The Effect Of Diosgenin On N2a Cell Apoptosis In Diabetic Peripheral Neuropathy

Purpose:In this study,N2 a cells cultured with high glucose and LPS were used to establish a cellular model of diabetic peripheral neuropathy.MTT assay was used to observe the effect of different concentrations of pangolin extract diosgenin on the survival rate of N2 a cells cultured with high glucose +LPS.Western blot and tunel assay were used to observe the effect of diosgenin extracted from pangolin on apoptosis of N2 a cells cultured with high glucose +LPS and the expression of its regulatory factors Bcl2,Bax and Cleaved Caspase-3.Material and method:1.N2 a cells were divided into a normal control group,a hyperglycemia +LPS model group(diosgenin intervention was not used),a hyperglycemia +LPS+ 5?g/ml diosgenin treatment group,a hyperglycemia +LPS+ 10?g/ml diosgenin treatment group,a hyperglycemia +LPS+20?g/ml diosgenin treatment group,a hyperglycemia +LPS+40?g/ml diosgenin treatment group,a hyperglycemia +LPS+ 80?g/ml diosgenin treatment group,a hyperglycemia +LPS+160?g/ml diosgenin treatment group,and a hyperglycemia +LPS+ 320?g/ml diosgenin treatment group.The normal control group:add normal culture solution to the culture flak and change the solution every other day.The high glucose +LPS model group:after 30 mM gluco-se and 1?g/ml LPS were added for 24 hours,the normal medium was added.The treatment groups:after 30 mM glucose and 1?g/ml LPS were added for 24 hours,diosgenin was added to obtain different concentrations(5?g/ml,10?g/ml,20?g/ml,40?g/ml,80?g/ml,160?g/ml,320?g/ml),the culture was continued for 24 hours.The survival rate of N2 a cells in each group was determined by MTT assay,to determine the optimal therapeutic concentration of the follow-up drug.2.N2 a cells were divided into three groups: a normal control group,a high glucose +LPS model group,a high glucose +LPS+ diosgenin treatment group.The normal control group:add normal culture solution to the culture flak and change the solution every other day.The high glucose +LPS model group:after 30 mM glucose and 1?g/ml LPS were added for 24 hours,the normal medium was added.The high glucose +LPS+ diosgenin treatment groups:after 30 mM glucose and 1?g/ml LPS were added for 24 hours,the optimal therapeutic concentration of diosgenin determined by MTT was added,the culture was continued for 24 hours.Thetunel assay was used to detect the apoptosis rate of N2 a cells in each group.The apoptotic proteins of B cl-2,Bax and Cleaved Caspase-3 in each group were detected by Western blot.Results:1.In each experimental group,compared with the high glucose + LPS model group,the survival rate of N2 a cells treated with different concentrations of diosgenin was from high to low: high glucose + LPS + diosgenin 40 ?g / ml experimental treatment group(P<0.001),high glucose+LPS+ diosgenin 80?g/ml experimental treatment group(P<0.01),high glucose+LPS+ diosgenin 160?g/ml experimental treatment group(P<0.05),high glucose+LPS+diosgenin 20 ?g/m experimental treatment group(P<0.05).2.Compared with the normal control,the expression of Cleaved Caspase-3 in the high glucose+ LPS model group was significantly increased(P< 0.001),and the expression of Cleaved Caspase-3 in the high glucose + LPS + diosgenin treatment group was increased(P< 0.01).Compared with the high glucose + LPS model group,the expression of Cleaved Caspase-3 in the high glucose + LPS + diosgenin treatment group was lower than that in the high glucose +LPS model group(P < 0.01).3.Compared with the normal control group,the expression of Bcl-2 was significantly decreased in the high glucose + LPS model group(P< 0.001),and the expression of Bcl-2 was decreased in the high glucose + LPS + diosgenin treatment group(P< 0.05).Compared with the high glucose + LPS model group,the expression of Bcl-2 in the high glucose + LPS +diosgenin treatment group was higher than that in the high glucose + LPS model group(P<0.001).Compared with the normal control,the expression of Bax in the high glucose + LPS model group was significantly increased(P< 0.001),and the expression of Bax in the high glucose + LPS + diosgenin treatment group was increased(P< 0.01).Compared with the high glucose + LPS model group,the expression of Bax in the high glucose + LPS + diosgenin treatment group was lower than that in the high glucose + LPS model group(P< 0.05).4.In the normal control group,scattered apoptotic cells were observed.Compared with the high glucose + LPS model group,the number of apoptotic cells in the normal control group was significantly lower than that in the high glucose + LPS model group.Compared with the high glucose + LPS model group,the number of apoptotic cells in the high glucose + LPS +diosgenin treatment group was lower than that in the high glucose + LPS model group.Compared with the normal control,the apoptotic rate of high glucose + LPS model group was significantly increased(P<0.01),and the apoptosis rate of high glucose + LPS + diosgenin treatment group was increased(P<0.01).Compared with the high glucose + LPS model group,the apoptotic rate of the high glucose + LPS + diosgenin treatment group was lower than that of the high glucose + LPS model group(P< 0.01).Conclusion:Diosgenin extracted from pangolin plays an anti-apoptotic role by regulating the pro-apoptotic protein of Bax and Cleaved Caspase-3 down and regulating the anti-apoptotic protein of Bcl-2 up in the N2 a cells.