| ObjectiveOsteoporosis(OP)is a chronic systemic diseases of skeletal system characterized by bone strength low,bone microstructure change and bone fragility increase,it is a special manifestation of physiological aging in bone.With the speeding up of old age and the extension of average life expectancy in our country,the incidence of OP is rising,our country will have the most osteoporotic patients in the world by 2020.Therefore,it is particularly important to seek the pathogenesis of OP and search the effective drugs.Bone mesenchymal stem cells(BMSCs)is a kind of pluripotent adult stem cells from bone marrow.The number of OB will reduce and the bone formation will low when the differentiation ability of BMSCs into osteoblast(OB)decline,this is one of the main pathogenesis of OP.Therefore,it is great significance to find the drugs that can accelerate the proliferation and osteogenetic differentiation of BMSCs.Traditional chinese medicine(TCM)theory say "the kidney stores essence,controls bone,and produces marrow",it means that bone marrow comes from kidney essence,if the adequate kidney essence,it can keep the bone normal growth,development,strong and powerful,on the contrary,it can cause OP.This theory is similar to the modern medicine theory that the cause of OP is for the decline of BMSCs to the osteogenetic differentiation.Therefore,Modern medicine thinks that BMSCs could be a form of kidney essence at the cellular level,they are looking for effective drugs from the chinese herbal medicine which can invigorate the kidney to delay the occurrence of osteoporosis.Catalpol is an effective ingredient of Rehmanniae,which has the effect of nourishing Yin and strengthening kidney.Catalpol can prevent and treat the nervous system disease,diabetes,cardiovascular disease,osteoporosis and other diseases.This study is to observe the effects of catalpol on the proliferation and differentiatial capacity to osteoblast of BMSCs,and observe the effects on Hedgehog signaling pathway which were closely related to differentiation of BMSCs.To define targets of catalpol on BMSCs,which can provide cellular and molecular evidences for catalpol preventing and curing osteoporosis from a new way,rich the TCM theory of "the kidney stores essence,controls bone,and produces marrow" and provide experimental basis for Chinese life-nurturing material medica.Methods1.BMSCs was isolated from Sprague-Dawley rats and purified by differential time adherent method.2.The morphology of BMSCs was observed by inverted microscope,the microstructure observed by electronic microscope,the growth curve observed by CCK-8 method,cell cycle and cell surface antigens CD29,CD90,CD45 and CD80 detected by flow cytometer,multidirectional differentiation detected by ALP and Alizarin dyeing.3.CCK-8 method was used to detect the proliferation of BMSCs after treating with different concentrations of catalpol(1×10-3mol/L?1 × 10-9mol/L).4.pNPP method was used to observe Alkaline phosphatase(ALP)activity of BMSCs after treating with different concentrations of catalpol(1×10-3mol/L?1×10-9mol/L,and confirm the best effective concentration of catalpol.5.The best effective concentration of catalpol was used to observe BMSCs activity in next experiment,experiment was divided into four groups:control group,catalpol group,classic group and combination group.Control group cells were cultured by culture medium of ?-MEM containing 15%fetal bovine serum;Catalpol group cells were cultured by culture medium of ?-MEM containing 15%fetal bovine serum and the best effective concentration of catalpol;classic group cells were cultured by osteogenic induction fluid containing 15%fetal bovine serum;combination group cells were cultured by osteogenic induction fluid containing 15%fetal bovine serum and the best effective concentration of catalpol.6.After treating with different groups,ALP kit was used to observe ALP activity,Alizarin dyeing was used to observe mineralization and ELISA method was used to observe osteocalcin activity of BMSCs.7.The mRNA expression levels of Shh and Ihh in the Hedgehog signal transduction pathway were measured by Real-time PCR after treating with different groups.8.The protein expressions of Ptchl?Smo and Gli1 in the Hedgehog signal transduction pathway were measured by Western blotting after treating with different groups.Results1.Cells isolated from SD rats showed the biological characteristics of BMSCs.The surface antigens of BMSCs showed the positive expressions of CD90 and CD29,the negative expressions of CD34 and CD45,BMSCs were induced successfully into osteoblasts and adipocytes.2.The results of CCK-8 method shows:OD values in defferent concentrations of catalpol(1×10-3mol/L?1×10-9mol/L)were significantly highter than the black group after 1,3 and 5 days(P<0.05);OD values in catalpol of 1×10-4mol/L?1×10-9mol/L were significantly highter than the black group after 7 days(P<0.05),it is no different with the black group in catalpol of 1×10-3mol/L group(P>0.05);OD values in catalpol of 1×10-5mol/L?1×10-9mol/L were significantly highter than the black group after 9 days(P<0.05),it is no different with the black group in catalpol of 1 × 10-3mol/L group and 1×14-4mol/L group(P>0.05);OD values in catalpol of 1×10-5mol/L and 1×10-6mol/L were significantly highter than the black group after 11 days(P<0.05),it is no different with the black group in other catalpol groups(P>0.05).3.The results of pNPP method shows:The ALP activity in defferent.concentrations of catalpol(1×10-3mol/L?1×10-9mol/L)were no different with the black group after 3,18 and 21 days(P>0.05).The ALP activity in catalpol of 1×10-4mol/L concentration were significantly highter than the black group after 6 days(P<0.05);The ALP activity in catalpol of 1×10-3mol/L to 1×10-5mol/L concentration were significantly highter than the black group after 9 days(P<0.05);The ALP activity in catalpol of 1×10-3mol/L and 1×10-4mol/L concentration were significantly highter than the black group after 12 and 15 days(P<0.05),especially 1×10-4mol/L catapol;the most effective concentration of catalpol on the differentiation of BMSCs into osteoblasts was 1×10-4mol/L and used to the the follow-up experiments.4.The results of ALP kit detention shows:Compared with control group,the ALP activity were significantly increased in catalpol group,classic group and combination group(P<0.05),the ALP activity in catalpol group was lower than the classic group(P<0.05),the ALP activity in combination group was no different with the classic group(P>0.05).5.The results of Alizarin dyeing shows:Compared with control group,the mineralization of BMSCs were significantly increased in catalpol group,classic group and combination group(P<0.05),the mineralization of BMSCs in catalpol group was lower than the classic group(P<0.05),the mineralization of BMSCs in combination group was no different with the classic group(P>0.05).6.The results of ELISA method shows:Compared with control group,the BGP content were significantly increased in catalpol group,classic group and combination group(P<0.05),the BGP content in catalpol group was lower than the classic group(P<0.05),the BGP content in combination group was no different with the classic group(P>0.05).7.The results of QPCR method shows:Compared with control group,the expression of Shh mRNA were significantly increased in catalpol group,classic group and combination group(P<0.05),the expression of Shh mRNA in catalpol group was lower than the classic group(P<0.05);the expression of Shh mRNA in combination group was no different with the classic group(P>0.05);the expression of Ihh mRNA in all groups were on different with the control group(P>0.05).8.The results of Western blotting method shows:Compared with control group,the protein expressions of Ptch1?Smo and Gli1 were increased in catalpol group,classic group and combination group(P<0.05),the protein expressions of Ptchl?Smo and Gli1 in catalpol group and combination group were all lower than the classic group(P<0.05),the protein expressions of Ptchl?Smo and Gli1 in catalpol group was no different with the combination group(P>0.05).Conclusions1.The obtained BMSCs by the differential time adherent method have good multiplication capacity and high degree of purity.2.Defferent concentrations of catalpol all can promote proliferation of BMSCs.3.The catalpol concentration of 1×10-3mol/L?1×10-5 mol/L could induce osteogenic differentiation of BMSCs,and the best effective concentration of catalpol was 1×10-4 mol/L.Catalpol could increase the ALP activity,the number of mineralizations and the BGP content of BMSCs.4.Ihh signaling molecules may do not participate in the process of osteogenic differentiation of BMSCs iduced by catalpol.5.Catalpol can increase the expression of Shh mRNA and improve the protein of Ptch1?Smo and Glil in Hedgehog signaling pathway.The results could say that Hedgehog signaling pathway plays a significant role on the process of BMSCs osteogenic differentiation. |
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