Effect of Huoxi Decoction(活膝汤)Drug-Containing Serum on Chondrocyte Pyroptosis Based on CCL2/CCR2/NF-κB Pathway
Objective:To investigate the protective effect of Huoxi Decoction(HXD)(活膝汤)drug-containing serum on lipopolysaccharide-in-duced chondrocyte pyroptosis.Method:CCL2 over-expression plasmid was constructed,HXD drug-containing serum was prepared,and the optimal concentration and intervention time on chondrocytes were determined through CCK8 test.The cultured chondrocytes were divided into 7 groups:blank control group,model control group,HXD 12.81 g/kg group,nuclear factor κ B(NF-κB)inhibition group,CCL2 over-expres-sion group,CCL2 over-expression+NF-κB inhibition group,and CCL2 over-expression+HXD 12.81 g/kg group.The expressions of inter-leukin-18(IL-18)and interleukin-1β(IL-1β)in the supernatant were detected by ELISA.The mRNA expressions of caspase-1,gasdermin-D(Gsdmd)and NOD-like receptor pyrin domain-containing protein 3(Nlrp3)were detected by RT-PCR.Western blot was conducted to de-tect the expressions of cleaved-caspase-1,GSDMD,NLRP3,and phosphorylated p65(Ser536)(p-p65(Ser536))proteins.The level of caspase-1 mediated chondrocyte pyroptosis was detected by immunofluorescence double staining.Result:Compared with the blank control group,the contents of IL-18 and IL-1β were increased in the model control group.Moreover,the expressions of NLRP3,GSDMD,caspase-1,cleaved-caspase-1,p-p65(Ser536)were significantly up-regulated(P＜0.01),accompanied by increased chondrocyte pyroptosis.Compared with the model control group,the HXD 12.81 g/kg group and NF-κB inhibition group showed a significant reduction in the contents of IL-18 and IL-1β in chondrocytes.The expressions of NLRP3,GSDMD,caspase-1,cleaved-caspase-1,p-p65(Ser536)were significantly down-regu-lated(P＜0.01),and chondrocyte pyroptosis was decreased.CCL2 over-expression group exhibited significantly elevated IL-18 and IL-1βcontents,significantly up-regulated levels of NLRP3,GSDMD,caspase-1,cleaved-caspase-1 and p-p65(Ser536)(P＜0.01),as well as boos-ted chondrocyte pyroptosis.Conclusion:HXD drug-containing serum may down-regulate caspase-1-mediated chondrocyte pyroptosis through inhibiting the signaling axis of CCL2/CCR2/NF-κB.
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