| Objective To investigate the effects of Astragaloside IV(AS-IV)on microglia activation and NLRP3/Caspase-1 pyroptosis pathway-related factors during neuroinflammation.Methods 1.Cell culture: Mouse microglia cell line(BV2)cultured in vitro.2.Drug concentration screening:(1)CCK-8 method was used to detect the effects of gradient concentration of thrombin(1,5,10,20,40,80,120 U/m L)on the viability of BV2 cells at different time nodes(6,12,24,48 h).(2)CCK-8 method was used to detect the effects of AS-IV(0.1,1,5,10,20,40,80 μmol/L)on the viability of BV2 cells at different time nodes(6,12,24,48 h).3.Experimental groups:(1)normal control group(Control group);(2)inflammation model group(20 U/m L thrombin);(3)low concentration AS-IV experimental group(1 μmol/L AS-IV+20 U/m L thrombin);(4)medium concentration AS-IV experimental group(5 μmol/L AS-IV+20 U/m L thrombin);(5)high concentration AS-IV experimental group(10 μmol/L AS-IV+20U/m L thrombin),all groups were incubated with drugs for 24 h.4.Cytomorphological observation: The morphological and cytomembrane changes of BV2 cells were observed using light microscopy and scanning electron microscopy to identify the activation and pyroptosis status of BV2 cells in each group.5.Protein level detection:(1)Western blot and Immunofluorescence were used to detect the changes of protein expression of pyroptosis related factors(NLRP3,ASC,Caspase-1,GSDMD-N)in each group of BV2 cells.(2)ELISA was used to detect the content of IL-18 and IL-1β,the downstream secretion of pyroptosis,in each group of cell culture medium.6.Gene level detection: q RT-PCR method was used to detect the changes of m RNA expression of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 in each group of BV2 cells.Results 1.Drug concentration screening:(1)CCK-8 results showed that in the thrombin concentration screening experiment,except for the 6h group,which showed no significant change in cell viability compared with the control group,the viability of BV2 cells at three time points(12,24 and 48 h)increased and then decreased with the thrombin concentration(all reached their respective peaks at 20 U/m L,P<0.05),with the highest viability at 20 U/m L after 24 h intervention.(2)In the AS-IV virulence assay,there was a significant decrease in cell viability in the 6h and 12 h groups at a concentration of 80 μmol/L of AS-IV(P<0.05),but no change in the other concentrations;both the 24 h and 48 h groups showed a significant decrease in cell viability after the AS-IV concentration was higher than10 μmol/L(P<0.05).2.Cytomorphological observation:(1)Light microscopy showed that BV2 cells in the model group had abnormal morphology and obvious cell aggregation compared with the control group,while in the experimental group with different concentrations of AS-IV,the aggregation phenomenon was reduced and the cell morphology tended to be resting.(2)Scanning electron microscopy showed that BV2 cells in the model group had severe membrane perforation and more vesicle-like projections on the membrane surface compared with the control group,while in the AS-IV group with different concentrations,the membrane perforation was reduced and the surface was smooth,with only a few projections.3.Protein level detection:(1)Western blot results showed that compared with the control group,the expression of intracellular pyroptosis related factors(ASC,NLRP3,Caspase-1,GSDMD-N)in BV2 cells in the model group increased significantly(P<0.05);compared with the model group,the expression of intracellular pyroptosis related factors in BV2 cells in different concentrations of AS-IV experimental group decreased to different degrees(P<0.05).(2)Immunofluorescence results showed that the number of staining cells of pyroptosis related factors(ASC,NLRP3,Caspase-1,GSDMD)in BV2 cells increased significantly in the model group compared with the control group(P<0.05),while the number of staining cells in the AS-IV group decreased to different degrees compared with the model group(P<0.05).(3)The ELISA results showed that the contents of IL-18 and IL-1β in the culture medium of the model group were significantly increased compared with those of the control group(P<0.05),while the contents of IL-18 and IL-1β in the culture medium of the experimental group with different concentrations of AS-IV were both reduced to different degrees compared with those of the model group(P<0.05).4.Gene level detection: q RT-PCR results showed that the m RNA expression of intracellular pyroptosis related factors(ASC,NLRP3,Caspase-1,GSDMD,IL-1β,IL-18)in BV2 cells was significantly increased in the model group compared with the control group(P<0.05),and the m RNA expression of intracellular pyroptosis in BV2 cells in different concentrations of AS-IV experimental group compared with the model group.The m RNA expressions of pyroptosis related factors in BV2 cells at different concentrations of AS-IV were all decreased to different degrees(P<0.05).Conclusion AS-IV can inhibit pyroptosis and anti-inflammatory effect by reducing the expression of pyroptosis related factors(ASC,NLRP3,Caspase-1,GSDMD-N,IL-18,IL-1β)in BV2 cells.AS-IV anti-pyroptosis effect was more pronounced with higher concentrations.It can participate in the anti-inflammatory effects by affecting the classical pathway of NLRP3 / Caspase-1 pyroptosis in microglia. |
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