Hepatopoietin Cn (HPPCn) Generates Protective Effects on Acute Liver Injury

Figure 1. rhHPPCn increased the survival of SMMC7721 cells injured by toxic substances. A CCK8 assay was used to evaluate the protective effect of rhHPPCn on SMMC7721 cells injured by different toxic chemicals. (A) Treated with different concentrations of rhHPPCn for 24 h after 10 mmol/L CCl4 injury. (B) Treated with different concentrations of rhHPPCn for 24 h after 0.6 mmol/L H2O2 injury. (C) Treated with different concentrations of rhHPPCn for 24 h after 150 mmol/L ethanol injury. *P < 0.05, ***P < 0.001 vs. Control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. Model.

Figure 2. Effects of rhHPPCn on the proliferation of SMMC7721 cells treated with CCl4 (100×). The BrdU incorporation and the PCNA expression in SMMC7721 cells treated with different concentrations of rhHPPCn after 10 mmol/L CCl4 injury detected by IHC. (A) The incorporation of BrdU in SMMC7721 cells treated with different concentrations of rhHPPCn after 10 mmol/L CCl4 injury. (B) The expression of PCNA in SMMC7721 cells treated with different concentrations of rhHPPCn after 10 mmol/L CCl4 injury.

Figure 3. rhHPPCn inhibits the apoptosis of SMMC7721 cells treated by CCl4. The cell apoptosis was detected using FITC-conjugated Annexin V and PI by flow cytometry. (A) Image of vehicle control group, HPPCn group (100 ng/ml HPPCn), CCl4 group (10 mmol/L CCl4), and CCl4+HPPCn group (10 mmol/L CCl4, 100 ng/ml rhHPPCn). (B) Statistical analysis of the apoptosis rate of the four groups (vehicle control group, HPPCn only group, CCl4 only group, and CCl4+HPPCn group). **P < 0.01, ***P < 0.001 vs. the vehicle control group; ### P < 0.001 vs. the CCl4 group.

Figure 4. rhHPPCn reduced CCl4-induced cytotoxicity in SMMC7721 cells. The levels of hepatocyte injury indicators, including GOT, GPT, MDA, LDH, GSH-PX, and SOD in the cell lysate were evaluated by ELISAs in cells treated with different concentrations of rhHPPCn for 24 h after 10 mmol/L CCl4-induced cell injury. (A–D) GOT, GPT, MDA, and LDH. (E and F) GSH-PX, SOD. rhHPPCn treatment significantly reversed the increased serum level of GOT, GPT, MDA, and LDH and significantly reversed the decreased serum level of GSH-PX and SOD in a dose-dependent manner after acute cell injury. ***P < 0.001 vs. Control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. Model.

Figure 5. The effects of rhHPPCn on the serum levels of GOT and GPT in CCl4-induced acute liver injury model mice. (A and C) The serum levels of GOT and GPT in CCl4-induced acute liver injury model mice treated with different concentrations of rhHPPCn for 24 were evaluated by ELISA kits. (B and D) The serum levels of GOT and GPT in CCl4-induced acute liver injury model mice treated with rhHPPCn for different time periods. The results showed that rhHPPCn could decrease the levels of GOT and GPT in the serum of acute liver injury model mice in a concentration- and time-dependent manner. *P < 0.05 vs. Control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. Model.

Figure 6. rhHPPCn reduced the acute liver injury detected by HE staining. (A) Control normal mice. (B) Acute liver injury model mice. (C) Acute liver injury model mice treated with rhHPPCn at 1.25 mg/12 h/kg body weight for 24 h. (D) Acute liver injury model mice treated with rhHPPCn at 2.5 mg/12 h/kg body weight for 24 h.

Figure 7. rhHPPCn promoted hepatocyte proliferation in CCl4-induced acute liver injury model mice. PCNA expression was detected by immunohistochemistry after acute injured liver treated with different concentrations of rhHPPCn. (A) Control normal mice. (B) Acute liver injury model mice. (C) Acute liver injury model mice treated by rhHPPCn at 1.25 mg/12 h/kg body weight for 24 h. (D) Acute liver injury model mice treated by rhHPPCn at 2.5 mg/1 2h/kg body weight for 24 h.