Protective Effects of Salidroside against Carbon Tetrachloride (CCl4)-Induced Liver Injury by Initiating Mitochondria to Resist Oxidative Stress in Mice

Abstract

The antioxidant effect of salidroside has been proven, but its role in liver injury is poorly understood. In this study, we aimed to evaluate the protective effects and mechanism of salidroside on liver injury induced by carbon tetrachloride (CCl₄) in vivo. Mice were pretreated with salidroside (60 mg/kg, intraperitoneally injected, i.p.) once per day for 14 consecutive days and then administered with CCl₄ (15.95 g/kg, i.p.) for 24 h to produce a liver injury model. Salidroside attenuated hepatic transaminase elevation in serum and ameliorated liver steatosis and necrosis, thereby suggesting its protective effect on the liver. Salidroside antagonized CCl₄-induced toxicity by equilibrating antioxidation system, thereby inhibiting reactive oxygen species accumulation, and restoring mitochondrial structure and function. Salidroside exerts antioxidant and liver-protective effects by selectively inhibiting the activation of genes, including growth arrest and DNA -damage-inducible 45 α (Gadd45a), mitogen-activated protein kinase 7 (Mapk7), and related RAS viral oncogene homolog 2 (Rras2), which induce oxidative stress in the mitogen-activated protein kinase pathway. These results revealed that salidroside can protect the liver from CCl₄-induced injury by resisting oxidative stress and protecting mitochondrial function.

Keywords: CCl4; Salidroside; liver injury; mitochondria; oxidative stress.

Figure 1

Chemical structural formula of salidroside (p-hydroxyphenethyl-β-D-pyran glucoside, Mr = 300.3). Aglycone denoted with the dotted box is the radical-scavenging active structure of salidroside in vitro.

Figure 2

Salidroside attenuated carbon tetrachloride (CCl₄)-induced liver steatosis and decreased hepatic transaminases in mice (n = 6). (A) Macroscopic characteristic of livers in control mice, model mice induced by CCl₄, and mice treated with single salidroside, salidroside+CCl₄, and diammonium glycyrrhizinate+CCl₄ (Con, Mod, Sal, Sal+CCl₄, and DG+CCl₄, respectively). Livers in the Con and Sal groups had normal gross morphology. However, the livers of mice in Mod, Sal+CCl₄, and DG+CCl₄ groups showed increased volumes, decreased glossiness, and smoothness. The most severe and slightest lesions were observed in the Mod and the Sal+CCl₄ groups, respectively. (B) Pathologic histology of livers in different groups. Mild steatosis can be observed in the liver of the Con group, and the Sal group showed a normal structure. Histopathological changes showed the fatty degeneration and necrosis of hepatocytes and infiltration of inflammatory cells, which were more and increasingly mild in the Mod, Sal+CCl₄, and DG+CCl₄ groups. Red arrows marked normal hepatocytes, green arrows marked cell nucleus pyknosis, black arrows marked cell nucleus broken, blue arrows marked vacuoles caused by steatosis, and dotted line denoted necrotic area. Magnification: 200×. (C) GOT and GPT activities in the serum of different groups. CCl₄ treatment increased the serum GOT and GPT levels (p < 0.05). Salidroside reduced the CCl₄-induced transaminase, and DG only alleviated GOT condition significantly (p < 0.05). The single-treatment of salidroside had no side-effect. p < 0.05 was considered statistically significant. **p < 0.05 (groups compared with the Con group) and ##p < 0.05 (groups compared with the Mod group). (GOT: glutamic oxalacetic transaminase; GPT: glutamic-pyruvic transaminase).

Figure 3

Salidroside balanced liver antioxidant system for oxidative stress resistance in mice. The vital antioxidases, namely, TSOD and CAT, nonenzymatic antioxidant GSH, and lipid peroxidative product MDA levels in the liver of different groups, showed that the model mice were induced by CCl₄ and mice treated with single salidroside, salidroside+CCl₄, and diammonium glycyrrhizinate+CCl₄ (Con, Mod, Sal, Sal+CCl₄, and DG+CCl₄ groups, respectively). In the Mod group, the SOD, CAT, and GSH levels were significantly reduced, but the MDA level increased (p < 0.05). The condition can be reversed in the Sal+CCl₄ and DG+CCl₄ groups compared with that in the Mod group (p < 0.05), but the TSOD level in the Sal+CCl₄ group and CAT/GSH level in the DG+CCl₄ group showed a weak remission_. The difference between the Sal and Con groups was insignificant (p > 0.05). p < 0.05 was considered to be statistically significant. **p < 0.05 (groups compared with the Con group) and ##p < 0.05 (groups compared with the Mod group). (CCl₄: carbon tetrachloride; TSOD: total superoxide dismutase; CAT: catalase; GSH: glutathione-SH; MDA: malonaldehyde).

Figure 4

Salidroside protected liver ultrastructure, especially mitochondria in mice. (A) Conditions of liver ultrastructure in different groups: model mice induced by CCl₄ and mice treated with single salidroside, salidroside+CCl₄, and diammonium glycyrrhizinate+CCl₄ (Con, Mod, Sal, Sal+CCl₄, and DG+CCl₄, respectively). In the Mod group, CCl₄ induced ultrastructural damage with decreased chromatin and organelles, partly dissolved ridges and membrane of swollen mitochondria, and increased secondary lysosomes. Salidroside and DG can remarkably alleviate ultrastructural damage, but the former was better than the latter. Single salidroside treatment had no side effect on liver ultrastructure. Red and green arrows marked mitochondria and hepatocytes, respectively. Red and green triangles marked nucleolus and endoplasmic reticulum, respectively. Red and green stars marked secondary lysosome and glycogen, respectively. Magnification: 6000× in the first row, 12000× in the second row. (B) Status of liver MMP in different groups. CCl₄ caused excessively low MMP, whereas salidroside effectively normalized the MMP level and even performed better in terms of the overall liver MMP protection ability than that of DG. p < 0.05 was considered statistically significant. **p < 0.05 (groups compared with the Con group) and ##p < 0.05 (groups compared with the Mod group). (CCl₄: carbon tetrachloride; MMP: mitochondrial membrane potential).

Figure 5

Salidroside inhibited liver reactive oxygen species (ROS) generations in the mice of different groups, that is, model mice induced by CCl₄ and mice treated with single salidroside, salidroside+CCl₄, and diammonium glycyrrhizinate+CCl₄ (Con, Mod, Sal, Sal+CCl₄, and DG+CCl₄ groups, respectively). CCl₄ caused excessively high ROS, whereas salidroside and DG effectively normalized the ROS levels. p < 0.05 is statistically significant. **p < 0.05 (groups compared with the Con group) and ##p < 0.05 (groups compared with the Mod group). (CCl₄: carbon tetrachloride).

Figure 6

Salidroside protected against carbon tetrachloride (CCl₄)-induced liver injury by downregulating oxidative stress-related gene Gadd45a, Mapk7, and Rras2. (A) Relative expression levels of Gadd45a, Mapk7, and Rras2 genes in different groups, that is, model mice induced by CCl₄, and mice treated with single salidroside, salidroside+CCl₄, and diammonium glycyrrhizinate+CCl₄ (Con, Mod, Sal, Sal+CCl₄, and DG+CCl₄ groups, respectively). p < 0.05 was considered to be statistically significant. **p < 0.05 (groups compared with the Con group) and ##p < 0.05 (groups compared with the Mod group). (B) Optical density statistics of Gadd45a and Rras2 proteins expression levels in different groups. (C) Immunohistochemical staining of Gadd45a and Rras2 proteins in different groups. Gadd45a was expressed and distributed in the cytoplasm surrounding the centrilobular veins of the liver in the CCl₄ group and seldom expressed in other groups. Rras2 protein was highly expressed in a sheet in CCl₄ and DG+CCl₄ groups and partially expressed in the hepatocyte of other groups. Brown-yellow or brown substances expressed in the cytoplasm were positive proteins (red arrows). Cell nucleus was stained blue (black arrows). Magnification: 200×. (Gadd45a: growth arrest and DNA damage-inducible 45 α; Mapk7: mitogen-activated protein kinase 7; Rras2: related RAS viral oncogene homolog 2).