The Function And Regulation Of Autophagy In Bone Marrow-derived Mesenchymal Stem Cells In Glucocorticoid-induced Osteoporosis Rats

Aims:Glucocorticoids are widely used in clinic and have various adverse effects on bone cells leading to glucocortico id-induced osteoporosis(GIOP).The manifestations of GIOP include decreased bone mineral density(BMD)and osteoporotic fractures,which occur in 30%–50% of adult patients.Excess glucocorticoids contribute to bone loss through their direct effects on osteoblasts,osteoclasts,and osteocytes.Previous studies have shown that excess glucocorticoids increase apoptosis of osteoblasts and osteocytes and prolong the life span of osteoclasts.Bone marrow-derived mesenchymal stem cells(BMSCs)are crucial in maintaining the dynamic homeostasis of bone tissue,and much attention has been paid to the role of BMSCs in the pathogenesis of osteoporosis in recent years.Given that the formation of new bone is primarily dependent on BMSCs and osteoblasts that arise from BMSCs,the negative effects on BMSCs caused by high doses of glucocorticoids is undoubtedly responsible for bone loss in GIOP.Therefore,the recovery of BMSCs in GIOP is essential for the clinical treatment of osteoporosis.Autophagy can be activated when cells are under stressful conditions and serves as a pro-survival mechanism.This process is essential for cell survival,development,and homeostasis and is widely implicated in many pathophysiological disorders.However,whether autophagy is involved in glucocorticoid-induced damage to BMSCs remains to be elucidated.Tetramethylpyrazine(TMP),an extract from one of the most recognized herbs in traditional C hinese medicine(C huanxiong),has been reported to have anti-inflammatory,anticancer,antioxidative,and antiapoptotic effects in many cell types.The current study hypothesized that autophagy was involved in the glucocorticoid-induced dysfunctions of BMSCs and we tried to use this small molecular compound to protect the defective BMSCs by regulating autophagy aiming to find a new strategy for preventing and treating GIOP.Methods:Here we tested whether autophagy was induced in BMSCs following exposure to glucocorticoids and could be regulated by TMP in vitro and in vivo.The primary BMSCs were isolated and treated with three concentrations of Dex(10-8 M,10-7 M and 10-6 M).Following 48 h culture with Dex,autophagy was detected by transmission electron microscopy,LC3 immunostaining and western blot analysis.3-MA(inhibitor of autophagy)and rapamycin(activator of autophagy)was used to test the function of autophagy in BMSCs exposed to 10-6 M Dex.Then we treated primary BMSCs with different concentrations of TMP(50,100,200 ?M)and exposed them to 10-6 M dexamethasone(Dex)for 48 h in vitro.The Cell Counting K it-8 was used to measure cell viability.Cell apoptosis was assessed by Annexin V/ PI double staining,TUNEL staining and Caspase-3 activity.After an osteogenic induction with 10-6 M Dex for 14 days,BMSCs were incubated with serum-free medium with or without TMP(50 ?M,100 ?M,or 200 ?M)for 48 h.Then the cells were stained for the ALP activity test.Total RNA was extracted from cells for quantitative real-time PCR analysis.After a 21-day osteogenic induction,Alizarin Red S staining was performed to detect calcium d eposition.Absorbance of the released Alizarin Red S was measured using an enzyme-linked microplate reader.We used Baf which block the fusion of autophagosomes with lysosomes and assessed the protein levels of LC3 and p62 to evaluate autophagic flux.Cells were treated with or without 10-6 M Dex and 50 ?M TMP in the presence or absence of 100 n M Baf.Then we determined the expression level of phospho-AMPK(p-AMPK),total AMPK,phospho-m TOR(p-m TOR),and total m TOR in BMSCs following administration of the AMPK inhibitor Compound C.Fifty 4-month-old female Sprague-Dawley rats weighing 223 ±18.5 g were obtained.Ten of them were used to isolate BMSCs.Forty of them were intraperitoneally administered either distilled water(as the control group,n=10)or 2.5 mg/kg prednisolone(as the GIOP group,n=30)da ily for 12 weeks.O ne week after the first administration,the 30 rats in the GIOP group were randomly divided into three experimental groups of ten rats per group.The rats were injected intraperitoneally with sesame oil(as a vehicle control),5 mg/kg or 20 mg/kg body weight of TMP daily for 12 weeks.Then,the first passage BMSCs were isolated from all four groups.Calcein double labeling and micro-CT scanning were used to monitor bone mass.Transmission electron microscopy and western blot analysis were used to assess autophagy in GIOP-BMSCs.Annexin V/ PI double staining and Caspase-3 activity analysis were performed to detect apoptosis.Results:1.Our data showed that autophagy was induced in BMSCs from GIOP rats.The number of BMSCs in the GIOP group was lower than in the placebo group,suggesting a reduced proliferative ability.2.Autophagy was induced by 10-8 M,10-7 M and 10-6 M Dex in BMSCs in vitro.The autophagy inhibitor 3-MA further reduced the relative growth rate and improved apoptosis rate of BMSCs compared with the Dex alone group,suggesting that autophagy maintained proliferation ability of BMSCs and played protective roles against apoptosis.3.TMP further promoted autophagy flux via AMP-activated protein kinase(AMPK)and mammalian target of rapamycin(m TOR)pathway and significantly improved viability of BMSCs and alleviated the Dex-induced apoptosis compared with the Dex alone group.And TMP alone did not induce autophagy in BMSCs exposed to Dex.In addition,TMP treatment groups displayed significant increase in ALP activity and mineralization as well as the expression levels of osteogenic genes including ALP,COL1A1,OCN and OSX in contrast to the Dex alone treated group.4.Calcein double labeling and micro-CT scanning indicated that 12 weeks of TMP administration augmented bone formation and protected the trabecular bone mass in GIOP rats.We also discovered that first-passage BMSCs isolated from the TMP treatment group had a lower rate of apoptosis and a higher LC3-II/LC3-I ratio than the GIOP group did,indicating that BMSCs responded to TMP in vivo.Conclusions:In summary,glucocorticoids induce autophagy in BMSCs and autophagy maintains the proliferative ability of BMSCs and protects BMSCs from Dex-induced apoptosis,suggesting that autophagy serves as a pro-survival mechanism.TMP could prolong BMSC survival following exposure to excess glucocorticoids by preserving cell viability and inhibiting apoptosis through the AMPK/m TO R-dependent promotion of autophagy in vitro.In vivo administration of TMP increased bone formation and prevented bone mass decrease in GIOP rats.Autophagy enhancement and protection of BMSCs against apoptosis in a GIOP state may be responsible for the anti-osteoporosis effects of TMP.Our study is the first to demonstrate that the regulation of autophagy flux is a promising strategy to improve rat BMSCs survival following excess exposure to glucocorticoids,and TMP may represent a new target drug for the prevention or treatment of GIOP.