The Role Of Autophagy In Cadmium Inhibiting Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells

OBJECTIVECadmium is a common environmental and industrial pollutant,which causes severe environmental pollution due to its huge emission.The International Agency for Research on Cancer(IARC)has classified cadmium as Class ? carcinogens.Cadmium is not easily biodegradable in the environment,and it can cause serious damage through biological amplification and accumulation because of exposing trace amounts of cadmium.People could be exposed to high concentration of cadmium due to their occupational needs,or they might also take in low amounts of cadmium through smoking and diet.Cadmium poisoning may cause osteoporosis,but its damage mechanism is still unclear.Therefore,an in-depth study of the mechanism of cadmium-induced bone damage can provide a scientific basis for effectively preventing the health hazards of cadmium and has important significance and value for the treatment of cadmium related osteoporosis.Our preliminary population survey found that osteoporosis patients living in a high-level cadmium area have significantly higher active glucocorticoids.Some studies suggested that glucocorticoids could activate autophagy of bone marrow mesenchymal stem cells(BMSCs)and then affect osteogenic differentiation.Given the fact the local glucocorticoids must be activated by 11?-hydroxysteroid dehydrogenase 1(11?-HSD1).Therefore,we proposed that cadmium could be elevating 11?-HSD1 to activate BMSCs autophagy,and in turn to cause its osteogenic differentiation.Such an effect may work through mTOR signaling pathway of autophagy,resulting in reduced bone formation and eventually causing osteoporosis.METHODSAlkaline phosphatase(ALP)staining solution was used to detect the ALP content after osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs);alizarin red staining solution was used to detect the formation of mineralized nodules;ALP activity test reagent was used to detect ALP activity;Cell Counting Kit-8(CCK-8)was used to detect the cytotoxicity of different concentrations of cadmium chloride(CdCl2)on hBMSCs;qRT-PCR was used to detected the mRNA expression of ALP,Runx2,Osterix and 11?-HSD1;Western blot was used to detect the protein expression of ALP,Runx2,Osterix,11?-HSD1,mTOR,p-mTOR,P70S6K,p-P70S6K,Beclin-1 and LC3B.RESULTSUnder 21 d osteoblasts differentiate induction of hBMSCs,the formation of ALP and mineralized nodule were increased.Moreover,CdCl2 exerted a dose-dependent cytotoxicity effect in hBMSCs.Low concentration(?3.5 ?M)did not inhibited cell viability up to 5.0 ?M.Compared with the control group,the group with treatment of 2.5?M and 5.0?M CdCl2 to hBMSCs during osteogenic differentiation for 1 d,4 d,7 d and 14 d caused the formation of ALP and mineralized nodules reduction,and the osteogenic markers of hBMSCs depletion with the dose of CdCl2 increased.Which indicated CdCl2 might inhibit the differentiation of hBMSCs into osteoblasts.Furthermore,compared with the control group,the expression of autophagy molecules and 11?-HSD1 elevated and the mTOR signaling pathway molecular decreased after the treatment of CdCl2 to hBMSCs during osteogenic differentiation.It indicated that CdCl2 up-regulated the expression of 11?-HSD1 and inhibited the mTOR pathway,thereby activating autophagy in hBMSCs.The combined treatment of 11?-HSD1 inhibitor BYT 2733 and CdCl2 increased the formation of osteogenic markers and reduced autophagy.However,it had no significant effect on the mTOR signaling pathway,the combined treatment of mTOR activator MHY 1485 and CdCl2 attenuated autophagy,while the expression of11?-HSD1 had no obvious change.Taken together,11?-HSD1 and mTOR signaling pathway parallelly played an important role on CdCl2 activated autophagy of hBMSCs during osteogenic differentiation.CONCLUSIONS1.CdCl2 inhibited the formation of ALP,mineralized nodules and the expression of osteogenic markers ALP,Runx2,Osterix,suggesting that CdCl2 inhibited the osteogenic differentiation of hBMSCs.2.CdCl2 up-regulated 11?-HSD1,inhibited mTOR signaling pathway and activated autophagy during hBMSCs osteogenic differentiation,indicating that CdCl2 may act on 11?-HSD1 and mTOR signaling pathway to activate autophagy,thereby inhibiting osteogenic differentiation.3.Inhibition of 11?-HSD1 had no significant effect on the mTOR signaling pathway;activation of the mTOR signaling pathway also had no significant effect on11?-HSD1.It meant that CdCl2 activated autophagy during differentiation of hBMSCs through the activation of 11?-HSD1,or inhibited the osteogenic differentiation of hBMSCs by activated autophagy via the mTOR signaling pathway.