环巴胺对佐剂性关节炎大鼠关节软骨细胞增殖凋亡的影响及其抗凋亡机制

Effects of cyclopamine on the proliferation and apoptosis of chondrocytes in adjuvant-induced arthritis rats and its anti-apoptotic mechanisms

目的研究Hedgehog(Hh)信号通路阻断剂环巴胺对佐剂性关节炎(AIA)大鼠关节软骨细胞增殖凋亡的影响及抗凋亡机制。方法弗氏完全佐剂诱导AIA大鼠模型,胰酶-胶原酶法分离培养关节软骨细胞,环巴胺体外给药处理AIA软骨细胞,MTT法检测细胞增殖,DNA电泳、Hoechst染色、Annexin V-FITC/PI双染检测细胞凋亡,RT-PCR检测Shh、Gli1、Ptch1、Bcl-2、Bax和Caspase-3 mRNA的表达。结果环巴胺(0.08、0.4、2、10、50μmol/L)剂量依赖性地提高AIA软骨细胞增殖。AIA组可见凋亡细胞DNA梯状条带,环巴胺(0.4、2、10μmol/L)给药组的梯状条带明显减少;AIA组存在核碎裂与染色质固缩,环巴胺给药组均匀蓝色荧光的细胞数量增多;流式分析结果表明AIA软骨细胞凋亡率显著升高,环巴胺明显减少细胞凋亡率;与正常组相比,AIA组软骨细胞中Hh信号通路相关基因(Shh、Ptch1、Gli1)mRNA表达显著升高,抗凋亡基因Bcl-2 mRNA表达显著下降,而促凋亡基因Bax、Caspase-3 mRNA表达明显升高。环巴胺体外给药能抑制Hh信号通路过度活化,提高Bcl-2 mRNA表达,并降低Bax、Caspase-3 mRNA表达。结论环巴胺体外给药能促进AIA软骨细胞的增殖,并抑制AIA软骨细胞的过度凋亡;该抗凋亡作用和调节Bcl-2、Bax和Caspase-3表达密切相关,提示干预软骨细胞Hh信号通路对保护类风湿关节炎关节软骨具有潜在的治疗意义。

Objective To explore the effects of cyclopamine, a hedgehog （ Hh） signal pathway inhibitor, on the proliferation and apoptosis of articular chondrocytes in adjuvant-induced arthritis （ AIA） rats and its anti-apoptotic mechanisms. Methods Freund’ s complete adjuvant was used to induce AIA. Articular chondrocytes were isolated and cultured by trypsin and collagenase digestion method. AIA articular chondrocytes were treated by cyclopamine in vitro. The cell proliferation was detected by MTT assay. The cell apoptosis was evaluated with DNA agarose gel electrophoresis, hoechst staining and flow cytometry analysis. The levels of Shh, Ptch1, Gli1, Bcl-2, Bax and Caspase-3 mRNA were detected by RT-PCR. Results Cyclopamine （0. 08, 0. 4, 2, 10, 50 μmol/L） could in-crease AIA articular chondrocytes proliferation in a dose-dependent. DNA ladder pattern was formed typically in AIA articular chondrocytes, while cyclopamine （0. 4, 2, 10μmol/L） treatment could reduce the formation of DNA ladder . AIA articular chondrocytes displayed nuclei fragmentation with condensed chromatin and cyclopamine could promote the number of uniform blue fluorescent cells. Flow cytometry analysis also indicated cyclopamine could vis-ibly reduce the rate of apoptotic cells in AIA articular chondrocytes. Compared with normal group, the levels of Hh pathway-related genes （ Shh, Ptch-1 and Gli1 ） mRNA apparently increased in AIA articular chondrocytes. Anti-apoptotic gene Bcl-2 mRNA level significantly decreased, while pro-apoptotic genes Bax and Caspase-3 mRNA lev-els were significantly increased. Cyclopamine treatment could inhibit the excessive activation of Hh pathway, up-regulate Bcl-2 mRNA expression, and reduce Bax and Caspase-3 mRNA expressions. Conclusion Cyclopamine could promote the proliferation and inhibit excessive apoptosis of AIA articular chondrocytes, which might be relat-ed to regulation of Bcl-2, Bax, and Caspase-3 expressions. Our results suggest that intervention of Hh signal in ar-ticular chondrocytes has potential therapeutic significance for articular cartilage protection in rheumatoid arthritis.