HMGB1 Mediated Paraquat-induced Autophagy Dysfunction Via Regulating α-synuclein Over-expression In Dopaminergic Cell Model

Objective In this study,we established an in vitro model of dopaminergic neurons(SH-SY5Y)and used related biological techniques to observe the effect of paraquat(PQ)on the autophagy function of dopaminergic neurons;To investigate whether high mobility group box-1 protein(HMGB1)and α-synuclein are the contributing factors in PQ affecting the autophagy function of neurons;Finally,elucidate the mechanism of HMGB1 involved in dopaminergic neuron autophagy inhibition by regulating α-synuclein expression.Methods Human neuroblastoma cell(SH-SY5 Y cell)was used as model of dopaminergic neurons in vitro.The cells were treated with different concentrations of PQ for 24 hours,and induced by 15 0 μmol/L PQ for different hours,The effect of PQ on the proliferation activity of SH-SY5 Y cells was detected by CCK8 assay;Lactate dehydrogenase(LDH)activity in supernatant was determined by spectrophotometry assay.The expression levels of autophagy-related proteins(LC3I,LC3 II,Beclin1,Vps34 and p62),α-synuclein and HMGB1 were detected by Western blot after PQ infected.The gene expression level of α-synuclein and HMGB1 was assayed by Real-time quantitative PCR;The changes of autophagosomes in cells were detected by MDC staining;The expression level and their positions of α-synuclein and HMGB1 was also evaluated by immunofluorescence.The cells were pretreated with 100 nmol /L autophagy inducer RAPA for 6 hours,The expression levels of autophagy-related proteins and α-synuclein were detected by Western blot;The interaction between HMGB1,Beclin1 and α-synuclein was detected by Co-Ip;Transfect HMGB1 shRNA lentivirus and over-express HMGB1 lentivirus into SH-SY5 Y cells,Western blot and IF were used to investigate the effects of HM GB1 on the expression levels of α-synuclein and autophagy-related proteins.Results(1)Effect of PQ on autophagy of dopaminergic neurons : PQ induced cell survival rate decrease in a time and dose dependent manner;The activity of LDH in the cell supernatant increased significantly after PQ exposure(P<0.05);Western blot analysis showed the ratio of autophagy-related protein LC3II/LC3 I,Beclin1 and Vps34 protein expression were significantly lower after PQ treatment while the expression of p62 prot ein was higher(P<0.05);MDC staining results showed that the formation of intracellular autophagosomes significantly reduced after PQ treatment(P<0.05);The expression levels of autophagy-related proteins LC3II/LC3 I,Beclin1 and Vps34 protein were significantly increased whlie there was no significant change in the expression level of p62 protein,α-synuclein protein level was decreased after RAPA induction(P<0.05);IF results showed that after RAPA intervention,the expression level of α-synuclein was significantly reduced(P<0.05).(2)α-synuclein and HMGB1 are key factors involved in PQ-induced abnormal autophagy of dopaminergic neurons: The results of qRT-PCR showed that the expression levels ofα-synuclein and HMGB1 genes in the PQ treatment grou p were significantly increased.Western blot analysis showed that α-synuclein and HMGB1 protein expression levels were markedly increased(P<0.05);IF results showed that the expression of α-synuclein and HMGB1 in the PQ treatment group were significantly increased,the immunofluorescence signal of α-synuclein was significantly enhanced and there was a trend of perinuclear aggregation,cytoplasmic translocation occurred in HMGB1,and the degree of co-localization of α-synuclein and HMGB1 increased;Co-Ip results showed that,compared with the control group,HMGB1 was dissociated from Beclin1 and HMGB1 was combined with α-synuclein after PQ treatment(P<0.05).(3)The mechanism of HMGB1 participating in dopaminergic neuron autophagy inhibition by regulating α-synuclein expression: Western blot results show that after the silencing of HMGB1,the expression of α-synuclein protein decreased(P<0.05),the expression level of autophagy-related proteins decreased,and the expression level of p62 protein increased(P<0.05).After overexpression of HMGB1,the expression level of positive α-synuclein protein significantly enhanced(P<0.05),while the autophagy-related protein decreased significantly(P<0.05),and the expression level of p62 protein increased(P<0.05).IF results showed that after silencing HMGB1,the fluorescence intensity of α-synuclein and HMGB1 both weakened,and their degree of colocalization decreased;the expression of LC3 decreased,and the fluorescence intensity decreased;after overexpression of HMGB1 the levels of α-synuclein and HMGB1 expression in cells increased significantly,cytoplasmic translocation occurred in HMGB1,and the degree of co-localization of α-synuclein and HMGB1 increased;the expression of LC3 decreased,and the fluorescence intensity decreased.Conclusion In an in vitro model of dopaminergic neurons,PQ induces autophagy dysfunction of dopaminergic neurons,and α-synuclein and HMGB1 are key factors involved in the abnormal autophagy of dopaminergic neurons induced by PQ.HMGB1 participates in PQ-induced dopaminergic neuron autophagy dysfunction by binding with α-synuclein,and inhibits autophagy degradation of abnormally aggregated α-synuclein.