O_(3)抑制Notch信号通路减少OA大鼠软骨细胞凋亡的实验研究

An experimental study on the reduction of chondrocyte apoptosis in OA rats by inhibiting the Notch signaling pathway with O_(3)

目的观察大鼠骨关节炎(OA)软骨细胞中Notch信号通路相关因子(Notch-1、Jagged1、keratin1、involucrin)及凋亡生物标志物(caspase-3、Bax、Bcl-2)的表达情况,探索臭氧(O_(3))作用于OA的具体机制。方法选取SD大鼠24只,按随机数字表分为空白组、模型组、模型+O_(3)治疗组,每组8只。使用木瓜蛋白酶合半胱氨酸混合液关节腔注射法复制OA模型(右膝),造模成功后,每周在模型+O_(3)治疗组大鼠右膝关节腔内注入2mLO_(3)(25μg/mL)1次,模型组注入等体积空气,空白组不做处理;治疗4周后处死大鼠,收集膝OA关节软骨组织,行HE染色观察软骨组织,免疫荧光检测caspase-3、Notch-1表达情况,WesternBlot检测Notch-1、Jagged1、Bcl-2、keratin1、involucrin的表达情况。结果HE染色观察显示,模型+O_(3)治疗组能有效改变软骨退变,使软骨细胞成簇增生,潮线间断。免疫组化及WesternBlot检测显示,与空白组相比,caspase-3、Notch-1、Jagged1和Bax蛋白含量在模型组中显著增高(P<0.05);模型+O_(3)治疗组与模型组比较,上述蛋白含量均有所下降,其中caspase-3和Notch-1蛋白含量与模型组相比下降程度更显著,差异有统计学意义(P<0.05);而Bcl-2、keratin1和involucrin蛋白含量在模型组显著降低(P<0.05),经O_(3)治疗后上述蛋白含量显著增高(P<0.05)。结论Notch信号通路通过调控OA软骨细胞凋亡参与OA发生发展的过程,而O_(3)能通过抑制Notch信号通路减少软骨细胞凋亡,保护关节软骨,延缓OA进展。

Objective To explore the specific mechanism of O_(3) acting on osteoarthritis(OA)by observing the expression of Notch signaling pathway-related factors(Notch-1,Jagged1,keratin1,involucrin)and apoptosis biomarkers(caspase-3,Bax,Bcl-2)in rat OA chondrocytes.Methods Twenty-four SD rats were selected and divided into blank group,model group and model+O_(3) treatment group randomly(8 rats in each group)before the reproduction of OA model(right knee)by intraarticular injection of papain-cysteine mixture.Every week after the successful modeling,2 mL of O_(3)(25μg/mL)was injected into the right knee joint cavity of the rats in the model+O_(3) treatment group,the model group was injected with an equal volume of air,and the blank group was not treated.After 4 weeks of treatment,the rats were sacrificed and the knee OA articular cartilage tissue was collected and observed by HE staining.The expression of caspase-3 and Notch-1 was detected by immunofluorescence and the expression of Notch-1,Jagged1,Bcl-2,keratin1,and keratin1 were detected by Western Blot.Results HE staining revealed that the model+O_(3) treatment group could effectively change the cartilage degeneration,make the chondrocytes proliferate in clusters and the tide line interrupted.Immunohistochemistry and Western Blot detection showed that,compared with the blank group,caspase-3,Notch-1,Jagged1 and the protein contents of Bax were significantly increased in the model group(P<0.05).Compared with the model group,the contents of the above proteins were decreased in the model+O_(3) treatment group,among which caspase-3 and the protein contents of Notch-1 decreased more substantially compared with the model group,which means the difference was statistically significant(P<0.05);Bcl-2,keratin1 and the protein contents of involucrin were greatly decreased in the model group(P<0.05),and the above protein contents were dramatically increased after O_(3) treatment(P<0.05).Conclusion The Notch signaling pathway participates in the development of OA by regulating the apoptosis of OA chondrocytes while O_(3) can reduce chondrocyte apoptosis by inhibiting the Notch signaling pathway and consequently protect articular cartilage,and delay the progression of OA.