The Interaction Between CKIP-1 And MTOR In Postmenopausal Osteoporosis And The Pharmacological Study Of Qiangguyin

Objective Observe the dual regulation of bone balance effect of Qiangguyin to postmenopausal osteoporosis and the relationship between CKIP-1 and PI3K/AKT/mTORC signaling pathway in bone balance,and to explore the intervention effect caused by Qiangguyin.Explore the molecular mechanism of Qiangguyin treating postmenopausal osteoporosis.Methods 1.We use ovarian surgery to establish a rat disease model of postmenopausal osteoporosis.2.We use micro-CT imaging analysis and Western-Blotting detection method to detect rat femur samples.3.Directly isolate,culture,and strengthen rat osteoblasts and osteoclasts.4.CKIP-1 overexpression lentiviral vector was established in vitro.5.Alizarin red staining detection of rat osteoblasts in vitro.6.Detection of TRACP activity of osteoclasts from rats in vitro.7.Western-Blotting in vitro to detect the content of p62 and LC3 ?/?.8.ELISA detection of osteoblasts in vitro.9.CKIP-1 and AKT immunoprecipitation detection.10.Western-Blotting detects the expression levels of atg5 and p62.11.Use a laser confocal microscope to observe the formation and changes of xi baLC3 point-like aggregate spots.Results 1.Both Qiangguyin and autophagy inhibitor 3-MA can inhibit the osteoporosis symptoms of model rats.Among them,Qiangguyin has two-way anti-osteoporosis effects of inhibiting bone destruction and promoting bone formation,while Qiangguyin and autophagy inhibitors both have synergistic effects.2.In the postmenopausal osteoporosis rat model group,the expression of CKIP-1 increased,the level of autophagy increased,the PI3K/AKT/mTOR pathway was down-regulated,the expression of RANKL was up-regulated,and the expression of RUNX2 and OPG was down-regulated.Down.Strong silver and 3-MA can inhibit various pathways and indexes of the model group.3.We successfully isolated and cultured rat osteoblasts and osteoclasts,completed cell identification,successfully constructed pLVX-IRES-ZsGreenl-Rat CKIP-1,completed transfection and completed sequencing.4.Both Qiangguyin and 3-MA can enhance the alizarin calcification nodules of CKIP-1 overexpression osteoblasts,and inhibit the alkaline phosphatase activity of CKIP-1 overexpression osteoblasts,and the two have a synergistic effect.5.Strong silver can inhibit the autophagy level of rat osteoblasts and the overexpression of CKIP-1 in osteoclasts.6.Simple inhibition of autophagy and specific inhibition of mTOR protein to promote autophagy can inhibit the osteogenic effect of Qiangguyin.Strong silver can also reverse the inhibitory effect of autophagy on the osteogenic ability of CKIP-1,that is,strong silver.The osteogenic effect on osteoblasts is related to the level of autophagy activity and related activities regulated by mTOR protein.7.CKIP-1 can promote the secretion of RANKL by osteoblasts and inhibit the secretion of OPG.Qiangguyin can reverse this process.8.The results of co-immunoprecipitation experiments show that CKIP-1 and AKT have an interaction,and strong silver treatment can reduce the combined amount of the two.9.CKIP-1 can promote the increase of autophagy level in osteoblasts and osteoclasts,and Qiangguyin treatment can inhibit the increase of autophagy level induced by CKIP-1.10.The results of confocal laser detection show that CKIP-1 can up-regulate the autophagy levels of osteoblasts and osteoclasts,while Qiangguyin can inhibit the increase in autophagy levels induced by CKIP-1.Conclusion Qiangguyin has the dual effects of regulating bone balance in postmenopausal osteoporosis,promoting bone formation and inhibiting bone resorption.In vitro rat osteoblasts and osteoclasts,CKIP-1 can promote autophagy levels in osteoblasts and osteoclasts,while Qiangguyin can inhibit the increase in autophagy levels induced by CKIP-1.Strong silver can interfere with the combination of CKIP-1 and AKT,which is a key factor in the PI3K/AKT/mTOR signal transduction axis,and up-regulate the PI3K/AKT/mTORC signal transduction pathway,thereby down-regulating the level of autophagy and maintaining skeletal balance.In vitro rat osteoblasts,CKIP-1 can promote RANKL secretion of osteoblasts and inhibit OPG secretion.Qiangguyin can reverse this process.