Enhancement Of Articular Chondrocytes Proliferation And Cellular Phenotype Maintenance By Icariin And Possible Mechanisms

Part1Icariin promotes articular chondrocytes proliferation and maintenance of chondrocytic phenotypeObjective The present study is aimed to determine the effects of icariin on articular chondrocytes proliferation and preservation of chondrocytic phenotype.Methods The effect of icariin on proliferation of chondrocytes was measured with CCK8 and flow cytometry. The expression of PCNA, Ki67, CyclinDl, SOX9, COL2 and Aggrecan in chondrocytes treated with icariinwas determined with RT-qPCR, Western Blot analysis or immunofluorescence.Results The proliferation of chondrocytes and the expression of PCNA, Ki67, CyclinD1, SOX9, COL2 and Aggrecan were improved by icariin.Conclusion Icariin significantly enhanced the proliferation of articular chondrocytes and preserves the chondrocytic phenotype through overexpression of SOX9, COL2 and Aggrecan.Part 2The role of YAP in the proliferation of articular chondrocytes promoted by icariinObjective This study was performed to explore the role of YAP in the proliferation of articular chondrocytes promoted by icariinMethods Expression of YAP and its target genes Ankrdl and CTGF in chondrocytes treated with icariin was detected withRT-qPCR and Western Blot analysis. Expression of Ankrdl, CTGF, PCNA, Ki67 and CyclinDl was measured with RT-qPCR and Western Blot analysis as YAP was knocked down via shRNA in chondrocytes with and without treatment of icariin. Expression of YAP and CTGF in articular cartilage was detected in vivo with immunohistochemistry after the rats were fed with icariin at 2mg per day for 30 days.Results Icariin enhanced the expression of YAP and its taget genes Ankrdl and CTGF in vitro. Expression of Ankrd1, CTGF, PCNA, Ki67 and CyclinDl decreased after the knockdown of YAP via shRNA whether the cells were treated by icariin or not. Icariin also enhanced the expression of YAP and CTGF in articular cartilage of rats. Conclusion Icariin promote the proliferation of articular chondrocytes via YAP. Oral application of icariin can achieve effective drug concentration in rat articular cartilage.Part 3The molecular mechanism on phosphorylation modification of YAP caused by icariinObjective The purpose of this study is to understand the mechanism of phosphorylation modification of YAP caused by icariin and to determine the relationship of F-actin and Hippo pathway in regulating the activity of YAP.Methods After treated by icariin, the levels of p-YAP, p-MYPT and p-Cofilin were measured by Western Blot analysis and the subcellular localization of YAP was determined with immunofluorescence, by which the activity of YAP and ROCK pathway was evaluated. The level of p-YAP was measured with Western Blot analysis and the structure of F-actin cytoskeleton was observed with immunofluorescence, after chondrocytes were treated with icariin, latrunculin B or the both of them. We utilized it to determine whether F-actin was the only target of icariin in regulating phosphorylation modification of YAP. Moreover, a technique of isobaric tags for relative and absolute quantitation (iTRAQ) was applied to search for another possible mechanism on phosphorylation modification of YAP caused by icariin.Results Icariin increased phosphorylation of MYPT and Cofilin and led to reduced phosphorylation and nuclear localization of YAP.Although it could not prevent the depolymerization of F-actin caused by latrunculin B, it was able to reduce phosphorylation of YAP resulting from depolymerization of F-actin. Further more, the level of SAV1 and p-Mob was decreased by icariin.Conclusion YAP can be activated by icariin via enhanced activity of ROCK pathway, increased polymerization of F-actin and reduced activity of Hippo pathway. F-actin is a possible upstream regulator of the Hippo pathway.