补阳还五汤对肺纤维化中HMGB1-RAGE信号通路的调控作用

Regulatory Effect of Buyang Huanwu Tang on HMGB1-RAGE Signaling Pathway in Pulmonary Fibrosis

摘要:

目的：观察肺纤维化中诱导免疫损伤和异常修复的高迁移率族蛋白Box-1（high mobility group Box-1 protein，HMGB1）及晚期糖基化终产物受体（receptor of advanced glycation end-product，RAGE）的表达，及补阳还五汤对HMGB1-RAGE信号通路的调控作用。方法：144只SD大鼠随机分为6组，分别为正常组，博莱霉素（BLM）模型组，补阳还五汤低、中、高剂量组（6.83，13.66，27.32 g·kg^-1），阳性药组（强的松，4.2 mg·kg^-1），动物实验采用博莱霉素气管注入法复制肺纤维化模型后，分别给予补阳还五汤高、中、低剂量和强的松灌胃，于造模后第7，14，28天分批提取样本。采用逆转录-聚合酶链式反应（RT-PCR）法检测肺组织中的HMGB1，RAGE mRNA的表达；采用蛋白质免疫印记法（Western blot）检测HMGB1，α-平滑肌肌动蛋白（alpha-smooth muscle action，α-SMA）的表达。细胞实验采用含药血清药理学的实验方法，以5%，10%，15%，20% 的补阳还五汤含药血清和空白血清来干预HMGB1诱导刺激的人肺成纤维细胞（HFL1），采用Western blot法检测α-SMA蛋白的表达。结果：动物实验中，与正常组比较，模型组在第28天显著上调HMGB1 mRNA（P<0.01），在第7天和第14天显著上调RAGE mRNA（P<0.01），在各时间点显著上调HMGB1，α-SMA（P<0.01）；与BLM模型组比较，补阳还五汤高剂量组在第14天和第28天显著下调HMGB1 mRNA（P<0.01），在第7天和第14天显著下调RAGE mRNA（P<0.01），在各时间点显著下调HMGB1和α-SMA蛋白（P<0.01）。细胞实验中，与空白组比较，20% 空白血清预培养加HMGB1组显著升高α-SMA蛋白（P<0.01）；与20% 空白血清预培养加HMGB1组比较，不同剂量的补阳还五汤含药血清显著下调细胞中α-SMA蛋白（P<0.01）。结论：补阳还五汤可抑制HMGB1在大鼠肺组织中和人肺成纤维细胞中所引起的RAGE，α-SMA的表达增加。提示补阳还五汤可能通过阻断HMGB1/RAGE信号通路，减少肌成纤维细胞生成，对肺纤维化的发生发展起到防治作用。

Abstract:

Objective: To observe abnormal immune injury and expressions of high mobility group Box-1 protein(HMGB1) and receptor of advanced glycation end-product(RAGE) in rat pulmonary fibrosis, and the regulatory effect of Buyang Huanwu Tang on HMGB1-RAGE signaling pathway. Method: A total of 144 SD rats were randomly divided into six groups, namely the normal group, the bleomycin (BLM) group, the low, middle and high-dose Buyang Huanwu Tang groups (6.83, 13.66, 27.32 g·kg^-1) and the positive drug group (prednisone, 4.2 mg·kg^-1). In the animal experiment, the bleomycin trachea injection method was used to reproduce the pulmonary fibrosis animal model, and given high, medium and low-dose Buyang Huanwu Tang by gavage. Samples were extracted on the 7th, 14th, 28th days after modeling.Reverse transcription PCR(RT-PCR) method was adopted to detect the mRNA expressions of HMGB1 and RAGE in lung tissues, and Western blot method was used to detect the protein expressions of HMGB1 and alpha-smooth muscle action(α-SMA). In the cell experiment, the drug-containing serum pharmacological methodology was adopted. Buyang Huanwu Tang -containing serum with concentrations of 5%, 10%, 15%, 20% and blank serum were adopted to intervene normal lung fibroblasts (HFL1) stimulated by HMGB1.Western blot method was used to detect the expression of α-SMA protein. Result: Compared with the blank control group, the mRNA expression of HMGB1 in the BLM model group was obviously up-regulated on the 28th day (P<0.01), the mRNA expression of RAGE was visibly up-regulated on the 7th and the 14th days (P<0.01), and the protein expressions of HMGB1 and α-SMA was significantly up-regulated at all times points (P<0.01) in animal experiments. Compared with the BLM model group, in the high-dose Buyang Huanwu Tang group, the mRNA expression of HMGB1 was significantly decreased on the 14th and 28th days (P<0.01), the mRNA expression of RAGE was significantly down-regulated on the 7th and the 14th days (P<0.01), and the protein expressions of HMGB1 and α-SMA was clearly down-regulated on all time points (P<0.01) in animal experiments. Compared with blank control group, the 20% serum added HMGB1 group showed a significant rise in the protein expression of α-SMA (P<0.01). Compared with the 20% serum added with HMGB1 control group, different concentrations of Buyang Huanwu Tang-containing serum significantly reduced the protein expression of α-SMA (P<0.01) in the cell experiment. Conclusion: Buyang Huanwu Tang can inhibit the expressions of RAGE and alpha SMA caused by HMGB1 in the rat lung tissues. The results suggest that Buyang Huanwu Tang may prevent and treat pulmonary fibrosis by blocking HMGB1/RAGE signaling pathways and reducing the growth of myofibroblasts.