| Objective:To investigate whether ligustilide(LIG),the active ingredient of Xionggui prescription,could reduce the inflammatory response and exert anti-inflammatory effects in an in vitro model of neuroinflammation through regulating the NLRP3/Caspase-1 pathway,in order to provide an experimental basis for the treatment of neuroinflammation associated with ischemic stroke.Methods:1.LPS-stimulated BV2 cells to establish an in vitro inflammatory model: The BV2 cells were cultured in 96-well plates with DMEM high sugar medium containing 10% fetal bovine serum for 24 hours and then replaced with serum-free DMEM high sugar medium.LPS was added to give a final concentration of 100 ng/ml and the inflammation model was prepared by intervening for 24 hours.For post-moulding cell viability assays and screening of drug concentrations.2.The experiments were grouped as follows:(1)Control group(control group): Cell culture in normal conditions;(2)Model group(LPS group): Cells treated with LPS at 100ng/ml for 24 hours;(3)ligustilide group(LIG group): We first pre-treated cells with different concentrations of LIG for 1 hours,followed by 24 hours of co-treatment with 100 ng/ml of LPS;(4)Activator group(ATP group): The cells were cultured for 24 hours,then 2.5 m M ATP was added for 30 minutes;(5)Inhibitor group(MCC950 group): We first pre-treated cells with 10 u M MCC950 for 1 hours,followed by 24 hours of co-treatment with 100 ng/ml of LPS;(6)LPS+ATP+LIG group: We first pre-treated cells with 40 u M LIG for 1 hours,followed by 24 hours of co-treatment with 100 ng/ml of LPS,then 2.5 m M ATP was added for30 minutes.3.The effect of different concentrations of LIG on BV2 cells and cell viability after modeling was measured using CCK-8,a cell proliferation activity assay kit,to screen the appropriate drug concentration of LIG.The effects of LIG on pro-inflammatory cytokines TNF-α,IL-1β and IL-6 in LPS-intervened BV2 cells were detected by qRT-PCR.The effects of LIG intervention on cell morphology as observed by phase contrast microscopy.4.The NLRP3 inflammasome activator ATP and inhibitor MCC950 were added,and the LIG concentration for the experiment was set to 40 u M.We detected the mRNA expression levels of IL-1β and NLRP3 by qRT-PCR.The expression levels of NLRP3 pathway-related proteins(NLRP3,ASC,pro-Caspase-1,Cleaved-Caspase-1)as well as IL-18,pro-IL-1β and mature-IL-1β proteins were measured by Western blot.Result:1.CCK-8 results showed that LIG concentration ≤ 40 um had no significant effect on the viability of BV2 cells(P>0.05).The results of qRT-PCR assay showed that the expression levels of TNF-α,IL-1β and IL-6 were significantly increased after LPS-induced activation of BV2 cells(P<0.0001),while LIG intervention could effectively inhibit TNF-α,IL-1β and IL-6 expression(P<0.01,P<0.001,P<0.0001),and there was dose-effect dependence.On microscopic observation of cell morphology,most of the cells in the normal control group were round or oval with small branches.After LPS stimulation,the branches increased and became elongated dendrites.LIG intervention improved the cell morphology to a certain extent.2.qRT-PCR assay showed that the mRNA expression levels of IL-1β and NLRP3 were raised significantly after LPS treatment in BV2 cells(P<0.0001).The LPS group showed higher mRNA expression levels of IL-1β and NLRP3 compared to the ATP group(P<0.0001).After LIG or inhibitor MCC950 intervention,IL-1β and NLRP3 expression was significantly downregulated compared to the LPS group(model group)(P<0.0001).In the LPS group,IL-1β and NLRP3 mRNA expression levels were higher than in the ATP group(P<0.0001).Compared with the LIG group and the inhibitor MCC950 group,the effect of the inhibitor group was better than in the LIG group(P<0.05).The mRNA expression levels of IL-1β and NLRP3 were increased in the LPS+ATP+LIG group compared with the LIG group(P<0.05,P<0.01).3.Western blot showed that the expression levels of NLRP3 pathway-related proteins(NLRP3,ASC,pro-Caspase-1,Cleaved-Caspase-1)and proteins of IL-18,pro-IL-1β,and mature-IL-1β were increased after LPS intervention(P<0.05).Protein expression decreased after intervention with LIG or the inhibitor MCC950(P<0.05).Among them,pro-IL-1βshowed a downward trend but no statistical difference after LIG intervention(P>0.05).The expression levels of pro-Caspase-1,Cleaved-Caspase-1,IL-18,and mature-IL-1β proteins were elevated in the LPS+ATP+LIG group compared with the LIG group(P<0.05).Compared to the ATP group,the LPS group showed a stronger stimulatory effect on NLRP3,ASC,pro-Caspase-1,and mature-IL-1β protein expression levels(P<0.05,P<0.01,P<0.0001).Conclusion:1.The LIG is effective in inhibiting LPS-stimulated BV2 cell activation,modifying the morphology of activated cells,and inhibiting inflammation.2.LIG inhibits mRNA expression of IL-1β and NLRP3 and the expression of NLRP3,ASC,pro-Caspase-1,Cleaved-Caspase-1,IL-18,and mature-IL-1β protein through NLRP3/Caspase-1 pathways,which reduces cellular inflammation and exerts an antiinflammatory effect. |
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