He-He-Shu-Yang formula alleviates liver fibrosis by inhibiting hepatic stellate cell activation in vivo and in vitro

Figure 1 High-Performance liquid chromatography chromatograms of He-He-Shu-Yang formula extracts. Peak 1: Paeoniflorin; Peaks 2: Isoflavone glucoside; Peak 3: Glycyrrhizin; Peak 4: Naringin; Peak 5: Hesperidin; Peak 6: Neohesperidin.

Figure 2 He-He-Shu-Yang formula alleviates carbon tetrachloride-induced liver fibrosis in Sprague-Dawley rats. ^aP < 0.05, ^bP < 0.01 vs control group; ^dP < 0.01 vs model group. Data are presented as median (inter quartile range), n = 9-10 rats/group. Rats were divided into five treatment groups: Control, model, low-dose He-He-Shu-Yang formula, high-dose He-He-Shu-Yang formula, and colchicine. A: Body weight in each group; B: Liver index in each group; C: Serum alanine aminotransferase level in each group; D: Serum aspartate aminotransferase level in each group; E: Representative images of liver pathology. Hematoxylin and eosin and Masson staining of liver tissues (magnification: 100’, scale bar: 100 μm). HHSY-L: Low-dose He-He-Shu-Yang formula; HHSY-H: High-dose He-He-Shu-Yang formula; CCl₄: Carbon tetrachloride; HE: Hematoxylin and eosin.

Figure 3 He-He-Shu-Yang formula inhibits hepatic stellate cell activation in rats with carbon tetrachloride-induced fibrosis. ^bP < 0.01 vs control group; ^dP < 0.01 vs model group. A: Representative images of α-smooth muscle actin immunohistochemical staining in the indicated treatment groups (bar = 50 μm); B: Average optical density of α-smooth muscle actin in each group. HSC: Hepatic stellate cell; α-SMA: Α-smooth muscle actin; HHSY: He-He-Shu-Yang formula; AOD: Average optical density; A.U.: Arbitrary unit.

Figure 4 He-He-Shu-Yang formula inhibits the nicotinamide adenine dinucleotide phosphate oxidase 4/reactive oxygen species/nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome pathway in rats with carbon tetrachloride-induced fibrosis. ^bP < 0.01 vs control group; ^dP < 0.01 vs model group. A: Representative images of nicotinamide adenine dinucleotide phosphate oxidase 4 immunohistochemistry staining in each group (bar = 100 μm); the brown area shows nicotinamide adenine dinucleotide phosphate oxidase 4 expression; B: Serum level of superoxide anion (O₂^-) in each group; C: Serum level of hydrogen peroxide (H₂O₂) in each group; D: Hydroxyl radical (OH) clearance rate in each group; E: Representative images of nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 immunohistochemistry staining (bar = 100 μm); F: Protein levels of nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 and cleaved caspase-1 in the different groups, as determined using western blotting. HHSY: He-He-Shu-Yang formula; NOX4: Nicotinamide adenine dinucleotide phosphate oxidase 4; NLRP3: Nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3.

Figure 5 He-He-Shu-Yang formula inhibits transforming growth factor-beta 1-induced activation of the human hepatic stellate cell line (LX2). ^bP < 0.01 vs control group; ^dP < 0.01 vs model group. Data are mean ± SD. A: Representative images of immunofluorescence staining of α-smooth muscle actin (α-SMA) in LX2 cells; B: Mean fluorescence intensity of α-SMA; C: Relative ACTA2 (which encodes α-SMA) mRNA levels. α-SMA: Α-smooth muscle actin; AU: Arbitrary unit.

Figure 6 He-He-Shu-Yang formula inhibits nicotinamide adenine dinucleotide phosphate oxidase 4 expression, intracellular reactive oxygen species generation, and nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome activation in transforming growth factor-beta 1-induced LX2 cells. ^bP < 0.01 vs control group; ^cP < 0.05, ^dP < 0.01 vs model group. A: Representative western blot of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) expression in each group. Protein levels were normalized to β-actin; B: Quantitation of NOX4 expression in each group based on the blot in panel A; C: Relative mRNA levels of NOX4 in the various groups; D: Intracellular reactive oxygen species levels as assessed using fluorescence microscopy; E: Mean fluorescence intensity of reactive oxygen species; F: Relative mRNA levels of nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 in each group; G: Relative mRNA levels of Interleukin-1β in each group. NOX4: Nicotinamide adenine dinucleotide phosphate oxidase 4; TGF-β: Transforming growth factor-beta 1; ROS: Reactive oxygen species; NLRP3: Nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3; IL-1β: Interleukin-1β.