normal conditions,the experiment was divided into a blank control group(SH-SY5 Y cells without paraquat-treated)and a paraquat-treated group(induced SH-SY5 Y cells with 1 mM paraquat for 2h),co-culture group(U87 cells co-cultured with SH-SY5 Y cells with 1mM paraquat for 2h).The expression of heat shock protein and TDP43 protein in SH-SY5 Y cells of the above groups was detected by Western blotting.The CCK8 method was used to detect the survival rate of SH-SY5 Y cells in each group.4.In order to study the effect of Paraqua-treated U87 cells on paraqua-treated SH-SY5 Y cells,the experiment was divided into a blank control group(SH-SY5 Y cells without paraquat-treated)and a co-culture group(SH-SY5 Y cells induced with paraquat for 24 hours).The expression of heat shock protein and TDP43 protein inSH-SY5 Y cells of the above groups was detected by Western blotting.The CCK8 method was used to detect the survival rate of SH-SY5 Y cells in each group.Results1.U87 cells and SH-SY5 Y cells were successfully co-cultured in the trans-well system,and a co-culture system was established.2.In paraquat model building experiment:(1)The CCK8 test results selected for the concentration of paraquat showed that with the increase of the concentration of paraquat,the survival rate of both cells decreased gradually(p<0.05),and the decreasing trend of SH-SY5 Y cells was more obvious than that of U87 cells.Compared with the 0 ?M paraquat group,there was no difference in the survival rates of SH-SY5 Y cells(p>0.05)and U87 cells(p>0.05)in the 100 ?M paraquat group.Compared with the 0 ?M paraquat group,when the paraquat concentration was 200 ?M,the survival rate of SH-SY5 Y cells decreased(P<0.05).When the concentration of paraquat was 300 ?M,the cell survival rate of U87 cells decreased,showing sensitivity to paraquat(P<0.05).(2)The immunofluorescence detection results of TDP43 protein aggregation expression in SH-SY5 Y cells showed that compared with the blank control group,green fluorescence of TDP43 protein appeared in the cytoplasm of the paraquat-treated group.The above results indicated that the paraquat disease model was established.3.During the trans-well co-culture,the effect of normal U87 cells on paraquat-treated SH-SY5 Y,in the experiment,after induced with 1mM paraquat for2h:(1)Western blotting detection results of heat shock protein expression experiments in SH-SY5 Y cells showed that there was no difference in the expression of HSF1,HSP70,and HSPB8 in SH-SY5 Y cells of each group(P>0.05).(2)Western blot detection results of TDP43 protein expression in SH-SY5 Y cells showed that there was no difference in the expression of TDP43 protein in SH-SY5 Y cells of each group(P>0.05).(3)The CCK8 test results for the survival rate of SH-SY5 Y cells in each group showed no difference between the paraquat-treated group and the blank control group(P>0.05).Compared with the paraquat-treated group,the SH-SY5 Y cell survival rate of the co-culture group was increased(P<0.05).4.In the experiment of trans-well co-culture,under the induction of paraquat,the effect of U87 cells on SH-SY5 Y cells:(1)Western blotting detection results of heat shock protein expression experiment in SH-SY5 Y cells showed:(1)After induced with 100?M paraquat for 24 h,compared with the blank control group,the expression of HSPB8 in SH-SY5 Y cells of paraquat treatment group increased(P<0.05),and there was no difference in the expression of HSF1 and HSP70(P>0.05).Compared with the paraquat treatment group,the expression of HSF1 in SH-SY5 Y cells of the co-culture group was reduced(P<0.01),the expression of HSPB8 was reduced(P<0.05),and the expression of HSP70 was not different(P>0.05).(2)After induced with 300?M paraquat for 24 h,compared with the blank control group,the expression of HSP70 in SH-SY5 Y cells of paraquat-treated group increased(P<0.001),and the expression of HSPB8 increased(P<0.05).There was no difference in the expression of HSF1 and HSPB8(P>0.05).Compared with the paraquat-treated group,the expression of HSF1 in SH-SY5 Y cells of the co-culture group was reduced(P<0.05),the expression of HSPB8 was reduced(P<0.05),and the expression of HSP70 was not different(P>0.05).(2)Western blotting detection results of TDP43 protein expression in SH-SY5 Y cells showed that:(1)After induced with 100?M paraquat for 24 h,compared with the blank control group,the expression of TDP43 protein in SH-SY5 Y cells of paraquat-treated group increased(P<0.05).There was no difference in the expression of TDP43 protein in SH-SY5 Y cells between the co-culture group and the paraquat-treated group(P>0.05).(2)After induced with 300?M paraquat for 24 h,compared with the blank control group,the expression of TDP43 protein in SH-SY5 Y cells of paraquat-treated group increased(P<0.01).There was no difference in the expression of TDP43 protein in SH-SY5 Y cells between the co-culture group and the paraquat-treated group(P>0.05).(3)The CCK8 test results for the survival rate of SH-SY5 Y cells in each groupshowed:(1)After 24 hours of induced with 100 ?M paraquat,there was no difference in cell survival rate between the blank control group,paraquat-treated group and co-culture group(P<0.05)(2)After being induced with 300?M paraquat for 24 h,compared with the blank control group,the survival rate of SH-SY5 Y cells in the paraquat-treated group decreased(P<0.001).Compared with the paraquat-treated group,the survival rate of SH-SY5 Y cells in the co-culture group was reduced(P<0.01).ConclusionUnder normal conditions,astrocytes have no effect on the expression of heat shock protein and TDP43 protein in paraquat-treated neurons,but they can promote neuronal survival.Astrocytes can reduce the expression of heat shock protein in PQ-treated neurons and promote cell death under paraquat-treated,but have no effect on the expression of TDP43 protein.