The Role Of Hedgehog Signaling Pathway In The Osteogenic Differentiation Of Bone Mesenchymal Stem Cells Stimulated By Simvastatin

Objectives To clarify the effects of simvastatin on rat BMSCs osteogenic differentiation and the expressions of the key molecules in Hedgehog signaling pathway,then investigate the role of Hedgehog signaling pathway in this process by using specific inhititor Cyclopamine.Methods Eight SPF 4-week-old Sprague-Dawley female rats were used in this study,all the rats were killed by cervical dislocation and removed bilateral femur and tibia under sterilization conditions.Whole bone marrow culture method utilized for primary and passaged cultures.The second generation of BMSCs under well growth state were randomly divided into four groups.CM group:the complete medium contains 10mmol/Lβ-glycerophosphate and 50μg/mL ascorbic acid as control group;SIM group:the osteogenic induction medium contains10^-77 mol/L simvastatin;SIM+Cpn:the BMSCs were blocked 2 hour beforehand by 5μmol/L Cpn and add 10^-7mol/L simvastatin simutaneouly with SIM group;Cpn:the osteogenic induction medium contains 10^-77 mol/L simvastatin and 5μmol/L Cpn.At 7d,we use alkaline phosphatase staining to test the capability of early stage of osteogenic differentiation and the level of Gli1,Col1 and OCN were evaluated by immunofluorescence.The level of Ihh,Ptc1,Gli1,Runx2,Smo,ALP,COL1A1,OCN mRNA were evaluated by using Real-time PCR at 7d and Ihh,Ptc1,Gli1,Smo,COL1A1,OCN mRNA at 14 d.The protein levels of Ihh,Gli1,ColΙand OCN were evaluated by using Western blot analysis both at 7d and 14 d.Alizarin Red S staining was performed at 28 d to detect the capability of matrix mineralization and measured asorbance at 570 nm by spectrophotometer.Results 1 ALP staining:SIM group ALP staining were stronger than CM group and positive cells were more than CM group,positive cells of SIM+Cpn group were less than SIM group but more than Cpn group.Cpn group ALP staining were lighter than SIM+Cpn group and positive cells were less than SIM+Cpn group.2 Immunofluorescence:Mean fluorescence intensity of Gli1 and ColΙin SIM group were significantly higher than CM group（P<0.05）,SIM+Cpn group were significantly lower than SIM group（P<0.05）and higher than Cpn group（P<0.05）,Cpn group were significantly lower than CM group（P<0.05）.Mean fluorescence intensity of Gli1 in SIM+Cpn group were significantly lower than SIM group（P<0.05）and higher than Cpn group（P<0.05）,Cpn group were significantly lower than CM group（P<0.05）in nucleus.Mean fluorescence intensity of OCN in SIM+Cpn group were significantly lower than SIM group（P<0.05）and higher than Cpn group（P<0.05）,Cpn group were significantly lower than CM group（P<0.05）.3 Real-time PCR:SIM could significantly promote expression of ALP、COL1A1 mRNA at7 days after treatment and up regulate key molecule of Hedgehog signaling pathway Ihh、Gli1 mRNA（P<0.05）,but this effect dosen not block by Cpn,an inhibitor of Hedgehog signaling pathway completely.At 14 days after treatment,SIM could significantly promote expression of Ihh、Gli1 mRNA and COL1A1 mRNA as an osteogenic maker（P<0.05）,but this effect dosen not block by Cpn completely.4 Western blot:SIM could significantly promote expression of Gli1 mRNA at 7 days after treatment（P<0.05）but this effect dosen not block by Cpn completely.The expression of Gli1 at 14 d in SIM group、SIM+Cpn group and CM group were significantly higher than the level of the same group at 7d（P<0.05）.2)Ihh was significantly down regulate by Cpn（P<0.05）and in SIM+Cpn group were significantly higher than Cpn group both at 7d and 14d（P<0.05）.3)SIM could significantly promote expression of ColΙ（P<0.05）and this effect dosen not block by Cpn completely.The expression of ColΙin SIM group、SIM+Cpn group and CM group were significantly higher than the level of the same group at 7d（P<0.05）.4)OCN was significantly down regulate by Cpn（P<0.05）and the expression of each group were significantly higher than the level of the same group at 7d（P<0.05）.5 Alizari Red S staining and calcium quantitation:Alizari Red S staining of CM group and SIM group were deeper than SIM+Cpn group and Cpn group.The OD values of SIM group were significantly higher than CM group（P<0.05）,SIM+Cpn group were significantly lower than SIM group（P<0.05）and higher than Cpn group（P<0.05）,Cpn group were significantly lower than CM group（P<0.05）.Conclusions Simvastatin could promote BMSCs osteogenic differentiation through upregulating the expression of ColⅠand OCN,coupled with enhanced expression of Ihh and Gli1 in Hedgehog signaling pathway,which has not been completely blocked by Cpn.The mechanism of BMSCs osteogenic differentiation induced by Simvastatin probably involved the interaction between Hedgehog signaling pathway and other signaling pathways or interlinked key molecules.