Effect Of Huoxue Decoction On The Skin Lesions Of Scleroderma Model Mice

Scleroderma is a self-immune connective tissue disease which can invade both skin or internal organs; the formation of fibrosis is the core of its pathological changes, transforming growth factor-β(TGF-β) is commonly known as one of the strongest fibrosis-inducing factor, it could activate the abnormal production of reactive oxygen spices(ROS) in human body and triggering oxidation process, and this process has been proven to be closely related to the abnormal proliferation of collagen. Platelet derived growth factor is a profibrotic factor which overly expressed in the serum of scleroderma patients, the combination of PDGF and its receptor can trigger the differentiation of fibroblast and myofibroblast and further lead to collagen deposition and fibrosis; at the same time, ROS can suppress the phosphatases activities to further promote combination of PDGF and its receptor to reinforce the process of fibrosis. PDGF also came across with TGF-β-mediated fibrosis process; and they could trigger the over-expression of Connective tissue growth factor (CTGF)and activate the fibrotic process.Fibrosclerosis is a relatively long process in scleroderma, as one of the key pathological feature of scleroderma, the treatment of fibrosis is crucial; however modern medicine has yet to develop a specifica for fibrosis. HuoXueChuBi decoction has satisfactory clinical effect, in order to reveal its mechanism in treating fibrosclerosis, we will mimic scleroderma by using animal model to observer HuoXueChuBi decoction’s effect in suppressing fibrotic process and the effect in regulating the profibrotic factors. In hope that we can provide laboratory evidence for HuoXueChuBi decoction in scleroderma treatment. This dissertation is consists of three parts:literature review, clinical case reports and laboratory experiment. The literature review parts contains 3 reviews, first one is the summary of the pathogenesis of scleroderma and its clinical diagnosis standards; second one is the summary of scleroderma therapeutic approaches and medicine used by modern medicine physicians; and the last review is about scleroderma pathogenesis in traditional Chinese medicine and the summaries of how TCM physicians treat scleroderma.Objective:Study the effect of HuoXueChuBi Decoction on scleroderma model mice and its possible mechanism.Method:Experiment 1:16 BALB/c mice were randomly assigned into 3 groups -① blank group ②model group low and ②model group high. ① received PBS 0.1ml/d intraermal(ID) injection, ②③ received 400μg/0.1ml and 800μg/0.1ml bleomycin hydrocholoride ID injection; after 4 weeks of ID injection, the mice were executed and skin samples were used in HE staining and Masson staining to observe and compare the degree of skin thickening and collagen proliferation in order to determine the potency of bleomycin solution to be used in experiment 2 for modeling. Experiment 2:30 BALB/c mice were randomly assigned into 5 groups-①blank group,②model group, ③traditional Chinese medicine high dosage group, ④traditional Chinese medicine middle dosage group and⑤ traditional Chinese medicine low dosage group.①was given PBS 0.1/d,②③④⑤ were given bleomycin hydrocholoride intradermal injection (400μg/d)for 4 weeks to esbalish scleroderma mouse model. From week 5 to 8, ①②were gavaged with equal amount of distilled water daily; ③④⑤ were gavaged with HuoXueChuBi Decoction in different concentration (44.8,22.4,11.2/g·kg) daily. At the end of 8th week, all mice were executed and skin sample were taken for HE staining, Masson staining and ROS quantity test. Experiment3: 30 BALB/c mice were randomly assigned into 5 groups -①blank group, ②model group,③ traditional Chinese medicine high dosage group, ④traditional Chinese medicine middle dosage group and⑤ traditional Chinese medicine low dosage group. Other conditions are the same with experiment 2.The pathological changes in the skin tissue samples taken from each group will be observed under microscope; the thickness of skin from each group will be measured and the expression of platelet-derived growth factor A(PDGF-A), Transforming growth factor-β(TGF-β), type-Ⅰ collagen (COL-Ⅰ) and type-Ⅲ collagen (COL-Ⅲ) were tested through immunohistochemical staining will be observed and compared.Results:(1) The result from experiment 1 shows that the thickness of skin and collagen indicator from②and③ are significantly higher compare to ①(P<0.01);the skin thickness and collagen indicator of ②are significantly higher than those of ③(P<0.01); the dermis tissue of model group was thicker than blank group, with the presence of thicker and greater number of collagen fibers; as well as infiltration of inflammatory cells; the pathological changes suits the description to that of scleroderma patietns. (2) The result from experiment 2 shows that the skin thickness, collagen indicator and ROS quantity are significantly higher in ②compare to ①(P<0.01); skin thickness, collagen indicator are significantly lower in ③④⑤compare to ②(P<0.01);and the skin thickess of ③is significantly lower than ④⑤ (P<0.01). ROS quantity test show that ③display significantly lower ROS quantity compare to ④⑤(P<0.01); ROS quantity in ④⑤ has no statistical significance(P>0.05). Correlation analysis shows that the quantity of ROS has positive correlation with thickness of the skin lesion (r=0.891, P<0.01);ROS quantity has positive correlation with collagen indicator (r= 0.866, P<0.01)and the thickness of dermis has positive correlation with collagen indiactor (r= 0.942, P<0.01).Experiment 3:The result of immunohistochemical staining shows the expression of PDGF-A, TGF-β,COL-Ⅰ and COL-Ⅲ in ② show significant increase than ②(P<0.01);Compare to ②,③④⑤shows significant less in TGF-β/PDGFA and COL-Ⅰ/Ⅲ (P<0.01). The expression of PDGF-A and TGF-βshows a positive correlation with the amount of COL-Ⅰ and COL-Ⅲ presented in each group. This result suggest that HuoXueChuBi decoction has the effect on reducing the expression of COL-Ⅰ/Ⅲ and FB related growth factors; thus HuoXueChuBi decoction can be used in clinical treatment of scleroderma.Conclusion:(1) The suitable modeling potency for bleomycin solution for ID injection for scleroderma modeling constructing is 400μg/d for 4 weeks; the pathological changes in the skin of model mice display same feature with that of a scleroderma patients. (2) The skin lesion in the modeling mice shows evidence of large quantity of ROS, and it display positive correlation with skin thickness and collagen indicator, it show evidence that oxidation reaction has strong connection with fibrosis; and as we intervened with HuoXueChuBi decoction the quantity of ROS, thickness of skin and collagen indicator became lower as the concentration of the decoction increase, suggested that HuoXueChuBi decoction’s effect in lowering fibrosis might be related to its efficacy in lowering the presence of ROS. (3) The profibrotic factors TGF-β/PDGFA and its product COL-I/III lower as the the concentration of the decoction increase, suggest that it could lower the fibrosis formation through suppressing the amount of TGF-β and PDGF, and it might have direct or (and) indirect effect in suppressing the formation of COL-I/III. (4) The quantity of ROS and the amount of TGF-β and PDGFA shows positive correlation to each other, suggest that they may have close interaction to each other in the formation of fibrosis. (5) HuoXueChuBi decoction is effective in lowering fibrosis and collagen formation in this experiment, and its effect increases as the concentration increases.