Salvianolate Protects Hepatocytes from Oxidative Stress by Attenuating Mitochondrial Injury

Abstract

Salvianolate is widely used to treat angiocardiopathy in clinic in China, but its application in liver diseases remains unclear. Our study aims to investigate the effect of Salvianolate on rat hepatic injury by protecting hepatocyte mitochondria. To evaluate the effects of Salvianolate on injured hepatocytes, alpha mouse liver 12 (AML-12) cells were induced with hydrogen peroxide (H2O2) and treated with Salvianolate. Cell viability and MitoTracker Green for mitochondria and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanide iodine (JC-1) levels and cytochrome C (Cyto-C) expressions were detected in vitro. To identify the effect of Salvianolate on protecting against mitochondria injury, male Wistar rats were injected with carbon tetrachloride (CCl4) and treated with Salvianolate (40 mg·kg(-1)). Serum liver function, parameters for peroxidative damage, hematoxylin and eosin (H&E) staining, and transmission electron microscope (TEM) of hepatocyte mitochondria were assayed. Our results showed that Salvianolate effectively protected hepatocytes, increased mitochondria vitality, and decreased Cyto-C expressions in vitro. Besides, Salvianolate alleviated the liver function, attenuated the indicators of peroxidation, and relieved the mitochondria injury in vivo. In conclusion, Salvianolate is effective in protecting hepatocytes from injury in vitro and in vivo, and the mechanism might be related to its protective effect on hepatocyte mitochondria against oxidative stress.

Figure 1

Cytotoxicity and proliferation of Salvianolate in AML-12 cell lines. (a) AML-12 cells were cultured in a 96-well plate at a density of 5,000 cells/well and incubated with Salvianolate with concentrations of 3.125–400 μg/mL for 24 h. (b) AML-12 cells were cultured in a 96-well plate at a density of 5,000 cells/well and exposed to 0.5 mM H₂O₂ for 20 min; then the cells were incubated with Salvianolate with concentrations of 6.25–25 μg/mL or NAC with concentration of 10 mM for 24 h. Both cytotoxicity of Salvianolate and proliferation of Salvianolate or NAC were detected by CCK8. ^∗ p < 0.05 and ^∗∗ p < 0.01 versus normal control; ^# p < 0.05 and ^## p < 0.01 versus model control.

Figure 2

Protective effects of Salvianolate on H₂O₂-induced mitochondrial injury in AML-12 cells in vitro. Hepatocytes injury model was established with 0.5 mM H₂O₂, and then the cells were incubated with Salvianolate with concentrations of 6.25, 12.5, or 25 μg/mL or NAC with concentration of 10 mM for 24 h. (a) Semiquantification data for expressions of viable mitochondria in AML-12 cells by quantifying the fluorescence intensity of mitochondria probe with MitoTracker Green kits. (b) Semiquantification data for expression of MMP in AML-12 cells by examining the fluorescence intensity ratio of JC-1 aggregation/JC-1 monomer. (c) Expression of Cyto-C (green fluorescence marked with red arrows) in cytoplasm was revealed by the images taken by Cellomics ArrayScan VTI HCS Reader. ^∗∗ p < 0.01 versus normal control; ^# p < 0.05 and ^## p < 0.01 versus model control.

Figure 3

Protective effects of Salvianolate on CCl₄-induced liver injury in rats. Male Wistar rats were treated as described in the legend. Serum ALT (b), AST (c), and TBA (d) were assessed by liver function tests kits, respectively. Levels of serum liver functions were decreased, and HSI (a) was alleviated by Salvianolate treatment with dose of 40 mg/kg in CCl₄-induced liver injury mice. (e) Histologic evaluation of liver tissues was stained with H&E, and the expression of Cyto-C and 4-HNE was assayed by IHC (×200). Semiquantitative analysis of liver injury was performed according to the nonalcoholic fatty liver disease activity score (NAS): (f) hepatic steatosis: 0 (<5%); 1 (5%–33%); 2 (34%–66%); and 3 (>66%) and (g) ballooning degeneration of liver cell: 0, none; 1, rare; and 2, many. ^∗∗ p < 0.001, versus normal control; ^# p < 0.05 and ^## p < 0.001, versus model control.

Figure 4

Effects of Salvianolate on CCl₄-induced liver oxidative stress injury in rats. Levels of parameters for peroxidative damage MDA, GSH, and ASAFR in hepatic homogenates were assayed according to the protocols of corresponding kits. ^∗∗ p < 0.01 versus normal control; ^# p < 0.05 and ^## p < 0.01 versus model control.

Figure 5

Salvianolate relieved mitochondrial injury in hepatocytes. Normal hepatocytes and mitochondria were observed in normal group. However, after stimulating with CCl₄, cellular nucleus concentrating, mitochondria swelling and mitochondria cristae fragmentation or disappearance, and so forth were commonly seen in model control (marked with red arrows). Meanwhile, all those pathological changes were ameliorated after the rats were given 40 mg/kg Salvianolate or NAC. Images of TEM for mitochondria in hepatocytes ×13800.