桂枝附子汤调控TXNIP/NLRP3/caspase-1通路抑制软骨细胞焦亡的机制研究  

Mechanism of Guizhi Fuzi Decoction on inhibiting chondrocytes pyroptosis by regulating TXNIP/NLRP3/caspase-1 pathway

作　　者：杨洋[1] 梅胜 辛龙[1] YANG Yang;MEI Sheng;XIN Long(Tongde Hospital of Zhejiang Province,Hangzhou 31000,Zhejiang,China)

机构地区：[1]浙江省立同德医院,杭州310000

出　　处：《现代实用医学》2021年第6期709-712,F0002,共5页Modern Practical Medicine

基　　金：浙江省中医药科技项目(2019ZB027、2019ZB029);浙江省卫计委科技计划基金(2020362988)。

摘　　要：目的探究桂枝附子汤(GZFZD)调控硫氧还蛋白互作蛋白(TXNIP)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸蛋白酶1(caspase-1)通路抑制软骨细胞焦亡的机制。方法不同浓度(0、10、20、50,100,200μmol/L)的桂枝附子汤干预软骨细胞24h后采用MTT法检测细胞活性并筛选药物浓度。通过氧糖剥夺/复氧(OGD/R)模型建立软骨细胞的细胞焦亡模型,将软骨细胞分为对照组、OGD/R组、低剂量桂枝附子汤组(10μmol/L)及高剂量桂枝附子汤组(100μmol/L)。采用微量酶标法检测各组软骨细胞乳酸脱氢酶(LDH)释放量采用Hoechst33342/碘化丙啶(PI)染色法检测软骨细胞PI阳性细胞数目,使用RT-PCR方法检测桂枝附子汤对软骨细胞TXNIP,NLRP3、caspase-1和白细胞介素(IL)-1β的mRNA表达,采用蛋白免疫印迹(Western blot)去检测各组软骨细胞TXNIP,NLRP3、caspase-1和IL-1β的蛋白表达水平。结果200μmol/L桂枝附子汤能明显降低软骨细胞活性(P<0.05)。与OGD/R组比较,低剂量桂枝附子汤组和高剂量桂枝附子汤组的LDH释放量、PI阳性细胞数量均明显降低(均P<0.05),TXNIP、NLRP3、caspase-1和IL-1β的mRNA、蛋白水平表达水平均明显降低改均(P<0.05)。结论桂枝附子汤能调控TXNIP/NLRP3/caspase-1通路抑制软骨细胞焦亡。Objective To investigate the mechanism of Guizhi Fuzi Decoction(GZFZD)on chondrocytespyroptosis by regulating thioredoxin interaction protein(TXNIP)/nucleotide binding oligomerization domain-like receptor protein 3(NLRP3)/caspase-1.Methods The cell viability was detected by MTT method after treated with GZFZD at different concentrations(0,10,20,50,100,200μmol/L)for 24 hous respectively,and the appropriate drug concentrations were selected.The pyrolysis model of chondrocytes was established by oxygen glucose deprivation/reoxygenation(OGD/R)model.Chondrocytes were divided into the control group,OGD/R group,low-dose GZFZD group(10μmol/L),high-dose GZFZD group(100μmol/L).Micro-enzyme standard method was used to detect the release of lactate dehydrogenase(LDH)of chondrocytes in each group.Hoechst33342/propidium iodide(PI)staining method was used to detect the numbers of PI positive cells in chondrocytes.RT-PCR method and western blot were used to detect the mRNA and protein expression levels of TXNIP,NLRP3,caspase-1 and IL-1βin chondrocytes in each group.Results After the intervention of 10,20,50,100μmol/L GZFZD,there was no significant difference in chondrocyte viability(all P>0.05),and 200μmol/L GZFZD could significantly reduce chondrocyte viability(P<0.05).Compared with the OGD/R group,the release of LDH and the number of PI positivessignificantly reduced in the low-dose GZFZD group and the high-dose GZFZD group(all P<0.05).The mRNA and protein expression levels of TXNIP,NLRP3,caspase-1 and IL-1βwere significantly reduced(all P<0.05).Conclusions GZFZD can inhibit the chondrocytes pyroptosis by regulating the TXNIP/NLRP3/caspase-1 pathway.

关 键 词：桂枝附子汤 软骨细胞 细胞焦亡 硫氧还蛋白互作蛋白 NLRP3炎症小体 半胱氨酸天冬氨酸蛋白酶1 

分 类 号：R684.3[医药卫生—骨科学]