Effects Of Astragalus Polysacharins On Brown Adipogenesis In C3H10T 1/2 Cells

Astragalus polysacharin(APS)is one of the most important natural active ingredients of Astragalus.It has a variety of biological functions,including immune regulation,antioxidants,anti-tumor,and anti-diabetics.It has been widely used in clinical practice.Brown adipose tissue(BAT)is a mammalian heat-producing organ,and its function is essential for the maintenance of the primary body temperature.There are a large number of long non-coding RNAs(lnc RNAs)and small RNAs(micro RNAs,miRNAs)in cells.Studies have shown that the differentiation of BAT is regulated by lnc RNA and miRNA.The relationship between APS and brown adipocyte differentiation has not been established.This experiment aims to explore the effect of APS on the differentiation of brown fat,and to clarify the role of lnc RNAs and miRNAs in this process.In this experiment,C3H10T 1/2 cells were used as the research object.0.4 mg/m L APS was added to the adipogenic differentiation medium.Oil red O staining,cellular immunofluorescence technology and Western Blotting were used to analyze the adipogenic differentiation ability of APS.Impact.By constructing a sequencing library and sequencing,the effects of APS on the expression profiles of mRNAs,lncRNAs and miRNAs in the differentiation of brown adipocytes were screened,and the target genes predicted by the cis and co-expression effects of differential m RNAs and differential lnc RNAs were analyzed.The dual luciferase reporter test detection system was used to analyze whether miR-6911 has a target relationship with PR domain zinc finger protein 16(PRDM16);transfection of miR-6911 mimics and inhibitors changes its expression,and studies The effect of miR-6911 on the key transcription factors in PRDM16 and brown adipogenesis.The expression levels of m RNA,lnc RNAs and miRNAs were randomly analyzed by q RT-PCR to verify the accuracy of the sequencing results.Research indicates:(1)APS promoted the brown adipogenesis of C3H10 T 1/2 cells.The addition of 0.4mg/m L APS promoted the formation of lipid droplets during brown adipose differentiation,and significantly increased the levels of transcription factors related to brown adipose cell differentiation,including adipogenic differentiation marker proteins C/EBPα,C/EBPβ,PPARγ and key thermogenic transcription factors UCP1,PRDM16,PGC-1α(P < 0.05).(2)APS enhances the insulin sensitivity of BAT cells.The addition of 0.4 mg/m L APS significantly improved the activity of the insulin signaling pathway and increased the expression of GLUT4 protein(P < 0.05).(3)The target relationship between miR-6911 and Prdm16 was verified.In the sequencing results,40 differentially expressed miRNAs were obtained.The q RT-PCR results showed that the expression levels of miRNAs were consistent with the sequencing results,which proved the accuracy of sequencing.Among them,the expression of miR-6911 in the experimental group was extremely significantly reduced(P < 0.01).It is predicted that miR-6911 targets the target gene Prdm16 related to brown fat differentiation.The results of dual luciferase report show that miR-6911 can significantly reduce the fluorescence intensity of the dual fluorescent reporter carrier containing Prdm16 3’UTR(P < 0.01),which proves that there is a target relationship between miR-6911 and Prdm16.(4)miR-6911 regulates the differentiation of brown adipocytes by targeting Prdm16.After transfection of miR-6911 mimics overexpression of miR-6911,PRDM16 protein expression decreased significantly compared with the control group,and the protein expression levels of PPARγ,UCP1,PGC-1α also decreased significantly;inhibition of miR-6911 expression showed the opposite Results(P < 0.05).Therefore,APS promotes the expression of PRDM16 by reducing the expression of miR-6911 and affects the brown fat differentiation of C3H10 T 1/2 cells.(5)APS changed the expression profile of lncRNAs in the differentiation of brown adipocytes.A total of 13450 lnc RNAs and 57776 m RNAs were obtained by sequencing.Through the analysis of its expression,length,number of exons and paired repeatability,the reliability of the sequencing data is proved to be good.A total of 153 differentially expressed lnc RNAs and 1238 differentially expressed mRNAs were screened.The results showed that the main GO annotations of differential mRNAs were enriched in 53 functional classifications,and KEGG analysis was enriched in 343 pathways.The target gene GO annotations predicted by differential lnc RNAs cis and co-expression were enriched in 34 and 33 functional categories,respectively,and the entries in molecular functions were consistent.KEGG analysis showed that multiple genes were enriched in the signaling pathways of fat metabolism and fat differentiation,especially in the glucagon and c AMP signaling pathways.In summary,APS changed the expression profile of lnc RNAs in the differentiation of brown adipocytes.In summary,the addition of APS effectively promoted the brown fat of C3H10 T 1/2 cells and increased insulin sensitivity.APS modulates the expression of miRNA-6911 to target Prdm16 to regulate brown fat.In addition,APS changed the expression profile of lnc RNAs in brown fat differentiation.The results of this study provide a theoretical basis for the rational use of APS to regulate BAT differentiation,which in turn affects heat production.