加味独活寄生合剂对膝骨关节炎模型兔膝关节软骨组织细胞自噬及凋亡相关蛋白的影响

Effect of Jiawei Duhuo Jisheng Mixture (加味独活寄生合剂) on Autophagy and Apoptosis-related Proteins of Articular Cartilage Tissue in Rabbit Model of Knee Osteoarthritis

目的 探讨加味独活寄生合剂治疗膝骨关节炎的可能作用机制。方法 46只雄性新西兰大白兔随机分为空白组(8只)、常规模型组(19只)和3MA自噬抑制剂造模组(19只)。常规模型组使用高分子树脂石膏绷带固定兔右侧后肢建立膝骨关节炎模型,3MA自噬抑制剂造模组在上述造模基础上加用自噬抑制剂3MA关节腔注射造模,造模周期为6周,两个造模组均造模成功各16只。将常规造模组分为模型组和加味独活寄生合剂组,3MA自噬抑制剂组分为3MA组和3MA+加味独活寄生合剂组,每组8只。加味独活寄生合剂组和3MA+加味独活寄生合剂组均给予加味独活寄生合剂单次生药量为2.26 g/kg灌胃,其余各组予以3.4 ml/kg蒸馏水灌胃,每日2次,持续4周。给药结束后观察各组兔膝关节软骨大体情况,苏木素-伊红(HE)染色检测软骨组织病理学变化,并分别通过Pelletier评分、Mankin评分进行量化评估;检测各组兔关节软骨组织中自噬相关蛋白[自噬相关基因7 (ATG7)、自噬关键分子酵母Atg6同系物1 (Beclin-1)、微管相关蛋白1轻链3 (LC3)]及凋亡相关蛋白[B淋巴细胞瘤-2 (Bcl-2)、半胱氨酸蛋白酶3 (Caspase3)]mRNA及蛋白表达水平。结果 与空白组比较,模型组兔膝关节软骨病理损伤明显,Pelletier评分及Mankin评分升高,软骨组织中Beclin-1、LC3、ATG7、Bcl-2、Caspase-3 mRNA表达均升高,Beclin-1、LC3、ATG7、Caspase-3蛋白表达升高(P<0.05或P<0.01)。与模型组比较,加味独活寄生合剂组关节软骨损伤改善,Pelletier评分及Mankin评分降低,软骨组织中Beclin-1、 LC3、 ATG7、 Bcl-2 mRNA及蛋白表达升高,Caspase-3 mRNA及蛋白表达降低(P<0.05或P<0.01)。与加味独活寄生合剂组比较,3MA+加味独活寄生合剂组Pelletier评分、Mankin评分升高,软骨组织中Beclin-1、LC3、ATG7、Bcl-2 mRNA及蛋白表达降低,Caspase-3 mRNA表达升高(P<0.05或P<0.01)。结论 加味独活寄生合剂对膝骨关节兔的关节软骨具有保护作用,其机制可能是通过诱导软骨组织自噬、抑制细胞凋亡实现的。

Objective To explore the possible mechanism of Jiawei Duhuo Jisheng Mixture(JDJM,加味独活寄生合剂)in the treatment of knee osteoarthritis(KOA).Methods Forty-six male New Zealand white rabbits were randomly divided into blank group(n=8),conventional model group(n=19)and 3MA autophagy inhibitor model group(n=19).The conventional model group used polymer resin plaster bandage for immobilization of the right hindlimb to establish the KOA model,while the 3MA autophagy inhibitor model group used the autophagy inhibitor 3MA by joint cavity injection additionally to establish the model,with a period of six weeks.After successful modeling,the conventional model group was divided into model group and JDJM group,while the 3MA autophagy inhibitor group was divided into 3MA group and 3MA+JDJM group,with eight rabbits in each group.Both the JDJM group and the 3MA+JDJM group were given 2.26 g/kg of JDJM by gavage,while the other groups were given 3.4 mL/kg of distilled water by gavage,twice daily for four weeks.The cartilage of the knee joints was observed,and the histopathological changes of cartilage were detected by hematoxylin-eosin(HE)staining and quantitatively evaluated by Pelletier's and Mankin's scoring criteria,respectively.The mRNA and protein expression levels of autophagy-related proteins including autophagy-related 7(ATG7),autophagy key molecule yeast Atg6 homolog 1(Beclin-1),microtubule-associated protein 1 light chain 3(LC3)and apoptosis-related proteins including B-cell lymphoma 2(Bcl-2)and cysteine protease 3(Caspase3)in cartilage tissues of rabbits were measured.Results Compared to those in the blank group,the cartilage damage in the model group was obvious;the Pelletier score and Mankin score significantly increased,and the mRNA expression levels of Beclin-1,LC3,ATG7,Bcl-2 and Caspase-3 as well as the protein expression levels of Beclin-1,LC3,ATG7 and Caspase-3 significantly increased(P<0.05 or P<0.01).Compared to those in the model group,the articular cartilage damage was improved in the JDJM group;the Pelletier score and Mankin score were reduced;the mRNA and protein expression levels of Beclin-1,LC3,ATG7 and Bcl-2 significantly increased,while those of Caspase-3 in the cartilage tissue were reduced(P<0.05 or P<0.01).Compared to the JDJM group,the 3MA+JDJM group had higher Pelletier score and Mankin score,lower mRNA and protein expression levels of Beclin-1,LC3,ATG7,and Bcl-2,and higher Caspase3 mRNA expression level(P<0.05 or P<0.01).Conclusion JDJM has a protective effect on the articular cartilage of knee osteoarthritic rabbits,which may be achieved by inducing cartilage autophagy and inhibiting apoptosis.