Effects Of YAP On Proliferation And Differentiation Of Sika Deer Antler Mesenchymal Cells And Chondrocytes

Antler horn is a organ in mammals that can be completely regenerated after loss.Therefore,antler horns provide an ideal model for studying mammalian organ regeneration.At the same time,antler is also a valuable traditional Chinese medicine with a long history of use.The antler horn growth process includes the differentiation of periosteal stem cells into mesenchymal cells and mesenchymal cells into chondrocytes.In this series of processes,various cells continue to proliferate and differentiate under the action of some growth factors.In the dynamic equilibrium,the growth and development of antler horns are maintained.YAP（Yes-associated protein）is a key switch protein in the Hippo signaling pathway.It regulates cell proliferation and survival during normal cell growth and development,and plays an important role in controlling organ growth,stem cell self-renewal,and cell differentiation.In this experiment,sika deer was used as the research object.The effects of YAP on sika deer antler mesenchymal cells and chondrocyte proliferation and differentiation were studied by EDU method,flow cytometry,and real-time PCR,and the regulation mechanism was discussed.Deer antler mesenchymal cells were isolated and cultured,and a certain concentration of YAP inhibitor（Verteporfin）was added.The results show,Verteporfin can significantly inhibit the expression of YAP in antler mesenchymal cells and significantly inhibit the proliferation activity of antler mesenchymal cells.It was found by flow cytometry that Verteporfin increased the percentage of G0 / G1 phase cells,decreased the percentage of S phase cells,and inhibited the proliferation of mesenchymal cells.At the same time,Verteporfin reduced the expression of Ccnd1,Ccnd2,Ccnd3,Ccne1,Cdk2,Cdk4 and Cdk6.Verteporfin can promote the expression of chondrocyte marker molecules COLⅡ,SOX9 and AGC in mesenchymal cells at 12 h,24 h,36 h,48 h and 72 h.The effect of YAP on ROS in mesenchymal cells was further studied,and the results showed that Verteporfin increased the ROS content in mesenchymal cells.Superoxide anion(O^2-)is one of the main components of ROS.Mitochondria are an important source of ROS.The results show that Verteporfin promotes the increase of O^2-and mitochondrial ROS（mtROS）content in mesenchymal cells.In order to investigate the effect of ROS on mesenchymal cell differentiation,2 hours after Verteporfin treatment,antioxidants GSH and NAC were added for 24 hours.The results showed that the levels of COLⅡ,SOX9 and AGC mRNA in mesenchymal cells were significantly reduced,indicating that ROS in mesenchymal cells play an important role in the differentiation process.Antler chondrocytes were isolated and cultured,and a certain concentration of Verteporfin was added.The results show,Verteporfin can significantly inhibit the expression of YAP in antler chondrocytes and promote the proliferative activity of chondrocytes.It was found by flow cytometry that Verteporfin can reduce the percentage of chondrocytes in GO/G1 phase,increase the percentage of S-phase cells,and promote the proliferation of chondrocytes.At the same time,Verteporfin increased Ccnd1,Ccnd2,Ccnd3,Ccne1,Cdk2,Cdk4 and Cdk6 mRNA levels in chondrocytes.Verteporfin treated chondrocytes for 6 h,12 h,24 h,36 h,and 48 h.The results showed that Verteporfin inhibited the expression of COLX,RUNX2 and ALPL,the marker molecules of hypertrophic chondrocytes in chondrocytes.The effect of YAP on ROS in velvet chondrocytes was further studied.It was found that Verteporfin increased the content of ROS and O^2-in chondrocytes,and increased the content of MDA,indicating that chondrocytes had oxidative stress and lipid oxidation.At the same time,Verteporfin reduced the levels of mtROS,ATP and mtDNA in chondrocytes and damaged mitochondrial function.The effects of YAP on the Nrfe-ARE signaling pathway and antioxidants in chondrocytes were further studied.The results showed that Verteporfin reduced the levels of Nrf2 and ARE,while the levels of SOD decreased significantly,and the levels of CAT,GPX,GR,GSSH and GSH significantly increased.In order to further investigate whether YAP regulates antler chondrocyte differentiation through mediating ROS,2 hours after Verteporfin treatment,antioxidants GSH and NAC were added for 24 hours.The results showed that the levels of COLX,RUNX2 and ALPL mRNA in chondrocytes increased.This indicates that YAP has an effect on antler chondrocyte differentiation through mediating ROS.