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目的探讨Hedgehog(Hh)信号通路抑制剂环巴胺体外对佐剂性关节炎大鼠(AA)模型的关节软骨细胞增殖的影响和部分机制。方法弗氏完全佐剂诱导AA大鼠,测量关节炎指数和继发性足肿胀度,HE染色观察两组软骨组织生长情况;取AA大鼠踝关节软骨组织,采用胰蛋白酶-胶原酶法分离、培养、鉴定,环巴胺(0、0.05、0.5、5、20μmol/L)体外给药,MTT法检测AA大鼠踝关节软骨细胞增殖,Annexin V-FITC/PI双染检测AA大鼠踝关节软骨细胞凋亡,Western blotting检测AA大鼠踝关节软骨细胞Shh、Ptch1、Gli1的蛋白表达。结果弗氏完全佐剂诱导后,与正常大鼠相比,AA大鼠关节炎指数和继发性足肿胀度明显升高,HE染色显示,AA大鼠踝关节软骨组织有破坏;甲苯胺蓝和Ⅱ型胶原鉴定体外成功培养AA大鼠踝关节软骨细胞;体外给药环巴胺(0.05、0.5、5、20μmol/L)可升高AA模型关节软骨细胞增殖,流式细胞检测结果显示,环巴胺能降低AA模型软骨细胞凋亡率;与未用环巴胺组相比,环巴胺(0.5、5、20μmol/L)给药对AA软骨细胞中Hh信号通路相关蛋白(Shh、Ptch1、Gli1)表达显著下降。结论弗氏完全佐剂诱导建立AA大鼠模型成立,体外给药环巴胺可抑制AA大鼠软骨细胞的增殖,抑制软骨细胞的凋亡,该作用与抑制AA大鼠软骨细胞Hh信号有关。
Objective To investigate the effect and mechanism of Hedgehog (Hh) signaling pathway inhibitor cyclopamine on articular chondrocyte proliferation in adjuvant arthritis (AA) model in vitro. Methods Freund’s complete adjuvant was used to induce AA rats. The arthritis index and secondary paw swelling were measured. The growth of cartilage tissue was observed by HE staining. The articular cartilage of AA rats was separated by trypsin-collagenase (0, 0.05, 0.5, 5 and 20μmol / L) in vitro. The proliferation of AA rat articular cartilage was detected by MTT assay and the ankles of AA rats were detected by Annexin V-FITC / PI double staining Articular chondrocyte apoptosis was detected by Western blotting. The protein expressions of Shh, Ptch1 and Gli1 in AA rat articular cartilage were detected by Western blotting. Results Compared with normal rats, arthritis index and secondary paraneoplasticity of AA rats were significantly increased after Freund’s complete adjuvant induction. Compared with normal rats, the arthritis cartilage of AA rats was damaged by HE staining. Toluidine blue And type Ⅱ collagen were used to culture AA rat articular chondrocytes in vitro. Cyclodamine (0.05, 0.5, 5 and 20μmol / L) in vitro could increase the proliferation of articular chondrocytes in AA model. Flow cytometry Cyclopamine decreased the rate of chondrocyte apoptosis in AA model rats. Compared with the non-cyclopamine group, cyclopamine (0.5, 5, and 20μmol / L) significantly decreased the expression of Hh signaling pathway related proteins (Shh, Ptch1, Gli1) expression was significantly decreased. Conclusions Freund ’s complete adjuvant induced establishment of rat AA model. Cyclodamine administered in vitro inhibits the proliferation of AA rat chondrocytes and inhibits the apoptosis of chondrocytes in AA rats. This effect is related to inhibition of Hh signaling in AA rat chondrocytes.