| Experiment I Effects of Salvianolic acid B on the expression profiles of mRNA,circRNA and lncRNA in white and brown adipose tissue of high fat diet induced obese miceObjectiveThis research used high fat diet-induced C57BL/6J obese mice to detect mRNA,circRNA,and lncRNA expression profiles using RNA sequencing in white and brown adipose tissue of Salvianolic Acid B intervention group and model group,and to find the potential biomarker for the role of salvianolic acid B in improving energy metabolism of adipose tissue at the RNA level.MethodsSixty 6-week-old C57BL/6J male mice were divided into normal group(n=10)and high fat diet group(n=50),the mice in the normal group were fed with normal diet and the high fat diet group fed the high fat for 12 weeks to induce obesity.Then 30 obese mice were randomly divided into the following groups:model group(n=10),which were given the same amount of sterile drinking water;metformin group(n=10),which were given oral administration by 75mg/kg/d volume;salvianolic acid B intervention group(n=10),which were administered with salvianolic acid B at a dose of 100 mg/kg/d.All mice were administered for 8 weeks and continued to be fed a high fat diet.After 8 weeks,the epididymal white adipose tissue and scapular area brown adipose tissue were harvested.Five samples from model and salvianolic acid B intervention group were randomly selected for RNA sequencing to analyze the mRNA,circRNA,and lncRNA expression profiles.Seven differentially expressed mRNAs were selected for validation by RT-PCR.Moreover,the GO and KEGG enrichment analyses were performed for differentially expressed mRNA to explore the possible targets and pathways of salvianolic acid B on adipose tissue.ResultsIn white adipose tissue,there were 132 differentially expressed mRNAs(24 upregulated and 108 downregulated),in the salvianolic acid B intervention group compared with the model group.We found that the upregulated Sfrp5,Adig and downregulated Saa3 play an important role in the obesity-induced adipose tissue inflammatory response.The bioinformatics analysis was performed on differentially expressed mRNA.GO analysis suggested that upregulated differentially expressed mRNAs were related to brown adipocyte differentiation,steroid biosynthesis,lipid transport,and lipid metabolism,while downregulated mRNAs were mainly involved in the immune system process and inflammatory response.KEGG analysis indicated that the upregulated mRNAs were enriched in insulin resistance and downregulated mRNAs were involved in IL-17 signaling pathway and Phagosome pathway.In brown adipose tissue,a total of 2532 differentially expressed mRNAs,including 275 upregulated and 2257 downregulated,were determined in the Salvianolic Acid B intervention group compared with the model group.GO analysis revealed that upregulated mRNAs were involved in the oxidation reduction process,metabolic process,and fatty acid metabolic process.Downregulated mRNAs were associated with the immune system process,inflammatory response,and innate immune response.KEGG analysis indicated that upregulated mRNAs were related to the metabolic pathway,propanoate metabolism,and oxidative phosphorylation.Moreover,downregulated mRNAs were enriched in osteoclast differentiation,systemic lupus erythematosus,and phagosome.In white adipose tissue,we found 19 differentially expressed circRNAs,with 9 upregulated and 10 downregulated in the Salvianolic Acid B intervention group compared with the model group.In brown adipose tissue,a total of 25 differentially expressed circRNAs included 10 upregulated and 15 downregulated ones.Among these,upregulated circRNAs such as Atp5o,Aldoa,and Bckdhb were involved in the process of energy metabolism,while downregulated circRNA Mthfd21 was related with mitochondrial function,but further investigation of their role in adipose tissue of obesity was needed.In white adipose tissue,we found 234 differentially expressed lncRNAs,with 87 upregulated and 147 downregulated ones,in Salvianolic Acid B intervention group compared with the model group.Among these,upregulated lncRNA-Rora was involved in lipid metabolism.Downregulated lncRNA-Dnm2 was involved in the mitochondrial function of muscle tissue.In brown adipose tissue,a total of 774 differentially expressed lncRNAs was found,which included 497 upregulated and 277 downregulated ones.Among these,we found upregulated lncRNA-Hsd11b1 was related to mitochondrial respiration and lipid metabolism.Downregulated lncRNA,such as Slc11a1and Sirpa,were associated with the development of diabetes.Conclusion1.The results of RNA sequencing showed that the expression profiles of mRNA,circRNA,and lncRNA were significantly different in the white and brown adipose tissue between the Salvianolic Acid B intervention group and model group.From the perspective of epigenetics,the possible targets for the anti-obesity effect of Salvianolic Acid B in white and brown adipose tissue were explained and provided a theoretical basis for further mechanism research.2.In white adipose tissue,differentially expressed mRNAs were mainly involved in adipocyte differentiation,lipid metabolism,and inflammation,and were related to insulin resistance and the IL-17 signaling pathway,suggesting that salvianolic Acid B mainly regulated anti-inflammatory related factors and signaling pathways.3.In brown adipose tissue,differentially expressed mRNAs participated in oxidation reduction process,fatty acid metabolic process,and were related to propanoate metabolism and oxidative phosphorylation,which indicated that salvianolic Acid B regulated the expression of mitochondrial function-related factors and enhanced the metabolic function of brown adipose tissue.Experiment Ⅱ Effects of Salvianolic acid B on mitochondrial function in 3T3-L1 adipocytesObjectiveIn this part,3T3-L1 preadipocytes were employed to analyze mitochondrial respiratory function,glycolysis capacity,mitochondria content and mitochondrial biosynthesis-related gene expression in differentiated adipocytes.In addition,the effects of salvianolic acid B on the mitochondrial function of adipocytes were investigated.MethodsThe differentiated adipocytes were divided into control,salvianolic acid B(50μM),GW9662(20μM),and GW9662+salvianolic acid B groups for 48 h.The mitochondrial oxygen consumption rate and extra cellular acidification rate in 3T3-L1 adipocytes were assessed by Seahorse Extracellular Flux Analyzer.Content of adipocytes’ mitochondria was stained using Mito-Tracker Green regeant and observed under fluorescent microscope.The expression of peroxisome proliferators-activated receptor y coactivator-1α(PGC-1α)wasdetermined through western blotting and immunofluorescence analysis.In addition,the mRNA expressions of NRF-1/2,UCP2,and PFKFB2 were detected by RT-PCR.ResultsSalvianolic Acid B could significantly increase the mitochondrial respiratory function including basal respiration,ATP production,proton leak,maximum oxygen consumption rate,and non-mitochondrial respiration capacity,of adipocytes.The difference in the results between this group and the control are statistically significant(P<0.05).However,the above parameters and spare respiration capacity were reduced after GW9662 administration(P<0.05).Compared with the GW9662 group,the salvianolic acid B group showed improved mitochondrial function,including mitochondrial basal respiration,maximum oxygen consumption rate,and non-mitochondrial respiration capacity(P<0.05),which were reduced on GW9662 treatment.In glycolytic function,compared with the control group,the salvianolic acid B group showed an increase in basal glycolysis rate and glycolytic capacity(P<0.05).However,GW9692 reduced the basal glycolysis rate and glycolytic capacity,and the differences were statistically significant(P<0.05).Compared with that in the GW9662 group,the basal glycolysis rate of adipocytes in the salvianolic acid B+GW9662 group was significantly increased(P<0.05).Regarding mitochondrial biosynthesis,compared with the control,salvianolic acid B could increase the fluorescence intensity and protein expression of PGC-1α,the key mitochondrial biosynthesis regulatory factor,but the difference was not statistically significant(P>0.05).The intensity and protein expression of PGC-1α were minimally reduced after GW9662 intervention.Compared with those in the GW9662 group,the intensity and protein expression of PGC-1α in salvianolic acid B+GW9662 group was significantly increased(P<0.05).Mito-Tracker Green was used to specifically label live cell mitochondria.Compared with that in the control group,the mitochondrial fluorescence intensity of adipocytes was significantly increased after salvianolic acid B treatment(P<0.01),but GW9662 downregulated the mitochondrial fluorescence intensity(P<0.01).Compared with the GW9662 group,the salvianolic acid B group showed an improvement in the inhibition and upregulation of mitochondrial fluorescence intensity(P<0.05).The effects of salvianolic acid B on mitochondrial-associated mRNA expression,mitochondrial synthesis genes NRF-1 and NRF-2,and glycolysis-related factors UCP2,and PFKFB2 after treatment with salvianolic acid B were compared with those of the control group.Compared with the GW9662 group,the salvianolic acid B+GW9662 group showed significantly upregulated the mRNA expression of NRF-2,and UCP2(P<0.05).Conclusions1.Salvianolic acid B could improve adipocyte mitochondrial respiratory function,including basal respiration,ATP production,proton leak,maximum oxygen consumption rate,spare respiration capacity,and non-mitochondrial respiration.2.Salvianolic acid B could enhance the adipocyte glycolytic capacity,which may be involved in upregulating the PPARy-mediated mRNA expression levels of UCP2 and PFKFB2.3.Salvianolic acid B promoted mitochondrial biosynthesis,increased the mitochondrial content of adipocytes and improved adipocytes energy metabolism by upregulating the PGC-1α and its related mRNA NRF-1and NRF-2 expression. |
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