Effects Of Autophagy On Differentiation Abilities Of Bone Marrow Mesenchymal Stem Cells In Estrogen Deficiency-induced Osteoporosis Mice

Osteoporosis is a common metabolic bone disease. Estrogen deficiency-induced osteoporosis is a type of osteoporosis which is frequently observed in postmenopausal women. Clinical manifestations include bone fragility and increasing incidence rate of fracture. Histology showed decreased bone density, reduction of trabecular bone and increased fat tissue in bone marrow.Currently, the development of treatment of estrogen deficiency-induced osteoporosis focues on inhibition of the activity of osteoclasts to slow down bone resorption. However, it doesn’t thoroughly resolve the problem of bone loss because it doesn’t restore the osteogenesis completely. Bone Marrow Mesenchymal Stem Cells(BMMSCs) are fibroblast-like cells derived from the mesodermal with self-renewal and multi-differentiation potentials,which play important roles in the research of bone diseases associated with osteogenesis dysfunction. Autophagy is a cellular process in which cellular components like aging, misfolded proteins and damaged organelles are engulfed by autophagysome and delivered to lysosome to be degraded and subsequently recycled to cytoplasm, to maintain cellular homeostasis. Autophagy is influenced by many factors, including nutrition deprivation, stress, hormones level change and inflammation. It is unclear that under estrogen deficient environment, whether the autophagy level in mouse BMMSCs will change and whether such change would influence the differentiation balance in BMMSCs. This study focuses on the abnormal autophagy in BMMSCs from estrogen deficiency-induced osteoporosis mice model and the effects of autophagy in BMMSCs on their differentiation abilities. It is intended to exploit the pathogenesis of estrogen-deficiency osteoporosis, and provide a new theoretical basis for treatments.Part1 The change of autophagy in BMMSCs from estrogen deficiency-induced osteoporosis miceAim:(1).To establish the estrogen deficiency induced osteoporosis and control mice model through ovariectomy and sham operation.(2)To compare the autophagy in BMMSCs from OVX mice with that from Sham mice.Methods:(1) OVX and Sham mice model were established by ovariectomy and Sham operation. Micro CT was used to assess the BMD and BV/TV in two models 2 months later.(2) OVX BMMSCs and Sham BMMSCs were isolated and purified through whole bone marrow culture. Western blot analysis was used to detect the expression of Beclin1 and LC3 proteins, while their m RNA expressions were revealed through RT-PCR. Immunofluorescence experiments were performed to compare the expression of LC3 in BMMSCs. The microscopic structures and autophagosomes of the cells from all models were observed with TEM.Results:(1) Micro CT showed the less BMD and BV/TV in OVX model.(2) Compared with Sham BMMSCs OVX, BMMSCs showed lower Beclin1, LC3 protein expression in Western Blot and m RNA expression in RT-PCR. Immunofluorescence staining showed that OVX BMMSCs had weaker LC3 fluorescence. And less autophagysomes were observed in OVX BMMSCs.Conclusion:(1) Estrogen deficiency-induced osteoporosis mice model was successfully established by OVX operation.(2) BMMSCs from OVX mice have lower autophagy level compared to BMMSCs from Sham mice.Part2 The effects of autophagy in mouse BMMSCs on its differentiation abilities in vitroAim:(1) To investigate the effects of activation of autophagy on the differentiation capabilities in OVX BMMSCs in vitro.(2) To expoler the effects of inhibition of autophagy on the differentiation capabilities in Sham BMMSCs in vitro.Methods:(1) Sham BMMSCs, OVX BMMSCs and OVX BMMSCs stimulated by autophagy activator Rapamycin(OVX+Rapamycin) were harvested after isolation and cultivation respectively. The expressions of osteogenic proteins Runx2 and Osterix and adipogenic protein PPAR-γ were detected by western blot analysis. Alizarin red staining and oil red O staining were used to examine the BMMSCs’ capabilities to form mineralize nodules and lipid droplets respectively.(2) Sham BMMSCs and Sham BMMSCs stimulated with autophagy inhibitor 3-methyl-adenine(3-MA)(Sham+3-MA) were cultured and the expressions of Runx2,Osterix, and PPAR-γ of each group were detected as well as the mineralization and lipid droplet formation by the same experimental techniques.Results:(1) Western Blot showed higher expressions of Runx2, Osterix and lower expression of PPAR-γ in OVX+Rapamycin BMMSCs compared with OVX BMMSCs. Alizarin red staining showed more mineralized nodule formation and oil red O staining showed less lipid droplet formation in OVX+Rapamycin BMMSCs.(2) Expression of Runx2, Osterix were less and PPAR-γ was more in Sham+3-MA BMMSCs compared with Sham BMMSCs. Decreased mineralized nodule formation and increased lipid droplet formation were observed in Sham+3-MABMMSCsConclusion:(1) Activation of autophagy in OVX BMMSCs increases osteogenic and reduces adipogenic differentiation in vivo(2) Inhibition of autophagy in Sham BMMSCs results in decreased osteogenic and increased adipogenic differentiation. This part implies that change of autophagy in BMMSCs effecs its differentiation capabilitiesPART3 Effects of change of systemic autophagy on the boss mass and differentiation abilities in BMMSCsAim:(1) To investigate the effects of activation of systemic autophagy on bone mass and differentiation abilities in BMMSC.(2) To expoler the effects of inhibition of systemic autophagy on bone mass and differentiation abilities in BMMSC.Methods:(1) Sham, OVX mice and OVX mice injected with Rapamycin(OVX+Rapamycin) mice model were established and analyzed a month later with micro CT for BMD, BV/TV in tibia. Calcein labeling was applied to detect and compare the bone formation rate. Oil O staining of bone frozen slice was used to compare the fat in bone marrow. RT-PCR was conducted to detect PPAR-γ m RNA expression in bone marrow. Alizarin red staining and oil red O staining were applied to assess mouse models-derived BMMSCs’ potentials in mineralization and lipid droplet formation.(2) Sham and Sham mice injected with 3-MA models were tested and compared for their BMD, BV/TV, bone formation rate, fat and PPAR-γ m RNA expression in bone marrow, mineralization and lipid droplet formation with the same experimental techniques.Results:(1) Micro CT results illustrated higher BMD and BV/TV of tibia in OVX+ Rapamycin mice compared with that in OVX mice. Calcein labeling demonstrated higher bone formation ratio in OVX+Rapamycin mice. Less Lipid droplets and lower expression of PPAR-γ m RNA were detected in the bone marrow from OVX+Rapamycin. The BMMSCs from OVX+Rapamycin mice showed more mineralized nodules with or without osteogenic induction and less lipid droplets with or without adipogenic induction compared with the BMMSCs from OVX mice.(2) BMD, BV / TV, bone formation rate decreased in Sham+3-MA mice.More lipid droplets and PPAR-γ m RNA expression were observed in bone marrow of the Sham +3-MA mice. The BMMSCs from Sham+3-MA mice demonstrated less mineralized nodules and more lipid droplets than BMMSCs from Sham mice.Conclusion:(1) Activation of autophagy in OVX mice could restore bone mass partly and reduce fat mass in bone.The BMMSCs from OVX+Rapamycin mice model showed increased osteogenic differentiation and decreased adipogenic differentiation abilities.(2) Inhibition of autophagy in Sham mice causes bone mass reduced and fat mass increased.The BMMSCs from Sham+3-MA mice model showed lower osteogenic differentiation and higher adipogenic differentiation abilities.