Expression And Some Function Research Of Hedgehog Signaling Pathway In Articular Cartilage Of Adjuvant-induced Arthritis In Rats

Objective We aimed to study the expression of hedgehog(Hh) signaling pathway in articular cartilage of adjuvant-induced arthritis(AIA) in rats and its pathologic significance. 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. The protein levels of Hh pathway related molecules(Shh, Ptch1, Smo and Gli1) in articular cartilage were observed by immunohistochemistry. The m RNA levels of Shh, Ptch1, Smo, Gli1, type-II collagen(COII) and aggrecan in articular cartilage were assayed by real-time quantitative PCR.In addition, we analyzed the possible relationships between Shh, Ptch1, Smo and Gli1 m RNA levels and the degree of articular cartilage damage in AIA rats including OARSI scores and COII, aggrecan m RNA levels. Articular chondrocytes were isolated and cultured by trypsin and collagenase digestion method. AIA rats 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 m RNA were detected by RT-PCR.Results 1.Expression of Hh pathway in articular cartilage of AIA rats and its pathologic significance We successfully established the AIA rat model.The protein and m RNA levels of Shh, Ptch1, Smo and Gli1 showed higher levels in articular cartilage of AIA rats compared with the normal rats and the m RNA levels of COII and aggrecan is apparently decreased in articular cartilage of AIA rats.The m RNA levels of Shh, Ptch1, Smo and Gli1 in articular cartilage of AIA rats significantly correlated positively with OARSI scores on articular cartilage, as well as significantly correlated negatively with COII and aggrecan m RNA level in articular cartilage.2.Effects of cyclopamine on the proliferation and apoptosis of chondrocytes in adjuvant-induced arthritis rats and its anti-apoptotic mechanisms Cyclopamine(0.08, 0.4, 2, 10, 50 μmol/L) could increase AIA rats articular chondrocytes proliferation in a dose-dependent. DNA ladder pattern was formed typically in AIA rats articular chondrocytes, while cyclopamine(0.4, 2, 10 μmol/L) treatment could reduce the formation of DNA ladder. AIA rats 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 visibly reduce the rate of apoptotic cells in AIA rats articular chondrocytes.Compared with normal group, the levels of Hh pathway-related molecules(Shh, Ptch-1 and Gli1) m RNA apparently increased in AIA rats articular chondrocytes. Anti-apoptotic gene Bcl-2 m RNA level significantly decreased, while pro-apoptotic genes Bax and Caspase-3 m RNA levels were significantly increased. Cyclopamine treatment could inhibit the excessive activation of Hh pathway, up-regulate Bcl-2 m RNA expression, and reduce Bax and Caspase-3 m RNA expressions.Conclusion 1.Hh signal pathway was over-activated in articular cartilage of AIA rats, and the elevated Hh signal was apparently associated with the pathologies of articular cartilage damage in AIA rats, our findings suggest that it is expected to become a new target in the treatment of rheumatoid arthritis. 2.Cyclopamine could promote the proliferation and inhibit excessive apoptosis of AIA rats articular chondrocytes, which might be related to regulation of Bcl-2, Bax, and Caspase-3 expressions. Our results suggest that intervention of Hh signal in articular chondrocytes has potential therapeutic significance for articular cartilage protection in rheumatoid arthritis.