The Effect And Mechanism Of BiochA On The Activation Of Microglia Induced By AngⅡ

Parkinsons disease(PD)is a kind of neurodegenerative disease with middle and old age as the main body.Its pathological characteristics are dopamine(dopamine,DA)neurons with type denaturation and apoptosis.So far,the pathogenesis of the disease is not clear,and there is no reliable and effective treatment in clinic.lt has been shown that brain renin-angiotensin system(RAS)may play an important role in the development of nenroinflammation5 oxidative stress and dopaminergic degeneration through?e’angiotensin 1(angiotensin 1 receptor^ATl)receptor.Different pathogenetic factors such as aging5 neurotoxin,and inflanunatory stimulating factors all lead to the activation of RAS.Local or paracrine ras is the activator of nicotinamide adenine dinucleotide phosphate oxidase,and is involved in oxidative stress and inflammatory reaction.A large number of experimental results showed that AngII(angiotensmII5AngII)induced neuroinflammation mediated by microglial activation is closely related to the course of PD.BiochA is a phytoestrogen of isoflavones.Previous studies have shown that BiochA can inhibit the activation of microglia induced by LPS,but its neuroinflammation mediated by Angll is not clear.Objective: To investigate the effect of BiochA on the activation of BV2 cell induced by Angll and its mechanism.Methods: 1.BV2 cells were divided into 6 groups :(1)control group;(2)AngII(25 nM)group;(3)Angll(50nM)group;(4)Angll(lOOnM)group;(5)AngII(200 nM)group;(6)Angll(400nM)group.Except for control,the corresponding concentration of Angll was added to the control group and the cells were treated for 24 h.The cytotoxicity of Angll to the cells was determined by MTT.The content of nitric oxide(NO)in supernatant was determined by Griess.2.BV2 cells were divided into 7 groups:(1)control group;(2)Angll(lOOnM)group;(3)losartan(luM)group;(4)AngII(100nM)+Losartan(luM)group;(5)Angll(100 nM)+ BiochA(1.25[iM)group;(6)Angll(100nM)+ BiochA(2.5pM)group;(7)AngII(100nM)+ BiochA(5.0p,M)group.Except for control group5 the corresponding drugs were added to each group for 24 h.The content of oxygen speciesrosins in the cells was measured by DCFH-DA probe method.3.BV2 cells were divided into 5 groups:(l)control group;(2)Angll(lOOnM)group;(3)BiochA(1.25jiM)group;(4)BiochA(2.5pM)group;(5)BiochA(5.0jiM)group.Except for control group,the corresponding drugs were added to each group for24 h.The ATI receptor was detected by Western blot.4.BV2 cells were divided into 5 groups:(l)control group;(2)Angll(lOOnM)group;(3)losartan(lpM)group;(4)Angll(100nM)+ losartan(IjjM)group;(5)Angll(lOOnM)-fBioA(2.5|j,M).Except for control group5 the corresponding drugs were added to each group for 24 h.The expression levels of NADPH subunits p47 phox and gp91phox5 inflammatory corpuscular associated proteins ASC、 Caspase-1 and NLRP3,interleukin-6(lL-6),interleukin-lp(lL-lp)and tumor necrosis factor-a(TNF-a)were detected by Western blot.5.BV2 cells were divided into 5 groups:(l)control group;(2)Angll(lOOnM)group;(3)losartangroup;(4)Angll(100nM)+ losartan(lpM)group;(5)Angll(lOOnM)+BioA(2.5|iM).Except for control group5 the corresponding drugs were added to each group for 24 h.The cell supernatant was collected and the content of TNF-a andIL-lp in the supernatant was determined by ELISA.Results: 1).MTT experiment: compared with the control group,the cell viability was significantly decreased when the concentration of Angll was higher than lOOnM,and the apoptosis was induced.The results of Griess assay showed that the content of NO in the supernatant of BV2 cells was significantly increased at lOOnM,and the concentration of Angll at lOOnM was the ideal drug concentration in this experiment.2).DCFH-DA probe assay : compared with Angll group,the expression of ROS in BiochA(2.5pM)-l-Angll(lOOnM)group was significantly lower than that in Angll group.The suitable concentration of BiochA was 2.5|j,M5 which indicated that BiochA decreased the expression of ROS induced by Angll.3).Westem blot test results:Compared with Angll group,Angll group did not decr^se the expression of ATI receptor protein,It suggests that BiochA does not decrease the production of proinflammatoiy cytokines through ATI receptors.4).Western blot test results: compared with Angll group,BiochA(2.5p,M)+AngII(lOOnM)group down-regulates the expression of p47 phox and gp91 phox subunits of NADPH oxidase,which indicated that BiochA down-regulated the expression of p47 phox and gp91 phox produced by BV2 activation.5).Westem blot test results: compared with Angll group,BiochA(2.5^M)+AngII(lOOnM)group down-regulates the expression of Caspase-l?ASCand NLRP3 protein 5and the expression of TNF-a,IL-lp and IL-6.6).PLISA test results showed that the content of TNF-a and IL_lp in the supernatant of BV2 cells in BiochA(2.5pM)+AngII(lOOnM)group was significantly lower than that in group Angll(lOOnM).Conclusion: In BV2,Angll can enhance the oxidative stress response mediated by NADPH oxidase by acting on ATI receptor.The mechanism of inhibiting the activation of microglia by Angll may inhibit the activity of NADPH oxidase and the activity of NLRP3 inflammatory bodies.