PTH1-34 Improves Osteoporosis In Ovariectomized Rats And Promotes Proliferation And Differentiation Of Osteoblasts By Enhancing Autophagy

Part Ⅰ: Changes in autophagy after PTH 1-34 treatment of osteoporosis in ovariectomized ratsObjective: To investigate the changes of autophagy level in ovariectomized rats treated with PTH 1-34.Methods: Twenty-four 3-month-old SD rats were randomly divided into sham operation group(sham),ovary removal group(OVX)and ovary removal and PTH 1-34 treatment group(OVX+PTH).After 8 weeks,through the micro-CT and DEXA bone microstructure and bone mass was measured.Eelisa kit was used to detect Procollagen type I N-peptide(PINP),C-terminal telopetide of type I collagen(CTX),estrogen(E2),superoxide dismutase(SOD),catalase(CAT).Western blot was used to detecte autophagy expression of Beclin 1 specific genes,LC3-Ⅱ and the p62 protein.Results: The bone metabolic indices PINP and E2 reduced,SOD,CAT,CTX increases in ovariotomy osteoporosis rats,autophagy related genes Beclin 1,LC3-Ⅱ and p62 protein expression increased in the OVX group.In OVX rats treated with PTH 1-34,bone density and bone microstructure were improved.PINP,SOD and CAT were increased,E2 was not changed,CTX was decreased,and autophagy related protein expression was further increased.Conclusion: PTH 1-34 treatment in postmenopausal osteoporosis of rats to promote bone microstructure is improved and increased bone mass,promote autophagy related genes Beclin 1,LC3-and Ⅱ p62 protein expression increased.PartⅡ: PTH 1-34 promotes the proliferation and differentiation of osteoblasts and inhibits apoptosis by improving autophagy.Objective: To study the process of PTH 1-34 promoting proliferation and differentiation of osteoblasts and inhibiting apoptosis by improving autophagy.Methods: MC3T3-E1 rat embryonic osteoblast precursors were cultured and divided into serum group,serum +PTH group,serum-free group and serum-free group.After the optimal concentration of PTH 1-34 in vitro was selected,MC3T3-E1 rat embryonic osteoblast precursors were divided into Control group(not treated),3-MA group(3-MA intervention group),PTH 1-34 group(PTH 1-34 intervention group)and 3-MA+PTH group.The morphology of autophagy body was observed by transmission electron microscopy,and the occurrence of autophagy was detected by immunofluorescence.The expression levels of autophagy related proteins becl-1,p62,ATG5,LC3-Ⅱ,cleaved caspase-3 and cleaved-PARP were detected by Western Blot,and apoptosis was detected by flow cytometry,while the expression levels of osteoblast proliferation-related proteins Runx2,OCN,COL 1,bmp-2 and Osterix were detected by Western blot and qRT-PCR.Results: PTH 1-34 promoted the proliferation of osteoblasts and the expression of osteoblasts anabolis-related proteins,and at the same time,the expression of autophagy related proteins increased and the formation of autophagic lysosomes increased.Apoptosis decreased.Western blot confirmed cleaved Caspase -3 and cleaved-PARP protein;Autophagy inhibitor alone(3-MA)has no significant effect on bone anabolism,but it can significantly change the changes of intracellular anabolism-related proteins under the action of PTH 1-34,and at the same time partially inhibit cell proliferation and differentiation.Conclusion: In addition to inhibiting apoptosis,PTH 1-34 also promotes the proliferation and differentiation of osteoblasts through autophagy and increases the indicators related to bone metabolism and synthesis.Part Ⅲ: The effect of Beclin 1 gene of MC3T3-E1 osteoblast proliferation,differentiation and apoptosis.Objective: To study the effect of Beclin 1 gene on proliferation,differentiation and apoptosis of MC3T3-E1 osteoblasts.Methods: Using siRNA technology to interfere with Beclin 1 gene vector,the Beclin 1 silencing cell line was established.The proliferation rate of osteoblasts was detected by cck-8 under PTH 1-34 intervention.Flow detection of Beclin 1 gene silencing on cell cycle;Osteoblast differentiation was induced at 4 weeks,and the mineralization ability of osteoblast was detected by alizarin red staining.Osteogenesis differentiation 48 HRS,western blot detection osteogenetic differentiation related Runx2,OCN,COL-1 protein expression level;Finally,AnnexinV-FITC/PI staining flow cytometry detection Beclin 1 gene effects on osteoblast apoptosis.Results: MC3T3-E1 cell lines were successfully constructed in stable Beclin 1 silencing.CCk-8 test results showed that Beclin 1 gene silencing led to cell proliferation inhibition.Beclin 1 gene silencing leads to cell cycle arrest in G1 phase.Osteogenesis differentiation 48 HRS,western  blot test results shown Beclin 1 inhibit Runx2 gene silencing,OCN,COL-1 gene and protein expression.PTH 1-34 process,the change has no obvious change.Conclusion: Autophagy genes Beclin 1 lead to silence suppression,osteoblast proliferation and differentiation and Beclin 1 lead to  PTH 1-34 gene silencing effect significantly diminished.Beclin 1 May be effective PTH 1-34 anti osteoporosis treatment targets.