Superior Renoprotective Effects Of The Combination Of Enalapril With Curcumin And Its Mechanism In Mice Liver Fibrosis

Objective: Hepatic fibrosis is a pathological process, that excessive deposition of extracellular matrix (ECM) because of chronic liver injury and imbalance of secretion and degradation of ECM. The activation and proliferation of hepatic stellate cell (HSC) play an important role in liver fibrosis, they are the main excreting cells of variety of inflammatory factors and ECM. In the physiological state, synthesis and degradation of ECM is in balance, in order to maintain the normal structure of the liver. However, excessive inflammatory response can increase hepatic injury and promote liver fibrosis. Cell adhesion molecules is closely related to inflammation and fibrosis, it play an important role between cells and cells or cells and ECM. NF-κB is a widespread signaling pathway in cells, which are critical steps in the process of liver fibrosis by various stimuli and the subsequent activation of gene expression such as intercellular adhesion molecule and vascular cell adhesion molecule. Therefore, we established a CCl4-induced liver fibersis model to explore the expression of NF-κB and its associated adhesion molecule ICAM-1 and VCAM-1 in liver tissue. To determine the molecule mechanism of NF-κB pathway in pathogenesis of hepatic fibrosis. And observe the protective effect of enalapril and curcumin on the liver injury and fibrosis, to clarify its mechanism and provide basis theory for the futher development of applications.Methods: Optional 8-week-old healthy male mice, were randomly divided into five groups (each group, n=10): normal control group, model group, curcumin treatment group, enalapril treatment group, curcumin combined with enalapril treatment group (combined group). Adaptive feeding after 1 week, the mice in model group and therapeutic groups were intraperitoneally injected with 10% CCl4 (5ul/g) two times a week for 10 weeks to induce liver fibrosis, the normal control group mice only received 0.9% sodium chloride by intraperitoneal injection. After 2 weeks, the therapeutic groups were given curcumin (100ug/g), enalapril (10mg/kg), and curcumin combined (100ug/g) with enalapril (10mg/kg) treatment respectively, 3 times a week, the mice in normal control group and model group were given 0.9% sodium chloride, all mice were sacrificed after 8 weeks treatment. Serum ALT, AST and HA levels were detected. Pathological changes in hepatic tissue were observed by HE and Masson. The expression of NF-κB P65, IκB, ICAM-1, VCAM-1 in hepatic tissue were examined by immunohistochemistry. The expression of ICAM-1, VCAM-1 mRNA were analyzed by RT-PCR.Results: 1 The general situation of mice: The mice in normal control group are in good spirits, actively, body weight increased gradually. The mice in model group were depressed, weight decreased significantly. The mice in all therapeutic groups were better than model group. The body weight of mice were as follows: model group (34.43±4.08g) enalapril treatment group (92.11±16.89U/L) >curcumin treatment group (83.75±14.54 U/L) >combined treatment group (47.50±9.17U/L) >normal control group (26.60±5.22U/L). ALT level in all treatment groups and normal control group were less than model group (P<0.05). ALT level in Combined treatment group mice was less than enalapril treatment group and curcumin treatment group (P<0.05). The difference between enalapril treatment group and curcumin treatment group was no significant difference (P>0.05). The serum AST levels in each group as followes: model group (205.29±15.86U/L) >enalapril treatment group (123.22±10.47U/L) >curcumin treatment group (98.38±12.41 U/L)>combined treatment group (79.88±9.83U/L) >normal control group (65.20±16.41U/L). The difference between each groups were statistically significant (P<0.05). The serum HA levels in each group as follows: model group (1680.22±94.19ng/ml) >enalapril treatment group (869.89±110.53 ng/ml) >curcumin treatment group (608.71±120.74ng/ml) >combined treatment group (396.13±78.17ng/ml) >normal control group (144.20±37.09 ng/ml). The difference between each groups were statistically significant (P<0.05).3 Liver pathological changes3.1 General form of liver: In normal control group, the liver was reddish-brown, smooth, soft, and shiny. The liver in model group was dark red, dull, there were many granular-nodules in the surface. General observation in the liver of treatment groups were better than the modle group.3.2 Observed under light microscope3.2.1 HE staining: The architecture of hepatic lobules of the normal control group mice was complete, and there was no fibroplasia and inflammatory cell infiltration. But in the mice of model group, the lobules of liver were destroyed, and there were severe necrosis, degeneration and inflammatory cell infiltration. Compared with model group, the liver injury in treatment groups were reduced, difference between model group and combined treatment group or normal control group has statistical significance (P<0.05); Difference between enalapril and normal control group has statistical significance(P<0.05).3.2.2 Masson staining: In normal control group only a small amount of collagen fibers at blood vessels and portal areas, no fibrous tissue hyperplasia and pseudolobule formation. But in the mice of model group, the lobules of liver were separated and surrounded by the collagen fibers, which resulted in apparent pseudolobules. The degree of fibrosis in treatment groups were mitigation than model group, difference between model group and combine treatment group or normal control group has statistical significance (P<0.05); Difference between enalapril and normal control group has statistical significance(P<0.05).4 Immunohistochemical staining4.1 The expression of NF-κB P65 protien in hepatic tissue: There were only few expression in normal control group; In model group, the expression of NF-κB P65 was significantly increased in liver nucleus, and its also expressed in cytoplasm. The NF-κB P65 expression in treatment groups were more than normal control group, but less than model group. The expression levels of NF-κB P65 in each group as follows: model group (2.20±0.10) >enalapril treatment group (1.18±0.13) >curcumin treatment group (0.95±0.07) >combined treatment group (0.62±0.06) >normal control group (0.42±0.06), The difference between each groups were statistically significant (P<0.05).4.2 The expression of IκB protien in hepatic tissue: In normal control group, there was weaker IκB expression in cytoplasm of liver cells but no expression in the nucleus. The expression level in model group and treatment groups were increased than in normal control group, in which the positive expression in combined treatment group was significantly increased and mainly in the cytoplasm; In the model group IκB protein were expressed in the cytoplasm and nucleus, mainly in nucleus, but the level was less than treatment groups. The expression levels of IκB in each group as follows: combined treatment group (2.20±0.14) >curcumin treatment group (1.35±0.12) >enalapril treatment group (1.08±0.07) >model group (0.94±0.13) >normal control group (0.50±0.06). The difference between each groups were statistically significant (P<0.05).4.3 The expression of ICAM-1 protien in hepatic tissue: There were few positive expressions of ICAM-1 in the hepatic sinusoid cells and vascular endothelial cells in normal control group. But in model group in addition to the positive expressions of ICAM-1 in above two types of cells, there were also positive expression in the hepatocytes. And the positive expression mainly located in the portal area, around the central vein, the area of necrosis and inflammatory cell infiltration in hepatic lobules. Compared with model group, the expression of ICAM-1 was significantly reduced in treatment groups. The expression levels of ICAM-1 in each group as follows: model group (2.02±0.09) >enalapril treatment group (1.61±0.08) >curcumin treatment group (1.11±0.14) >combined treatment group (0.75±0.13) >normal control group (0.36±0.1). The difference between each groups were statistically significant (P<0.05).4.4 The expression of VCAM-1 protien in hepatic tissue: There were almost no VCAM-1 expression in normal control group. Follow the development of inflammation and fibrosis, positive expression are visible in liver sinus endothelial cells and hepatocytes. In model group the positive expression increased in vascular endothelial cells around central vein, portal area, and inflammatory cell infiltration in hepatic lobules, the expression in hepatocytes also increased. The expression levels of VCAM-1 in each group as follows: model group (1.66±0.11) >enalapril treatment group (1.12±0.10) >curcumin treatment group (1.01±0.05) >combined treatment group (0.69±0.05) >normal control group (0.26±0.08). The diffenence between each groups were statistically significant (P<0.05).5 RT-PCR detection5.1 The expression of ICAM-1 mRNA in hepatic tissue: Compared with normal control group the expression of ICAM-1 mRNA was significantly increased in the model group. The expression levels in all the treatment groups are more than normal control group but less than model group. The expression levels of ICAM-1 mRNA in each group as follows: model group (1.10±0.16) >enalapril treatment group (0.79±0.11) >curcumin treatment group (0.69±0.13) >combined treatment group (0.42±0.08) >normal control group (0.29±0.11). Compared with model group, the expression level of the normal control group and all treatment groups were significant difference (P<0.05). The expression of normal control group and combined treatment group were lower than the expression of curcumin or enalapril treatment group (P<0.05). There were no significant difference between normal control group and combined treatment group or curcumin treatment group and enalapril treatment group (P>0.05).5.2 The expression of VCAM-1 mRNA in hepatic tissue: The expression levels of VCAM-1 mRNA in each group as follows: model group (1.39±0.21) >enalapril treatment group (0.89±0.12) >curcumin treatment group (0.81±0.12) >combined treatment group (0.54±0.10) >normal control group (0.28±0.10). The expression level of normal control group and treatment groups were lower than model group (P<0.05). Expression levels of combined treatment group lower than curcumin or enalapril treatment group, the difference was statistically significant (P<0.05); There was no significant difference between curcumin treatment group and enalapril treatment group (P>0.05).6 Correlation analysis about liver fibrosis stage and ICAM-1 or VCAM-1: The expression level of ICAM-1 and VCAM-1 protein has linear correlation with fibrosis stage. The more severe of the degree of fibrosis, the higher level of expression of ICAM-1 and VCAM-1 protien.Conclusion:1. The hepatic febrosis model could be established successfully by intrapertioneal injection with CCl4 to mice for 10 weeks. And its pathological changes consistent with the characteristics of human liver fibrosis.2. Occurrence and development of hepatic fibrosis was closely related to the activity of NF-κB pathway and high level expression of its downstream gene ICAM-1 and VCAM-1. High expression of IκB could inhibit the progress of liver fibrosis.3. Enalapril and curcumin can improve liver function and lessen liver inflammation and fibrosis.4. Enalapril and curcumin may be through inhibiting NF-κB activity and thus reduce its downstream gene ICAM-1, VCAM-1 mRNA expression, reducing liver inflammation and liver fibrosis, to play a role in the treatment of liver fibrosis.5. Enalapril combined with curcumin are more effective on the treatment of liver fibrosis than either single drug treatment, its mechanism may be related to synergistic inhibitory effect on NF-κB activation, reducing ICAM-1, VCAM-1 mRNA expression.