Fig 1: Expression and characterization of recombinant human hepassocin produced by CHO cells. (A) SDS-PAGE analysis of purity of 3 batches of HPS protein. The HPS molecular weight of the naturally active form (dimer form) is 68 KD and the molecular weight of the monomer is 34 KD. (B) Immunoblotting analysis of HPS protein using human HPS antibody. (C) WRL68 cells and primary hepatocytes were treated with 50 or 100 ng/mL HPS as indicated for 24 h. MTT was performed to detect cell proliferation. Data are represented the results of three independent experiments and expressed as mean ± SEM. A Student’s t-test was used to compare the mean relative values between groups. (** p < 0.01, *** p < 0.001). (D) WRL68 cells and primary mouse hepatocytes were serum starved overnight, following by treated with 100 ng/mL of HPS for indicated times. Immunoblotting was performed to detected ERK phosphorylation. Total ERK was used as control. For panel (E) and panel (F) BALB/c male mice at 8 weeks of age were intraperitoneally injected with various amounts of HPS (0.2 or 1.0 mg/kg) or PBS 12 h before and 24 and 48 h after 100 µL/kg of CCl4 (1:100 diluted in corn coil) injection as indicated. (E) Mice sera were harvested, and ALT levels were detected at indicated time points after CCl4 injection. (n = 5/group). (F) Liver tissues were fixed, sectioned, and stained with H&E for histopathological and morphological analysis at indicated time points after CCl4 injection. Scale bar, 200 µm. The part enclosed by a dashed line represents the necrotic area. Part of the necrotic area in the 24-h group and 36-h group is enlarged and displayed below. The percentage of necrotic area was quantitated using ImageJ software and values are the mean ± SD of five fields of measurements. For panel (E) and panel (F), data are represented as mean ± SD. A Student’s t-test was used to compare the mean values between groups. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (G) BALB/c male mice at 8 weeks of age were intraperitoneally injected with various amounts of HPS (0.2 or 1 mg/kg) or PBS 12 h before and 24 and 48 h after 2.5 mL/kg CCl4 (1:100 diluted in corn coil) injection. Cumulative survival analysis was determined with a Kaplan–Meier diagram (n = 10/group). Log rank test was used to compare the survival distributions between groups (*p < 0.05).
Fig 2: HPS treatment attenuated the damage of D-GalN/LPS-induced liver injury in mice. (A) BALB/c male mice at 8 weeks of age were intraperitoneally injected with various amounts of HPS (0.2, 1.0, or 5.0 mg/kg) or PBS 12 h before and 24 and 48 h after D-GalN (1000 mg/kg) and LPS (50 µg/kg) injection (n = 10/group). Cumulative survival analysis was determined with a Kaplan–Meier diagram. Log rank test was used to compare the survival distributions between groups (* p < 0.05, *** p < 0.001). For panel (B) and panel (C), BALB/c male mice at 8 weeks of age were intraperitoneally injected with various amounts of HPS (0.2, 1.0, or 5.0 mg/kg) or PBS 12 h before D-GalN (500 mg/kg) and LPS (20 µg/kg) injection (n = 5/group). (B) Mice serum ALT levels were detected at indicated time points after D-GalN/LPS injection. Data are represented as mean ± SD. A Student’s t-test was used to compare the mean values between groups. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (C) Liver tissues were fixed, sectioned and stained with H&E for histopathological and morphological analysis at indicated time points after D-GalN/LPS injection. Scale bar, 100 µm. The part enclosed by a dashed line represents the necrotic area. Part of the necrotic area in the 8-h group and 12-h group is enlarged and displayed on the right. The percentage of necrotic area was quantitated using ImageJ software and values are the mean ± SD of five fields of measurements. A Student’s t-test was used to compare the mean values between groups. (**** p < 0.0001).
Fig 3: The pharmacokinetics of HPS. (A) Plasma concentration-time profiles after intravenous or subcutaneous administration of HPS (3.5 mg/kg) in rats (n = 3/group). Data are presented as the mean ± SD. (B) Tissue distribution of HPS after intravenous administration in rats (n = 5/group). (C) Cumulative urinary excretion and fecal elimination as a percent of dose administrated (n = 6/group).
Fig 4: Peripheral delivery of recombinant human HPS prevents acute liver injury in in nonhuman primates. Cynomolgus monkey were injected intravenously with 300 mg/kg D-GalN. HPS (0.25 mg/kg) or PBS were injected intravenously into the monkey at 12 h before and 24, 48, 72 h after D-GalN injection (n = 5/group). (A) Serum ALT, AST, ALP, TBIL, and DBIL levels at 96 h after D-GalN injection were detected. (B) Representative images of H&E stained-liver sections of the monkey at 96 h after D-GalN injection. The part enclosed by a dashed line represents the necrotic area, which is enlarged and displayed on the right. Scale bar, 50 µm. (C) Representative images of immunohistochemistry analysis of TUNEL stained liver sections of the monkey at 96 h after D-GalN injection and the number of TUNEL positive cells of five fields of measurements. Scale bar, 50 µm. (D) Serum levels of IL-6, IFN?, and TNF-a were determined at 96 h after D-GalN injection by FACS using nonhuman primate Th1/Th2 Cytokine Kit (BD Cytometric Bead Array (CBA)). (E) Serum levels of 4-HNE were quantified at 96 h after D-GalN injection by colorimetric detection using a competitive ELISA Kit. (F) Representative images of immunohistochemistry analysis of 4-HNE-stained liver sections of the monkey at 96 h after D-GalN injection and the number of 4-HNE positive cells of five fields of measurements. Scale bar, 50 µm (G) Representative images of immunohistochemistry analysis of PCNA stained liver sections of the monkey at 96h after D-GalN injection and the number of PCNA positive cells of five fields of measurements. Scale bar, 50µm. All data are represented as mean ± SD. For panel (A), panel (D), and panel (E), Mann–Whitney test was used to compare the mean relative values between groups (* p < 0.05). For panel (C), panel (F), and panel (G), Student’s t-test was used to compare the mean relative values between groups. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Fig 5: Effects of a single dose of HPS on tissue injury in mice. BALB/c male mice at 8 weeks of age were intravenously injected with various amounts of HPS (5.0, 10.0, or 20.0 mg/kg) or PBS (n = 5/group). Seven days later, primary safety evaluation was analyzed. (A) Body weight. (B) Serum ALT, AST, BUN, CREA, and CK levels. (C) Routine blood tests. (D) Liver tissues were fixed, sectioned and stained with H&E for histopathological and morphological analysis. Scale bar, 200 µm.
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