| Source of the research work: this research work was funded by Chinese Medicine Bureau of Guangdong province.Objectives and purposes: to investigate the Molecular Mechanism of anti-liver fibrosis treatment by eliminating heat and wetness evil(EHAWE) therapy, thus providing a theoretical foundation for applying EHAWE therapy to the clinic, and establishing a solid theoretical basis for developing the traditional Chinese medicine on anti-liver fibrosis.Methods: The fibrosis rat model was induced by multiple factors and was given QingXiangSan(QXS) which has the function to eliminate heat and wetness evil and was consist of Chinese Drug xiangru,didancao,chaihu,huangqin,yinchen,huzhang, danpi,chishao,shanzha,baizhu,fulin,et al and,in the same time the effects of QXS on fibrosis rat model was studied.Fifty Wistar rats were randomly divided into normal group(A), model group(B), Colchicine group(C),High dose QingXiangSan(QXS) group(D) and middle QXS group(E).All rats except rats in group A were subcutaneous injected with a dose of 0.5ml/100g of body weight 40% carbon tetrachloride in peanut oil once on the first day, then with a dose of 0.3ml/100g once every three days for six weeks. In those days they were fed with fodder mixed 79.5% corn powder with 20% lard and 0.5%Cholesterol.30% alcohol was used as drinking water. Rats in group A were subcutaneous injected with equal peanut oil and fed with normal fodder and water. Rats in group C, D and E were respectively given Colchicine(0.011mg/100g),QXS (2g/100g) and QXS (1g/100g) every day for six weeks via gastrogarage. Rats in group A and B were served with distilled water at the same time. At the end of the sixth week, blood and tissue specimens were taken from all the rats. Some serum were used for analysis of aspartate transaminase (AST) and alanine transaminase (ALT) activities according to the direction of the kit and others for analysis of total protein (TP), Albumin (ALB) and globulin (GLB) with an automatic biochemical machine and interieukin 6(IL-6) by radioimmunoassay(RIA). Some liver specimens were made into liver homogenate for analysis of hydroxyproline (Hyp) ,superoxide sidmutase (SOD) and malondialdehyde (MDA) according to the direction of the kit. Some were fixed in a 2.5% solution of glutaraldehyde for electron microscope observation. Some were mixed in a 10% solution of formaldehyde in O.lmol/L phosphatobuffered saline, and embedded in paraffin .Five-micron thick sections were prepared to evaluate the fibrosis degree under a light microscope, observe the heptocyte apoptosis in situ hybridization(ISH) and analyze the expression of transforming growth factor-beta 1(TGF- β1), TGF-beta type I receptor (T P R I ), Smad4and connective tissue growth factor(CTGF) in immunohistochemistry (IH) methods . As to statistical analysis, data were analyzed with SPSS 11.0 software. Quantitative data were presented as mean±SD and compared using one way ANOVA procedure. Comparisons of frequency data were conducted using Ridit procedure.Content and conclusions:1. To observe the influence of EHAWE therapy on symptoms, physical signs and indices (liver wet weight/100g body weight) of liver and spleen (spleen wet weight/100g body weight) in liver fibrosis rat modelRats in model group displayed lassitude, thinness, anorexia and their stool was loose, the urine was yellow and less. The surface of livers in model group was not smooth, the colour was lightly red, the edge was blunt and the quality was hard. Even there were some little tubercles on the liver surface. The spleens were obviously swelling. Compared with rats in model group, rats in the treatment groups were in better condition. High and middle dosage QXS were better than colchicine in improving the symptoms including water, food intake and the quality of liver and spleen. High and middle dosage QXS both significantly decreased the indices of liver and spleen (P<0.01 in both groups) .There was no difference between High, middle dosage QXS and colchicines (P>0.05) .2. To observe the influence of EHAWE therapy on liver morphology in liver fibrosis rat modelUnder a light microscope in the model group damaged lobules of liver degeneration, necrosis of liver cells, lots of inflammatory cells, collage fibers and even pseudolobules were observed, while in the treatment groups there were less damaged lobules of liver degeneration, necrosis of liver cells, inflammatory cells and collage fibers. But there were still incomplete pseudolobules in very few rats. High and middle dosage QXS significantly decreased the grades of liver fibrosis (P<0.05) , and no difference was found between High,middle dosage QXS and colchicines. (P>0.05) .Under an electron microscope in the model group the typical structure of damaged hepatocyte, hepatic sinusoidal capillarization, activated hepatic stellate cells (HSCs) in Disse internal and lots of collage fibers were discovered. In the treatment groups the degree of damaged liver cells were variously decreased, there were less hepatic sinusoidal capillarization, coUagenous fibres and activated hepatic stellate cells in Disse internal. In high and middle dosage QXS groups some HSCs contain plenty of fat drops and lots of apoptosis HSCs were detected and obvious proliferation of HSCs did notappear.3. The influence of EHAWE therapy on serum hepatic function and IL-6 in liver fibrosis rat modelCompared with the normal group, levels of ALT, AST, AST/ALT and IL-6 in the model group went up strikingly (P<0.01); levels of TP, ALB, GLB and A/G fell, but there was no difference between them. Compared with the model group, levels of ALT, AST and IL-6 in high and middle dosage QXS groups went down obviously (all P <0.01, AST/ALT also significantly was decreased (P<0.01,P <0.05); The effects on decreasing the levels of ALT of high and middle dosage QXS were better than colchicines (all P<0.05); The effects on decreasing the levels of AST of high dosage QXS were also better than colchicines (P<0.05); Ratio of AST/ALT and levels of IL-6 remained the same among the treatment groups (P>0.05) .The treatment groups had no significant effects on the levels of TP, ALB, GLB (allP>0.05) .4. The influence of EHAWE therapy on liver tissue Hyp, SOD and MDA in liver fibrosis rat modelCompared with normal group contents of liver tissue Hyp and MDA increased significantly, SOD decreased considerably (P<0.01); Compared with model group contents of Hyp and MDA in high and middle dosage QXS groups dropped obviously(P<0.01) ,SOD went up strikingly (P<0.01,P<0.05) ;The effects on decreasing MDA and increasing SOD of high dosage QXS were better than colchicines (P<0.05,P<0.01), the effects on increasing SOD of middle dosage QXS was also better than colchicines(P<0.01) .5. The influence of EHAWE therapy on hepatocyte apoptosis in liver fibrosis rat modelThere was more hepatocyte apoptosis in the model group .The apoptotic liver cells were located in liver parenchyma especially in the area of massive hepatic necrosis.Expression of apoptotic liver cells exhibited as brown particles in nucleus. Compared with model group the apoptosis index in high and middle dosage QXS groups went down obviously (P<0.01 > P<0.05); The effects of high dosage QXS outperformed colchicines (P<0.05).6. The effects of EHAWE therapy on the expression of TGF- P 1 and its signal pathway in liver tissue in fibrosis rat modelOnly a mild Positive staining of TGF-|3l was found at central vein and Disse's areas but none at hepatocytes on sections of normal controls, whereas on sections of the model group, the positive staining was seen at interstitial cells, inflammatory cells, impaired hepatocytes as well as normal hepatocytes. Fibrous c septa were only slightly stained. In the treatment groups the positive staining cells decreased and the staining was evidently light.Normal liver tissue hardly expressed Tp*R I or only a mild positive staining was found at portal vein, central vein and portal tract. Whereas in the model group T|5R I mostly was located in the portal tract, around sinusoid and fibrotic septa .Expression of T|5R I exhibited as brown particles predominantly in cytoplasm or cell membrane. A few positive expressions were found in nucleus. Contrast to the model group.the positive staining cells and the staining degree in the treatment groups was not obviously changed.Normal liver tissue hardly expressed Smad4, only a mild positive staining was found at matrix in portal tract and at cytoplasm in interstitial cells, whereas in the model group Smad4 dominated. It was detected in interstitial cells at fibrous septa, portal tract, portal vein, central vein and around sinusoid, especially at fibrous septa. In the treatment groups the number of positive staining cells decreased, mainly expressed at portal tract, portal vein and central vein.Statistical data analysis demonstrated that the expression level of TGF-J31, T(3R Iand Sma4 were increased significantly in the model group(p<0.01). Compared with the model group, the Immunostaining score of TGF-J31 and Smad4 in high and middle dosage QXS groups was remarkably decreased (P<0.01 in both groups), Compared with colchicine group, all these effects in high and middle dosage QXS groups saw no statistical difference(P>0.05);TPR I was not noticeably decreased((P>0.05 in all treatment groups).7. The effects of EHAWE therapy on the expression of CTGF in liver tissue in fibrosis rat modelCTGF in normal liver tissue was barely detected, whereas in the model group CTGF expressed strongly. Expression of CTGF exhibited as brown particles predominantly in cytoplasm. It was mainly seen at portal tract and fibrous septa. It was mostly located in interstitial cells at fibrous septa but not in hepatocytes. In the treatment groups the number of positive staining cells decreased and the staining was noticeably light.Statistical data analysis showed that the expression level of CTGF in model group was significantly enhanced (P<0.01) . Compared with the model group CTGF in high and middle dosage QXS groups was markedly decreased (P<0.05 in both groups). Though there was no statistical difference between the colchicine group and the high dosage QXS group(P>0.05), the effect of decreasing the expression of CTGF of middle QXS was significantly inferior to colchicine (P<0.05) .Results: EHAWE therapy inhibited the process of liver fibrosis in rats model, the mechanisms were as follows.1.EHAWE therapy can improve the life quality through improving the spirits, increasing the body weight and food, water intake.2.EHAWE therapy can significantly decrease the levels of serum ALT, AST and IL-6, which indicates EHAWE therapy can decrease the inflammation, protect theliver cells and inhibit the start of the liver fibrosis.3.EHAWE therapy can increase the activities of SOD and decrease the contents of MDA in liver tissue; as a result EHAWE therapy has the function to eliminate the free radicals and anti-oxidation.4.Morphological observation shows EHAWE therapy can improve the damaged structure of heptocyte, decrease the fibrosis tissue, it can also inhibit the HSCs to activate and induce the HSCs to apoptosis.5.EHAWE therapy can suppress the hepatic cells apoptosis and increase the hepatic cells regeneration.6.EHAWE therapy can reduce the expression of TGF- P 1, and its signal transduction molecule Smad4 in the liver tissue, so the signal transduction of TGF- P 1 is inhibited by the EHAWE therapy.7.EHAWE therapy can lower the expression of CTGF in the liver tissue, which means CTGF is a good target for EHAWE therapy to anti-fibrosis.In conclusion, EHAWE therapy has the complicated pharmacological action on preventing the liver fibrosis. It has multi-way and multi-target anti-fibrosis mechanisms. One of the molecular mechanisms is to inhibit the expression of CTGF, TGF- P 1 and its signal transduction which is concerned to inhibit the inflammation and apoptosis, decrease the waterfall action of the cytokine and inhibit the start of the fibrosis. |
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