| ObjectiveTo explore the anti-PF mechanism of Yiqihuatanquyu capsule and provide experimental evidence for its extensive clinical application and patent application by observing the effect of Yiqihuatanquyu capsule on the related cytokine system in rats with bleomyc in-induced pulmonary fibrosis such as serum type Ⅲ collagen, type IV collagen, laminin, hyaluronic acid, tumor necrosis factor-a and transforming growth factor β1.MethodsSixty healthy wistar rats weighing240±20g were randomly divided into six groups:normal control group, model group, positive control group, Yiqihuatanquyu capsule high-dose group, Yiqihuatanquyu capsule medium-dose group and Yiqihuatanquyu capsule low-dose group, n=10. With10%chloral hydrate skin anesthesia, the neck skin of supine rats with head and limbs fixed on the bench was incised and the tracheae were exposed. The tracheae of normal control group were received saline infusion, and the remaining five groups of rats were used by tracheal instillation of bleomyc in solution to establish the model of pulmonary fibrosis, and then each rat was daily treated with intramuscular injection of80,000units of penicillin to prevent infection in continuing three days. The general status of rats including activity, size, fur, mental state, feeding and feces was observed in the experiment. After48h of modeling, Yiqihuatanquyu capsule5.625g/kg,2.8125g/kg and1.40125g/kg equivalent to20,10and5times clinical daily dose according to body weight ratio were administered respectively to high-dose group, medium-dose group and low-dose group by gavage. The rats of positive control group were given prednisone acetate suspension0.25mg/kg equivalent to7.5times clinical daily dose according to body weight ratio by gavage once a day. The rats of normal control group and model group were given normal saline10ml/kg by gavage once per day. The six groups were all administered for28days, and after that we killed all the rats and compared the changes of lung index of each group, and observed the appearance of their lungs. The middle lobe of the right lung of each rat was fixed in10%formalin and then the paraffin-embedded samples were sliced into the paraffin sections of4m. After HE staining, we observed the morphological changes of lungs with light microscopy. We detect the contents of type Ⅲ collagen, Ⅳ collagen, laminin and hyaluronic acid with the automatic biochemical analyzer, and the expression of cytokines TGF-β1and TNF-a in serum by ABC-ELISA assay.Results(1) Observation of the general condition:there was no death during the experiment; the rats of normal control group were lively and active, and their furs were bright; the symptoms of the model group were cough and asthma which lasted about seven days, squinting, dull and poor mental, and there were five thin rats whose backs were arched, whose bodies were curled up, and whose furs were dull in;three days after modeling; the initial symptoms of the positive control group were similar to that of the model group, and the general states of rats were slightly better after one week of treatment, but their poor appetite did not improve; the maniFes-tations of Yiqihuatanquyu capsule groups were similar to that of model group, and their general condition were significantly improved after treatment, and their mental appetite was better. Observation of body weight:body weight of the model group was not significantly increased; that of the positive control group and Yiqihuatanquyu capsule groups was significantly increased; compared with the positive control group, body weight of Yiqihuatanquyu capsule high-dose group was significantly increased (P<0.05).(2) Comparison of lung coefficient:after modeling and administration, lung coefficient of Yiqihuatanquyu capsule high-dose, medium-dose and low-dose groups and positive group were significantly lower than that of the model group (P<0.05).(3) Comparison of serum PF marker:the content of type Ⅲ collagen, Ⅳ collagen, laminin and hyaluronic acid of the positive control group and Yiqihuatanquyu capsule high-dose, medium-dose and low-dose groups were significantly lower that those of the model group (P<0.05); the content of laminin and hyaluronic acid of Yiqihuatanquyu capsule high-dose and Medium-dose groups were lower than those of the positive control group, but there was no statistics significance (P>0.05).(4) Results of pulmonary tissue pathology:alveolar wall thickening, pulmonary focal fibrosis with diffuse congestion and hemorrhage, alveolar epithelial cell degeneration, a large area of interstitial fibrosis, hemosiderin lymphocyte infiltration and a large number of lymphocyte infiltration in the model group; alveolar wall thickening, a large area of fibrosis, interstitial lymphocyte infiltration, epithelial degeneration and necrosis, and lymphocyte containing hemosiderin in the positive control group; alveolar wall focal thickening, mild interstitial fibrosis, lymphocyte infiltration and containing hemosiderin in Yiqihuatanquyu capsule high-dose group; alveolar wall thickening, mild interstitial fibrosis in some rats in Yiqihuatanquyu capsule high-dose group; thickening, some alveolar wall broken, a large area of fibrosis, and hemosiderin lymphocyte infiltration in Yiqihuatanquyu capsule low-dose group.(5) Comparison of TNF-a and TGF-β1:serum TGF-β1levels of the model group were significantly higher than those of the normal group (P<0.01); serum TGF-β1levels of Yiqihuatanquyu capsule high-dose, medium-dose and low-dose groups were significantly lower than those of the model group (P<0.05); serum TNF-a levels of the model group and Yiqihuatanquyu capsule low-dose group were significantly higher than the normal group (P<0.05); the difference between serum TNF-a levels of Yiqihuatanquyu capsule high-dose and medium-dose groups and those of the normal group was not significant (P>0.05); serum TNF-a levels of Yiqihuatanquyu capsule medium-dose groups were lower than those of the positive control group, but there was no statistics significance(P>0.05).(6) Comparison of HYP pulmonary tissue:the model group and Yiqihuatanquyu capsule positive control group of HYP were significantly higher that those of the normal control group (P<0.05); the normal group Yiqihuatanquyu capsule high-dose,medium-dose and low-dose groups of HYP were significantly lower that those of the model control group (P<0.05); the normal group Yiqihuatanquyu capsule high-dose groups of HYP were significantly lower that those of the positive control group (P<0.05);Conclusions1. Lung tissue pathological changes of the model group is consistent with those reported in the literature, indicating that it is a successful model replication.2. Yiqihuatanquyu capsule can significantly improve the pulmonary fibrosis, and this effect may be achieved through the following means:regulating the balance of the oxidant/antioxidant system in the pulmonary fibrosis, reducing the degree of oxidative damage in the pulmonary tissue which induced by oxygen free radicals; inhibiting the synthesis of collagen in lung tissue, thereby reducing the structural damage of bleomycin to lung tissue, reducing the degree of pulmonary fibrosis. In summary, Yiqihua-Tanquyu capsule can play a preventive and treatable effect, and has a significant protective effect on bleomycin-induced pulmonary fibrosis.3. Yiqihuatanquyu capsule may play an anti-fibrosis role by partly regulating the expression of TNF-a and TGF-β1. |
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