Effect Of Cyclopamine On The Articular Cartilage Cell Proliferation Of Adjuvant Arthritis Rat In Vitro

Background Destruction of articular cartilage is disabled node of pathological changes in rheumatoid arthritis(RA),effective prevention and treatment of the destruction of articular cartilage and bone tissue is a key link in the process of treating RA,which may be impedance or even inverse RA pathology process,reduce RA morbidity.Hedgehog(Hh)signaling pathway is common in animals which has evolutionary conservative,and adjustable tissue differentiation,tumor formation,etc.,studies have shown that articular cartilage cells in patients with osteoarthritis have activatein Hh signaling pathways,and Hh signaling pathway inhibitor(cyclopamine)specific blocking or cyclopamine gene silence,can effectively alleviate the condition of patients with osteoar thritis,prevention and treatment of joint disease obviously,but research of this pathway in RA is less.Objective To establish adjuvant arthritis(AA)animal model with SD rats,and to observe the effect of Hh pathway inhibitorscyclopamine on articular cartilage cell activation effect of AA rats in vitro,and to provide new scientific basis for the pathogenesis of RA.Methods SD rats were randomly divided into 2 groups,control group and model group,20 in each group.(1)Evaluation model AA rats after articular cartilage damage: freund's complete adjuvant foot intradermal injection induced animal model of AA rats,control group foot intradermal injection amount of normal saline,tested secondary arthritis foot swelling degree periodically;(2)HE staining: observation of normal group and model group,compared two groups of joint cavity synovial tissue and subsynovial cartilage growth;(3)Culture of articular cartilage cells in vitro,identification and cell proliferation experiment: take AA rats ankle joint cartilage,the pancreatic enzyme-collagen enzymatic isolation,culture,cell chemical toluidine blue staining and immunohistochemical type II collagen appraisal culturing synovial cells;(4)Electron microscope observation: the morphological characteristics of chondrocytes in normal and model groups were observedwith transmission electron microscopy.(5)Cell proliferation experimentof administration withcyclopaminein vitro: cyclopamine(0,0.03,0.3,3,10)administionin vitro,CCK-8 was used to detect the effect of cyclopamine treatment on the proliferation of chondrocytes in AA rats;(6)Effect of cyclopamine on the apoptosis intervention of cartilage cells in vitro: chondrocytes culturedin vitro,cyclopamine(0,0.03,0.3,3,10 mu mol/L)in vitro,application of Hoechst 33258 testingcyclopamine effect on AA rat chondrocytes apoptosis;(7)Cyclopamine intervention of Hh signaling pathwaysin vitro: the influence of the exponential phase of chondrocytes,joinedcyclopamine with different concentrations(end concentrationof cyclopamine 0,0.3,3,10 ?mol/L)in vitro,the proteins expression of Shh,Ptch1 and Gli1 were detected and image analyzedby Western blot in vitro Hh signaling pathways blocking of cyclopamine in ankle joint chondrocytes of AA rats.Result(1)Compared with normal rats,the arthritis index and secondary foot swelling degree increased significantlyin AA rats;(2)HE staining showed the AA rats ankle joint lumen,articular synovial hyperplasia and pannus formation,inflammatory cells infiltration,synovial membrane under cartilage destruction;(3)Chondrocytes identified,and the coloration of both methylaniline blue and type II collagen stained chondrocytes,indicating the success cultured of AA rat's ankle chondrocytes in vitro;(4)Under the electron microscope,there were swelling of rough endoplasmic reticulum and golgi complex in the cytoplasm of the rat chondrocyte in AA model,and the loss of mitochondrial ridge.(5)The proliferation of articular chondrocyteswas observedby CCK-8 method,indicating that cyclopamine(0.3,3,10?mol/L)could increase the proliferation of articular chondrocytes in AA modelin vitro.(6)Hoechst 33258 testingshowed that cyclopamine could reduce the apoptosis rate of chondrocytes in AA model;(7)Compared with normal group rats,Western blot test showed that Shh,Ptch-1,Gli1 protein expression declined significantlyby cyclopamine(0.3,3,10 ?mol/L)in vitro in AA model group rats articular cartilage.Conclusion: This experiment was established successfullyby freund's complete adjuvant induced AA rat model,AA rats ankle joint cartilage cells in vitro culture,cyclopamine can increase cartilage cell proliferationin vitro,inhibit the apoptosis of cartilage cells,the inhibition of AA rat chondrocytes Hh signal.