Intragastric Safflower Yellow Alleviates HFD Induced Metabolic Dysfunction-Associated Fatty Liver Disease in Mice through Regulating Gut Microbiota and Liver Endoplasmic Reticulum Stress

Figures

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  Figure 1. Intragastric safflower yellow (SY) ameliorated diet-induced obesity (DIO) and metabolic dysfunction in mice. DIO mice were treated with SY by gavage for 12 weeks. (A) Graphical protocol of animal experiment. (B) The body weight of mice from beginning to 9th week. (C) The body weight gains and body weight gain percentage of mice in 9th week. (D) Food intake of mice from beginning to 9th week. (E) The WAT (g) and WAT (%) of mice in 12th week. (F) The oral glucose tolerance test (OGTT), (H) intraperitoneal oral glucose tolerance test (PIGTT) and (K) the intraperitoneal insulin tolerance test (IPITT) in 8th week. (G,I,L) Areas under the curve (AUCs) of the OGTT, IPGTT and IPITT. (J) the comparison of the AUCs of OGTT and IPGTT. (M) H&E staining images of epididymal WAT sections of mice at ×200 magnification (scale bars, 50 μm). The data were represented as the mean ± S.E.M. (n = 10 in each group). *: vs. SF group, p < 0.05; #: vs. HFD group, p < 0.05.

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  Figure 2. Intragastric SY increased the energy expenditure of DIO mice. Mice were treated with SY by gavage for 10 weeks, then (A,B) Oxygen consumption (VO2) of mice, (C,D) CO2 production (VCO2) of mice, (E,F) Respiratory exchange ratio (RER) of mice, (G,H) Energy expenditure (EE) of mice, (I) Food intake of mice, (J) Pedmeters, (K) Wheelmeters and (L) Allmeters of mice, (M,N) The mRNA levels of uncoupling protein 1 (Ucp1), peroxisome proliferative activated receptor, gamma, coac-tivator 1 alpha (Ppargc1a) in subcutaneous adipose tissue, (O,P) The mRNA levels of Ucp1 and Ppargc1a in brown adipose tissue. The data were represented as the mean ± S.E.M. (n = 5 in each group). *: vs. SF group, p < 0.05; #: vs. HFD group, p < 0.05.

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  Figure 3. Intragastric SY improved the hepatic steatosis and reduced the expression of lipogenesis-related genes in liver of DIO mice. Mice were treated with SY by gavage for 12 weeks. (A) The picture of the whole liver. (B) The liver weight (g) and liver index (%) of mice. (C)The serum ALT and AST levels of mice. (D) H&E staining images of liver sections of mice at ×100 (scale bars, 50 μm). (E) The MAFLD score of mice. (F–I) The mRNA levels of sterol-regulatory element binding proteins (Srebp1), acetyl CoA carboxylase (Acc), fatty acid synthetase (Fasn), stearyl CoA desaturase 1 (Scd1), tumor necrosis factor (Tnf), interleukin 1b (Il 1b), activating transcription factor 4 (Atf4), activating transcription factor 6 (Atf6), DNA-damage inducible transcript 3 (Ddit3), X-box binding protein 1 (Xbp1), heat shock protein 5 (Hspa5) and Xbp1s, peroxisome proliferator activated receptorα (Ppara) and carnitine palmitoyl transferase 1α (Cpt1a) in liver of mice. The data were represented as the mean ± S.E.M. (n = 10 in each group of animal experiments). *: vs. SF group, p < 0.05; #: vs. HFD group, p < 0.05.

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  Figure 4. Intragastric SY altered the composition of gut microbiota in DIO mice. Mice were treated with SY by gavage for 12 weeks. (A) Differential bacteria in top 10 abundance at the phylum levels. (B) Differential bacteria in top 10 abundance at the genus levels. (C) Differential bacteria in top 10 abundance at the species levels. The data were represented as the mean ± S.E.M. (n = 5 in each group). *: vs. SF group, p < 0.05; #: vs. HFD group, p < 0.05.

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  Figure 5. Intragastric SY changed the function of the gut microbiota of DIO mice. Mice were treated with SY by gavage for 12 weeks. (A) Violin plot of α diversity index; (B) Violin plot of β diversity index; (C) Principal co-ordinates analysis (PcoA) plot; (D) Linear discriminate analysis effect size (LEfSe) analysis. (E) Association map of metabolic phenotypes and differential gut microbiota. (F) Heatmap of the predicted function of the gut microbiota (n = 5 in each group). (G) Heatmap of the correlation of metabolic phenotype and differential gut microbiota (n = 5 in each group). The data were represented as the mean ± S.E.M. (n = 5 in each group). *: vs. SF group, p < 0.05; #: vs. HFD group, p < 0.05.

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  Figure 6. The effect of different dose of SY on the expression of fatty acid-related and endoplasmic reticulum stress-related genes in the PA-induced MAFLD cell model. (A) The oil red O (ORO) staining of HepG2 cells. (B) the quantify of ORO dye. (C–F) The mRNA level of SREBP1, FASN, ACC, SCD1, TNF, IL1b, ATF4, ATF6, DDIT3, XBP1, PPARA and CPT1A in HepG2 cells. The data were represented as the mean ± S.E.M. (n = 10 in each group of animal experiments, n = 3 in each group of cell experiments). *: vs. SF group or control cells, p < 0.05; #: vs. HFD group or PA-treated group, p < 0.05.