基于NLRP3/Caspase-1/GSDMD信号通路研究生骨再造丸对BMSCs焦亡水平的干预效应

Based on the NLRP3/Caspase-1/GSDMD signaling pathway intervene effect of Shenggu Zaizao Pills on pyroptosis of BMSCs

目的研究生骨再造丸通过NLRP3/Caspase-1/GSDMD信号通路对BMSCs焦亡水平的影响。方法购买原代兔BMSCs,制备焦亡BMSCs模型,采用慢病毒技术及siRNA技术转染焦亡的BMSCs;制备生骨再造丸含药血清,对焦亡的BMSCs进行干预。将上述细胞分为空白组、模型组、慢病毒组、siRNA组、含药血清组。通过PCR检测各组BMSCs中GSDMD基因的表达水平;WB检测各组BMSCs中NLRP3、Caspase-1、GSDMD的表达水平;ELISA检测各组BMSCs中IL-1β、IL-18水平;通过CCK-8检测BMSCs活力。结果与空白组相比,模型组GSDMD基因表达增加(P<0.01),NLRP3、Caspase-1、GSDMD蛋白水平显著升高(P<0.05)(P<0.01)(P<0.01),IL-18、IL-1β水平上升(P<0.01)(P<0.01)。与模型组相比,慢病毒组BMSCs中GSDMD基因表达升高(P<0.01),NLRP3、Caspase-1、GSDMD蛋白水平增加(P<0.05)(P<0.05)(P<0.05),IL-18、IL-1β水平明显增加(P<0.01)(P<0.05);siRNA组BMSCs中GSDMD基因表达显著降低(P<0.01),NLRP3、Caspase-1、GSDMD蛋白水平降低(P<0.01)(P<0.05)(P<0.05),IL-18、IL-1β水平明显降低(P<0.01)(P<0.01);含药血清组BMSCs中GSDMD基因表达降低(P<0.05),NLRP3、Caspase-1、GSDMD蛋白水平降低(P<0.05)(P<0.05)(P<0.01),IL-1β、IL-18水平降低(P<0.05)(P<0.05)。结论NLRP3是调控BMSCs焦亡的关键靶点,生骨再造丸能通过降低NLRP3表达,抑制NLRP3/Caspase-1/GSDMD信号通路,调控BMSCs焦亡水平。

Objective To study the effect of bone reconstruction pills on the level of pyroptosis in BMSCs through NLRP3/Caspase-1/GSDMD signaling pathway.Methods Pyroptosis BMSCs models were prepared,and lentivirus and siRNA technologies were used to transfect pyroptosis BMSCs.The medicated serum of raw bone reconstruction pills was prepared to intervene in the BMSCs of pyroptosis.The above cells were divided into blank group,model group,lentivirus group,siRNA group and drug-containing serum group.CCK-8 was used to detect the activity of BMSCs in each group,and the expression level of GSDMD gene in BMSCs was detected by RT-qPCR.WB was used to detect the expression levels of NLRP3,Caspase-1 and GSDMD in BMSCs.ELISA was used to detect the levels of IL-1βand IL-18 in BMSCs.Results Compared with the blank group,the expression of GSDMD gene in the model group increased(P<0.01),the protein levels of NLRP3,Caspase-1 and GSDMD were significantly increased(P<0.05)(P<0.01)(P<0.01),and the levels of IL-18 and IL-1βincreased(P<0.01)(P<0.01).Compared with the model group,the expression of GSDMD gene in BMSCs in the lentivirus group increased(P<0.01),the protein levels of NLRP3,Caspase-1 and GSDMD increased(P<0.05)(P<0.05)(P<0.05),the levels of IL-18 and IL-1βincreased significantly(P<0.01)(P<0.05),and the expression of GSDMD gene in BMSCs in the siRNA group de-creased significantly(P<0.01),NLRP3,The protein levels of Caspase-1 and GSDMD decreased(P<0.01)(P<0.05)(P<0.05),the levels of IL-18 and IL-1βdecreased significantly(P<0.01)(P<0.01),the expression of GSDMD gene in BMSCs in the drug-containing serum group decreased(P<0.05),NLRP3,Caspase-1,The levels of GSDMD protein decreased(P<0.05)(P<0.05)(P<0.01),and the levels of IL-1βand IL-18 decreased(P<0.05)(P<0.05).Conclusion NLRP3 is a key target for regulating pyroptosis of BMSCs,and bone reconstruction pills can regulate the level of pyroptosis of BMSCs by reducing the expression of NLRP3 and inhibiting the NLRP3/Caspase-1/GSDMD signaling pathway.