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Combined use of astragalus polysaccharide and berberine attenuates insulin resistance in IR-HepG2 cells via regulation of the gluconeogenesis signaling pathway

ABSTRACT: Insulin resistance (IR) is likely to induce metabolic syndrome and type 2 diabetes mellitus (T2DM). Gluconeogenesis (GNG) is a complex metabolic process that may result in glucose generation from certain non-carbohydrate substrates. Chinese herbal medicine astragalus polysaccharides and berberine have been documented to ameliorate IR, and combined use of astragalus polysaccharide (AP) and berberine (BBR) are reported to synergistically produce an even better effect. However, what change may occur in the GNG signaling pathway of IR-HepG2 cells in this synergistic effect and whether AP-BBR attenuates IR by regulating the GNG signaling pathway remain unclear. For the first time, we discovered in this study that the optimal time of IR-HepG2 cell model formation was 48 hours after insulin intervention. AP-BBR attenuated IR in HepG2 cells and the optimal concentration was 10mg. AP-BBR reduced the intracellular H2O2 content with no significant effect on apoptosis of IR-HepG2 cells. In addition, a rapid change was observed in intracellular calcium current of the IR-HepG2 cell model, and AP-BBR intervention attenuated this change markedly. The gene sequencing results showed that the GNG signaling pathway was one of the signaling pathways of AP-BBR to attenuate IR in IR-Hepg2 cells. The expression of p-FoxO1Ser256 and PEPCK protein was increased and the expression of GLUT2 protein was decreased significantly in the IR-HepG2 cell model, and both of these effects could be reversed by AP-BBR intervention. AP-BBR attenuated IR in IR-HepG2 cells, probably by regulating the GNG signaling Pathway. Overall design: HepG2 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in 1640 medium (Hyclone, Beijing, China) containing 10% fetal bovine serum (FBS, Hyclone, Beijing, China) and 1 × streptomycin in a 37 ℃ 5% CO2 saturated humidity incubator. The normally cultured HepG2 cell lines in log phase were centrifuged at 100grpm for 5 min, and 20000 cells/well were placed in a 96-well plate and incubated at 37°C.The experiment was performed in 24-h control group, 24-h model group, 36-h control group, 36-h model group, 48-h control group, 48-h model group, 72-h control group and 72-h model group. Insulin (Gibco, NY, USA) was diluted to a final concentration of 10-6 mol/L in complete medium. 200µl insulin preparation was added into each well for the model group and an equal amount of complete medium was added into each well for the control group. Culture was performed in a 37 ℃ 5% CO2 and saturated humidity incubator. The supernatant of the corresponding medium was collected according to the time point by centrifugation at 3000 r/min for 5 min and stored at-80 ℃ for use. RNA isolation, purification and quantification: Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Agilent 2100 with RIN number >7.0.cDNA Library Construction: Poly(A) RNA was purified from total RNA (5ug) using poly-T oligo-attached magnetic beads using two rounds of purification. Then, the poly(A) RNA was fragmented into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA, which was subsequently used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP. An A-base was then added to the blunt ends of each strand, which were prepared for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, the ligated products were amplified by PCR under the following conditions: initial denaturation at 95℃ for 3 min, 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, extension at 72℃ for 30 sec, and then final extension at 72℃ for 5 min. The mean insert size for the final cDNA library was 300 bp (±50 bp). Finally, 150bp paired-end sequencing was performed on an Illumina Hiseq 4000 (LC Bio, China) following the vendor's recommended protocol.Pathway enrichment analysis: Using the DAVID database and mouse genome as background control, the differentially expressed genes were analyzed by gene ontology under "FunctionalAnnotation Chart" functional module. The differentially expressed genes were divided into three categories according to their functions: the biological process, the cell component and the molecular function. Pathway analysis was carried out by KEGG analysis function.

INSTRUMENT(S): Illumina HiSeq 4000 (Homo sapiens)

ORGANISM(S): Homo Sapiens

SUBMITTER: Yinan Liu  

PROVIDER: GSE139929 | GEO | 2019-11-14


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