The Clinical And Experimental Research Of Ruangan Granule On Liver Fibrosis Induced By Chronic Hepatitis B

AimThe Rangan Granule (RGG) was composed of huang qi (radix astragali), chuan shan jia (squama manitis), tian qi (radix notoginseng), dan shen (radix salviae miltiorrhizae), tao ren (semen persicae), bie ja (carapx trionycis), ye xia zhu (phyllanthus urinaria L), fu lin (poria), and zhi ke (fructus aurantii), which based on the theory of TCM and optimized according to the perennial clinical experiences of treating chronic hepatitis and liver fibrosis. It has the effects of anti-inflammatory, anti-necrosis, regulate liver function and prevent liver fibrosis. This experimental research is to study the influence of RGG on the TGFβ1 mRNA expression in the liver of rats with liver fibrosis, the serum levels of TGFβ1, collagen type Ⅰ and Ⅲ and the liver content of oxyproline hydr-oxyproline (Hyp) and to explore the mechanism of protecting against liver fibrosis at the molecular level by observing the effects of RGG on rats with immune liver fibrosis and experimental liver fibrosis induced by Carbon Tetrachloride (CC14). It aimed to look for the target on inhibiting liver fibrosis of RGG and lay a foundation of treating liver fibrosis with RGG in clinic. Correspondingly this clinical research was to observe the influence of RGG on the clinical symptoms and signs, the indexes of liver function and serum indexes of liver fibrosis in patients with liver fibrosis and early stage hepatocirrhosis in order to evaluate the therapeutic effect of RGG on liver fibrosis and to provide the evidence of its wider Clinical applications.Method1. Experimental research1.1 To study the effect and mechanism of RGG on rats with experimental liver fibrosis induced by CC14100 SD rats were randomly divided into five groups: the control group, the group treated with CC14, the group treated with CC14 and given a low dosage of RGG, the group treated with CC14 and given a high dosage of RGG and the group treated with CC14 and given colchicines. Each group contained 20 rats. On the first day, all the groups, except for the control group, were given a subcutaneous injection of 40% CC14 mixed in 5ml/kg of castor oil. Thereafter, 3-4 days per week, they were injected with a 40% CC14 mixed in 3ml/kg of castor oil. All the groups were given the formula once a day from the onset of the experiment. The rats were given distilled water in accordance with the formula of 10ml*kg-l*d-l; in addition, for the low dose group, RGG (6.46g/kg); for the high dose group, RGG (12.92g/kg) and for the colchicines group, 0.001% of colchicines; every group for a total of 90 days. At day 45 and 90, 10 animals per group were killed. Excise the eyes to take blood, and remove partial right liver lobe which would be fixed with formaldehyde, packed with paraffin wax or quick-frozen with liquid nitrogen. The liver function was detected by auto biochemist analytic machine; the serum levels of TGF P l, Collagen type I and III and the liver content of oxyproline hydroxyproline( Hyp) were detected with the Rennard's methods; and the TGFI3 1 mRNA expression of different phases were detected with the Northern- blot methods. We removed the left liver lobe to observe the pathological structure with HE coloring method and to observe the ultramicrostructure by the H-600 transmission electron microscope with lead uranium coloring method.1.2 To study the effect and mechanism of RGG on rats with immune hepatic fibrosis Liquid was mixed with 8g/L BSA in normal sodium and same volume of FO-ICadjuvant. 50 rats were given five injections in several locations via a 0.5 ml hypodermic injection. First and second injection had a 2 week interval and after that injections were given every other week. Later IV BSA was administered twice a week, the first containing 5g/L, increasing the concentration by 0.25 g/L each time, last injection being 8.0g/L, for a total 12 times. After 8 injections, we followed the method of division by body weight and randomly divided the rats into 1) a model group, 2) a low dose RGG group, 3) a high doseRGG group and 4) a colchicines group, with 10 rats in each group. For the next three weeks for a total 45 days, the rats were given distilled water in accordance with the formula of 10ml*kg-l*d-l; in addition, for the low dose group, RGG(6.46g/kg); for the high dose group, RGG(12.92g/kg) and for the colchicines group, 0.001% of colchicines. 10 rats were also set up as normative control group and given distilled water with the same dose at the same time. At day 45, all animals were killed. Excise the eyes to take blood, and remove partial right liver lobe which would be fixed with formaldehyde, packed with paraffin wax or quick-frozen with liquid nitrogen. The liver function was detected by auto biochemist analytic machine; the serum levels of TGF P 1, Collagen type I and III and the liver content of oxyproline hydroxyproline( Hyp) were detected with the Rennard's methods; and the TGF 3 1 mRNA expression of different phases were detected with the Northern- blot methods. We removed the left liver lobe to observe the pathological structure with HE coloring method and to observe the ultramicrostructure by the H-600 transmission electron microscope with lead uranium coloring method.2. Clinical Studies120 cases of the chronic hepatitis B and hepatocirrhosis with 2 or over abnormal serum indexes of liver fibrosis among 6 indexes were observed. They were randomly divided into therapeutic group and control group, with 60 cases in each group. The former was treated by RGG, lOg per time, 3 times per day, for a total 3 months. The latter was treated with a-2blntron A, 5000 thousands units per time, intramuscular, once two days, for a total 3 months. The severe cases in two groups were both given the 15—Amino acid, vitamins, energy injections and other support treatments. The patients' symptoms and signs before and after treatment were separately observed. And the levels of HA, PIIIP, CIV, LN were detected with radioimmunoassay; the level of TEVIP1, TGF pi with ELISA method; the diameter of hepatic portal vein, splenic vein and spleen width were detected by color Doppler ; and the serum levels of ALT, A/ G, and ?- G were also detected. Results1. Experimental research1.1 To study the effect and mechanism of RGG on rats with experimental liverfibrosis induced by CC14At day 45 and 90 of the experiment, the serum level of ALT, AKP obviously increased while that of total protein and albumin decreased in the CC14 group, and were significantly different from the normative control group(P<0.01). The RGG and colchicines obviously decreased the serum level of ALT and AKP while increased that of total protein and albumin, with a significant difference in comparison to the control group (P<0.01). The herbal groups' results of decreasing the serum ALT level were both significantly better than that of the colchicines group (P<0.01 or P<0.05). At day 45 of the experiment, The herbal large dosage group' s result of decreasing the serum ALT level was significantly better than that of the colchicines group( P<0.05) but there was no significant difference between those of the herbal large dosage group and the colchicines group( P>0.05). At day 90 of the experiment, The herbal large dosage group and the low one' s results of decreasing the serum ALK level were both significantly better than that of the colchicines group( P<0.05) while herbal large dosage group' s result of increasing the serum total protein level was significantly better than that of the colchicines group (PO.05).At day 45 and 90 of the experiment, the serum levels of TGFpi, I - And III-collagen and the liver content of oxyproline hydroxyproline (Hyp) obviously increased in the CC14 group, and were significantly different from the normative control group(P<0.01). The RGG and colchicines obviously decreased the serum levels of TGFpi, Collagen type I and III and the liver content of oxyproline hydroxyproline( Hyp), with a significant difference in comparison to the control group( P<0.01 —0.05). The herbal large dosage group's result of decreasing the serum levels of TGF was significantly better than those of the low dose group and the colchicines group (P<0.01) but there was no significant difference between the results of herbal large dosage group and the colchicines group (P>0.05). Except for the 90th day, the herbal group's results of decreasing collagen type I and III were both significantly better than those of the colchicines group(P<0.01-0.05), those of the herbal large dosage group were significantly better than the low one (P<0.01). As for the liver content of oxyproline hyp, there were no significant differences among thethree groups (P<0.05).When viewed histologically, the normal liver displays a uniform arrangement of portal tracts separated by parenchyma composed of hepatocellular plates and sinusoids. Terminal hepatic venules are equidistant from portal tracts. Without fibrous tissue deposition. In the model group, hepatocellular necrosis and inflammation were present, the construction of hepatic lobules was destroyed , the injury is accompanied by fibrosis and formation of pseudolobuli. In the other groups, all of those injuries were decreased significantly by light microscope. When viewed by electron microscope, In the control group, The stellate cells were located within the spaces of Disse. Their cytoplasm contained abundant droplets. There was few fibrous tissue deposition surrounding the stellate cells. In the model group, The counts and squares of the droplets were decreased significantly in the stellate cells. There was abundant collagen deposition surrounding cells. In the other groups, that abnormal appearance was improved significantly comparing with the model group.Immunohistochemical studies have shown that the amount of positive stains for collagen type I and III surrounding the sinusoidal spaces, the portal vessels and central veins were increased significantly after being challenged for 45 or 90 days comparing with the control group. But in the other groups, the collagen type I and III depositions were decreased significantly. Result of Northern blot hybridization shows that at day of 45 and 90 of the experiment the rats' liver TGF 1 mRNA expression in the model group, both the herbal groups and colchicines group were all significantly higher than that of control group( P<0.01). But the rats' liver TGFpi mRNA expression of both herbal group and colchicines group were all significantly lower than the model group (p<0.01). The expression of herbal large dose group was lower than herbal low dose group and colchicines group (p<0.01).1.2 To study the effect and mechanism of RGG on rats with immune liver fibrosis The serum level of ALT, AKP obviously increased while that of total protein andalbumin decreased in model group with immune liver fibrosis, and were significantly different from the normative control group(P<0.01). The RGG and colchicines obviously decreased the serum level of ALT and AKP, with a significant difference in comparison to the model group (P<0.01). The herbal large dosage groups' results of decreasing the serum ALT and AKP level were significantly better than those of the colchicines group( P<0.01 or P<0.05). The herbal low dosage group's result of decreasing the serum AKP level was significantly better than that of the colchicines group (P<0.05).The serum levels of TGF01, collagen type I and III and the liver content of oxyproline hydroxyproline (Hyp) obviously increased in the model group, and were significantly different from the normative control group(P<0.01). The RGG and colchicines obviously decreased the serum levels of TGFpi, collagen type I and III and the liver content of oxyproline hydroxyproline(Hyp),with a significant difference in comparison to the model group(P<0.01or P<0.05). The herbal large dosage group's result of decreasing the serum levels of TGF, collagen type I and III and the liver content of oxyproline hydroxyproline( Hyp) was significantly better than those of the low one and the colchicines group( P<0.01 or P<0.05) but there was no significant difference between the results of herbal low dosage group and the colchicines group( P>0.05).When viewed histologically, in the model group, typical of the common forms of spotty necrosis and focal necrosis could be seen, mostly in the limiting plate, but there was few in hepatic lobules. The construction of hepatic lobules was irregular and fibrous septum was infiltrated with inflammatory cells. Like as Lymphocytes, eosinophils and neutrophils. The results of the other groups were the same as the model group. The results by electron microscope have shown that hepatocytes of the normal group were polyhedral with a central spherical nucleus. They were arranged in plates one cell in thickness with blood-filled sinusoids on each side of the plates. The cytoplasmic membrane has specialized domains that provide a canalicular region on the lateral walls and numerous microvilli on the sinusoidal (basolateral) surfaces. In the model group, the granules were ballooning. The endoplasmic reticulumdilated like as large balloon, irregular arrangement, glycoproteins decreased. A number of Kupffer cells and fibroblasts proliferated, and so on. But in the other groups, those appearances were improved significantly. Immunohistoche -mical studies have shown that the amount of positive stains for collagen type I and III surrounding the sinusoidal spaces, the portal vessels and central veins in the model group were increased significantly comparing with the control group. But in the other groups, the collagen type I and III depositions were decreased significantly. Result of Northern blot hybridization shows that the rats' liver TGFpi mRNA expression in the model group, both the herbal groups and colchicines group were all significantly higher than that of control group (P<0.01). But the rats' liver TGFpi mRNA expression of both herbal group and colchicines group were all significantly lower than that model group (p<0.01). The expression of herbal high dose group was lower than herbal low dose group and colchicines group (p<0.01).2. Clinical researchAfter the treatment 2 month, 80% of the therapeutic group patients' hepatitis conditions were obviously alleviated or disappear while the control group also had the certain improvement. Before and after treatment the symptoms or signs as follow: tired (54/6), had a poor appetite (55/17), abdominal distension (36/4), liver area was painful (53/14), swollen spleen (36/15), swollen liver (42/16). The results of therapeutic group were significantly better than the control group (P<0.01 or P<0.05). About 50% of the therapeutic group patients' spider birthmark and liver palm were obviously alleviated or disappear but the control group had no improvement.After treatment the indexes of liver function of two groups were both improved obviously, such as ALT, ? - G and A/ G, with a significant difference than before (P<0.01 or P<0.05). As for the decrease of ALT, the result of therapeutic group was significantly lower than the control group (P<0.01). As for the increase of ? - G and A/ G, the results of therapeutic group were significantly higher than the control group (P<0.05).After treatment, the figures of HA, LN, PIIIP and TGFpi in therapeutic group were significantly lower than before (P<0.01) while those of C-IV and TIMPl were significantly...