荣筋拈痛方抑制PARP1延缓软骨细胞衰老治疗膝骨关节炎OA

Objective To investigate the mechanism of Rongjin Niantong Formula(RJNTF)in treating knee osteoarthritis(KOA)via in delaying chondrocyte senescence through the modulation of Poly(ADP-ribose)polymerase 1(PARP1).Methods This study was divided into two experiments.In experiment one,chondrocytes were divided into the si-NC group(negative control virus),the si-PARP1 group(PARP1 knockdown),the D-galactose(D-gal)+si-NC group(negative control virus+D-gal)and the D-gal+si-PARP1 group(PARP1 knockdown+D-gal).In experiment two,chondrocytes were divided into the normal group(10％blank serum),the model group(10％blank serum+10 g/L D-gal),the treatment group(10％RJNTF drug-containing serum+10 g/L D-gal)and the inhibitor group(10％blank serum+10 g/L D-gal+10 μmol/L PJ34).Senescence was assessed by senescence-associated β-galactosidase(SA-β-gal staining),and cell proliferation was evaluated by the EdU assay.NAD+levels and the NAD+/nicotinamide adenine dinucleotide(NADH)ratio were measured.Interleukin-6(IL-6),high mobility group box 1(HMGB1),tumor necrosis factor-alpha(TNF-α)were quantified by ELISA.The mRNA and protein expression of Poly ADP-ribose 1(PARP1),multiple tumor suppressor 1(p16)and matrix metallopeptidase 13(MMP-13)were detected by RT-qPCR and Western Blot,respectively.Results Results of experiment 1:Compared with the si-NC group,the positive rate of SA-β-gal,the expressions of IL-6,HMGB1,TNF-α,and the mRNA and protein expressions of PARP1,p16,and MMP-13 in the D-gal+si-NC group increased(P＜0.01).The content of NAD+,the ratio of NAD+/NADH,and the positive rate of EdU decreased(P＜0.05,P＜0.01).Compared with the D-gal+si-NC group,the positive rate of SA-β-gal,the expressions of IL-6,HMGB1,TNF-α,and the mRNA and protein expressions of PARP1,p16,and MMP-13 in the D-gal+si-PARP1 group decreased(P＜0.05,P＜0.01).The content of NAD+,the ratio of NAD+/NADH,and the positive rate of EdU increased(P＜0.05,P＜0.01).Results of experiment 2:Compared with the model group,the positive rate of SA-β-gal,the expressions of IL-6,HMGB1,and TNF-α in the treatment group and the inhibitor group decreased(P＜0.05,P＜0.01),while the content of NAD+,the ratio of NAD+/NADH,and the positive rate of EdU increased(P＜0.05,P＜0.01).The mRNA expressions of PARP1,p16,MMP-13 in the treatment group and p16,MMP-13 in the inhibitor group decreased(P＜0.05),and the protein expressions of PARP1,PAR,p16,MMP-13 in the treatment group and PAR,p16,MMP-13 in the inhibitor group decreased(P＜0.01).Conclusions RJNTF ameliorates chondrocyte senescence in KOA by inhibiting PARP1 expression,reducing the positive rate of SA-β-gal in chondrocytes,decreasing the consumption of NAD+in chondrocytes,promoting the proliferation of chondrocytes,down-regulating the expression of IL-6,HMGB1 and TNF-αcytokines and mRNA and protein expression of PARP1,p16 and MMP-13.