Mangiferin Ameliorates HFD-Induced NAFLD through Regulation of the AMPK and NLRP3 Inflammasome Signal Pathways

Figure 1. Man reduces weight and lowers blood glucose and blood lipids in HFD-induced NAFLD mice. (a) The body weight change of mice during experimental period. (b) The body weight of mice after a 12-week treatment. (c) The last FBG before the finished experiment. (d–g) Serum levels of TG (d), LDL-c (e), CHO (f), and HDL-c (g). The results represent as the mean ± SD, n = 8. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the normal group. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. the model group.

Figure 2. Man improves hepatic fatty deposition and reduces liver damage. (a) Liver weight and liver index of animals. (b) TG levels of liver. (c, d) The levels of AST and ALT in serums. All results represent as the mean ± SD, n = 8. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the normal group. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. the model group.

Figure 3. Man ameliorates insulin resistance and glucose tolerance in NAFLD mice. (a) The curve of OGTT. (b) The area under the curve of OGTT. (c) The curve of ITT. (d) The area under the curve of ITT. Data represent as the mean ± SD (n = 8). ∗∗p < 0.01 vs. that of the normal group. #p < 0.05 and ##p < 0.01 vs. that of the NAFLD model group.

Figure 4. Man improves hepatic steatosis in HFD-induced NAFLD mice. (a) The H&E staining, one representativeness of pathological images of each group. (b) The steatosis scores of liver pathological changes. Data represent as the mean ± SD (n = 8). ∗∗∗p < 0.001 vs. that of the normal group. ##p < 0.01 vs. that of the NAFLD model group.

Figure 5. RNA-seq assay of mouse hepatocytes between Man treatment and NAFLD model groups. (a) The Pearson correlation between samples. (b) Differentially expressed gene volcano map (∣log2 (fold change)∣>1 and padj ≤ 0.05). (c) Differentially expressed genes clustering heat map of 1601 (∣log2 (fold change)∣>1 and padj ≤ 0.05). (d, e) The Gene Ontology (GO) biological process enrichment of upregulation (d) or downregulation (e) of differentially expressed genes, and the threshold value for significant enrichment of GO function was padj ≤ 0.05. (f, g) One of the representations of KEGG pathway (f) about upregulation of differentially expressed genes and its ID, description, p value, and padj value (g). (h, i) One of the representations of KEGG pathway (h) about downregulation of differentially expressed genes and its ID, description, p value, and padj value (i). (j) The NLRP3/Casp-1/GSDMD-mediated inflammatory signal pathway in (h), which is closely related to the effects of Man treatment.

Figure 6. Man activates AMPK and regulates the NLRP3 inflammasome signaling pathway to exert anti-inflammatory effects in vivo. The total protein was extracted from animal liver of experiment. The p-AMPK, NLRP3, pro-caspase-1, caspase-1 (p10), IL-1β, and GSDMD-N protein levels were detected by Western blots and quantified by using ImageJ software. (a) Western blots. (b) The protein levels of p-AMPK were normalized to those of β-actin, respectively, and plotted as fold of the normal group. (c) The protein levels of GSDMD-N were normalized to those of β-actin, respectively, and plotted as fold of the normal group. (d) The protein levels of NLRP3, caspase-1 (p10), and IL-1β were normalized to those of β-actin, respectively, and plotted as fold of the normal group. (f–h) The NLRP3 and ASC caspase-1 mRNA levels were detected by quantitative real-time PCR in animals' liver. The data are expressed as the mean ± SD (n = 8). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the normal group. #p < 0.05 and ##p < 0.01 vs. the NAFLD model group.

Figure 7. Man effects on cell viability. HepG2 cells were incubated with Man at indicated concentrations for 24 h; cell viabilities were tested by MTT assay. All result values are represented as the mean ± SD (n = 5). ∗∗∗p < 0.001 vs. DMSO.

Figure 8. Man modulates cell glycolipid metabolism by activating the AMPK signal pathway. (a) Glucose consumptions. All result values are represented as the mean ± SD (n = 5). ∗∗p < 0.01 or ∗∗∗p < 0.001 vs. DMSO. (b) Glucose consumptions with compound C pretreatment. Values are represented as the mean ± SD (n = 5). ∗∗∗p < 0.001 vs. DMSO, ##p < 0.01 vs. Man, and &&p < 0.01 vs. Met. (c) After Man treatment, the total cell proteins of every well were extracted; p-AMPKα (Thr172) (p-AMPKα), AMPKα, p-ACC (Ser79) (p-ACC), ACC, and β-actin were determined by western blot. Representative blot pictures as presented. (d) Normalizing the level of p-AMPKα and p-ACC to those of AMPKα and ACC, respectively, and plotted as fold of DMSO-treated cells. The values are represented as the mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. DMSO. (e) HepG2 cells were pretreated with cc for 60 min; then, Man was added and incubated for 24 h; total cell proteins were extracted; p-AMPKα (Thr172) (p-AMPKα) and AMPKα were detected by western blot. Representative blot pictures as presented. (f) The protein level of p-AMPKα was normalized to AMPKα, respectively, and plotted as fold of DMSO-treated cells.…

Figure 9. The effects of Man on NLRP3 inflammasome signal axis and its inhibition of NLRP3-mediated pyroptosis in vitro. (a) After serum starvation treatment, cells were untreated or treated with PA-BSA or PA-BSA+Man or PA-BSA+Met as indicated for 24 h. Then, total cell proteins were made; p-AMPKα (Thr172) (p-AMPKα), NLRP3, caspase-1, caspase-1 (P10), GSDMD-N, IL-1β, and β-actin were detected by western blot. Representative blot pictures as shown. The protein levels of p-AMPKα (b), GSDMD-N (c), and NLRP3/caspase-1 (p10)/IL-β (d) were normalized to β-actin, respectively, and plotted as fold of the DMSO+10% BSA-treated group. Values are represented as the mean ± SD (n = 3). ∗∗∗p < 0.001 vs. DMSO+10% BSA and ##p < 0.01 and ###p < 0.001 vs. DMSO+PA-BSA.