Huangqin decoction alleviates lipid metabolism disorders and insulin resistance in nonalcoholic fatty liver disease by triggering Sirt1/NF-κB pathway

Figure 1 High-performance liquid chromatography fingerprint spectrum of Huangqin decoction and structures of the major phytochemicals in Huangqin decoction. A and B: High-performance liquid chromatography profiles of Huangqin decoction (HQD) and the standard mixture: (1) Gallic acid, (2) paeoniflorin, (3) scutellarin, (4) liquiritin, (5) baicalin, (6) scutellarein, (7) wogonoside, (8) baicalein, (9) wogonin, and (10) chrysin; C: Structures of the major phytochemicals in HQD.

Figure 2 Huangqin decoction improves adiposity and hepatic histological abnormalities in high-fat diet-induced nonalcoholic fatty liver disease rats. Data are presented as mean ± SD (n = 4–8). ^aP < 0.01 vs the normal group, ^bP < 0.05 and ^cP < 0.01 vs the high-fat diet group. A: Workflow of the experimental design; B: Dynamic curve of the body weight; C-E: Barcharts showing the effects of different treatments on body weight (C), liver weight at the termination of the experiment (D), and liver index (liver weight/body weight) (E) at the termination of the experiment; F and G: Representative micrographs of Hematoxylin and eosin and Oil Red O stained liver sections; H: Quantitative analysis of positive areas in Oil Red O staining. CMC-Na: Sodium carboxymethylcellulose; Fen: fenofibrate; HQD: Huangqin decoction; I.G.: Intragastric administration.

Figure 3 Huangqin decoction alleviates liver damage and lipid metabolism disorders in high-fat diet-induced nonalcoholic fatty liver disease rats. Data are presented as mean ± SD (n = 8). ^aP < 0.01 vs the normal group, ^bP < 0.05 and ^cP < 0.01 vs high-fat diet group. A-J: Effects of different treatments on levels of alanine transaminase (A); aspartate transaminase (B); total cholesterol (TC) (C); triacylglycerol (TG) (D); free fatty acid (FFA) (E); low-density lipoprotein cholesterol (F); high-density lipoprotein cholesterol (G); TC (H); TG (I); FFA (J) levels in rats are shown. ALT: Alanine transaminase; AST: Aspartate transaminase; Fen: Fenofibrate; FFA: Free fatty acid; HDL-c: High-density lipoprotein cholesterol; HFD: High-fat diet; HQD: Huangqin decoction; LDL-c: Low-density lipoprotein cholesterol; TC: Total cholesterol; TG: Triacylglycerol.

Figure 4 Huangqin decoction suppresses hepatic inflammation and insulin resistance in high-fat diet-induced nonalcoholic fatty liver disease rats. Data are presented as mean ± SD (n = 4–8). ^aP < 0.01 vs the normal group, ^bP < 0.05 and ^cP < 0.01 vs the high-fat diet (HFD) group. A: Representative images of immunohistochemical staining of interleukin (IL)-1β in liver sections; B: Quantitative analysis of staining intensity of IL-1β; C-E: Levels of IL-6 (C), IL-1β (D), and tumor necrosis factor-α (E) in the liver of HFD-fed rats; F: Oral glucose tolerance test (OGTT); G: Area under the curve from OGTT; H: Level of fasting serum insulin; I: Values of homeostasis model assessment of insulin resistance. AUC: Area under the curve; Fen: Fenofibrate; HFD: High-fat diet; HOMA-IR: Homeostasis model assessment of insulin resistance; HQD: Huangqin decoction; IL: Interleukin; TNF: Tumor necrosis factor.

Figure 5 Huangqin decoction activates the Sirt1/NF-Κb pathway in high-fat diet-induced nonalcoholic fatty liver disease rats. Data are presented as mean ± SD (n = 3–4). ^aP < 0.01 vs the normal group, ^bP < 0.05 and ^cP < 0.01 vs the high-fat diet group. A: Expression levels of Sirt1, p-NF-κB/NF-κB, p-IRS-2/IRS-2, sterol regulatory element-binding protein (SREBP)-1c, fatty acid synthase, and cluster of differentiation 36; B-G: Semi-quantitative analysis of these proteins; H-J: Representative images of immunohistochemical staining of Sirt1 (H), NF-κB (I), and SREBP-1c (J) in liver sections; K-M: Quantitative analysis of the staining intensity; N: Representative images of immunofluorescent staining of Sirt1 and NF-κB in liver sections; O and P: Quantitative analysis of the fluorescence intensity. CD36: Cluster of differentiation 36; DAPI: 4',6-diamidino-2-phenylindole; FAS: Fatty acid synthase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HFD: High-fat diet; HQD, Huangqin decoction; SREBP: Sterol regulatory element-binding protein.

Figure 6 Huangqin decoction improves lipotoxicity, inflammation, and insulin resistance in palmitic acid-induced HepG2 cells. Data are presented as mean ± SD of at least three independent experiments. ^aP < 0.01 vs the control group, ^bP < 0.05 and ^cP < 0.01 vs the palmitic acid (PA) group. A: Viability of HepG2 cells following incubation with different concentrations of Huangqin decoction (HQD); B: Viability of HepG2 cells following co-culture with PA and different concentrations of HQD; C and D: Levels of triacylglycerol and total cholesterol in HepG2 cells; E: Representative micrographs of Oil Red O staining of HepG2 cells; F–H: Levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α in HepG2 cells; I: Level of glucose uptake in HepG2 cells. 2DG: 2-deoxydglucose; HQD, Huangqin decoction; IL: Interleukin; TC: Total cholesterol; TG: Triacylglycerol; TNF: Tumor necrosis factor; PA: Palmitic acid.

Figure 7 Huangqin decoction activates the Sirt1/NF-κB pathway in palmitic acid-induced HepG2 cells. Data are presented as mean ± SD of at least three independent experiments. ^aP < 0.01 vs the control group, ^bP < 0.05 and ^cP < 0.01 vs the palmitic acid group. A: Expression levels of Sirt1, p-NF-κB/NF-κB, p-IRS-2/IRS-2, sterol regulatory element-binding protein 1, fatty acid synthase, and cluster of differentiation 36 in HepG2 cells; B–G: Semi-quantitative analysis of these proteins; H: Representative images of immunofluorescent staining of Sirt1 and NF-κB in HepG2 cells. CD36: Cluster of differentiation 36; DAPI: 4',6-diamidino-2-phenylindole; FAS: Fatty acid synthase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HQD, Huangqin decoction; PA: Palmitic acid; SREBP: Sterol regulatory element-binding protein.

Figure 8 Huangqin decoction protects hepatocytes by triggering Sirt1/NF-κB pathway in palmitic acid-induced HepG2 cells. Data are presented as mean ± SD of at least three independent experiments. ^aP < 0.01 vs the control group, ^bP < 0.05 and ^cP < 0.01 vs the palmitic acid group, ^dP < 0.05 and ^eP < 0.01 vs the Huangqin decoction group. A and B: Levels of triacylglycerol (A) and total cholesterol (B) in HepG2 cells co-cultured with or without EX-527; C-E: Levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α in HepG2 cells co-cultured with or without EX-527; F: Level of glucose uptake in HepG2 cells co-cultured with or without EX-527; G: Representative micrographs of Oil Red O staining of HepG2 cells co-cultured with or without EX-527; H: Expression levels of Sirt1, p-NF-κB/NF-κB, p-IRS-2/IRS-2, sterol regulatory element-binding protein-1c, fatty acid synthase, and cluster of differentiation 36 in HepG2 cells co-cultured with or without EX-527; I-N: Semi-quantitative analysis of these proteins; O: Representative images of immunofluorescent staining of Sirt1 and NF-κB in HepG2 cells co-cultured with or without EX-527. 2DG: 2-deoxydglucose; CD36: Cluster of differentiation 36; DAPI: 4',6-diamidino-2-phenylindole; FAS: Fatty acid synthase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HQD: Huangqin decoction; IL: Interleukin; PA: Palmitic acid; SREBP: Sterol regulatory element-binding protein; TC: Total cholesterol; TG: Triacylglycerol; TNF: Tumor necrosis factor.

Figure 9 Schematic diagram illustrating the effects of Huangqin decoction on Sirt1/NF-κB pathway-modulated hepatic steatosis and insulin resistance. CD36: Cluster of differentiation 36; FAS: Fatty acid synthase; HFD: High-fat diet; HQD: Huangqin decoction; IL: Interleukin; SREBP: Sterol regulatory element-binding protein; TNF: Tumor necrosis factor.