Protective Effects And Mechanism Of Buyanghuanwu Decoction On Idiopathic Pulmonary Fibrosis Rats Induced By Bleomycin

Objective:Based on the key mediators of HMGB1 pro-fibrotic generate, to probe Buyanghuanwu Decoction on bleomycin-induced idiopathic pulmonary fibrosis in rats pharmacodynamic mechanism of action.Method:1.144 male SD rats which weighted 200 ± 20g gramme were randomly divided into control group, model group, prednisone group, Buyanghuanwu decoction high, medium and low dose groups. In addition to the control group the rest of the groups were intratracheal injection of bleomycin-induced pulmonary fibrosis neomycin law. after the model given appropriate medication, in each group were randomly selected in the first eight days of 14th and 28th rat femoral artery were put the death, bronchoalveolar lavage fluid (Broncho alveolar fluid, BALF) and lung tissue with HE, Masson staining alveolar lung tissue inflammation, fibrosis, enzyme-linked immunosorbent assay (ELISA) to detect early inflammatory factor in bronchoalveolar lavage fluid TNF-a, the level of IL-1β, IL-6, and the late inflammatory factor HMGB1 expressing reverse transcriptase polymerase chain reaction (RT-PCR) assay lung HMGB1, TLR2mRNA of the HMGB1 Western-blot method to detect lung tissue, the content of a-SMA immunohistochemical staining (streptomycin avidin a peroxidase, SABC method) lung tissue sections HMGB1, distribution and expression of a-SMA protein.2. A549 purchase from the Shanghai Institute of Life Sciences, Institute of Cell Resource Center, the culture spread to the third generations were based on experimental needs in different kinds of Petri dishes, collect the logarithmic growth phase cells used in the experiment. Was added to DMEM/F12 medium diluted solution Buyanghuanwu Decoction; added HMGB 1 protein to stimulate the culture supernatants were harvested 24h after dispensing to-80℃ cryopreservation standby; consistent conditions before, added to DMEM/F12 medium diluted Buyanghuanwu soup solution were added HMGB1 protein to stimulate 0,6,24,72h total protein was extracted. The expression of A549 cell proteins STATS, P-STAT3 in Western-blot method.1. result:The general situation:the control group rats body sturdy, responsive, smooth and supple fur, normal appetite, weight gain significantly. Model rats showed listlessness, eyes dull, unresponsive, breathing breathlessness, individual red nose and mouth secretions, lack of exercise, dry dull fur, arched back straight, reducing food intake than the normal group, slow weight gain. The rest of the treatment groups rats spirit better, more rapid response, breathing more stable, good gloss coat, with the advance of time, activity, food intake decline improved to varying degrees are degrees, weight increased.2 pathological: ①HE staining:control group rat lung tissue structure clear, no obvious morphological changes; compared with the control group, the alveolar structure model rats in varying degrees of damage, inflammatory cell infiltration and collagen fibers hyperplasia; compared with the model group, alveolitis and pulmonary fibrosis each administration group at different time points were improved to varying degrees. ②Masson staining control group of rats group bronchial wall collagen layer is thin, a small amount of dyed blue alveolar region of collagen fiber distribution. Model group were significantly widened alveolar Inter 7d, in the bronchial wall, the vessel wall and the alveolar septa are visible blue stained collagen fibers; within the first outer wall 14d bronchial and vascular levels, thickening of the alveolar septa are visible blue collagen fibers; Article 28d in the bronchioles, low adventitia and fibrosis consolidation area shows a large number of blue collagen fibers, generate diffuse pulmonary fibrosis. Prednisone group and traditional Chinese medicine in each dose group could reduce pulmonary fibrosis;3 TNF-α, IL-1β, IL-6, HMGB1 ①TNF-α:Compared with the control group, alveolar lavage fluid at each time point in the model group (BALF) in TNF-α levels increased (P<0.05).28 days decreased TNF-α, and 7 and 14 days and the difference was significant (P<0.05); Compared with the model group, prednisone group and each time point Buyanghuanwu decoction high, medium and low dose group were lower alveolar lavage fluid (BALF) in TNF-α content (P<0.05); compared with the prednisone group,7 and 14 days Buyanghuanwu Decoction; each dose group were significantly different (P<.0.05), the 28th day Buyanghuanwu Decoction; each dose group no significant difference (P> 0.05); comparison between Buyanghuanwu Decoction; each dose group at each time point of the high-dose group and low, there are differences (P<0.05) between dose group ② IL-1β:compared with the control group, alveolar lavage fluid at each time point in the model group (BALF) of IL-1β levels increased (P<0.05). Its IL-1β content was progressively decreasing trend, the seventh day of the experiment reached a peak and then gradually decline, but still at a high level. Compared with the model group, prednisone group and each time point Buyanghuanwu decoction high, medium and low decreased alveolar lavage fluid (BALF) of IL-1β content (P<0.05); And Strong loose group compared to the first seven days Buyanghuanwu decoction high, medium and low-dose group had significant difference (P<0.05), compared with prednisone,14 and 28 days Buyanghuanwu no significant difference (P>0.05) soup each dose group. No significant difference (P> 0 05.) Between Buyanghuanwu Decoction; each dose group ③IL-6:blank group, model group alveolar lavage fluid (BALF) of IL-6 levels increased (P< 0.05). Model group at each time point,14 and 28 days and on day 7 than there are differences (P <0.05), which IL-6 levels showed a progressive decrease. Compared with the model group, prednisone group and each time point Buyanghuanwu decoction high, medium and low decreased alveolar lavage fluid (BALF) in IL-6 levels (P<0 05.); And Strong loose group compared to the first seven days of high, medium and low dose group had significant difference (P<0.05). No significant difference (P>0.05) 14 and 28 days Buyanghuanwu soup each dose group. No between Buyanghuanwu Decoctionxach dose group was significantly difference (P>0.05) ④HMGB1:Compared with the control group, alveolar lavage fluid at each time point in the model group (BALF) of HMGB1 content increased (P<005), comparison between the model group at each time point, no significant difference (P.> 0.05);. compared with the model group, prednisone group and each time point Buyanghuanwu decoction high, medium and low are reduce alveolar lavage fluid (BALF) of HMGB1 content (P<0.05); compared with prednisone, in the 14th and 28th day Buyanghuanwu Decoction; each dose group were significantly different (P<0.05).4 the expression of HMGB1, α-SMA:①Compared with the control group, the expression of lung tissue of rats at each time point HMGB1 in model group increased (P<0.01). Model group at each time point, no significant difference (P>0.05); Compared with the model group, predmsone group and Buyanghuanwu decoction high, medium and low at each time point were lower in lung tissue over-expression of HMGB1(P<0.05); compared with the prednisone group, the 14th and 28th day Buyanghuanwu Decoction; each dose group were significantly different (P<0.05)(2) with the control group, the expression at each time point model lung tissue a-SMA increased (P<0.01). There was no significant difference (P>0.05) in the model group at each time point. Compared with the model group, prednisone group and each time point Buyanghuanwu decoction high, medium and low expression in lung tissue decreased over the a-SMA (P<0.05); Compared with the prednisone group, the 28 days Buyanghuanwu Decoction; each dose group were significantly different (P<0.05);5 HMGB1, TLR2mRNA expression: ① in the control group had no significant difference (. P> 0 05) HMGB1mRNA of expression at each time point, compared with the control group, the expression at each time point HMGB1mRNA lung tissue of rats in the model group increased (P<0 05), model group at each time point, no significant difference (P>0 05);. compared with the model group, prednisone group and each time point Buyanghuanwu decoction high, medium- and low decreased lung overexpression of tissue HMGB1mRNA (P<0 05.); compared with the prednisone group, a significant difference (P<0 05.) at 14 and 28 days Buyanghuanwu Decoction each dose group; ② the control group no significant difference (P>0 05) TLR2mRNA expression at each time point, the comparison with the control group, the expression of lung tissue in rats at each time point TLR2mRNA model group increased (P<0 05); the model group at each time point There was no significant difference between (P> 0 05); compared with the model group, prednisone group and Buyanghuanwu decoction high, medium and low decreased lung TLR2mRNA overexpression (P<0 05.); complement no Yang Huan Wu Tang each dose group and prednisone group compared significant difference (P> 0.05).Conclusions:1 Buyanghuanwu Decoction can improve BLM-induced lung inflammation in rats with pulmonary fibrosis and pulmonary fibrosis scarring.2. Buyanghuanwu Decoction; can reduce the BLM-induced pulmonary fibrosis in rats bronchoalveolar lavage inflammatory markers TNF-a, IL-1β, IL-6, IL-8, concentration of HMGB1, which may be one of the mechanisms which play a role in anti-fibrosis.3. BuyanghuanwuDecoction can effectively inhibit the expression of lung tissue HMGB1, TLR2mRNA of Buyanghuanwu decoction can inhibit lung tissue HMGB 1, the expression of α-SMA protein Buyanghuanwu Decoction containing serum can inhibit STAT3, P-STAT3expression was confirmed Buyanghuanwu Decoction can play better anti pulmonary fibrosis related to regulating HMGB1 signaling pathways, HMGB1 may be generated to promote a key mediator of fibrosis.