Study On Maclurin Promote Chondrogenic Differentiation Of BMSCs By Regulating MiR-203a-3p/Smad1

ObjectiveOsteoarthritis(OA)is a common chronic and frequently-occurring disease in clinic.It is common in the elderly.The main manifestations of OA are joint capsule thickening,bone tissue dysplasia,cartilage wear and tear,often leading to joint pain,joint damage and functional loss.Because the etiology and pathogenesis of OA is not yet fully clear,there is no safe and effective treatment method.At present.the main clinical treatment of OA is to relieve pain,reduce weight,reduce activity,pay attention to diet and other auxiliary relief of joint pain.In order to find a better way to treat OA,some researchers have proposed that the method of repairing cartilage injury by cell transplantation has been w idely recognized.Because cartilage tissue is composed of chondrocyte and collagen Ⅱ,Ⅸ,Ⅸ and proteoglycan,autologous chondrocyte transplantation has limitations,and in vitro chondrocyte has few sources,weak proliferation ability and limited survival time.The traditional cartilage transplantation is not ideal.Bone marrow mesenchymal stem cells(BMSCs)are a kind of stem cells existing in bone marrow.BMSCs can differentiate into osteocytes.chondrocytes,adipocytes and so on under appropriate conditions,and participate in the repair and regeneration of human tissues.Therefore.BMSCs-derived chondrocytes as seed cells are an ideal method to repair damaged cartilage of OA.Cytokines have been found to promote chondrogenic differentiation of BMSCs,but they can not be used in clinical work because of their high cost,short half-life,biological safety and other factors.Therefore,finding a safe and effective inducer is the key to solve the problem.According to TCM,OA belongs to "bone arthralgia" and originally belongs to"kidney".Chinese herbal medicine for tonifying kidney can treat OA by nourishing liver and kidney.Previous studies of this project group have shown that some phenols of Chinese herbal medicine for tonifying kidney can promote the differentiation of BMSCs into chondrocytes.Mulberry,a traditional Chinese medicine for tonifying kidney,is used for both medicine and food to nourish liver and kidney.Maclurin is an effective active ingredient in mulberry.It contains phenols which are highly oxidative.Maclurin can promote chondrocyte differentiation of BMSCs.However,the mechanism of Maclurin promoting chondrocyte differentiation of BMSCs is still unclear.MicroRNA(microRNA.microRNA)is a kind of endogenous non-coding single-stranded RNA that exists in eukaryotic organisms.It can affect protein expression by regulating the expression of RNA and plays an important role in the maturation of cell formation and development.MicroRNA is also an important regulator of cartilage formation.It has been reported that microRNA-203a-3p is involved in regulating the differentiation of BMSCs osteoblasts.Therefore,this study is to explore whether BMSCs also participate in the differentiation of BMSCs chondrocytes.It has been successfully established that the Chinese medicine monomer of BMSCs osteogenic differentiationmicroRNA-target gene(mRNA)system.The aim of this study was to investigate whether small Chinese medicine molecule Maclurin could regulate the differentiation of BMSCs chondrogenic cells through the mechanism of influencing the target gene of microRNAs.Methods1.BMSCs of SD rats was obtained by whole bone marrow culture method,the methods of BMSCs culture,passage,freezing and resuscitation were used to observe the cell growth under the inverted optical microscope.The cells in good condition were used in the follow-up experiment.2.By consulting the literature,the best method of inducing chondrogenic differentiation of BMSCs in vitro was selected.Then the experiment was divided into blank control group and induction group,and the inducer acted on BMSCs in good condition for 7 days.The fluorescence intensity of Sox9,Coll2a associated with early chondrogenic differentiation was observed by immunofluorescence,and then the expression of Sox9,Coll2a protein was detected by Western Blot.3.The cytotoxicity of BMSCs was detected by CCK 8 assay.Then the experiment was divided into three groups:control group.DMSO group,induction group and maclurin group.After 7 days of exposure,the fluorescence intensity of Sox9,Coll2a was observed by immunofluorescence and the expression of Sox9.Coll2a protein was detected by Western Blot.4.The total RNA,of the blank group,induction group and hesperidin group were extracted by RNA extraction reagent and then the expression of miR-203a-3p was detected by RT-qPCR.5.The mimic,m-NC,inhibitor,i-NC of miR-203a-3p was transfected into BMSCs.for 7 days.The fluorescence intensity of Sox9.Coll2a was observed by immunofluorescence,and then Sox9,was detected by Western Blot.The expression level of Coll2a protein.6.The target gene of miR-203a-3p was predicted by biological information website,the expression of target protein was detected by immunofluorescence,Western Blot,and the relationship between miR-203a-3p and target gene was verified by constructing double luciferase reporter gene.7.Three interfering fragments of the target gene were constructed,and the best interfering fragments were screened by Western Blot.The screened interfering fragments were transfected into BMSCs,and detected by Western Blot assay and the expression level of early chondrogenic differentiation protein Sox9,Col12a was detected by Western BlotResults1.The technique and method of BMSCs culture in whole bone marrow were explored by our research group,and the cells obtained by this method were detected by flow cytometry and confirmed to be BMSCs.2.TGF-β3 was used to induce chondrogenic differentiation of BMSCs in vitro.The results of immunofluorescence assay showed that the fluorescence of Sox9.Coll2a protein in the induction group was stronger than that in the control group,and the Sox9,Coll2a protein in the induction group was higher than that in the control group(P<0.01).The results show that the induction method can be used in subsequent experiments.3.The results of CCK 8 showed that 25 μg/mL mulberry maclurin had no cytotoxicity to BMSCs(P>0.05).The results of immunofluorescence and Western Blot test showed that maclurin could promote chondrogenic differentiation of BMSCs by synergetic with inducer.4.The results of RT-qPCR showed that miR-203a-3p changed in the process of BMSCs chondrogenesis promoted by maclurin,and the expression level decreased(P<0.05).5.After BMSCSs was transfected with mimic,m-NC,inhibitor.i-NC of miR-203a-3p and cultured in induction medium for 7 days,the fluorescence intensity of Sox9.Coll2a protein in the mimic group was lower than that in the induction group,but the Sox9.in the inhibitor group was lower than that in the inhibitor group.The fluorescence intensity of Coll2a protein was higher than that of induction group.The results of Western Blot showed that the expression of Sox9.Coll2a protein in mimic group was lower than that in induction group,but the expression of inhibitor was lower in inhibitor group.The expression of Sox9(P<0.05).Coll2a(P<0.05)protein in the induction group was higher than that in the induction group.6.Smadl was predicted to be the target gene of miR-203a-3p by bioinformatics website.The results of immunofluorescence showed that the fluorescence intensity of Smad1 protein in mimic group was weaker than that in m-NC group(P<0.05).The fluorescence intensity of Smadl protein in inhibitor group was higher than that in i-NC group(P<0.05).Western Blot assay showed that the expression of Smadl protein in mimic group was lower than that in m-NC group(P<0.05).The expression of Smadl protein in inhibitor group was higher than that in i-NC group(P<0.05).The results of double luciferase reporter gene showed that miR-203 in wild plasmid The fluorescence intensity of mimic group was lower than that of m-NC group,while that of inhibitor group was higher than that of i-NC group(P<0.05),but there was no difference among the four groups in mutant plasmid(P>0.05).7.The results of Western Blot experiment show that siR-Smadl-002.siR-Smadl-003 has good inhibition effect.The results of transfection of siR-Smad1-003 fragment into BMSCs,Western Blot showed that the decreased expression of Smadl could affect the expression of early chondrogenic differentiation protein Sox9,Coll2a(P<0.05).ConclusionIn the experiments,we found that maclurin can co-promote the chondrogenesis of BMSCs with TGF-β3,and miR-203a-3p is involved in this process,and the expression of Smadl protein is down-regulated in BMSCs chondrogenesis.In BMSCs,miR-203a-3p targets Smadl protein.