Study On Sinapine Inhibits H₂O₂-induced Adipogenic Differentiation Of MSCs By Regulating MiR-330-5p

ObjectiveAccording to the reports of Chinese residents’nutrition and chronic disease were released in 2015,nearly 10 years,residents of overweight obesity are on the rise,diseases caused by obesity problem are still.It is well known that obesity increases the risk of heart disease,stroke and diabetes and other diseases,and for obesity,fundamentally a lack of long-term effective drug treatment,while Chinese medicine treatment for obesity is given priority to with spleen and eliminating phlegm to clearing damp.Most studies indicate that Chinese medicine can change obesity phenotypes,but fewer researches on the mechanism.In this study,Sinapine,the effective ingredient of Semen Raphani,was investigated to be observed the effect of its monomer on obesity.Other studies have demonstrated that heart disease,diabetes,and other diseases are related to lipid peroxidation.Oxidative stress,obesity,fat cells are closely associated.The increase of oxidative stress will accelerate the accumulation of fatty and then contribute to metabolic syndrome.Thus,redox status in adipose tissue is a feasible therapeutic target for the obesity-related metabolic syndrome.Based on the points of view,in this study,an oxidative stress-promoting fat model was established and to be used for the entire experimental designs.And all the studies have shown that adipocytes derive from bone marrow mesenchymal stem cells（MSCs）.In this study,the cell model of MSCs into adipocyte was induced by H₂O₂and was treated with Sinapine thiocyanate,using CCK8,Oil Red O and molecular biology techniques,a preliminary study on the regulative effect of the Sinapine thiocyanate on the adipogenic differentiation of MSCs were induced by oxidative stress.At the same time,experimentally observing the effect of Sinapine on miR-330-5p and screening miR-330-5p target genes to explore its mechanism of regulating the differentiation of MSCs into adiposity.Further,enrich the contemporary biological interpretation of the spleen-like Chinese medicine.Methods1.The study on H₂O₂ induces MSCs adipogenic differentiation as a model.The undifferentiated MSCs was divided into the Blank group and the H₂O₂-induced group.In the cell fusion to about 80%,using 100μM H₂O₂ to induce 1 h as the H₂O₂-induced group,after the end,replacement of complete medium for continuous cultivation of 24 h.The changes of cell morphology and lipid droplets in two groups were observed by Oil Red O and the expression of fat-related genes was detected by Western Blot.Rely on them,the feasibility of the model was estimated.2.The study on the intervention of Sinapine thiocyanate.The undifferentiated MSCs was intervened with different concentrations of Sinapine for24 h,using CCK8 assay to find the concentrations of without obvious toxicity to MSCs.MSCs are divided into Blank group,H₂O₂-induced group,Sinapine group.After H₂O₂ induce,different concentrations of Sinapine intervened for 24 h.Oil Red O assay was used to observe cell morphology and lipid droplets.RT-qPCR to detect the fat-related gene from mRNA level,and Western Blot to observe the effect of Sinapine on adipogenic differentiation of MSCs from the level of protein.According to the early laboratory studies and the literature supports,the different expression of miR-330-5p was observed before and after under the oxidative stress.Experiments are divided as above.Using RT-qPCR and Western Blot to observe the change of miR-30-5p and RXRγafter H₂O₂ induce,at the same time,to observe the expression of miR-330-5p under the action of Sinapine for 24 h.3.The study on adipogenic differentiation of MSCs by miR-330-5p and its target gene.Predicting the possible targets of miR-330-5p on the bioinformatics website,a specific expression of rno-miR-330-5p mimics were constructed and transfected into MSCs,then were induced by H₂O₂.Compared with the Blank group,the negative control group and the H₂O₂-induced group,the effect of overexpression miR-330-5p on the adipogenic differentiation of MSCs was detected.Mimic,inhibitor and their respective negative controls were transfected into the MSCs 48 h,which specifically suppressed the expression of rno-miR-330-5p.Then using Western Blot to detect expression of candidate target genes of RXRγ.Further,the relationship between RXRγand miR-330-5p was verified by constructing a luciferase report plasmid which containing RXRγ-3’UTR and rno-miR-330-5p binding site.Constructing the RXRγinterference fragment,and the best fragment was transfected into MSCs,after that,H₂O₂ was used to induce cells.Compare to the Blank group,the negative control group and the H₂O₂-induced group,the effect of RXRγon adipogenic differentiation of MSCs was detected.Co-transfecting the rno-miR-330-5p mimic and RXRγinterference fragments and detecting the expression of RXRγ.Subsequently,H₂O₂ induction was performed on the above groups.Western Blot to detect the expression of related genes,to further verify whether miR-330-5p depends on RXRγto influence on adipogenic differentiation of MSCs induced by H₂O₂.Results1.H₂O₂ induces MSCs to adipogenic differentiation.Oil Red O showed that the fusion of MSCs to 80%with 100μM H₂O₂ induce for 1 h,compared with the Blank group,the H₂O₂-induced group increased perinuclear vacuoles,the number of lipid droplets increased significantly,while the expression of adipose-related genes PPARγand aP2 increased with statistical significance（P<0.01）.2.Sinapine thiocyanate inhibits MSCs adipogenic differentiation and promotes the expression of miR-330-5p.The CCK8 assay showed that the concentration of Sinapine was under 40μM has no toxicity to MSCs.Oil Red O semi-quantitative assay suggested that the inhibition of MSCs to adipogenic differentiation was more noticeable with the increase of the concentration of Sinapine.Combining with the results of the CCK8 assay,the concentration of 15μM was considered as a follow-up study.Oil Red O,Western Blot,and RT-qPCR results indicated that Sinapine thiocyanate can inhibit MSCs into adipogenic differentiation by H₂O₂,and down-regulate PPARγ,aP2 and Glut4 expression（P<0.05）.In addition,RT-qPCR and Western Blot demonstrated that compared with the Blank group,the miR-330-5p expression in H₂O₂-induced group decreased（P<0.05）,while RXRγexpression increased（P<0.01）.After Sinapine intervention 24 h,the function of H₂O₂ will be weakened and the expression of miR-330-5p will be promoted（P<0.01）.3.MiR-330-5p inhibits adipogenic differentiation of MSCs through targeting RXRγ.Transfection of miR-330-5p mimic showed that compared with the H₂O₂-induced group,overexpression of miR-330-5p decreased the number of lipid droplets,as well as decreased expression of PPARγ,aP2,C/EBPα,etc.（P<0.01）.Bioinformatical predicts show that one of the potential targets of miR-330-5p is RXRγ.It noted that overexpression of miR-330-5p remarkably inhibited the luciferase reporter activity of the WT RXRγ-3’UTR（P<0.01）,but not that of the MUT RXRγ-3’UTR.Supplementary experiments confirmed that miR-330-5p overexpression markedly suppressed the expression of RXRγat both the mRNA and protein levels compared with m-NC group（P<0.05）.These findings indicated that miR-330-5p may directly regulate RXRγexpression.When silencing RXRγ,the adipogenic differentiation function of MSCs was inhibited,compared with the H₂O₂-induced group,the number of lipid droplets and the expression of PPARγ、C/EBPαwere reduced（P<0.05）.Results were more noticeable after co-transfecting miR-330-5p mimics and RXRγinterference fragment into MSCs（P<0.01）.ConclusionIn the experiments,we found that H₂O₂ can induce MSCs into adipocytes and Sinapine can inhibit H₂O₂ induce MSCs differentiation by regulating miR-33-5p.The explicit mechanism is to enhance the expression of miR-330-5p,and down-regulate the expression of RXRγ.It further affects the expression of PPARγ,aP2,and other downstream genes,then inhibit the process of oxidative stress-inducing MSCs differentiate into adipocytes.