藿香蓟叶提取物对大鼠创伤性骨关节炎的治疗作用

Therapeutic Effect of Leaf Extract of A geratum conyzoides L.on Post-traumatic Osteoarthritis in Rats

目的:探究藿香蓟叶提取物(ACLE)对创伤性骨关节炎(PTOA)模型大鼠的治疗作用及其可能的分子机制。方法:SPF级雄性SD大鼠采用前交叉韧带切断法构建PTOA大鼠模型,然后将建模成功的大鼠随机分为模型组、藿香蓟叶提取物2.5 g组(ACLE2.5 g组)、藿香蓟叶提取物5 g组(ACLE 5 g组)和藿香蓟叶提取物10 g组(ACLE 10 g组),每组21只。另取21只SD大鼠设为假手术组,大鼠只执行前交叉韧带暴露手术,但是不切断。ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠在造模结束后分别灌胃给药,剂量分别为2.5 g/kg、5 g/kg和10 g/kg,持续给药处理8周。假手术组和模型组大鼠给予等量生理盐水每日灌胃处理,同样持续干预处理8周。给药结束后,收集各组大鼠的血清、膝关节灌洗液和膝关节软骨组织。采用HE染色(Mankin’s评分)和番红O+固绿染色(OARSI评分)评估大鼠关节损伤情况。采用ELISA试剂盒检测大鼠血清中ALP、BGP和CTX-Ⅰ水平,以及大鼠血清和关节灌洗液中TNF-α、IL-6和IL-1β水平。采用试剂盒检测大鼠血清和关节灌洗液中MDA、SOD、GSH-Px和ROS水平。采用Western blot检测大鼠软骨组织中Aggrecan、SOX-9、ColⅡ、MMP-13、Wnt1、β-catenin、Bcl-2和Bax蛋白表达水平。结果:模型大鼠的膝关节软骨组织损伤严重,软骨层状组织结构遭受破坏;ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠膝关节软骨组织结构和病理状态均得到不同程度的恢复。与模型组比较,ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠Mankin’s和OARSI的评分值均降低(P<0.01)。与模型组比较,ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠血清中ALP和BGP水平升高,CTX-Ⅰ水平降低(P<0.05)。与模型组比较,ACLE 2.5 g组、ACLE 5g组和ACLE 10 g组大鼠血清和膝关节灌洗液中IL-6、IL-1β、TNF-α、NO和MDA水平均降低(P<0.05),SOD和GSH-Px水平均升高(P<0.05)。与模型组比较,ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠关节软骨组织中Aggrecan、SOX-9、ColⅡ和Bcl-2蛋白表达水平均升高(P<0.05),MMP-13、Wnt1、β-catenin和Bax蛋白表达水平均降低(P<0.05)。结论:ACLE能够降低PTOA大鼠膝关节软骨组织中炎症反应水平,增强抗氧化能力;抑制Wnt/β-catenin通路的过度激活,通过升高Aggrecan、ColⅡ、SOX-9和Bcl-2的表达,促进软骨细胞外基质的恢复,并抑制软骨细胞的凋亡,从而改善PTOA大鼠软骨组织损伤程度。

Objective:To explore the therapeutic effect of A geratum conyzoides L.leaf extract(ACLE)on rats with post-traumatic osteoarthritis(PTOA)and its possible molecular mechanism.Methods:PTOA rat model was established by anterior cruciate ligament amputation in SPF male SD rats.Then,the PTOA rats were randomly divided into model group,ACLE 2.5 g group,ACLE 5 g group and ACLE 10 g group,with 21 rats in each group.Another 21 SD rats were selected as sham group,and only the anterior cruciate ligament was exposed,but not severed.The rats in ACLE 2.5 g group,ACLE 5 g group and ACLE 10 g group were administrated by intragastric administration at doses of 2.5 g/kg,5 g/kg and 10 g/kg,respectively,and the treatment was continued for 8 weeks.Rats in sham group and model group were given the same amount of normal saline intragastric treatment every day for 8 weeks.After the administration,the serum,knee lavage fluid and knee cartilage tissue of each group were collected.The joint injury of rats were evaluated by HE staining(Mankin's score)and saffron O+solid green staining(OARSI score).The levels of ALP,BGP and CTX-I in rat serum,as well as the levels of TNF-α,IL-6 and IL-1βin rat serum and decoction were detected by ELISA kit.The levels of MDA,SOD,GSH-Px and ROS in serum and knee lavage solution of rats were determined by the kit.The protein expression levels of Aggrecan,SOX-9,Col II,MMP-13,Wntl,β-catenin,Bcl-2 and Bax in rat cartilage were detected by Western blot.Result:The knee cartilage tissue of model rats was seriously damaged and the layered structure of cartilage was destroyed.The knee cartilage tissue structure and pathological status of rats in ACLE 2.5 g,ACLE 10 g and ACLE 2.5 g groups were recovered to different degrees.The knee cartilage tissue of model rats was seriously damaged and the layered structure of cartilage was destroyed.The knee cartilage tissue structure and pathological status of rats in ACLE 2.5 g,ACLE 10 g and ACLE 2.5 g groups were recovered to different degrees.Compared with model group,Mankin's and OARSI scores of rats in ACLE 2.5 g,ACLE 5 g and ACLE 10 g groups were decreased(P<0.01).Compared with model group,ALP and BGP levels in serum of ACLE 2.5 g,ACLE 5 g and ACLE 10 g groups were increased,while CTX-I levels were decreased(P<0.05).Compared with model group,the levels of IL-6,IL-1β,TNF-α,NO and MDA in serum and knee lavage solution of rats in ACLE 2.5 g,ACLE 5 g and ACLE 10 g groups were decreased(P<0.05),while the levels of SOD and GSH-Px were increased(P<0.05).Compared with model group,Aggrecan,SOX-9,Col II and Bcl-2 protein expression levels in articular cartilage of rats in ACLE 2.5 g ACLE 5 g and ACLE 10 g groups were increased(P<0.05).The protein expression levels of MMP-13,Wntl,β-catenin and Bax were decreased(P<0.05).Conclusion:ACLE can decrease the level of inflammatory response and enhance the antioxidant capacity in the cartilage tissue of PTOA rats.The over-activation of Wnt/β-catenin pathway was inhibited by ACLE,and the recovery of extracellular matrix of chondrocytes was promoted and the apoptosis of chondrocytes was inhibited by increasing the expression levels of Aggrecan,Col II,SOX-9 and Bcl-2,thus improving the degree of cartilage tissue injury in PTOA rats.