| Postmenopausal osteoporosis(PMOP)is one of the most common kind of osteoporosis.At present,the main drugs for the treatment of PMOP include anti-resorptive agents,such as bisphosphonates,and bone formative drugs,such as teriparatide(PTH1-34).Intermittent PTH(1-34)administration can promote bone formation,but its mechanism has not been fully elucidated.Mesenchymal stem cell(MSCs)is one kind of pluripotent stem cell with multi-differentiation potential.MSCs mainly exists in bone marrow and they can be mobilized into the blood under some disease conditions,reaching to injury site with the circulation to participate in the disease response and the repair of tissue damage.The circulating MSCs has potential implications for the treatment of PMOP.Current studies have confirmed that the number of MSCs in PMOP patients decreased and their osteogenic activity was impaired.The effect of intermittent PTH(1-34)treatment on circulating MSCs in peripheral blood of patients with PMOP has not been reported.PTH(1-34)can promote the osteogenic differentiation of BMSCs,but the mechanism has not been fully elucidated.Autophagy is an intracellular degradation system in eukaryotic cells.Autophagic dysfunction is associated with a variety of diseases.Increasing numbers of studies have shown that autophagy plays an important role in maintaining bone homeostasis.However,the role of autophagy in osteogenic differentiation of BMSCs induced by PTH(1-34)has not been reported.This study will be divided into the following three parts: Part Ⅰ Purpose: To study the effect of intermittent PTH(1-34)treatment on the number of circulating MSCs in peripheral blood of patients with PMOP.Methods: Fifty-four untreated PMOP patients were enrolled and randomly divided into two groups to receive teriparatide(PTH 1–34)or alendronate treatment for 12 months,respectively.Peripheral blood samples were obtained at 0,1,3,6,12 months after initiation of treatment.The number of circulating MSCs in peripheral blood was identified by flow cytometry.Changes of serum bone turnover markers were detected by enzyme-linked immunosorbent assay(ELISA),and dual energy X-ray absorptiometry(DXA)was used to analyse the bone mineral density(BMD)of lumbar vertebrae,femoral neck and hip.The statistical differences between the two groups were compared.Results: Results of flow cytometry showed that the number of circulating MSCs in peripheral blood of PMOP patients increased significantly after 1 month of PTH(1-34)treatment,which was 141±96% higher than that before treatment(P<0.001),and persisting until month 12.However,there was no significant change in the number of MSCs in alendronate group during study period.Pearson correlation analysis showed that the increase of circulating MSCs in PTH group was positively correlated with serum bone turnover markers and lumbar BMD(P1NP:r=0.67,P<0.001;OCN:r=0.54,P=0.003;β-CTX:r=0.57,P=0.002;Lumbar spine BMD: r=0.61,P=0.01).Conclusions: Intermittent PTH(1-34)administration increased number of circulating MSCs in peripheral blood of PMOP patients.The increase of MSCs were positively correlated with the changes of bone turnover markers and BMD,indicating that these cells may contribute to bone remolding.Part Ⅱ Purpose: To study the changes of osteogenic differentiation ability of circulating MSCs in patients with PMOP after intermittent PTH(1-34)treatment.Methods: Six PMOP patients treated with PTH(1-34)in the first part were selected.Peripheral blood samples were collected from each patient before initiation and after 1 month of PTH(1-34)treatment,respectively.Circulating MSCs were isolated from blood samples.After osteogenic induction culture,ALP staining,ALP activity analysis,and alizarin red staining were performed.RT-PCR were used to detect the expression of osteogenic related genes.Results: Compared with the control group,the ALP activity of MSCs,number of ALP stained cells and the amount of calcium deposition in the PTH(1-34)treated group were significantly higher.RT-PCR results showed that the osteogenic related gene Runx-2,OSX,COL-1a1 and OCN were significantly upregulated in the intermittent PTH treated cells than control.Conclusions: The osteogenic differentiation of circulating MSCs of PMOP patients increased significantly after intermittent PTH(1-34)treatment.Part Ⅲ Purpose: To study the mechanism of autophagy in intermittent PTH(1-34)promoting osteogenic differentiation of BMSCs.Methods: Ten female patients undergoing spinal surgery in orthopaedic department of our hospital were enrolled,including 5 PMOP patients and 5 normal patients.Bone marrow was collected when iliac bone graft was taken during the operation.BMSCs were isolated from the bone marrow and cultured in vitro.1.Comparison of autophagy activity and osteogenic differentiation between the two groups: The expression of autophagy associated proteins Beclin-1,p62 and LC3 was detected by Western blot,and the expression of LC3-II was detected by immunofluorescence.After 21 days of osteogenic induction,the activity of ALP was detected by ALP kit,the expression of osteogenic related protein was detected by Western blot,and the expression of osteogenic activity related genes was detected by RT-PCR.2.The influence of PTH(1-34)on the autophagic activity of BMSCs: BMSCs derived from PMOP patients were divided into 4 groups,and treated with PTH1-34(PTH group),rapamycin(RAP group),PTH1-34 and rapamycin(PTH+RAP group)and PBS(control group),respectively.Autophagic activity and osteogenic differentiation ability of each group cells were measured.3.The role of autophagy mediated by AMPK/mTOR/ULK1 pathway in osteogenic differentiation of BMSCs induced by PTH(1-34): BMSCs derived from PMOP patients were divided into three groups,and treated with PTH1-34(PTH group),PTH1-34 and AMPK inhibitor Compound C(PTH+CC group)and PBS(control group),respectively.The expression of AMPK/mTOR/ULK1 signaling associated proteins AMPK,p-AMPK,mTOR,p-mTPR,ULK1 and p-ULK1 were detected by Western blot.The autophagic activity and osteogenic differentiation ability of each cell group were also compared.Results: 1.Compared with the normal group,BMSCs derived from PMOP patients showed decreased expression of Beclin-1 and LC3-II/I,while p62 protein was increased.Immunofluorescence analysis showed that the number of LC3-II fluorescence in PMOP group was significantly lower than that in normal group.After osteogenic induction,the alkaline phosphatase activity,osteogenic related protein and osteogenic related gene expression in PMOP group were significantly decreased than those in normal group.2.Compared with the control group,the autophagic activity and osteogenic differentiation ability of PTH,RAP and PTH+RAP groups were significantly increased.3.Compared with the control group,the expression of p-AMPK and p-ULK1 increased,but the expression of p-mTPR decreased in PTH group,and the expression of p-AMPK and p-ULK1 decreased after pretreatment with AMPK inhibitor(CC).However,the expression of p-mTPR was increased,and the autophagy activity and osteogenic differentiation ability of CC pretreatment group were lower than those of PTH group.Conclusions: The autophagy activity of BMSCs in PMOP patients was decreased.Intermittent PTH(1-34)stimulation in vitro promoted the osteogenic differentiation of BMSCs by inducing autophagy mediated by AMPK/mTOR/ULK1 pathway. |
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