Mechanism Of Icariin Inhibits Osteoclast Differentiation Through LAP Pathway Inhibits Typical Autophagy

Objective:To study the mechanism of multiple autophagy pathways（LAP,typical autophagy）in the process of osteoclast differentiation induced（physiological）and hormone intervention（pathological）,and the mechanism of icariin in inhibiting osteoclast differentiation by up-regulating LAP autophagy and inhibiting typical autophagy.Methods:RAW264.7 cells with good growth were selected and cultured in 20ng/ml RANKL（RA）and complete medium（CM）to induce osteoclasts.10-10,10-9,10-8,10-7,10-6,10-5,10-4 M icariin（ICA）or 10-12,10-11,10-10,10-9,10-8 According to the research purpose,the experiment was divided into physiological stage（RA induced for 1 day,3 days and 5 days）,pathological stage（RA induced for 5 days with 10-10,10-9,10-8 and 10-7 M DEX intervention）,the RApeutic stage（ra induced for 5 days with 10-10 M DEX and 10-6 M ICA intervention）.There are eight groups of RA-1D,RA-3D,RA-5D,RA-DEX10-10-5D,RA-DEX10-9-5D,RA-DEX10-8-5D,RA-DEX10-10-ICA10-6-5D and CM-5D.Identification of osteoclast differentiation by TRAP staining.Proteins were extracted from 8 groups of cells,separated by SDS-PAGE,transferred to PVDF membrane,and incubated with primary and secondary antibodies of corresponding proteins and factors.Western-blot technique was used to detect the expression levels of actin,Cathepsin K,Rubicon,LC3B,p65,p-p65 and p62.The expression levels of Cathepsin K,Rubicon,LC3B,p65,p62,TNF-α,p16 and GAPDH were detected by RT-q PCR.The results and data were statistically analyzed,and the difference was statistically significant when P was less than 0.05.Results:The results of CCK8 showed that when DXE interfered osteoclast differentiation for 24H and 72H,with the increase of DEX concentration(10^-12-10^-8M),the overall cell proliferation ability increased.At120H,in a certain concentration range(10^-10-10^-7M),with the increase of DXE concentration,the overall cell proliferation ability decreased;When the concentration of ICA intervened osteoclast differentiation for 72H and 120H,the overall cell proliferation ability increased with the increase of ICA intervention concentration.The whole cell proliferation ability of RAW264.7cells induced by osteoclast for 120H was significantly lower than that induced by osteoclast for 24 h.The results of induced groups（RA-1D,RA-3D,RA-5D）showed that during the osteoclast induction of RAW264.7,Rubicon decreased gradually,the expression of LC3B increased,the expression of p62protein increased in RA-1D,but there was no obvious change in RA-3 and RA-5D,and p65,p-p65,Cathepsin K increased significantly,p16 Pathological group(RA-DEX10^-10-5D,RA-DEX10^-9-5D,RA-DEX10^-8-5D)showed that with the increase of DEX concentration,Rubicon decreased obviously,the conversion rate of LC3B did not change obviously,the absolute expression level increased obviously,and the p62 protein expression of RA-DEX10^-10-5D increased.There is no obvious change in RA-DEX10^-9-5D and RA-DEX10^-8-5D,but p-p65 and Cathepsin K are significantly increased,p16 gene expression is significantly decreased in RA-DEX10^-9-5D and RA-DEX10^-8-5D groups,and TNF-αgene expression level in RA-DEX10^-10-5D is obvious Compared with RA-DEX10^-10-ICA10^-6-5D,Rubicon in treatment group(RA-DEX10^-10-5D)increased significantly,absolute expression and transformation rate of LC3B decreased significantly,p62 increased significantly,p65 and Cathepsin K decreased significantly,and p16 and TNF-αgene expression levels increased significantly.Conclusions:The results showed that RANKL osteoclast inducing factor and dexamethasone inhibited LAP autophagy pathway by inhibiting Rubicon in the process of osteoclast induction（physiology）and pathological differentiation,and promoted typical autophagy and osteoclast differentiation.Icariin activates LAP pathway by up-regulating Rubicon,and inhibits typical autophagy and hyperfunction of dexamethasone on osteoclasts.Icariin may promote osteoclast aging by regulating autophagy,but not osteoclast necrosis,so as to achieve the ultimate osteoclast inhibition effect.