The Study Of BMSCs Osteogenic Differentiation Via MiR-155 In Cadmium-induced Osteoporosis And The Protective Effect Of Prunella Vulgaris L.

PurposeCadmium(Cd)accumulation in bone can induce osteoporosis which results serious endangering human health.However,the mechanism of Cd-induced osteoporosis is not clear.Osteogenic differentiation inhibition of bone marrow mesenchymal stem cells(BMSCs)is the main link in the occurrence of osteoporosis.There is few study to explore the effect of Cd on the osteogenic differentiation of BMSCs.Cd exposure can cause changes in the expression profile of miRNAs,and miRNAs has been verified can regulate osteogenic differentiation.but there is no study on the regulation of miRNAs in Cd induced bone injury.At present,there are no effective prevention and treatment measures for Cd-induced osteoporosis.Prunella vulgaris L.(PV)has been indicated hac a protective effect on osteoporosis,but there is no evidence shown the protective effect of PV on Cd-induced osteoporosis.In this study,we established a rat model of osteoporosis induced by Cd to explore the effect of Cd on the osteogenesis differentiation of BMSCs and the expression of miR-155.Then combined the Cd-incued rat model and human BMSCs(hBMSCs)to explore the mechanism of miR-155 in Cd-inhibited bone formation and differentiation.Finally,the protective effect and potential mechanism of PV on Cd-induced osteoporosis were clarified through the intervention of PV in rats with Cd-induced osteoporosis.To reveal the molecular mechanism of osteoporosis caused by Cd,and provide new clues for finding the prevention and treatment measures of Cd related bone injury.Method1.Effects of Cd on the differentiation of BMSCs and miR-155 in Cd-induced osteoporosis ratsThe female SD rats aged 6-8 weeks were treated with cadmium chloride(CdCl2)at 0,25,50 and 100 mg/L for 16 weeks in drinking water to establish a Cd-induced osteoporosis model.And the primary BMSCs of femur were isolated and cultured for osteogenic induction and differentiation.Serum bone transformation index,osteogenic differentiation markers in bone tissue and BMSCs,BMP/Smad signaling pathway were detected.The expression of miR-155 in serum,bone tissue and BMSCs was detected.2.The role and mechanism of miR-155 in Cd-induced osteogenic differentiation of BMSCsPrimary human BMSCs(hBMSCs)were treated with different concentrations of CdCl2(0,2.5,and 5 μmol/L)to detect osteogenic differentiation indexes,clarify the dose-effect relationship of Cd regulation on bone formation of hBMSCs,detect the expression of intracellular miR-155 to clarify the dose-effect relationship of cadmium regulation on these indexes.Dual luciferase reporter genes were used to detect the direct regulation of miR155 and target gene transcription.The expression of miR-155 in hBMSCs was downregulated by miR-155 inhibitors,and then hBMSCs were treated with CdCl2 to detect intracellular miR-155,Smad5,Runx2 and osteogenic differentiation indexes.The role of miR-155 in the regulation of Cd on the osteogenic differentiation of hBMSCs was clarified through the inverse intervention experiment.3.The protective effect and potential mehcanism of Prunella axella on Cd-induced osteoporosisFemale SD rats aged 6-8 weeks were treated with CdCl2(50 mg/L),CdCl2+lowdose water extract of Prunilla subtilis(125 mg/kg bw)and CdCl2+high-dose water extract of Prunilla subtilis(250 mg/kg bw)for 16 weeks.Primary BMSCs of rat femur were isolated and cultured for osteogenesis induction and differentiation.miR-155 and BMP/Smad signaling pathways in bone tissue and BMSCs were detected.Result1.Effects of Cd on BMSCs differentiation and miR-155 in Cd-induced osteoporosis in rats(1)Cd inhibited bone formation in ratsmicro-CT showed that the number of bone trabeculae in the CdCl2 exposed group was reduced,sparse and broken,and quantitative analysis showed that the bone volume fraction(BV/TV),the number of bone trabeculae(Tb.N)and the connection density(Con.D)in the 50 and 100 mg/L CdCl2 exposed groups were significantly lower than those in the control group.The trabecular separation degree(Tb.Sp)was higher than that of the control group.HE staining showed that the area of bone trabeculae decreased significantly,the area of fat cells increased and the area of fat cells increased in Cd group.Serum BALP and BGP decreased significantly in 50 and 100 mg/L CdCl2 groups,and TRACP-5b increased with the increase of CdCl2 concentration.(2)Cd inhibited BMP/Smad signaling pathway bone tissue in ratThe mRNA and protein expression of Runx2 and Osterix in bone tissue of rats exposed to 50 and 100 mg/L CdCl2 were significantly decreased.And there is a dosereactivity of CdCl2 to decrease the mRNA expression of BMP/Smad signaling pathway molecules.The protein expression of BMP/Smad signaling pathway were decreased by 50 and 100 mg/L CdCl2,especially in 50 mg/L CdCl2 group.(3)Cd inhibited osteogenic differentiation of BMSCsDuring osteogenic differentiation of BMSCs,mRNA expressions of ALP,OCN,Runx2 and Osterix genes was decreased.Alizarin red staining showed that the number of calcified nodules decreased significantly in 50 and 100 mg/L CdCl2 groups.(4)Cd promoted the expression of miR-155 in ratsCdCl2 exposure increased the expression of miR-155 in serum,bone tissue and BMSCs of rats which more obvious in the 50 mg/L CdC12 exposure group.2.The role of miR-155 in Cd regulation of BMSCs osteogenic differentiation(1)Cd inhibited the activity and osteogenic differentiation of hBMSCs.Treatment with 40 and 80 μmol/L CdCl2 inhibited the proliferation of hBMsCs for 24 h.CdCl2 treated with 10-80 μmol/L inhibited cell proliferation for 7 days.In the subsequent study,cells treated with no proliferation inhibition were selected,namely 0,2.5,5.0 μmol/L CdCl2 treatment of hBMSCs.Compared with the control group,ALP staining gradually became lighter with the increase of dose,while alizarin red staining gradually reduced the number and volume of calcified nodules.(2)Cd promoted the expression of miR-155 in hBMSCs2.5 and 5.0 μmol/L CdCl2 promoted the mRNA expression of miR-155 and downregulated Smad5,and only 5.0 μmol/L CdCl2 down-regulated Smad5 and Runx2.(3)miR-155 targets Smad5Dual luciferase gene verification showed that miR-155 could simultaneously bind to the 735-741 and 830-835 bases of Smad5 3 ’-UTR to inhibit its expression.(4)Inhibiting the expression of miR-155 effectively reversed the inhibitory effect of Cd on hBMSCsmiR-155 inhibitor treatment of hBMSCs,results showed that CdCl2 combined with miR-155 inhibitor treatment enhanced ALP staining,and the calcified nodules were larger and more numerous than the CdCl2 exposed group.3.Protective effect of Prunella axella on Cd-induced osteoporosis in rats(1)PV can relieve bone trabecular injury caused by CdBone trabecular parameters BMD,BV/TV,Tb.N,Tb.Th increased significantly,while Tb.Sp decreased significantly.H&E staining showed that the bone trabecular area in the PV intervention group was significantly increased compared with that in the Cd group.TRAP staining showed that the number of TRAP positive cells decreased significantly in PV treatment group.Serum ALP and BGP were increased and TRACP5b was decreased in the PV treated group.(2)PV inhibited the expression of miR-155 and activated BMP/Smad signaling pathway in ratsThe expression of miR-155 in serum,bone tissue and BMSCs of rats was decreased by PV intervention,and the expression of Osterix,Runx2,and BMP/Smad signal-related genes in bone tissue was effectively up-regulated.(3)PV can promote bone differentiation of BMSCsCompared with the Cd group,mRNA expression of osteogenic genes Runx2,Osterix,BMP4,Smad1,Smad5 and Smad9 were increased in the PV treatment group.Calcium nodules in BMSCs were stained with alisarin red.PV promoted the formation of calcium nodules in Cd-treated BMSCs.conclusion1.Cd exposure can promote the occurrence and development of osteoporosis and inhibit the osteogenic differentiation of BMSCs in rats with a dose-reactivity,which may be related to the activation of miR-155 and the inhibition of BMP/Smad signaling pathway in osteogenic differentiation pathway,leading to reduced bone formation and thus promoting the development of osteoporosis.2.Cd up-regulates miR-155,which inhibits the expression of Smad5 and negatively regulates the hBMSCs osteogenic differentiation by complementary binding with the 3’UTR base of the target gene Smad5.3.PV can promote BMSCs osteogenic differentiation to improve Cd-induced osteoporosis in rats,which may be related to BMP/Smad signaling pathway activated by miR-155/Smad axis.