Mechanism Of HA15 Targeting HSPA5 Inhibiting ERS Pathway To Alleviate Osteoporosis

Objective: The purpose of this study is to explore whether HA15 could change the expression of osteogenic / adipogenic related indicators by affecting the Heat Shock Protein 5(HSPA5),the key protein of endoplasmic reticulum stress(ERS)signaling pathway in vivo and in vitro experiments on ovariectomized mice.Methodologies:(1)Mouse marrow mesenchymal stem cell line C3H10 was cultured,and the expression of HSPA5 was knocked down and overexpressed by transient transfection.Immunofluorescence staining and real-time staining were used to test the expressions of Osteogenic differentiation indexs such as ALP,BMP2,Col1a1,Osterix,RUNX2,OCN and those of adipogenic differentiation indexes such as a P2,C/EBPα,PPARγ,adiponectin after cultured under a standard condition.Meanwhile,and the mineralization ability was evaluated by alizarin red staining;(2)C3H10 cells in the control group were intervened through HA15.Immunofluorescence staining,alizarin red staining and real-time PCR were used respectively to detect the expressions of the above osteogenic / adipogenic indexes again.Then,the related effects of HA15 were analyzed;(3)26 4-week-old C57 BL / 6 female mice were randomly divided into sham-operation group and ovariectomized group.After 12 weeks,5 mice were randomly sampled from each group for detecting the success of modeling by micro CT.And the rest of 16 ovariectomized mice were randomly divided into saline injection group and HA15 injection group(normal saline and HA15 was separately injected into the mouse marrow cavity in two groups).Each group was arranged the injection one a week for totally 8times.Two months later,26 mice were treated with HE staining,micro CT detection,immunofluorescence test,and so on.The changes of histopathology,ERS key protein,downstream related osteogenic/adipogenic differentiation and other related indicators were observed.Results:(1)The knockdown of HSPA5 gene could promote the osteogenic differentiation of ALP,BMP2,Col1a1,Osterix,Runx2,OCN and inhibit adipogenic differentiation of AP2,C/EBPα,PPARγ,adiponectin,while overexpression of HSPA5 had the opposite effect;(2)HA15 could inhibit the expression of HSPA5 in C3H10 cells,promote osteogenic differentiation and hold-up adipogenic differentiation;(3)In OVX group,the expression of HSPA5 was increased in the process of osteoporosis modeling,and HSPA5 could be expressed in osteoblasts and adipocytes.After injecting with HA15,the osteoporosis symptoms of ovariectomized mice were significantly improved,while the control group injected with normal saline had no significant change and the autophagy and apoptosis of osteoblasts may be involved.Conclusion: The expression of HSPA5,the key protein of ERS pathway,is significantly increased in osteoporosis.The expression of HSPA5,the key protein of ERS pathway,is significantly increased in osteoporosis.HA15 treatment can reduce the expression of HSPA5,inhibit ERS pathway,and then affect the osteogenic / adipogenic differentiation ability of bone marrow mesenchymal stem cells.Besides,this kind of treatment could reduce their withering effect by increasing the autophagy of osteoblasts,so as to alleviate the occurrence and development of osteoporosis to a certain extent.