Changes of the Cytoplasmic Proteome in Response to Alcoholic Hepatotoxicity in Rats

Abstract

Proteomic analyses have already been used in a number of hepatological studies and provide important information. However, few reports have focused on changes in the cytoplasmic proteome. The present study therefore aimed to evaluate changes in cytoplasmic proteome of rats in response to alcoholic hepatotoxicity. Rats were fed a Liber-DeCarli liquid diet containing ethanol for four weeks. Cytoplasmic proteins except mitochondrial proteins from the livers of these animals were investigated using two-dimensional gel electrophoresis and mass spectrometry. Alcohol induced a decrease in body weight gain and an increase in alanine transaminase (ALT), cholesterol, and phospholipid levels. Histopathological observations revealed hepatic damage characterized by necrosis and fatty change in alcohol-treated group at week 2, which continues until week 4. Our proteomic analysis revealed that 25 proteins were differentially expressed in the ethanol-fed group. Of these, 12 cytoplasmic proteins are being reported for the first time. Taken together, our results provide further insights into the disease mechanism and therapeutic information of alcoholic liver disease.

Keywords: alcohol; cytoplasm; hepatotoxicity; proteomics.

Figure 1

Histopathological and immunohistochemical changes in ethanol fed rat liver. (A) Normal control diet-fed rat liver; (B) Two weeks after an ethanol diet was fed to the rats. Hepatic fatty change and necrosis were visible in the cytoplasm of hepatocytes in the centrilobular areas of ethanol-fed rats; (C) Four weeks after the ethanol diet was fed to the rats. Hepatic fatty change and necrosis were more severe than that observed in B, and prominent fatty changes were observed in hepatocytes (H&E staining, ×132); (D) Normal control diet fed rat liver. CYP 2E1 immunoreactivity was relatively weak; (E) Two weeks after the rats were fed an ethanol. CYP 2E1 expression increased in pericentral hepatocytes at two weeks compared to D; (F) Four weeks after the rats were fed an ethanol. Expression of CYP 2E1 was stronger than that observed in E (×66); (G) Normal control diet fed rat liver. GS expression was week; and (H) Four weeks after the rats were fed an ethanol. GS strongly expressed in perivenous hepatocytes. (×50 and ×400).

Figure 2

Two-dimensional electrophoretic gel images of cytoplasmic proteins. Spots indicated by spot number showed statistically significant differences in expression between the pair-fed and ethanol-fed group (n = 5, p < 0.05). (A,B) pair-fed rat liver; and (C,D) ethanol-fed rat liver; down (blue in B), down-regulated protein by ethanol consumption; up (red in D), up-regulated protein by ethanol consumption.

Figure 3

Protein spots and immunoblots of Cu/Zn-SOD, transferrin, and GS in control and ethanol-fed groups. (A) Cu/Zn-SOD decreased significantly in ethanol-fed rat liver compared with the pair-fed control. Comparing the Cu/Zn-SOD enzyme expression by spot volume using ImageMaster II, Cu/Zn-SOD in ethanol-fed rat liver was 0.68-fold that of control rat liver in proteomics (n = 5). Transferrin and GS were significantly increased in ethanol-fed group. Transferrin and GS in ethanol-fed group were 2.1-f and 2.18-fold that of the control group, respectively (n = 5); and (B) In the immunoblotting analysis (n = 3), the expression of Cu/Zn-SOD, transferrin, and GS in the ethanol-fed group were 0.81-, 1.2-, and 2.15-fold that of the control group, respectively. β-actin was used as a control in immunoblot. Data are shown as the mean ± SEM. (* p < 0.05, ** p < 0.01).

Figure 3

Protein spots and immunoblots of Cu/Zn-SOD, transferrin, and GS in control and ethanol-fed groups. (A) Cu/Zn-SOD decreased significantly in ethanol-fed rat liver compared with the pair-fed control. Comparing the Cu/Zn-SOD enzyme expression by spot volume using ImageMaster II, Cu/Zn-SOD in ethanol-fed rat liver was 0.68-fold that of control rat liver in proteomics (n = 5). Transferrin and GS were significantly increased in ethanol-fed group. Transferrin and GS in ethanol-fed group were 2.1-f and 2.18-fold that of the control group, respectively (n = 5); and (B) In the immunoblotting analysis (n = 3), the expression of Cu/Zn-SOD, transferrin, and GS in the ethanol-fed group were 0.81-, 1.2-, and 2.15-fold that of the control group, respectively. β-actin was used as a control in immunoblot. Data are shown as the mean ± SEM. (* p < 0.05, ** p < 0.01).

Figure 4

The illustration of proposed changes in cytoplasmic proteins and pathophysiology in the livers of ethanol-fed rats. “↑” and “↓” indicates the up- and down-regulation by chronic ethanol consumption, respectively. Chronic ethanol ingestion induced up-regulation of LACS2 that was followed by increase of lipogenesis in hepatocytes. Down-regulation of PBE, FABP, and ubiquinone by ethanol induced a decrease of β-oxidation. The increase of lipogenesis and decrease of β-oxidation led to fat accumulation and fatty liver. Chronic ethanol consumption also induced increase in PRBP and decrease in α-TTP, CAIII, Cu/Zn-SOD, rhodanese, and peroxiredoxin 1, which may cause of oxidative stress. Microtubular impairment in the cell membrane by ethanol induced up-regulation of transferrin, TBPA, and PRBP. These changes, in addition to the down-regulation of sideroflexin 1 and up-regulation of transferrin, can lead to the increase in iron storage in hepatocytes. Excessive iron can create a hypoxic state in liver cells and induce oxidative stress. All of these changes attack liver cells chronically and induce hepatocyte injury. Mildly injured liver tissue can produce GS and CDC2 for restoration and hepatocyte proliferation. However, a hypoxic state may induce extramedullary hematopoiesis by increase of β-globin and PADII and hypoxic preconditioning state by down-regulation of KCIP-1, which can lead to PKC secretion.