补肾活血法对类风湿关节炎软骨细胞焦亡及HMGB1/NLRP3信号通路的影响

Effects of tonifying kidney and promoting blood circulation method on rheumatoid arthritis chondrocyte pyrolysis and HMGB1/NLRP3 signal pathway

目的:探讨补肾活血法对类风湿关节炎(RA)软骨细胞焦亡的影响及其分子机制。方法:取胶原诱导型关节炎小鼠膝关节软骨进行体外细胞培养,予补肾活血法的代表方断藤益母汤低中高剂量进行干预。CCK8法检测软骨细胞活力,微板法检测乳酸脱氢酶(LDH)释放,TUNEL法检测细胞焦亡,RT-PCR法检测caspase-1、IL-1β、IL-18 RNA表达情况,Western Blot法检测HMGB1、NLRP3蛋白表达水平。结果:断藤益母汤冻干粉稀释液在6、12、24μg/mL的浓度下对RA软骨细胞的活力没有明显的影响。与空白组比较,断藤益母汤各剂量组均可不同程度地抑制RA软骨细胞LDH释放和caspase-1、IL-1β、IL-18 mRNA表达(P<0.05,P<0.01)。空白组RA软骨细胞细胞核浓缩,TUNEL染色明显呈阳性,细胞焦亡明显。经断藤益母汤干预后,RA软骨细胞发生焦亡比率明显减少(P<0.05,P<0.01)。与空白组比较,断藤益母汤可显著抑制RA软骨细胞HMGB1、NLRP3蛋白表达水平(P<0.05,P<0.01)。结论:补肾活血法能减轻RA软骨细胞焦亡,其机制可能与抑制HMGB1/NLRP3信号通路有关。

Objective:To investigate the effects and molecular mechanism of tonifying kidney and promoting blood circulation method on rheumatoid arthritis(RA)chondrocyte pyrolysis.Methods:The knee joint cartilage of mice with collageninduced arthritis was cultured in vitro and treated with Duanteng Yimu Decoction(DTYMD),a representative of the method of tonifying kidney and promoting blood circulation,with low,medium,and high doses for intervention.The CCK8 method was used to detect the viability of chondrocytes,the microplate method was used to detect the release of lactate dehydrogenase(LDH),the TUNEL method was used to detect pyrolysis,the RT-PCR method was used to detect the mRNA expression of caspase-1,IL-1β,IL-18,and the Western Blot method was used to detect the protein expression levels of HMGB1 and NLRP3.Results:The dilution of the freeze-dried powder of DTYMD had no obvious effect on the viability of RA chondrocytes at the concentrations of 6,12,24μg/mL.Compared with the blank group,the low,medium,and high dose groups of DTYMD could inhibit the LDH and mRNA expressions of caspase-1,IL-1β,IL-18 release of RA chondrocytes to varying degrees(P<0.05,P<0.01).The nuclei of RA chondrocytes in the blank group were concentrated,TUNEL staining was obviously positive,and the pyrolysis was obvious.After the intervention of DTYMD,the rate of scorch death of RA chondrocytes was significantly reduced(P<0.05,P<0.01).Compared with the blank group,DTYMD can significantly inhibit the expression levels of HMGB1 and NLRP3 in RA chondrocytes(P<0.05,P<0.01).Conclusion:Tonifying kidney and promoting blood circulation method can reduce the pyrolysis of RA chondrocytes,and its mechanism may be related to the inhibition of the HMGB1/NLRP3 signaling pathway.