Based On The Regulation Of IKK/IκB/NFκB Pathway, Fuzheng Tongluo Xiaoji Recipe In The Treatment Of Pulmonary Fibrosis And Its Long-term Effect Mechanism

ObjectiveTo screen and optimize the pulmonary fibrosis animal model, intratracheal aspiration and installation of bleomycin (5 mg/kg) in rats were observed in this study. Vital parameters,including histomorphology, pulmonary function and HYP contents were used to evaluate effects of Fuzheng Tongluo Xiaoji formulae. Parameters of inflammatory response and IKK/IκB/NFκB signal pathway were used to illustrate the mechanism.MethodsExperiment 1: 200 rats were randomly divided into control group (CG), intratracheal aspiration of saline group (IA+ NS), intratracheal aspiration of bleomycin group (IA+ BLM),intratracheal installation of saline group (IT+ NS), intratracheal installation of bleomycin group (IT+ BLM). BLM (5 mg/kg) and NS were used to establish models on day 0. The rats were sacrificed on day 7, 14, 28, 56 and 84, pulmonary fibrosis, blood cells examination,histomorphology, HYP contents and lung coefficient were tested on each time point.Experiment 2: 280 rats were randomly divided into control group (Control), model group(BLM), Yangqing Kangxian formula treated group (YKF), Baofei Huaxian formula treated group (BFF), Jinshui Huanxian formula treated group (JSF), prednisone treated group (PD)and pirfenidone treated group (PF). Intratracheal installation of bleomycin (5 mg/kg) was used to establish models on day 0, drug treatments were from day 1 to 42, and interval observations were from day 43 to 70. The rats were sacrificed on day 7, 14, 28, 42 and 70, pulmonary fibrosis, blood cells examination, histomorphology, HYP contents and lung coefficient were tested on each time point.Experiment 3: Based on the method of experiment 2, serum and BALF IL-1β contents were tested by ELISA; TNF-α, IFN-γ, IL-1β and IL-13 expression in lung tissue were tested by inmunohistochemical staining; TNF-a, IFN-y, IL-13, NF-κB, IKKβ and IκBα mRNA expression in lung tissue were assayed by RT- PCR; Total protein of p- IKKβ and p-IκBα and nuclear protein of p-P65 expression were tested by Western blotting .ResultsExperiment 1: Compared with CG group, pulmonary function of IT+ BLM group significantly decreased from day 7 to 70, inflammation infiltration and fibrosis focus were observed in lung tissue, and aggravated from day 28 to 84. The Ashcroft score was significantly increased from day 7 to 84. HYP contents and lung coefficient were significantly increased from day 28 to 84. Blood leukocyte, neutrophil, lymphocyte and monocyte numbers were increased at different levels, especially on day 7 and 14. Compared with CG group,pulmonary function of IT+ NS group significantly decreased on day 7 and recovered from day 14 to 84, inflammation infiltration and tight fibrosis focus were observed in lung tissue, and alleviated from day 28, with the similar variation trend in Ashcroft score. HYP contents significantly increased on day 28. Blood leukocyte and neutrophil obviously increased on day 7 and 14, and lung coefficient significantly increased on day 7Experiment 2: Compared with Control group, BLM group FVC markedly decreased and Ashcroft score and lung coefficient significantly increased after bleomycin challenged. Blood inflammatory cell numbers increased from day 7 and 84 at different levels, especially leukocyte and lymphocyte. Compared with BLM group, all treatment group FVC increased from day 7 to 70 at different degree. HYP contents, lung coefficient and blood inflammatory cell level were also decreased at different levels. The amelioration of these parameters were occurred earlier in BLM+ YKF group than other treatment groups.Experiment 3: Compared with Control group, BLM group serum IL-1β contents significantly increased after bleomycin challenged,expression of TNF-α.IL-1β、IL-13 in lung tissue were also increased from day 7 to 70. BALF IL-1β contents significantly increased from 7 to 28.The expression of IFNy was slightly increased on day 7 and 14, and decreased from day 28 to 70. The expression of NFκB mRNA, IκBα mRNA, IKKβ mRNA, nuclear protein p-P65, total protein p-IκBα and p-IKKβ were increased after bleomycin challenged. Compared with BLM group, all treatment group serum and IL-1β contents decreased at different degree, the BALF IL-1β level of BLM+YKF, BLM+BFF and BLM+ JSF were slightly lower than the other two treatment groups on day 28, the serum IL-1β level of BLM+YKF and BLM+ JSF group still decreased on day 70. The expression of TNF-a, IL-1β, IL-13 mRNA and protein in lung tissue also decreased at different degree, the expression of TNF-a protein in BLM+YKF and BLM+JSF group significantly decreased on day 7, and still markedly decreased in BLM+BFF and BLM+ JSF group on day 70. The expression of TNF-a mRNA significantly decreased in BLM+JSF group on day 14. The expression of NFκB,IκBα and IKKβ mRNA decreased at different degree in all treatment groups. IκBα mRNA expression of BLM+ JSF group was slightly lower than other treatment groups on day 7, 14 and 28; IKKβ mRNA expression of BLM+ YKF group was slightly lower than other treatment groups on day 14 and 42, and was still significantly decreased on day 70. The expression of total protein p-IκBαand p-IKKβ in all treatment groups decreased at different degree from day 7 to 70. The expression of p-IκBα in BLM+ YKF group was decreased on day 14, 28, 42 and 70 at different degree. The expression of p-IKKβ in BLM+ JSF was less than other treatment groups on day 7. The expression of nuclear protein p-P65 were decreased in all treatment groups, the expression of p-P65 in BLM+YKF were less than other treatment group on day 7,14 and 28.Conclusion1. Pulmonary fibrosis model could be established by intratracheal aspiration and installation of bleomycin (5 mg/kg) in rats. More typical and stable fibrosis pathological changes were induced by intratracheal installation of bleomycin on day 28, and these changes were still observed on day 84. This model is suitable for further study on IPF pathogenesis mechanisms and evaluation on drug long-term effects.2. Fuzheng Tongluo Xiaoji Formulae have beneficial therapeutic effective and long-term effects on IPF by improving pulmonary function, alleviating inflammatory response. The stable effects of Yangqing Kangxian formula on inhibiting HYP contents, Ashcroft score,leukocyte, neutrophil, lymphocyte and lung coefficient occurred earlier than the effects of the other treatment groups.3. Fuzheng Tongluo Xiaoji Formulae developed anti-fibrosis effects by suppressing inflammatory response, ameliorating lung injury via down-regulating. IKKβ/ IκBα/ NF-κB signal pathway.