| Hepatic fibrosis, the final stage of which is cirrhosis, can result from several chronic liver diseases caused by many pathogenic factors. Hepatic stellate cell (HSC), a main liver fibrogenic cell, plays a critical role in hepatic fibrosis. 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, a new non-selectiveβreceptor blocker with function of vasodilatation, has similar blocking effect onα1 receptor. The beneficial effect of Carvedilol on myocardial infarction (MI) and chronic cardiac failure (CCF) has already been confirmed by large-scale clinical trials. 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, few researches were reported on influences of development of hepatic fibrosis by blocking classical neurotransmitter (NE) of SNS to combine with specific adrenoceptor. Objective:To investigate the influence of Carvedilol on HSC proliferation and apoptosis as well as collagen metabolism in rat hepatic fibrosis tissue.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; hydroxyproline assay was employed to detect liver collagen synthesis; immunohistochemistry and TUNEL were applied to detect HSC activation and apoptosis; Western Blot and Real-time Q-PCR were used to measure the dynamic expression of MMP-13 and TIMP-1 during hepatic fibrosis.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 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 high dose groups(16.56%±2.11%, 16.17%±1.25%, 14.19%±1.41%, 12.87%±1.11%) were significantly higher than that of the sham operation group(3.4%±0.24%), 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 high dose groups(34.35%±3.27%, 31.02%±2.75%, 28.64%±2.44%, 24.18%±2.49%) were significantly higher than that of the sham operation group(3.46%±0.17%), P<0.01; collagen area density of low dose, middle dose and high dose groups decreased significantly compared with that of model group, P<0.05, P<0.01, P<0.01 respectively; collagen area density of high 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 The effects of Carvedilol on the proliferation and apoptosis of HSC in liver tissue of hepatic fibrosis rats4.1 The inhibition effects of Carvedilol on HSC activation and proliferationImmunohistochemistry stain ofα-SMA showed that it only expressed weakly positive in vessel wall smooth muscle cells of normal rats hepatic tissues; with the development of hepatic fibrosis,α-SMA positive cells obviously increased in rats liver, distributing mainly in header area, fiber compartment, hepatic sinusoid surroundings, and proliferated bile duct surrounding cells.After 2 wk of BDL,α-SMA positive area density in the model group, low dose, middle dose and high dose groups (23.77%±2.43%, 21.96%±2.03%, 19.95%±2.02%, 18.77%±2.15%)were significantly higher than that of the sham operation group(5.69%±1.15%), P<0.01;α-SMA positive area density of middle dose and high dose groups have significantly decreased compared with that of model group, P<0.01; but no significant differences were found between low dose group and model group, P>0.05;α-SMA positive area density of high dose groups have also significantly decreased compared with that of low dose group, P<0.05, 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.After 4 wk of BDL,α-SMA positive area density in the model group, low dose, middle dose and high dose groups (36.88%±2.83%, 33.86%±2.23%, 30.77%±2.17%, 28.72%±1.74%) were significantly higher than that of the sham operation group(5.43%±1.03%), P<0.01;α-SMA positive area density of low dose, middle dose and high dose groups decreased significantly compared with that of model group, P<0.05, P<0.01, P<0.01 respectively;α-SMA positive area density of middle dose and high dose groups decreased significantly compared with that of low dose group, P<0.05, P<0.01 respectively; no significant differences were found between middle dose and high dose groups, P>0.05.4.2 The enhancement effects of Carvedilol on HSC apoptosisApoptotic HSC was determined by dual staining, which containing both the terminal deoxynucleotidy transferrase UTP-nick end labeling (TUNEL) andα-SMA immunohistochemistry stain after 2 wk of BDL. The result showed that no apoptotic HSC were found in control group; HSC apoptosis index in middle and high dose groups (6.45%±1.12%, 7.89%±1.06%) were significantly higher than that in model group(4.95%±0.95%), P<0.05, P<0.01 respectively; no significant differences were found between low dose group (5.19%±1.09%) and model group, P>0.05. HSC apoptosis index in high dose groups significantly increased compared with that in low and middle dose groups, P<0.01, P<0.05 respectively; and no significant difference were found between low dose and middle dose groups, P>0.05.After 4 wk of BDL, the result showed that no apoptotic HSC were found in control group; HSC apoptosis index in middle and high dose groups(4.63%±1.06%,6.17%±1.27%) were significantly higher than that in model group(2.35%±0.94%), P<0.05, P<0.01 respectively; no significant differences were found between low dose group(3.12%±1.04%) and model group, P>0.05. HSC apoptosis index in middle and high dose groups significantly increased compared with that in low dose groups, P<0.05, P<0.01 respectively; HSC apoptosis index in high dose groups significantly increased compared with that in middle dose groups, P<0.05.5 The effects of Carvedilol on Collagen expression of liver in hepatic fibrosis rats5.1 Carvedilol can degrade the content of hydroxyproline in hepatic tissueAfter 2 wk of BDL, hepatic tissue hydroxyproline content in the model group, low dose, middle dose and high dose groups(0.363±0.027, 0.338±0.028, 0.315±0.034, 0.279±0.031)μg·mg-1 were significantly higher than that of the sham operation group(0.163±0.007), P<0.01; hydroxyproline content of middle dose and high dose groups decreased significantly compared with that of model group, P<0.05, P<0.01, respectively; and no significant difference were also found between low dose group and model group, P>0.05. hydroxyproline content of high dose group decreased significantly compared with that of low dose and middle dose groups, P<0.05, P<0.01, respectively; and no significant difference were also found between low dose and middle dose groups, P>0.05.After 4 wk of BDL, hepatic tissue hydroxyproline content in the model group, low dose, middle dose and high dose groups(0.778±0.052, 0.719±0.024, 0.654±0.048, 0.605±0.034)μg·mg-1 were significantly higher than that of the sham operation group(0.164±0.006), P<0.01; hydroxyproline content of low dose, middle dose and high dose groups decreased significantly compared with that of model group, P<0.05, P<0.01, P<0.01 respectively; hydroxyproline content of middle dose and high dose groups decreased significantly compared with that of low dose group, too. P<0.05, P<0.01 respectively; hydroxyproline content of high dose groups decreased significantly compared with that of middle dose group, too, P<0.05.5.2 Carvedilol inhibit Collagen I mRNA expression in hepatic tissuesAfter 2 wk of BDL, Collagen I mRNA relative expression content in the model group, low dose, middle dose and high dose groups(6.07±0.51, 5.64±0.43, 5.1±0.49, 4.44±0.62) were significantly higher than that of the sham operation group(1.00±0.14), P<0.01; Collagen I mRNA relative expression of middle dose and high dose groups decreased significantly compared with that of model group, P<0.01; and no significant difference were found between low dose group and model group, P>0.05. Collagen I mRNA relative expression of high dose group decreased significantly compared with that of low dose and middle dose groups, P<0.01, P<0.05 respectively; but no significant difference were also found between low dose and middle dose groups, P>0.05.After 4 wk of BDL, Collagen I mRNA relative expression content in the model group, low dose, middle dose and high dose groups(11.59±1.1, 10.26±0.99, 9.73±1.3, 8.25±0.91) were significantly higher than that of the sham operation group(1.00±0.18), P<0.01; Collagen I mRNA relative expression of low,middle and high dose groups decreased significantly compared with that of model group, P<0.05, P<0.01, P<0.01; Collagen I mRNA relative expression of high dose group decreased significantly compared with that of low dose and middle dose groups, P<0.01, P<0.05 respectively; but no significant difference were also found between low dose and middle dose groups, P>0.05.6 The effects of Carvedilol on TIMP-1 and MMP-13 protein expression of liver in hepatic fibrosis rats6.1 Carvedilol can promote MMP-13 protein expression of liver in hepatic fibrosis rats After 2 wk of BDL, Western Blot showed that the relative expressions of MMP-13 protein in the model group, low dose, middle dose and high dose groups (1.1±0.11, 1.27±0.11, 1.30±0.13, 1.43±0.1) were significantly higher than that of the sham operation group (0.72±0.09), P<0.01; the relative expression of MMP-13 protein in the low dose, middle dose and high dose groups increased significantly compared with that of model group, P<0.05, P<0.05, P<0.01 respectively; MMP-13 protein level of high dose group increased significantly compared with that of low dose groups, P<0.05; but no significant differences compared with that of middle dose group, P>0.05; no significant differences were found between low dose and middle dose groups, P>0.05.After 4 wk of BDL, Western Blot showed that the relative expressions of MMP-13 protein in the model group, low dose, middle dose and high dose groups (1.73±0.14, 1.85±0.15, 2.05±0.20, 2.28±0.13) were significantly higher than that of the sham operation group (0.75±0.06), P<0.01; MMP-13 protein level of middle dose and high dose groups increased significantly compared with that of model group, P<0.01; no significant differences were found between low dose and model groups, P>0.05. MMP-13 protein level of middle dose and high dose groups increased significantly compared with that of low dose group, too, P<0.05, P<0.01 respectively; high dose group increased significantly compared with that of middle dose groups, P<0.05. 6.2 Carvedilol inhibit TIMP-1 protein expression of liver in hepatic fibrosis ratsAfter 2 wk of BDL, Western Blot showed that the relative expression of TIMP-1 protein in the model group, low dose, middle dose and high dose groups (1.51±0.18, 1.4±0.15, 1.22±0.19, 1.12±0.16) were significantly higher than that of the sham operation group (0.5±0.08), P<0.01; TIMP-1 protein level of middle dose and high dose groups decreased significantly compared with that of model group, P<0.01; no significant differences were found between low dose and model groups, P>0.05; TIMP-1 protein level of high dose groups decreased significantly compared with that of low dose group, P<0.01, but no significant differences were found compared with that in middle dose group, P>0.05; no significant differences were found between low dose and model groups, P>0.05.After 4 wk of BDL, Western Blot showed that the relative expression of TIMP-1 protein in the model group, low dose, middle dose and high dose groups(2.53±0.21, 2.33±0.16, 2.15±0.19, 1.94±0.13) were significantly higher than that of the sham operation group(0.55±0.05), P<0.01; TIMP-1 protein level of middle dose and high dose groups decreased significantly compared with that of model group, P<0.01; no significant differences were found between low dose and model groups, P>0.05; TIMP-1 protein level of high dose groups decreased significantly compared with that of low dose and middle dose groups, too. P<0.05, P<0.01 respectively; TIMP-1 protein level of high dose group decreased significantly compared with that of middle dose groups, P<0.05, though no significant differences were found between low dose and model groups, P>0.05.6.3 Carvedilol regain the dynamic balance between MMP-13 and TIMP-1 on protein level of liver in hepatic fibrosis ratsAfter 2 wk of BDL, MMP-13/TIMP-1 ratio in the model group, low dose, middle dose and high dose groups(0.74±0.08, 0.91±0.10, 1.07±0.18, 1.29±0.11) were significantly lower than that of the sham operation group(1.42±0.06), P<0.01, P<0.01, P<0.01, P<0.05 respectively; MMP-13/TIMP-1 ratio of middle dose and high dose groups have significantly increased compared with that of model group, P<0.01; no significant differences were found between low dose and model groups, P>0.05. MMP-13/TIMP-1 ratio of high dose group increased significantly compared with that of low dose and middle dose groups, P<0.01; no significant differences were found between low dose and middle dose groups, P>0.05.After 4 wk of BDL, MMP-13/TIMP-1 ratio in the model group, low dose, middle dose and high dose groups(0.69±0.09, 0.79±0.10, 0.95±0.05, 1.18±0.08) were significantly lower than that of the sham operation group(1.37±0.10), P<0.01; MMP-13/TIMP-1 ratio of middle dose and high dose groups have significantly increased compared with that of model group, P<0.01; no significant differences were found between low dose and model groups, P>0.05. MMP-13/TIMP-1 ratio of middle dose and high dose groups increased significantly compared with that of low dose group, P<0.05, P<0.01 respectively; MMP-13/TIMP-1 ratio of high dose group increased significantly compared with that of middle dose groups, P<0.01.7 The effects of Carvedilol on TIMP-1 and MMP-13 mRNA expression of liver in hepatic fibrosis rats7.1 Carvedilol can promote MMP-13 mRNA expression of liver in hepatic fibrosis ratsAfter 2 wk of BDL, MMP-13 mRNA relative expression content in the model group, low dose, middle dose and high dose groups(4.89±0.54, 5.19±0.46, 5.6±0.64, 6.05±0.68) were significantly higher than that of the sham operation group(1.38±0.22), P<0.01; MMP-13 mRNA content of middle dose and high dose groups increased significantly compared with that of model group, P<0.05, P<0.01 respectively; no significant differences were found between low dose and model groups, P>0.05; MMP-13 mRNA content of high dose group increased significantly compared with that of low dose group, P<0.05, 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.After 4 wk of BDL, MMP-13 mRNA relative expression content in the model group, low dose, middle dose and high dose groups(6.25±0.63, 7.27±0.89, 7.91±0.98, 8.92±0.83) were significantly higher than that of the sham operation group(1.4±0.15), P<0.01; MMP-13 mRNA content of low, middle and high dose groups decreased significantly compared with that of model group, P<0.05, P<0.01, P<0.01; MMP-13 mRNA content of high dose groups increased significantly compared with that of low dose and middle dose groups, P<0.01, P<0.05 respectively; no significant differences were found between low dose and middle dose groups, P>0.05.7.2 Carvedilol can inhibit TIMP-1 mRNA expression of liver in hepatic fibrosis ratsAfter 2 wk of BDL, TIMP-1 mRNA relative expression content in the model group, low dose, middle dose and high dose groups(7.56±0.98, 6.72±0.97, 5.85±0.79, 5.13±0.97) were significantly higher than that of the sham operation group(1.00±0.17), P<0.01; TIMP-1 mRNA content of middle dose and high dose groups decreased significantly compared with that of model group, P<0.01; no significant differences were found between low dose and model groups, P>0.05; TIMP-1 mRNA content of high dose group decreased significantly compared with that of low dose group, P<0.01, while no significant differences compared with that of middle dose group, P>0.05; no significant differences were found between low dose and middle dose groups, P>0.05.After 4 wk of BDL, TIMP-1 mRNA relative expression content in the model group, low dose, middle dose and high dose groups(11.72±1.03, 10.34±1.08, 9.39±1.13, 8.11±1.06) were significantly higher than that of the sham operation group(1.00±0.21), P<0.01; TIMP-1 mRNA content of low, middle and high dose groups decreased significantly compared with that of model group, P<0.05, P<0.01, P<0.01 respectively; TIMP-1 mRNA content of high dose group decreased significantly compared with that of low dose and middle dose groups, P<0.01, P<0.05 respectively; but no significant differences were found between low dose and middle dose groups, P>0.05.7.3 Carvedilol regain the dynamic banlance between MMP-13 and TIMP-1 on mRNA level of liver in hepatic fibrosis ratsAfter 2 wk of BDL, MMP-13/TIMP-1 ratio in the model group, low dose and middle dose groups(0.66±0.16,0.78±0.09,0.96±0.09, 1.19±0.12) decreased significantly compared with that of the sham operation group(1.37±0.17), P<0.01, P<0.01, P<0.01, P<0.05 respectively; middle dose and high dose groups increased significantly compared with that of model group, P<0.01; no significant differences were found between low dose and model groups, P>0.05; MMP-13/TIMP-1 ratio of middle dose and high dose groups increased significantly compared with that of low dose group, P<0.05, P<0.01 respectively; MMP-13/TIMP-1 ratio of high dose group increased significantly compared with that of middle dose group, P<0.05.After 4 wk of BDL, MMP-13/TIMP-1 ratio in the model group, low dose, middle dose and high dose groups(0.54±0.09, 0.71±0.12, 0.83±0.13, 1.07±0.1) decreased significantly compared with that of the sham operation group(1.39±0.19), P<0.01; middle dose and high dose groups increased significantly compared with that of model group, P<0.01; no significant differences were found between low dose and model groups, P>0.05; MMP-13/TIMP-1 ratio of high dose groups increased significantly compared with that of low dose and middle dose group, P<0.01; no significant differences were found between low dose and middle dose groups, P>0.05.Conclusions:1 Carvedilol can inhibit HSC activation in hepatic fibrosis rats, and promote HSC apoptosis.2 Carvedilol can inhibit TIMP-1 expression and promote MMP-13 expression in hepatic fibrosis tissues of rat, thus keep dynamic balance of TIMP-1 and MMP-13, and then degrade liver collagen.3 Carvedilol can inhibit the development of hepatic fibrosis in experimental rats in a dose-dependant manner.
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