| Nrf2acts as a key antioxidant to participate in hepatic defence fibrotic mechanism, in response to oxidative stress stimulated by hepatocytes repeatedly phlegmonosis and necrosis, undergo persistent insults to liver, including drugs and/or ethanol abuse, viral infection, chemical carcinogen etc, Nrf2dissociates from Kelch-like ECH-associated protein1(Keapl) anchoring Nrf2in the cytoplasm in homeostatic condition, and translocates to the nucleus, and then combine with the promoter region of anti-oxidant response element (ARE) sequences and coordinates induction of its target gene transcription. Traditional Chinese medicinal herbs FZH capsule is used for anti hepatic fibrosis. Furthermore, multiple studies have demonstrated that FZH capules have been characterized as a key to promote the resolution of fibrosis through against HSC activation. The ability of Nrf2to act as important character for anti-inflammatory and antioxidant, as well as the hepatoprotective effects of FZH capsule have been previously demonstrated to antifibrosis in liver. The purpose of the present study was to evaluate the effect of Fuzhenghuayu capsules on expression of Nrf2of hepatocytes in an hepatoxic chemical fibrotic model of CCL4in mice.Objective1. To made an hepatic fibrosis model by CCL4intraperitoneal injection, and proved by pathological histology detection;2. To investigate expression of nuclear factor-E2-realated factor2(Nrf2), NAD(P)H quinine oxidoreductase1(Nqo1), α-Smooth muscle actin(α-SMA), Fibronectin(FN) in hepatic fibrosis in mice;3. To observe the effect of Fuzhenghuayu capsule on the changes of degrees of inflammation and fibrosis indexes; 4. To study the influention of Nrf2、 Nqo1、 α-SMA、 FN expression with Fuzhenghuayu compound administration, and explore the most possible protective mechanisms of Fuzhenghuayu capsule to antifibrosis in liver.Methods70mice were randomly divided into2groups:control group and model group. The control group was composed of groupAl:mice were nontreated; group A2:mice were given mineral oil alone once three days; group A3:mice were given daily Fuzhenghuayu compound at2mg/g body weight (BW)(the concentration of Fuzhenghuayu capsule powder diluted with double-distilled water is0.1g/ml) for4weeks by gastric perfusion since the7th week. Mice in the model group were received CCL4(diluted in mineral oil, volume ratio1:10) at dose of0.25mg/g BW respectively by intraperitoneal injection once three days for10weeks. Random samples,9mice, were killed to be group B1at end of6weeks. The rest mice were divided into group B2:8mice, were injected with CCL4intraperitoneal continually for4weeks; group Cl:10mice were intragastric administrated with lower-dose FZHc at1mg/g BW for4weeks, and group C2:9mice, began to received high-dose FZHc at2mg/g BW for4weeks. At the end of6th and10th week, the specimens were respectively collected and detected as follows:1. The degrees of hepatic fibrosis and inflammation ware judged by routine haematoxylin-eosin staining and Masson staining.2. Immunohistochemistry staining was used to measure the expression of Nrf2、 Nqo1、 a-SMA、 FN of hepatocytes in mice.3. Western-blotting was used to detect Nrf2and Nqol total protein expression and Nrf2nuclear translocation.4. Expression of Nrf2mRNA was determined using the real-time fluorescence quantitative PCR.Results1. Histopathological examination showed, in a time-dependent manner from6to10weeks, the severe inflammation of the liver and collagen fibers accumulation of gourp B1compared with group A1, and more aggravation in group B2(P<0.05);2. Immunohistochemisty staining paralleled to histopathological examination, gourp B1with positive expression of Nrf2、 Nqo1、 α-SMA、 FN, and more obviously observation in group B2in a time-dependent manner from6to10weeks (P<0.05);3. Western blot and RT-PCR proved coordinately, compared separately with group A1, Nrf2nuclear translocation and Nrf2mRNA, as well as Nqo1protein of group A2、 A3have no statistically changes at the end of10weeks time point; with persistently CCL4intraperitoneal injection for10weeks, the expressions of Nrf2protein, Nrf2nuclear translocation and Nrf2mRNA were all increased statistically (P<0.05), as well as Nqo1protein were also significantly positive expression in group B2(P<0.05).4. Compared with group B2, after administration of Fuzhenghuayu compound, the degrees of hepatic inflammation and fibrosis in groups L-FZHc C1and H-FZHc C2were all significantly decreased (P<0.05), and group H-FZHc C2has more obviously antiinflammation impact in liver fibrosis(P<0.05); and FZH compound treatment reduced CCL4-induced a-SMA and FN expression at the10weeks time-point, and in a does-dependent manner from1to2mg/g BW(P<0.05);5. At the10-weeks time point, compared with group B2, western blot and immunohistochemisty staining proved paralleled, after administration of Fuzhenghuayu compound, the expressions of Nrf2protein, Nrf2nuclear translocation, and Nqol protein in group L-FZHc C1and H-FZHc C2were increased statistically in a does-dependent manner from1to2mg/g. kg (P<0.05);6. RT-PCR found that although there was no statistical difference between groups B1and B2; the expressions of Nrf2mRNA in group L-FZHc C1comparing with group B2was increased(P<0.05), and more statistically increasedly in group H-FZHc C2(P<0.05).ConclusionsWith persistently CCL4intraperitoneal injection for6weeks, hepatocytes has obviously inflammatory response and extracellular matrix deposition, and with modeling process lengthened, hepatic stellate cells activation marker a-SMA expression positively, as well as extracellular matrix FN accumulation, and hepaticfibrosis pathologic changes exacerbation.Fuzhenghuayu capsule could increase the expression of Nrf2protein of hepatic fibrosis in mice and induce Nrf2transport into nuclear, following by increasing the expression of target gene Nqo1, suppressing the activity of HSCs and decreasing the deposition of FN. Therefore, Fuzheng huayu capsule can restrain the injury of hepatocytes in hepatic fibrosis in mice by exerting an anti-hepatic fibrosis effect. |
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