| Objective: Hepatic fibrosis (hepatic fibrosis, HF) is a chronic liver disease due to various reasons. Hepatitic fibrosis is a common pathological process for chronic liver disease progression to cirrosis. Reverse liver fibrosis in early stage is extremely important for blocking liver cirrosis. Hepatic fibrosis related to a variety of cells, cytokines and extracellular matrix changes of liver tissue. Nuclear factor-κB (nuclear factor-kappaB, NF-κB) is a broad variety of cells in the body of nuclear transcription factor, the body's immune and inflammatory responses is closely related to apoptosis regulation, Bcl-2 family is its downstream gene, in the process of gene regulation of apoptosis plays a crucial role. Bcl-2 family members decided to constitute a percentage of cell survival. Curcumin (curcumin) the main pharmacologically active ingredient is isolated from Curcuma medicine, has anti-inflammatory,anti-lipid peroxidation, anti-tumor, nourish the liver and a variety of pharmacological effects. Recent studies found that angiotensin-converting enzyme inhibitors (ACEI) inhibit hepatic fibrosis effectivly. In this study, by detecting NF-KB and related apoptotic protein Bcl-2, Bax expression in the CCL4-induced model of liver fibrosis, to explore the effect of combination therapy of curcumin and enalapril therapy, determine the molecule mechanism. Provide a new target for treatment hepatic fibrossis.Methods: Forty-five 8-week-old male Kunming mice were randomly divided into five groups, the normal control group(n=10), model group(n=10), curcumin treatment group(n=10), enalapril treatment group (n=10), and curcumin combined with enalapril treatment group(n=10). In addition to the normal control group, mice in each group were treated with carbon tetrachloride 5ul/g body weight intraperitoneal injection to establish hepatic fibrosis model, two times a week, a total of 10 weeks. Curcumin treatment group, enalapril treatment group and the curcumin combined with enalapril treatment group (after here referred to as combination therapy group) were from the second week to 10th week to give a gavage treatment curcumin 100ug/g, enalapril 10mg/kg, and curcumin 100ug/g combined with enalapril 10mg/kg, 3 times per week, the normal control group was given distilled water, 3 times a week. All mice were killed at end of 10 weeks. Blood and liver tissue were collected for test serum ALT, AST and HA levels. Part of liver tissue werefixed in 10% neutral formalin, paraffin-embedded. Hematoxy lin-eosin (HE) staining and Masson staining to observe histopathological changes,immunohistochemical staining analyze IKB, NF-kBP65, Bcl-2, Bax protein expression. Reverse transcription polymerase chain reaction (RT-PCR) detect liver tissue Bcl-2, Bax mRNA expression.Resultsl General situations of mice:The mice in normal control group are in good spirit,hair shiny,weekly weight gain gradually.Model group of mice injected with carbon tetrachloride,become irritability, apathetic,loss of appetite, weight gain is slow or no increase. The treated mice were better than the model group mice. The body weight of mice in model group, enalapril treatment group, curcumin-treated group, combination therapy group and the normal control group were followed: 34.43±4.08,43.56±2.88,43.38±4.75,49.75±3.62,53.60±3.51. Compared with the normal control group, the body weight in other four groups were lower. The difference was statistically significant (P﹤0.05). Compared with the model group, the other four groups of mice gained weight significantly, the difference was statistically significant (P﹤0.05). The control group and combined treatment group body weight were higher than curcumin treatment group and the enalapril treatment group (P﹤0.05); The difference between curcumin treatment group and enalapril treatment group, normal control group and combined treatment group have no significant difference (P﹥0.05).2 The serum levels of ALT, AST and HA: serum ALT levels of experimental mice, model control group, enalapril treatment group, curcumin treatment group, combination therapy group and normal control group, followed by:155.29±21.56U/L, 92.11±16.89U/L, 83.75±14.54U/L, 47.50±9.17U/L, 26.60±5.22U/L. The treatment group and the control group, serum ALT were lower than model group (P﹤0.05), combination therapy more effective than enalapril and curcumin treatment group (P﹤0.05), the serum ALT levels between the enalapril treatment group and the curcumin treatment was no significant difference (P﹥0.05). Serum AST levels, model control group, enalapril treatment group, curcumin treatment group, combination therapy group and normal control group, followed by:205.29±15.86U/L, 123.22±10.47U/L, 98.38±12.41U/L, 79.88±9.83U/L, 65.20±16.41U/L, the AST levels of between each groups are statistically significant, P﹤0.05. Experimental mice serum HA levels, model group, enalapril treatment group, curcumin-treated group, combination therapy group, normal control group, serum HA levels followed:1680.22±94.19ng/ml, 869.89±110.53ng/ml,608.71±120.74ng/ml, 396.13±78.17ng/ml, 144.20±37.09ng/ml, the HA levels of between two groups are statistically significant,P﹤0.05.3 Liver tissue Pathological detect3.1 Pattern of liver tissue: In normal control group, the liver of mice is reddish-brown, soft, bright and shiny. In model group, the liver of mice is darker then the normal control group, smooth surface, due to the size decrease compared with the normal control group, the edge of liver become blunt and texture is solid, it can be seen yellow fat particle scattered at the surface of liver. Enalapril-treated group and curcumin treatment group livers of mice in general liver's color was a little darker, liver size was slight decrease compared with the normal control group, the combination therapy group of mice liver reddish-brown, shiny, size no significantly reduced compared with the normal control group.3.2 liver pathology HE staining: in normal control group(G0), the liver structure is organizational, hepatic lobule structure was normal, liver cells cord orderly arranged, the size of liver cells were orderly and uniform, no degeneration and necrosis. In model group of mice(G3-G4), the normal lobule structure was damaged or disappeared, the portal area and central vein out of thick collagen fiber cable divided and surrounded the hepatic lobule, the liver cell cord disarrangement, hepatocellular obvious necrosis, a large number of inflammatory cell infiltration in fiber interval and can see the false lobules formationing. Enalapril treatment group and curcumin treatment group(G2-G3) shows a small number of fibrous tissue, no pseudolobule formation and see infiltration of inflammatory cells, compared with model group, the liver cell's edema reduced; combined treatment group(G1-G2) was better than enalapril treatment group and the curcumin treatment group.3.3 Liver pathology Masson staining:we focuse on the distribution of green collagen fibers under microscope, in normal control group(S0), only a small amount of green perivascular fibers, thin and short, the structure of liver unit is normal, no fibrosis was seen; In the model group(S3-S4), liver fibers can be seen increased significantly, widely distributed and interconnected to form larger coarse fiber spacing, most of which located in the portal areas and perivascular tube; In enalapril treatment group and the curcumin treatment group(S2-S3) smaller green fibers can be seen around the blood vessels. Compared with enalapril treatment group and the curcumin treated group fibrosis in combined treatment group(S1-S2) was slightly.4 Liver tissue immunohistochemical staining study4.1 Bcl-2 immunohistochemical staining: The positive expression of Bcl-2 protein was stained brown in cells and tissues compared with the blank negative control. In normal control group, there was a low level expression in Sinusoids, liver cytoplasm and the central vein. In the model group Bcl-2 protein are widely distributed in liver tissue, mainly in periportal areas, fiber spacing, sinusoids, liver cell membrane, the central vein and Liver cytoplasm. The sequence of Bcl-2 expression in five groups are : Combined treatment group(1.31±0.51)%﹥curcumin treatment group(1.17±0.41)%﹥enalapril treatment group(0.97±0.25)%﹥Model group(0.70±0.36) %﹥normal control group(0.58±0.34)%. two two compared among the groups,P <0.05, differences with statistical significance.4.2 Bax immunohistochemical staining: The positive expression of bax protein was stained brown in cells and tissues compared with the blank negative control. In normal mice liver tissue, the central vein and its surrounding sinusoid was low-level expression. In the model group mainly expressed in liver cytoplasm, mainly in degeneration of liver cytoplasm, in severe cases can diffuse distribution, followed by the sinusoid, fiber spacing and central venous. Bax expression in five groups are: model control group(1.52±0.20)%﹥enalapril treatment group ( 0.88±0.27 ) %﹥curcumin treatment group(0.85±0.31)%﹥combination therapy(0.63±0.22)%﹥normal control group(0.25±0.20)%. two two compared among the groups, P<0.05, differences with statistical significance.4.3 NF-κBp65 immunohistochemical staining: NF-κB p65 positive substance showed brown particles, expression in the cytoplasm and was activated in the nucleus. In normal mice liver tissue, less expression of NF-κBp65. In the model group, there are strong expression, Mainly distributed in liver cells, HSC cells, the cytoplasm was also expressed. Compared with the control group, the expression in treatment groups were increased, and the positive expression mainly located in the cytoplasm of liver cells. NF-κBp65 expression in five groups:model group(2.20±0.10)%﹥enalapril treatment group(1.18±0.13)%﹥curcumin treatment group(0.95±0.07)%﹥combination therapy(0.62±0.06) %﹥normal control group(0.42±0.06)%. two two compared among the groups, P<0.05, differences with statistical significance.4.4 IκB immunohistochemical staining: IκB in liver tissue expression was similar to NF-κBp65, In normal mice liver tissue, there was a weak expressin in liver cells cytoplasm, no expression within the nucleus. In the model group and the treatment groups, liver cytoplasm and nucleus were expressed brown-yellow granules, Combination therapy group mainly expression in cytoplasmic, the nucleus and HSC also see the expression. Compare to the Combined treatment group, the expression in model group expressed in cytoplasm and nucleus significantly reduced. IκB expression of five groups: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)%> control group(0.50±0.06)%. two two compared among the groups, P <0.05, differences with statistical significance.5 Liver tissue RT-PCR detect5.1 The expression of Bcl-2 mRNA in liver tissue: Combination therapy group, curcumin treatment group, enalapril treatment group, model group, normal control group, Bcl-2mRNA expression was significantly decreased. The expression of liver tissue Bcl-2mRNA level order: combined treatment group(1.40±0.09)%>curcumin treatment group(1.19±0.15)%>enalapril treatment group(0.87±0.12)%>model group(0.56±0.07)%> control group(0.30±0.09)%. two two compared among the groups, P <0.05, differences with statistical significance.5.2 The expression of BaxmRNA in liver tissue: Model control group, enalapril treatment, curcumin treated group, combination therapy group, normal control group, Bax mRNA expression was significantly decreased, the expression of liver tissue Bax mRNA level order: model group(1.06±0.14)%>enalapril treatment group(0.92±0.07)%>curcumin treatment group(0.79±0.08)%>combined treatment group (0.67±0.09)%>normal control group(0.51±0.10)%, two two compared among the groups, P <0.05, differences with statistical significance.6 corelationship analysis of liver firosis stage and liver tissue Bcl-2 and Bax protein levels, Bax was positively correlated with the degree of fibrosis, fibrosis was heavier and Bax expression more; Bcl-2/Bax ratio was negatively correlated with the degree of fibrosis, fibrosis was heavier the ratio was smaller.Conclusion1 The hepatic fibrosis models could be established successfully by intraperitoneal injection with the mixture of 10% CCL4 and olive oil for 10 weeks. The Serum biochemical and pathological features are consistent with hepatic fibrosis in human. 2 Apoptosis is one of the important mechanism for the pathogenesis of liver fibrosis, Nuclear factor NF-κB and Bcl-2 gene family is most closely related to hepatocyte apoptosis, Bcl-2 is the most important anti-apoptotic factor, also known as the "survival genes", Bax was homologous gene of Bcl-2, it was the most important anti-apoptotic factor, The ratio of Bcl-2/Bax decided cell's survival or apoptosis,Bcl-2/Bax ratio increased and apoptosis reduced, Bcl-2/Bax ratio decreased and apoptosis increased. Bax was positively correlated with the degree of fibrosis, Bcl-2/Bax ratio was negatively correlated with the degree of fibrosis.3 Curcumin and enalapril alone can prevent carbon tetrachloride-induced liver fibrosis in mice, Curcumin combined with enalapril prevent carbon tetrachloride-induced liver fibrosis is the best. The mechanism may be related with inhibiting NF-κB activity and promoting the expression of Bcl-2, reducing the expression of bax, thus inhibit liver cell apoptosis and reduce liver fibrosis.
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