| ObjectiveOsteoarthritis(OA)is the most common joint disease among people over 60 years old in the world,and has become the fourth most disabling disease,seriously affecting the quality of life of patients.With the increase of the elderly population in China,it will greatly increase the economic burden of the entire society.At present,the clinical treatment plans of OA are mainly to relieve joint pain,delay the development of OA,and fail to fundamentally cure OA.In recent years,some researchers have proposed to transplant chondrocytes instead of apoptotic chondrocytes to treat OA,but due to their own limitations such as difficulty in obtaining chondrocytes and no longer producing cartilage-specific matrix in vitro,limits the clinical application of this strategy.With the application of stem cell therapy in the treatment of a variety of clinical diseases,researchers began to explore its application in the treatment of OA.Bone mesenchymal stem cells(BMSCs)are a kind of adult stem cells derived from mesoderm.It not only participates in the occurrence and formation of cartilage during embryonic period,but also BMSCs still retain the ability to differentiate into chondrocytes after birth.The occurrence of OA is related to the degradation of BMSCs’ ability to differentiate into chondrocytes.Therefore,promoting chondrogenic differentiation of BMSCs and using bmscs-derived chondrocytes to repair damaged cartilage of OA is a new therapeutic approach.It has been found that cytokines can promote the differentiation of BMSCs into chondrocytes,so adding growth factors in the culture system or transfecting genes can promote the differentiation of BMSCs into chondrocytes.However,the problems such as high price,short half-life and biosafety of transgene limited its further application.Finding a safe and effective way to induce chondroblast differentiation of BMSCs is the key to solve the problem.Kidney-tonifying herbs can promote the differentiation of BMSCs into chondrocytes by regulating micro RNAs(miRNAs).Traditional chinese medicine(TCM)believes that OA belongs to the category of "bone paralysis",which is originally in the kidney.Kidney-tonifying Chinese medicine can nourish the liver and kidneys and nourish bones to treat OA.BMSCs belong to kidney essence and are important target cells of traditional chinese medicine for tonifying kidney.Traditional Chinese medicine has the advantages of multiple components and multiple targets for the treatment of diseases.This effect itself has positive and negative feedback regulation,which can promote the differentiation of BMSCs into chondrocytes without excessive.Current research has confirmed that miRNA promotes or inhibits the differentiation of BMSCs into chondrocytes by regulating target mRNA.Therefore,the study on the regulation of chondroblast differentiation of BMSCs by TCM for kidney tonifying through affecting the expression of miRNAs can be regarded as an OA treatment method with the characteristics and advantages of TCM.Early preliminary experiments found that the aqueous extract of mulberry fruit,a representative Chinese medicine for kidney supplementation,can promote the differentiation of BMSCs into chondrocytes and affect the expression of some miRNAs.Based on the previous research,the most significant down regulated miR-139 was selected in this study to further study the regulatory mechanism of differentiation of BMSCs into chondrocytes,and to explore the effect of miR-139 on the differentiation of ectopic cartilage in BMSCs.Methods1.Femur and tibia of male SD rats were taken and BMSCs were obtained by bone marrow culture for culture.BMSCs with good growth were selected for proliferation,passage and cryopreserved for subsequent experiments.2.Cell Counting Kit-8(CCK 8)method was used to detect the effect of aqueous extract of mulberry fruit on the proliferation of BMSCs.The experiments were grouped into the Blank group and the mulberry water extract group with different concentrations.After 48 hours,the microplate reader detected the absorbance of each group.3.The effect of aqueous extract of mulberry fruit on the differentiation of BMSCs into chondrocytes was preliminary explored.The experiment was divided into the Blank group and the aqueous extract of mulberry fruit with different concentrations(30 μg/mL,60μg/mL,120 μg/mL).After 7 days of induction,immunofluorescence experiment and Western Blot experiment were respectively used to observe the expressions of Sox9 and Collagen Ⅱ,proteins related to early chondroblast differentiation.4.miRNAs with different expressions were screened by RT-qPCR.The experiment was divided into the Blank group and the 60 μg/mL aqueous extract of mulberry fruit.After the treatment for 7 days,the total RNA of the cells was extracted,and RT-qPCR were used to detect the level of miR-139.5.miR-139 mimic,inhibitor and its corresponding NC were transfected into BMSCs with lip3000,and the aqueous extract of mulberry fruit was used for 7 days.Then immunofluorescence assay was used to observe and Western Blot assay,RT-qPCR assay were used to quantify the expressions of Sox9 and Collagen Ⅱ,which the proteins related to early chondroblast differentiation.6.Biological information analysis was used to predict the target genes of mir-139,construct wild-type or mutant luciferase reporter plasmids with MMP-14-3 ’UTR binding sites to mir-13 9,which was transfected to BMSCs with miRNA.The luciferase activity was detected to verify the targeting relationship between miR-139 and MMP-14.Then transfect miRNA,extract protein and RNA respectively,and detect whether over-expression of miR-139 and inhibition of miR-139 have an effect on the predicted target gene MMP-14.7.Western Blot experiments were used to screen the best interfering fragments of MMP-14,which were transfected into BMSCs.After being treated with aqueous extract of mulberry fruit for 7 days,the total cell protein Western Blot was extracted to detect the expression of early chondrocyte differentiation-related proteins Sox9 and Collagen Ⅱ.Total RNA was extracted and detected.RT-qPCR was used to detect whether the expression level of miR-139 had changed after knocking down the target gene MMP-14.8.BMSCs transfected with miR-139 mimic and inhibitor were transplanted to the above sites of 6-8 weeks nude mice.After embedding the sections,the expression of Sox9 and Collagen Ⅱ,proteins related to early chondrocyte differentiation,was detected by immunohistochemistry.Results1.The BMSCs purified by whole bone marrow adherence method,the BMSCs of P3 generation have a long spindle shape and vortex arrangement under a microscope,and their sizes are uniform and good,which can be used for subsequent experiments.2.The results of CCK 8 toxicity test showed that the aqueous extract of mulberry fruit at different concentrations had no toxicity to BMSCs for 24 h(P>0.05).3.Immunofluorescence and Western Blot showed that the expression of Sox9 and Collagen Ⅱ,which the proteins related to early chondroblast differentiation,could be improved after the aqueous extract of mulberry fruit of different concentrations acted on BMSCs 7 d(P<0.05).The results suggested that the aqueous extract of mulberry fruit could promote chondrogenic differentiation of BMSCs.4.The results of RT-qPCR experiments showed that miR-139 showed the most significant change among the differentially expressed miRNAs,showing a downregulated trend(P<0.001).5.The results of immunofluorescence and Western Blot experiments showed that compared with their respective NC groups,protein levels were earlier in the expression of chondroblast differentiation-associated proteins Sox9 and Collagen Ⅱ showed a downward trend in the miR-139 mimic group and an up-regulation trend in the miR-139 inhibitor group(P<0.05).RT-qPCR results showed that the expression of Sox9 and Collagen Ⅱ at the mRNA level was consistent with the protein level(P<0.05).The above results suggest that miR-139 inhibits the differentiation of BMSCs induced by aqueous extract of mulberry fruit into chondrocytes.6.Dual luciferase reporter gene results showed that compared with their respective NC groups,the luciferase activity of the wild-type plasmid and miR-139 mimic co-transfection group was down-regulated,while the luciferase activity of the wild-type plasmid and miR-139 inhibitor co-transfection group was up-regulated(P<0.01).There was no significant change in luciferase activity in the four groups co-transfected with the mutant plasmid and miR-139(P>0.05).The results of Western Blot experiments showed that compared with the respective NC group,the expression level of MMP-14 protein decreased in the mimic group that overexpressed miR-139,while inhibitor group that inhibited miR-139 increased the expression of MMP-14 protein(P<0.05).7.SiMMP-14-3 was selected as the best interference fragment by Western Blot and RT-qPCR(P<0.01).SiMMP-14-3 was transfected into BMSCs and treated with aqueous extract of mulberry fruit for 7 days before Western Blot analysis.The results showed that MMP14 knockdown could down-regulate Sox9 expression,while aqueous extract of mulberry fruit could reverse the effect of knockdown MMP-14 on Sox9 to some extent.After transfection of si-MMP-14-3 into BMSCs,RT-qPCR was used to detect the expression of miR-139 after MMP-14 knockdown.The results showed that the expression of miR-139 increased after MMP-14 knockdown(P<0.05).8.The results of immunohistochemistry in the in vivo ectopic cartilage differentiation experiment showed that compared with the Blank group,the expression of early chondrocyte differentiation-related proteins Sox9 and Collagen Ⅱ in the miR-139 mimic group decreased significantly,while that in miR-139 inhibitor group showed an opposite trend.ConclusionThe aqueous extract of mulberry fruit,a Chinese herbal medicine for kidney,does not need to add TGF-β to directly induce the differentiation of BMSCs into chondrocytes,and has a significant effect of promoting the differentiation of BMSCs.The expression of miR-139 decreased significantly during the chondrogenic differentiation of BMSCs induced by the aqueous extract of mulberry fruit.MMP-14 was the target gene of miR-139.The aqueous extract of mulberry increased the expression of MMP-14 by inhibiting the expression of miR-139,which promoted the expression of Sox9,Collagen Ⅱ of early chondrogenic differentiation related proteins,and further promoted the differentiation of BMSCs chondrocytes.miR-139/MMP-14 is a potential new target for the treatment of OA. |
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