Research On The Intervention Effect Of Buyanghuanwu Decoction To HMGB1 Which Induced Immune Injury In Pulmonary Fibrosis

Pulmonary fibrosis (PF) is fibrous proliferative diseases which characterizes to destroy parenchyma, create fiber spot and fibroblast foci, and also to make excessive deposition extracellular matrix. A variety of interstitial lung disease can develop to diffuse pulmonary fibrosis in advanced stage of illness and in this case, idiopathic pulmonary fibrosis (IPF) is the most common. There is lack of effective treatment because the etiology and pathogenesis of advanced stage IPF has still been unclear. Recent evidence suggests that high mobility group protein (HMGB1), one kind of important inflammatory factors, can be used as a transcriptional activator of a-SMA gene in the nucleus. It can induce process of epithelial mesenchymal transition (EMT) in that epithelial cells become important source of myofibroblasts in pulmonary fibrosis fibroblast foci. According to the characteristics clinical features and pathogenesis changes of IPF. In Chinese traditional medical theories, it is similar diseases with "Pulmonary atrophy", "Pulmonary paralysis", "Pulmonary Swell" and it locates at lung and kidney. It is classified the deficiency fundamental with excess disease factors in TCM. Pathogenies are disease invasion, lung injury by chronic illness, Qi of body virtual. Those create many pathological products such as phlegm, blood stasis and pyrotoxin which cause many pathological changes such as deficiency syndromes of the lung and kidney, deficiency Qi and stasis blood and block of channels. The treatments of this disease could supplement Qi, invigorate the circulation of blood, dredging collaterals and channels, eliminating phlegm, supplement Yin, warm Yang.Objective:This study chose HMGB1 as target spot, human lung HFL1, non-small cell lung cancer cells A549 and rats with pulmonary fibrosis by bleomycin as study subjects. The researching began from centre pathogenesis of deficiency Qi and blood stasis to combine with serum pharmacology and intervene by BuYangHuanWu decoction internal and external body. To discuss the molecular mechanism of TCM method "supplement Qi, invigorate the circulation of blood" to cure pulmonary fibrosis.Methods:1. The effects of different concentrations BuYangHuanWu decoction containing serum intervened ?-SMA?ERK1/2?NF-?Bp65 protein expression on HFL1 and A549 cells after HMGB1 stimulation.First, determined by MTT and ELISA to detect the influence of HFL1, A549 cell and proliferation of IL-6 by 0,10,50,100,100 ng/ml HMGB1 stimulation. According to the results, choosing the best concentration of HMGB1 as induction dose to stimulate HFL1, A549 cells. Then using 5%,10%,15%,20% BuYangHuanWu decoction containing serum effected the cells. After 24h, to extract the function proteins of cells by Western blot for detecting the function of drug-containing serum to influence the protein expression of ?-SMA?ERK1/2? NF-?Bp65, etc. To detect the factor changes of IL-6?IL-1? in cell supernatant by ELISA.2. To detect the influence of optimal concentration of BuYangHuanWu decoction containing serum to impact proteins of ?-SMA?ERK1/2?p-ERK1/2?STAT3?p-STAT3 in HFL1 and A549 which stimulated by HMBG1 in different time points.According the experiment's result to choose the best concentration drug-containing serum, after added HFL1, A549 cells for 0.5 h, added HMGB1 stimulation the cells. Then in 0,6,24,72 h, total protein was extracted. To detect the influence of optimal concentration of BuYangHuanWu decoction containing serum to impact proteins of ?-SMA?ERK1/2?p-ERK1/2?STAT3?p-STAT3, etc. by Western blot.3. Impact of BuYangHuanWu decoction to pulmonary HMGB1 and its receptor RAGE in rats with pulmonary fibrosis.The model of pulmonary fibrosis was copied by bleomycin instillation. In 7 days, 14 days,28 days, to collect lung tissues. Take the left lung tissue were fixed in 4% paraformaldehyde and detect the replication model by HE and Masson staining. Lung homogenates were extracted RNA and protein and detect the influence of Buyanghuanwu decoction to HMGB1 mRNA, RAGE mRNA expression by PCR, and assay HMGB1, ?-SMA protein expression by Western blot.Result:1. The results of MTT and ELISA showed that to compare with the control group, the effects of cell proliferation and promote IL-6 secretion was the most obvious with 100 ng/ml of HMGB1(P<0.01). After using different concentrations of BuYangHuanWu decoction containing serum to intervene HFL1, A549 cells for 0.5 h and added 100ng /ml HMGB1 as inducer to stimulate the cells for 24 h, the intra-cellular protein expression showed that HMGB1 could increase a-SMA expression in HFL1, A549 cells to compare with the control group (P<0.01).Compared 20% of drug-containing serum with HMGB1 stimulation group, drug-containing serum could reduce two intra-cellular a-SMA expression (P<0.01). After HMGB1 (100ng/ml) stimulated HFL1 and A549 for 24 h, ERK1/2 expression increased in cells and the rising trend of HFL1 was more obvious. Compare 20% of drug-containing serum with HMGB1 stimulation group, the drug serum could reduce ERK1/2 expression in HFL1 and A549 (P<0.01).After HMGB1 (100ng/ml) stimulated HFL1 and A549 for 24 h, expression of NF-KBp65 increased in cytoplasm and nucleus. The NF-?Bp65 in nucleus of HFLl was higher than it in cytoplasm. In A549 cell, the NF-?Bp65 expression in cytoplasm was higher than it in the nucleus. The results of compared 20% the drug-containing serum with HMGB1 group in HFL1 cells (P<0.05) and 15% the drug-containing serum with HMGB1 group in A549 cells (P<0.01) showed that BuYangHuanWu decoction containing serum could reduce the expression of NF-?Bp65 in two cell nucleus.After HMGB1 (100ng/ml) stimulated HFL1 and A549 for 24 h, IL-6 and IL-1? expression increased in cells. BuYangHuanWu decoction containing serum could decrease the IL-1? expression and increase IL-6. Compare with HMGB1 stimulation group, this trend was obvious in 20% of drug-containing serum with statistically significant.2. Compare with control group, after HMGB1 (100ng/ml) stimulated of A549, expression of ?-SMA, ERK1/2, STAT3, p-STAT3 increased (P<0.01) but increasing of p-ERK1/2 was not obvious. ?-SMA kept increasing for 24 hours and ERK1/2 reached the peak at 72h. After using 20% BuYangHuanWu decoction containing serum intervention, the expression of ?-SMA, ERK1/2 STAT3 could be inhibited and p-STAT3 was promoted with statistically significant.Compared with control group, expressions of ?-SMA?ERK1/2?p-ERK1/2? STAT3?p-STAT3 all increased in cells after HMGB1 (100g/ml) stimulation (P<0.01). ?-SMA?ERK1/2 increased obviously at it working until 72h. p-ERK1/2 kept increasing from the beginning to 72h. After using 20% BuYangHuanWu decoction containing serum intervention, the expression of a-SMA% ERK1/2-p-ERK1/2?STAT3?p-STAT3 could be decreased with statistically significant. 3. With bleomycin instillation, lung of the model group appeared typical fibrosis change. The results of HE and Masson showed that in 7-28 days, alveolitis was reduced and fibrosis increased gradually and to combined Szapiel grading criteria for judge the animal model of pulmonary fibrosis.The expressions of HMGB1 mRNA, RAGE mRNA and HMGB1 ?-SMA proteins in pulmonary fibrosis lung tissue of model group rats were increased. HMGB1 mRNA, HMGB1 and a-SMA proteins was gradually increasing. It reached a peak at No.28 day with a statistically significant result. After reached the peak at No.7 day, RAGE mRNA decreased gradually and continued declining but to compare with the control group, results of each time points were statistically significant (P<0.01). Compared with model group, after intervention with different doses of Buyanghuanwu decoction could reduce HMGB1 mRNA, RAGE mRNA, HMGB1 and a-SMA expression as different level. The obvious effects was high dose group of BuYangHuanWu decoction (P<0.01).Conclusion:Buyanghuanwu decoction could inhibit the expression of RAGE and a-SMA in lung tissues of pulmonary fibrosis rats and block many signal pathways of inflammation and fibrosis which activated by HMGB1 in vitro. It could reduce immune injury and inhibit the transformation of the epithelial cells and fibroblasts to myofibroblasts. It also could produce multi-target control to pulmonary fibrosis.