The Mechanism Of Adipogenic Differentiation Of BMSCs By AtractylodeⅢ Targeting MicoRNA-466c-5p

Objective:To investigate the mechanism of the adipogenic different-iation of bone marrow mesenchymal stem cells in rats by traditional C-hinese medicine AtractylodeⅢregulating the expression of micoRNA-466c-5p.Bone marrow mesenchymal stem cells（BMSCs）are stem cells with multiple di fferentiation potential.It can be induced to differ-ent kinds of cells in vitro under s pecific conditions,including osteoblasts,adipocytes and chondrocytes.The osteogen ic and adi-pogenic differentiation of BMSCs are balanced mutually under physiologi cal condition.However,the various pathological metabolic diseases such as osteopor osis,age-related bone loss,obesity,diabetes and cardiovascular and cerebrovasc ular diseases arise when the differentiations out of balance.In this study,the regulat ion mechanism of AtractylodeⅢon adipogenic differentiation of BMSCs as well as the regulatory role of AtractylodeⅢwere mainly studied.Our team experiments h ad showed that oxidative stress can promote the adipogenic differentiation of BMSC s and 100μM hydrogen peroxide（H₂O₂）can induce adipogenic differentiation of BM SCs,and AtractylenolideⅢcan promote chondrog-enic differentiation of BMSCs wh ich may attribute to the inhibition of the oxidative stress promoting chondrogenic di fferentiation of BMSCs.Our group also proved that some micoRNAs might involve in the adipogenic differentiation of BMSCs through regulating their target genes.Th erefore,we guess that AtractylodesⅢand miRNAs may play an important role in the physiology and pathology process of adipogenic differentiation of BMSCs.Methods:1.Whole bone marrow culture method was used to isolate and culture the BMS Cs of rats,and the flow cytometry was used to identify the primary cells.The oil r ed O staining,qRT-PCR and Western Blot were used to identify the ability of adi pogenic differentiation after the induction of H₂O₂ on BMSCs 1h.CCK8 and oil red O staining were used for screening the best concentration of atractylactoneⅢon a dipoge-nic differentiation.qRT-PCR and Western Blot were used to demonstra-te the regulatory effect of atractylodeⅢon the adipogenic differe-ntiation of BMSCs ind uced by H₂O₂.The qRT-PCR technique was used to determine the changes in the c ontent of the miR-466c-5p in the model group and the atractylodeⅢmedicine grou p.2.Predicting the target gene of miR-466c-5p in the software of Bioinformatics.C onstructing the mimics and inhibitors of rno-miR-466c-5p specifically expressed and transfected into BMSCs.Detecting the expression of target protein by Western Blot while constructing the target gene 3,UTR region of dual luciferase reporter gene co ntainingrno-miR-466c-5p binding sites to clarify the target gene miR-466c further.3.The rno-miR-466c-5p mimic was transfected into BMSCs which had induced by H₂O₂.In order to detect whether miR-466c could regulate adipogenic differentiatio n on BMSCs,the oil red O test and Western Blot technology were used to examin e the change of differentiation ability on BMSCs compared with the blank group an d model group.4.qRT-PCR method was used to Screen the best target gene interf-erence frag ment in 3 synthesized siRNA fragments and then transfectedthe optimal siRNA into test group.Oil red O test and Western Blot were used to examine the change of di fferentiation ability on BMSCs aimed to determine whether the target gene was invo lved in adipogeni-c differentiation of BMSCs.5.After co-transfection of target gene siRNA and miR-466c-5p mimics into B MSCs,oil red O test and Western Blot were used to examine the changes of adipo genic differentiation function of BMSCs in each group to illustrate the mechanism o f target gene involved in the process of adipocyte differentiation by miR-466-c-5p.Results:1.After the method of whole bone marrow culture,the cells were identified as BMSCs by flow cytometry.BMSCs were induced to adipoge-nesis by H₂O₂（100μM,1h）.Adipocyte differentiation of BMSCs induced by H₂O₂ was inhibited by 20μM AtractylactoneⅢ.The results of qRT-PCR showed that the expression of miR-466c-5p in the model group was decreased in the model group while was increased in the Atractyl-actoneⅢmedicine group（P<0.05）,differences all being signifi ca-nt.2.The target gene of miR-466c-5p might be plin1 protein predicted by Bioinf-or matics.Compared with negative control,the expression of plin1 dete-cted by western blot was decreased in the group of miR-466c mimics while increased in inhibitor gr oup after transfection of miR-466c-5p mimics and inhibitors into BMSCs（P<0.05）,differences all being significant.The results of report gene were same as those show ed in Western Blot test（P<0.05）,differences all being significant.3.Oil red O staining indicated that the lipid droplet content wasdecreased in the miR-466c mimics group and blank compared with the model group and negative c ontrol group（P<0.05）,differences all being significant.Western blot results showe d the proteins related to adipocyte dif-ferentiation like PPARgama,AP2,GLUT4were decreased in the group of miR-466c mimics and blank compared with the mo del group and negative control group（P<0.05）,differences all being significant.4.The best interference fragment was GAACCATGAAGACCAGACA selectedby qRT-PCR.Oil red O staining indicated that the lipid droplet conte-nt was decreased in the plin1-siRNA group and blank group compared with the model group and ne gative control group（P<0.05）,differenc-es all being significant.Western blot resul ts showed PPARgama,AP2,GLUT4 proteins were decreased in the group of plin1-siRNA-miR-466c group and blank compared with the model group and negative c ontrol group（P<0.05）,differences all being significant.5.After co-transfection into BMSCs.Oil red O staining showed that the lipid droplet content was decreased in the cotransfection of plin1-siRNA-miR-466 group a nd blank compared with the model group and negative control group（P<0.05）,d ifferences all being significant.Western blot results indicated the PPARgama,AP2,GLUT4 proteins were decreased in the cotransfection of plin1-siRNA-miR-466 mimi cs group and blank group compared with the model group and negative control gro up（P<0.05）,differences all being significant.Conclusion:AtractylactoneⅢinhibits adipogenic differentiation of BMSCs deduced by H₂O₂ which may be regulated by the different expressions of miR-466c-5p in the adipo genic differentiation process.MiR-466c-5p involves the adipogenic differentiation pro cess by regulateing the expression of the target protein plin1.This study provides a theore-tical basis for the inhibition of adipogenic differentiation in BMSCsby Atract ylactoneⅢand provides a new way for the prevention and treatment of diseases r elated to the lipid metabolism.