| Echinops latifolius polysaccharide B(ETPB)is a polysaccharide component extracted from the Chinese herbal medicine Echinops latifolius.Preliminary studies have confirmed that ETPB can improve liver insulin resistance(IR)in Type 2 diabetes mellitus(T2DM)rats under a high-fat diet.But the mechanism of ETPB is still unclear.In this study,we constructed the insuin-resistant HepG2 cells in vitro to explore whether ETPB can improve the insulin resistance effect of IR-HepG2 cells induced by high fat.Methods:(1)Human-derived hepatocyte cell line HepG2 was used as the research object,MTT method was used to screen the safe concentration of ETPB intervention in HepG2 cells.IR-HepG2 cells was induced by 0.25mM palmitic acid(PA)for 24 h.The improvememt effect of ETPB on the glucose and lipid metabolism disorder of IR-HepG2 cells were evaluated.(2)Sequencing was performed by the Illumina Hi Seq 2500 platform,and differentially expressed miRNAs(DEMs)and m RNAs(DEGs)were screened.The filter conditions were fold change>1.5 and p-value<0.05.We performed bioinformatics analysis on DEMs and DEGs and used qPCR to verification them.We also constructed miRNAs-m RNAs regulatory network about them.(3)Cell transfection,qPCR and Western blot to were used to verify the regulatory effect of miR-494-3p on the FOXO1/PGC1α signaling pathway.(4)Cell transfection,qPCR and Western blot were used to verify the regulatory effect of ETPB on miR-494-3p/FOXO1/PGC1α signaling pathway.(5)High performance gel permeation chromatography(HPGPC)was used to determine the molecular weight of ETPB.High performance liquid chromatography(HPLC)PMP pre-column derivatization was used to analyze the monosaccharide composition of ETPB.Results:(1)The result of MTT method showed that 3mg/mL ETPB was the maximum safe dose for HepG2 cells.The IR-HepG2 cells model was successfully constructed after treating HepG2 cells with 0.25mM PA for 24 h.Compared with the model group,the glucose consumption and glycogen synthesis of IR-HepG2 cells increased significantly after treatment with 1mg/mL ETPB for 24h(p<0.05,p<0.01).Compared with the normal control group,the content of FFAs and TG in the model group was significantly increased(p<0.01),and this situation was reversed by 1mg/mL ETPB treatment for 18h(p<0.01).After oil red O staining,the normal groups only had a few small lipid droplets,while the situation of model groups were completely opposite.The results showed that HepG2 cells had steatosis after PA treatment.After treatment with 3mg/mL ETPB for 24 h,the amount of lipid droplets in the model group cells were decreased.(2)Sequencing and bioinformatics analysis were performed on the normal group(NC),model group(MC),1mg/mL ETPB treatment for 12h(ETPB1)and 24h(ETPB2)groups,which identified DEGs and DEMs in the comparison between groups.We selected8 DEGs and 5 DEMs for qPCR detection.After Short Time-series Expression Miner(STEM)analysis of all DEGs,we performed GO and KEGG enrichment analysis on genes in 6 profiles.And we also constructed a regulatory network between DEGs in insulin resistance signaling pathway and DEMs that have a targeted relationship with them.(3)In HepG2 cells,miR-494-3p targeted FOXO1,which in turn regulated molecules such as PGC1α,PCK2 and G6 PC to inhibit the gluconeogenesis of IR-HepG2.(4)ETPB inhibited the gluconeogenesis of IR-HepG2 by regulating miR-494-3p/FOXO1/PGC1α.(5)ETPB was composed by lucose(Glc),galacturonic acid(Gla UA),galactose(Gal),arabinose(Ara),mannose(Man),xylose(Xyl),rhamnose(Rha),glucosamine(Glc N)and glucuronic acid(Glc UA)with a molar ratio of 0.406∶0.19∶0.145∶0.137∶0.058∶0.027∶0.021∶0.01∶0.006.And it was a homogeneous heteropolysaccharide with a molecular weight of 9KDa.Conclusion:(1)ETPB can improve the glucose and lipid metabolism of IR-HepG2 cells induced by PA.(2)The regulation mechanism of ETPB to improve the IR of HepG2 cells maybe through miR-494-3p/FOXO1/PGC1α signaling pathway to inhibit the gluconeogenesis of IR-HepG2.(3)ETPB is a homogeneous heteropolysaccharide with a molecular weight of 9KDa. |
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