川续断总皂苷调节miR-19a对大鼠骨关节炎软骨细胞SOX9/NF-κB信号通路的影响

Effects of Total Saponins of Dipsacus Asperoides on SOX9/NF-κB Signaling Pathway in Rat’s Osteoarthritic Chondrocytes through Regulating miR-19a

目的:探讨川续断总皂苷调节miR-19a对大鼠骨关节炎软骨细胞SOX9/核因子κB(NF-κB)信号通路的影响。方法:120只SD大鼠随机分成6组:正常对照组、模型组、地塞米松组(30 mg·kg-1)、川续断总皂苷低、中、高剂量组(10,20,40 mg·kg-1),每组20只。除正常对照组外,其余各组制备大鼠膝关节骨性关节炎模型,地塞米松组、川续断总皂苷各剂量组于造模成功第1天开始给予相应剂量药物灌胃(灌胃体积1.0 ml/100g),持续给药4周,正常对照组和模型组给予等体积生理盐水。给药结束后,进行大鼠关节炎症积分评分,测定机械痛阈(PWT)及热刺激潜伏期(PWTL),实时荧光定量PCR(RT-PCR)法及Western-Blot法测定膝关节软骨细胞miR-19a基因及SOX9、NF-κB基因和蛋白水平。结果:与正常对照组比较,模型组PWTL、PWT评分、miR-19a mRNA表达水平显著降低,关节炎症积分、SOX9、NF-κB mRNA及蛋白表达水平显著升高(P<0.05);与模型组比较,地塞米松组、川续断总皂苷各剂量组PWTL、PWT评分、miR-19a mRNA表达水平显著升高,关节炎症积分、SOX9、NF-κB mRNA及蛋白表达水平显著降低(P<0.05),且川续断总皂苷组给药剂量与各指标变化呈正相关(P<0.05);川续断总皂苷组低、中剂量组与地塞米松组比较,各指标差异有统计学意义(P<0.05)。正常对照组关节软骨结构完整,未见炎症细胞浸润;模型组软骨层明显增厚,软骨细胞疏松紊乱,大量炎症细胞浸润及软骨成纤维细胞增生;地塞米松组及川续断总皂苷高剂量组关节软骨结构较为完整,纤维组织增生、炎症细胞浸润及软骨细胞坏死均减少;川续断总皂苷低、中剂量组具有少量炎症细胞浸润,但软骨结构疏松紊乱,剂量依赖效应明显。结论:川续断总皂苷能明显减轻骨关节炎软骨损伤,减轻骨关节炎软骨炎症反应;其机制与川续断总皂苷可上调miR-19a的表达进而激活SOX9 mRNA和蛋白表达,抑制NF-κB mRNA和蛋白表达有关。

Objective:To investigate the effects of total saponins of Dipsacus asperoides on SOX9/NF-κB signaling pathway in rat’s osteoarthritic chondrocytes through regulating miR-19a.Methods:Totally 120 SD rats were randomly divided into 6 groups:the normal control group,the model group,dexamethasone group(30 mg·kg-1),total saponins of Dipsacus asperoides low,medium and high dose groups(10,20 and 40 mg·kg-1)with 20 rats in each group.Except the normal control group,the other groups were used to establish the model of knee osteoarthritis in rats.The dexamethasone group and the total saponins of Dipsacus asperoides groups were given corresponding dose of drug(1.0 ml/100 g of gastric volume)by gavage on the first day of successful modeling and treated for 4 weeks.The normal control group and the model group were given the equal volume of normal saline.After the administration,the arthritis score,mechanical pain threshold(PWT)and thermal stimulation latency(PWTL)were measured.RT-PCR and Western blot were used to detect the levels of miR-19a gene,SOX9,NF-κB gene and protein in chondrocytes of knee joint.Results:Compared with those in the normal control group,PWTL,PWT and miR-19a mRNA expression levels decreased significantly in the model group,and joint inflammation score,SOX9,NF-κB mRNA and protein expression levels were increased significantly(P<0.05)as well.Compared with those in the model group,PWTL,PWT and miR-19a mRNA expression levels in the dexamethasone group and the total saponins of Dipsacus asperoides groups were increased significantly,and the joint inflammation score,SOX9,NF-κB mRNA and protein expression levels were decreased significantly(P<0.05).There was a positive correlation between the dose of total saponins of Dipsacus asperoides and the changes of various indicators(P<0.05).There were statistically significant differences in the indices between the total saponins of Dipsacus asperoides low-dose group and medium-dose group(P<0.05).In the normal control group,the articular cartilage structure was intact without inflammatory cell infiltration.In the model group,the cartilage layer was significantly thickened,and the chondrocytes were loose and disordered,a large number of inflammatory cells infiltrated and cartilage fibroblasts proliferated.In the dexamethasone group and the high-dose total saponins of Dipsacus asperoides group,the articular cartilage structure was relatively complete,and the proliferation of fibrous tissue,inflammatory cell infiltration and chondrocytes necrotic were reduced.In the low and middle dose of total saponins of Dipsacus asperoides groups,there were a small amount of inflammatory cell infiltration,but the cartilage structure was loose and dose-dependent effect was obvious.Conclusion: Total saponins of Dipsacus asperoides can significantly reduce osteoarthritiscartilage injury and reduce osteoarthritis cartilage inflammation. Its mechanism is related to the up-regulation of the expression of miR-19a,the activation of SOX9 and protein expression,and the inhibition of the expressions of NF-κB and protein.