| Hepatic fibrosis, the final stage of which is cirrhosis, can result from several chronic liver diseases caused by many pathogenic factors and can be classified as a wound-healing response to a variety of chronic stimuli. It is characterized by an excessive deposition of extracellular matrix (ECM) proteins which mainly includes collagens. The activated hepatic stellate cell (HSC) is the primary cell type responsible for the excessive production of collagen. Following the fibrogenic stimulus, HSCs change from a quiescent state to an activated status which can produce collagen-producing cell.Sympathetic nervous system (SNS), which take part in the regulation of liver function and metabolism, is an important component of liver nerves. Recently, more and more studies indicate that SNS influences the development of hepatic fibrosis by regulating proliferation and apoptosis of HSC. Carvedilol is a kind of sympathetic adrenergic receptor blockers with multiple actions including non-selectiveβreceptor block anα1 receptor block, and is also a kind of strong antioxidant and oxygen free radical scavenger with strong reaction. It is reported that Carvedilol might directly inhibit fibroblast proliferation by blocking SNS receptor, and inhibit extracellular matrix (ECM) synthesis, lessen heart matrix remodeling and prevent development of heart failure (CF). However, to the best of our knowledge, whether at home or at abroad, few researches were reported on influences of Carvedilol in the development of hepatic fibrosis, especially in the process of collagen metabolism and oxidative stress. Our preliminary studies have shown that carvedilol can inhibit HSC activation and proliferation and promote apoptosis of activated HSC in liver tissues of rats with hepatic fibrosis. In this study, we will further investigate the influence of Carvedilol on typeâ… ,â…¢,â…£collagen metabolism as well as its antioxidation effect in liver tissues of rats with hepatic fibrosis.Objective: To investigate the influence of Carvedilol on collagen metabolism and its antioxidation effects in rat liver tissues with hepatic fibrosis.Methods: Rat hepatic fibrosis models were established by applying bile duct ligation (BDL) and adenoreceptor blocker Carvedilol was administered as drug intervention. HE, Masson staining were used to determine hepatic fibrosis degree; Hepatic oxidative status was estimated on liver homogenates by measuring super oxide dismutase(SOD) and malonyldialdehyde (MDA) with the xanthine oxidase method and Thiobarbituric acid method (TBA), respectively; immunohistochemistry was applied to detect the expressions of typesâ… ,â…¢,â…£collagen and TGF-β1; Western blot and real time Q-PCR were used to measure the dynamic expressions of MMP-2 and TIMP-2 during hepatic fibrosis. Fifty male Sprague Dawley rats were randomly divided into five groups, sham operation control group, bile duct ligation (BDL) model group and BDL+ low dose Carvedilol (0.1mg·kg-1·d-1) group, middle dose Carvedilol (1mg·kg-1·d-1) group and large dose Carvedilol (10mg·kg-1·d-1) group, with 10 rats in each group. Then each group is stochastic divided into two sub-groups, and the specimens were harvested at 2 wk and 4 wk after BDL, respectively.Results:1 General condition in rat hepatic fibrosis modelsAt 1-2 h after BDL, rats recovered motion again; at about 48 h, urine became yellow; 3-4 d later, their skin and hair began to turn yellow and they gradually became weak, ate less than the control group, with insignificant increasing or slight decreasing of body weights. 8-9 d later, the general state became poorer, with sleepy, slow response, less activity, significant jaundice and grey feces. 20 d later, their food-intake reduced significantly, body weights decreased significantly compared with that of control group, and some rats emerged abdominal bulge gradually.2 Hepatic fibrosis models were successfully established by bile duct ligationThe liver of modeling rats was brownish green or brown observed by naked eyes, with fine granule and hard texture on surface. HE and Masson trichrome stain showed that the liver tissue of sham operation group rats had complete hepatic lobule, well arranging hepatic plate, no swollen hepatocytes, no bile duct proliferation, no silt chole or lymphocytes infiltration, clear nucleous, and small quantity of connective tissue limited in portal area. Two weeks after modeling, the model group showed disappeared normal arrangement of hepatic plates, disordered lobule structure, extensive proliferation small bile duct in portal areas extending to lobule, chaplet like hepatic lobule, fibrous tissues around portal areas, enlarging portal areas, fibrous tissues proliferation also in hepatic lobule. Four weeks after modeling, the liver tissue of model group demonstrated extensive fibrous connective tissue proliferation, and the proliferated fibers connected, enclosed and divided with each other to change the original hepatic lobule, even forming pseudo lobule.3 The effects of Carvedilol on liver pathohistologyTwo weeks after modeling, HE stain showed that the liver tissue of treatment group had small amounts of fibrous tissue and small bile duct proliferation, slight circuity of hepatic cord, little damage of hepatic lobule structure. Four weeks after modeling, HE stain showed that the normal structure of hepatic lobule in treatment group was partly destroyed; fibrous tissues around lobule and header areas had slight proliferation; hepatic obviously lowered by 48.73% than that in Con group, P<0.01; cord disorder was slighter than that of the model group; hepatocytes proliferation was also present.Masson trichrome stain collagen area density measurement demonstrated that 2 weeks after BDL, collagen area density in the model group, low dose, middle dose and large dose groups were all significantly higher than that of the sham operation group P<0.01; no significant difference was found between low dose group and model group, P>0.05; collagen area density of middle dose and high dose groups decreased significantly compared with that of model group, P<0.05, P<0.01 respectively; collagen area density of middle and high dose groups decreased significantly compared with that of the low dose group, too, P<0.05, P<0.01 respectively; no significant differences were found between middle dose and high dose groups, P>0.05.After 4 wk of BDL, collagen area density in the model group, low dose, middle dose and large dose groups were all significantly higher than that of the sham operation group, P<0.01; collagen area density of low dose, middle dose and large dose groups decreased significantly compared with that of model group, P<0.05, P<0.01, P<0.01 respectively; collagen area density of large dose group decreased significantly compared with that of low and middle dose group, too, P <0.01, P<0.05 respectively; no significant differences were found between low dose and middle dose groups, P>0.05.4 Carvedilol down-regulated the expressions of collagens in liver tissues of hepatic fibrosis rats4.1 Carvedilol down-regulated the expressions of typeâ… ,â…¢,â…£Collagens at 2 wk after BDLImmunohistochemistry stain showed that at 2 wk after BDL, typeâ… Collagen positive area density in the model group, low dose, middle dose and large dose groups(15.99%±1.47%, 12.66%±1.89%, 11.24%±0.60%, 10.58%±3.74%) were significantly higher than that of the in sham operation group(3.05%±1.02%), P<0.01; typeâ… collagen positive area density of low dose group, middle dose group and large dose group decreased significantly compared with that of model group, P<0.05, P<0.05, P<0.01 respectively. No significant differences were found in the three Carvedilol dose groups, P>0.05. Typeâ…¢Collagen positive area density in the model group, low dose group, middle dose group and large dose group(17.85%±1.92%, 14.14%±0.64%, 11.96%±2.01%, 10.27%±0.99%) were significantly higher than that in the sham operation group (5.18%±0.79%), P<0.01; typeâ…¢Collagen positive area density of low dose group, middle dose group and large dose group decreased significantly compared with that of model group, P<0.01; typeâ…¢Collagen positive area density of large dose group have also significantly decreased compared with that of low dose group, P<0.01 while no significant differences compared with that of middle dose group, P>0.05, and no significant difference were found between low dose and middle dose groups, P>0.05. Typeâ…£Collagen positive area density in the model group, low dose group, middle dose group and large dose group(11.89%±1.45%, 9.30%±1.01%, 9.14%±1.48%, 8.80%±1.21%) were significantly higher than that in the sham operation group (1.82%±0.77%), P<0.01;typeâ…£collagen area density of low dose group, middle dose group and large dose group decreased significantly compared with that of model group, P<0.05. However, no significant differences were found in the three Carvedilol dose groups, P>0.05.4.2 Carvedilol reduced the expressions of typeâ… ,â…¢,â…£Collagens at 4 wk after BDLImmunohistochemistry stain showed that at 4 wk after BDL, typeâ… Collagen positive area density in the model group, low dose group, middle dose group and large dose group(23.69%±2.95%, 15.74%±0.51%, 14.57%±1.55%, 12.54%±3.64%) were significantly higher than that in the sham operation group(4.14%±0.98%), P<0.01; typeâ… collagen positive area density of low dose group, middle dose group and large dose group decreased significantly compared with that of model group, P<0.01; no significant differences were found in the three Carvedilol dose groups, P>0.05. Typeâ…¢Collagen positive area density in the model group, low dose group, middle dose group and large dose group(21.53%±0.76%, 16.84%±1.99%, 16.03%±0.42%, 11.17%±2.30%) were significantly higher than that of the sham operation group(5.48%±0.79%), P<0.01; typeâ…¢Collagen positive area density of low dose group, middle dose group and large dose group decreased significantly compared with that of model group, P<0.01; typeâ…¢Collagen positive area density of large dose group have also significantly decreased compared with that of low dose group and of middle dose group , P<0.01; while no significant differences were found between low dose group and middle dose group, P>0.05. Typeâ…£Collagen positive area density in the model group, low dose group, middle dose group and large dose group(18.05%±1.15%, 12.02%±1.50%, 11.41%±1.65%, 9.84%±2.08%)were significantly higher than that in the sham operation group (2.01%±0.49%) , P<0.01; typeâ…£collagen area density of low dose group, middle dose group and large dose group decreased significantly compared with that of model group, P<0.01. However, no significant differences were found in the three Carvedilol dose groups, P>0.05.5 Effects of Carvedilol on protein and mRNA expressions of MMP-2 and TIMP-2 in liver fibrosis tissues in ratsWestern blot showed that at 2 wk after BDL, the relative expressions of MMP-2 protein in the model group, low dose group, middle dose group(1.99±0.13, 1.87±0.12, 1.88±0.04)were significantly higher than that in the sham operation group(1.24±0.09), P<0.01, while no significant differences were found between large dose group (1.33±0.04) and the sham operation group, P>0.05; the relative expressions of MMP-2 protein of large dose group decreased significantly compared with that of model group, middle dose group as well as low dose group, P<0.01; no significant differences were found in low dose group, middle dose group and model group, P>0.05. At 4wk after BDL, the relative expressions of MMP-2 protein in the model group(1.49±0.22) was significantly lower than that in 2 wk; the relative expressions of MMP-2 protein in low dose, middle dose group and large dose group(1.74±0.17, 1.99±0.16, 2.15±0.24) were significantly higher than that in the sham operation group(1.26±0.13),P<0.01; the relative expressions of MMP-2 protein of middle dose group and large dose group increased significantly compared with that of model group, P<0.01; however, no significant difference were found between the model group and sham-operated control group, low dose group and middle dose group, as well as that between middle dose group and large dose group, P>0.05.Real time Q-PCR showed that at 2 wk after BDL, suppose that the mRNA expression level in the sham-operated control group was 1, then the relative expression level of MMP-2 mRNA in the model group, low dose group, middle dose group and large dose group were 1.83, 1.58, 1.53 and 1.12 respectively. The MMP-2 mRNA expressions in the model group, small dose group and middle dose group were significantly higher than that in sham-operated control group, P<0.01. The MMP-2 mRNA expression in large dose group decreased significantly compared with those in model group, middle dose and low dose group, P<0.05. No significant differences were found between low dose group and middle dose group, and between large dose group and the sham operation group, P>0.05. At 4 wk after BDL, the relative MMP-2 mRNA expression in the model group was 1.28, which decreased significantly compared to that in model group at 2wk, and the relative MMP-2 mRNA expressions in low dose group, middle dose group and large dose group were 1.66, 1.92 and 1.98 respectively, which were significantly higher than that in sham-operated control group, P<0.01. The relative expression of MMP-2 mRNA in middle dose group and large dose group increased significantly compared with that in model group, P<0.01. However, no significant difference were found between the model group and sham-operated control group, between low dose group and middle dose group, as well as between middle dose group and large dose group, P>0.05.6 TIMP-2 protein and mRNA expression in fibrogenic liver tissues are down-regulated by Carvedilol in ratsWestern blot showed that at 2 wk after BDL, the relative expressions of TIMP-2 protein in the model group, low dose, middle dose groups(1.41±0.09, 1.32±0.05, 1.27±0.23, 0.78±0.06)were significantly higher than that in the sham operation group(0.53±0.05), P<0.01, P<0.01, P<0.01, P<0.05 respectively; the relative expressions of TIMP-2 protein of large dose group decreased significantly compared with that of model group, middle dose group as well as low dose group, P<0.01; no significant differences were found in low dose group, middle dose group and model group, P>0.05. At 4 wk after BDL, the relative expressions of TIMP-2 protein in the model group, low dose group, middle dose group and large dose group(1.42±0.07, 1.12±0.13, 1.06±0.15, 0.92±0.07) were significantly higher than that in the sham operation group(0.56±0.09), P<0.01; the relative expressions of TIMP-2 protein of low dose group, middle dose group and large dose group decreased significantly compared with that of model group, P<0.01; TIMP-2 protein level of large dose group decreased significantly compared with that of low dose groups, P<0.05; however, no significant differences were found between low dose and middle dose groups as well as that between middle dose group and large dose group, P>0.05.The expressions of TIMP-2 mRNA in the hepatic tissues of rats were detected by real time Q-PCR. At 2 wk after BDL, suppose that the mRNA expression level in the sham-operated control group was 1, then the relative expression of TIMP-2 mRNA in the model group and the low dose group, middle dose group and large dose group were 2.73, 2.56, 2.38 and 1.70 respectively.Tthe relative expressions of TIMP-2 mRNA in the model group, small dose group and middle dose group were significantly higher than that in sham-operated control group, P<0.01, P<0.01, P<0.01, P<0.05 respectively. The relative expression of TIMP-2 mRNA in large dose group decreased significantly compared with those in model group, middle dose group as well as low dose group, P<0.01, P<0.05, P<0.05 respectively, no significant differences were found among low dose group , middle dose group and model group, P>0.05. At 4 wk after BDL, suppose that the mRNA expression level in the sham-operated control group was 1, then relative expressions of TIMP-2 mRNA in the model group and the low dose group, middle dose group and large dose group were 2.67, 1.97, 1.89 and 1.54 respectively. The relative expressions of TIMP-2 mRNA in the model group, small dose group and middle dose group and large dose group were significantly higher than that in sham-operated control group, P<0.01, P<0.01, P<0.01, P<0.05 respectively, the relative expressions of TIMP-2 mRNA in low dose group, middle dose group and large dose group were decreased significantly compared with that in model group, P<0.05, P<0.01, P<0.01, respectively. However, no significant difference were found among the low dose group, middle dose group as well as large dose group, P>0.05.7 Carvedilol reduced the expression of TGF-β1 in liver tissues of hepatic fibrosis ratsImmunohistochemistry stain showed that at 2 wk after BDL, TGF-β1 positive area density in the model group, low dose group, middle dose group and large dose group(10.10%±1.14%, 9.51%±1.07%, 7.14%±1.60%, 6.44%±3.64%)were significantly higher than that of the sham operation group(4.14%±0.82%), P<0.01; TGF-β1 positive area density of middle dose group and large dose group decreased significantly compared with that of model group, P<0.05, and middle dose, large dose group was significantly lower than that of low dose group, P<0.05; no significant differences were found between low-dose treatment group and model group, as well as that between middle dose and large dose groups, P>0.05. At 4wk after BDL, TGF-β1 positive area density in the model group, low dose group, middle dose group and large dose group(14.00%±1.81%, 7.35%±0.68%, 7.04%±0.33%, 6.53%±0.81%) were significantly higher than that in the sham operation group(4.64%±0.34%), P<0.01, P<0.01, P<0.05, P<0.05, respectively; TGF-β1 positive area density of low dose group, middle dose group and large dose group decreased significantly compared with that of model group, P<0.01; no significant differences were found in the three Carvedilol dose groups, P>0.05.8 The effect of Carvedilol on oxidative stress of hepatic fibrosis tissues in rats8.1 The effect of Carvedilol on MDA (nmol/mgprot) of hepatic fibrosis tissues in ratsThiobarbituric acid method (TBA) showed that at 2 wk after BDL, MDA contents in the model group, low dose group, middle dose group and large dose group (0.94±0.19, 0.73±0.09, 0.67±0.62, 0.63±0.09) were significantly higher than that in the sham operation group (0.41±0.06), P<0.01. MDA contents in low dose group, middle dose group and large dose group decreased significantly compared with that in model group, P<0.01. No significant differences were found among the three Carvedilol treatment groups, P>0.05. At 4 wk after BDL, the MDA contents in the model group, low dose group, middle dose group and large dose group (1.21±0.23, 0.98±0.13, 1.00±0.03, 1.10±0.14) were significantly higher than that in the sham operation group (0.43±0.07), P<0.01. The MDA contents in low dose group, middle dose group and large dose group decreased significantly compared with that in model group, P<0.01. No significant differences were found among the three Carvedilol treatment groups and the model group, P>0.05.8.2 The effect of Carvedilol on SOD(U/mgprot)of hepatic fibrosis tissues in ratsXanthine oxidase method showed that at 2 wk after BDL, SOD activities in the model group, low dose group, middle dose group and large dose group (1.48±0.14, 1.73±0.13, 1.78±0.11, 1.84±0.16)were significantly lower than that in the sham operation group(2.24±0.11), P<0.01. SOD activities in low dose group, middle dose group and large dose group increased significantly compared with that in model group, P<0.01. No significant differences were found among three Carvedilol treatment groups, P>0.05. At 4 wk after BDL, SOD activities in the model group, low dose group, middle dose group and large dose group (0.84±0.06, 1.50±0.10, 1.66±0.14, 1.67±0.19) were significantly lower than that in the sham operation group (2.32±0.14), P<0.01. SOD activities in low dose group, middle dose group and large dose group increased significantly compared with that in model group, P<0.01. No significant differences were found among three Carvedilol treatment groups, P>0.05.Conclusions:1 Carvedilol can reduce typeâ… ,â…¢andâ…£collagen deposition and inhibit the development of hepatic fibrosis in experimental rats in a dose-dependant manner.2 Carvedilol regulate the expression of MMP-2 and TIMP-2 in gene and protein level in hepatic fibrosis rat liver tissue, and promote the restoration of MMP2 / TIMP-2 dynamic balance; reduce the expression of TGF-β1, it may be one mechanisms of Carvedilol reduce the expression of typeâ… ,â…¢,â…£collagen and slow down the process of hepatic fibrosis. 3 Carvedilol may reduce the expression of MDA and improve SOD activity in hepatic fibrosis rat liver tissue to reduce oxidative stress.
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