The Mechanism Of Melatonin Regulating The Differentiation Of Antler Mesenchymal Cells Through MT1 Activation Of PI3K/AKT/mTOR

The quality and yield of the antler is an important indicator to evaluate the performance of deer.The medicinal value of antler is one of the main economic indicators of sika deer breeding,and antler is also the only regenerative organ in mammals.The growth of the antler is influenced by photoperiod and endogenous hormone levels,and shows periodic growth and shedding.Melatonin(MLT),an important hormone regulating circadian rhythm in animals,is shown to play an important role in the growth of antlers.The rapid growth of antler depends on the proliferation and differentiation of mesenchymal cells.Therefore,in the present study,deer antler mesenchymal cells were treated in vitro with melatonin to investigate cell proliferation,cycle,and secretion levels of relevant growth factors.At the same time,RNA interference technology was used to specifically knock down melatonin receptor1A(MTNR1A,MT1)to explore the effect of melatonin and its receptor on antler mesenchymal cells differentiation and the related regulatory mechanism.The main findings were as follows:(1)After treating deer antler mesenchymal cells with melatonin at concentrations of 0,1,5,10,20,and 50 μM for 24,48,and 72 hours,cell viability was measured.The results showed that the cell viability of deer antler mesenchymal cells in the 1 μM treatment group was significantly increased(p<0.001);By detecting the proliferation of EDU cells,the positive rate of EDU in the 1 μM melatonin treated group was significantly higher than that in the control group(p<0.01)(2)Anttake mesenchymal cells were treated with melatonin(1 μM)to detect the expression of mesenchymal markers and cell cycle genes.The results showed that the expressions of mesenchymal markers and cell cycle-and proliferation-related genes were significantly increased in the treatment group.The expression of chondrocyte markers Col2a1,Col1 and Aggrecan in the mesenchymal cells was significantly increased(p<0.01),and the gene expression of TGF-β1 was also significantly increased(p<0.01).(3)Western blot results showed that the expression of p-m TOR and p-4EBP1 was significantly up-regulated in the process of melatonin promoting the proliferation of deer antler mesenchymal cells(p<0.05).(4)Differentiation experiments were performed by adding 1 μM melatonin and differentiation induction medium to the cell culture medium of antler mesenchymal cells.The results of staining showed that mesenchymal cells were differentiated.The q RT-PCR results showed that the gene expression of Aggrecan and Col2a1 was up-regulated,and the gene expression of Cyclin D1 and TGF-β1 was also up-regulated.(5)Western blot results showed that the protein expressions of PI3 K,p-AKT,p-m TOR and p-4EBP1 were significantly increased during the differentiation of antler mesenchymal cells induced by melatonin(p<0.001).(6)MT1 si RNA was used to interfere the expression of MT1 in antler mesenchymal cells,and the results showed that the gene expression of BMP2 and Runx2 in si-MT1+MLT group was down-regulated(p<0.001),and the protein expression of Col2a1 in antler mesenchymal cells was significantly decreased(p<0.05).The protein expressions of PI3 K and p-AKT in PI3K/AKT signaling pathway were significantly decreased(p<0.01).In summary,melatonin accelerates the cyclic progression of antler mesenchymal cells,thereby promoting their proliferation,and it also accelerates their differentiation.Melatonin enhances the expression of the chondrocyte markers Col2a1 and Aggrecan in velvet antler mesenchymal cells by activating the PI3K/AKT/m TOR signaling pathway via MT1,which accelerates the chondrocyte development process in antlers.