脂氧素受体激动剂BML-111抑制小鼠肺纤维化及相关机制研究

发布时间：2018-05-11 17:13

本文选题：BML-111 + 肺纤维化　；参考：《华中科技大学》2013年博士论文

【摘要】：第一部分脂氧素受体激动剂BML-111抑制博来霉素诱导的小鼠肺纤维化及其相关机制目的：研究脂氧素受体激动剂BML-111对博来霉素（belymicn, BLM）诱导肺纤维化小鼠生存率、肺组织总胶原蛋白、羟脯氨酸和TGFβ1含量、以及肺组织α-平滑肌肌动蛋白（α-SMA）和纤维连接蛋白1（Fn1）表达的影响，以探讨BML-111对肺纤维化的干预作用及相关机制。方法：通过气管内一次性给予BLM2.5mg或2mg/kg复制小鼠肺纤维化模型。用于生存率研究的小鼠气管内给予BLM的剂量为2.5mg/kg，动物随机分为三组：Sham组（生理盐水（saline, SAL））、BLM组（BLM+SAL）、BML-111组（BLM+BML-111），连续21天每天记录动物生存情况用于生存率研究。评估BML-111干预效果使用的BLM为2mg/kg，动物随机分为四组：Sham组、BLM组（BLM+SAL）、 BML-111组（BLM+BML-111）、 BOC-2组（BLM+BML-111+BOC-2）。Sham组气管内给予SAL，给予SAL后2h腹腔注射SAL1ml，以后隔天注射。其余三组均气管内给予BLM，BLM组2h后腹腔注射SAL，以后隔天注射。 BML-111组和BOC-2组给予BLM后2h腹腔注射BML-111（1mg/kg），BOC-2组在每次给予BML-111前30min给予B0C-2（50μ g/kg）。而其余三组也于相同时间给予含0.125%DMSO的SAL1ml作为溶剂对照。21天后取肺组织标本， HE染色及Massion染色用于观察小鼠肺损伤及纤维化程度；化学比色法检测肺组织羟脯氨酸含量；染料结合法检测肺组织总胶原蛋白浓度；ELISA法检测肺组织标本中TGFβ1水平；Western blotting检测α-SMA, Fn1蛋白表达。结果：BML-111可以显著改善BLM诱导肺纤维化小鼠的生存情况（P0.01）；HE及massion染色显示，Sham组肺组织结构无明显病理改变，BLM组、BML-111组以及BOC-2组都有不同程度的损伤和纤维化，与BML-111组相比，BLM组和BOC-2组损伤和纤维化更加明显；与Sham组相比，其余三组肺组织总胶原蛋白水平、羟脯氨酸含量、TGFβ1明显升高(P0.05或P0.01)， BLM组和BOC-2组又高于BML-111组(P0.05或P0.01)。结论：BML-111可以减轻BLM诱导肺纤维化小鼠肺组织胶原蛋白沉积，降低α-SMA的表达，抑制TGFβ1的产生，减轻小鼠肺纤维化程度，而BOC-2可以拮抗BML-111的这种作用。第二部分脂氧素受体激动剂BML-111抑制TGFβ1诱导的胚胎肺成纤维细胞激活目的：研究脂氧素受体激动剂BML-111对TGFβ1诱导的小鼠胚胎肺成纤维细胞α-SMA和Fn1的表达、总胶原蛋白的合成，以及细胞增殖能力的影响，以探讨BML-111是否可以抑制TGFβ1诱导的成纤维细胞激活。方法：以5ng/ml的TGFβ1刺激体外培养的NIH3T3细胞，首先用不同浓度的BML-111(1nM，10nM，100nM，200nM，500nM)进行干预，，选出BML-111作用浓度（200nM）。根据处理药物的不同将细胞分为五组：（1）对照组：用含0.028%甲醇的无血清培养基处理；（2）TGFβ1组：用含0.028%甲醇的无血清培养基处理后加终浓度为5ng/ml的TGFβ1；(3) BML-111组：用终浓度为200nM的BML-111的无血清培养基处理；(4) BML-111+TGFβ1组：用终浓度为200nM的BML-111的无血清培养基处理30min后加终浓度为5ng/ml的TGFβ1处理；（5）BOC-2+BML-111+TGFβ1组：在用BML-111和TGFβ1处理前加10μM BOC-2处理30min。TGFβ1处理24h后进行相应检测。逆转录PCR检测NIH3T3细胞脂氧素受体表达；采用MTT法检测细胞增殖情况；Western blotting和免疫荧光技术检测成纤维细胞激活标记物α-SMA蛋白的表达；Western blotting检测Fn1的表达；染料结合法检测细胞培养基中总胶原蛋白的含量。结果： NIH3T3内检测到mRNA水平的脂氧素受体（ALX1/FPR-rs1和ALX2/FPR2）；BML-111可以抑制TGFβ1诱导的NIH3T3细胞的增殖效应(P0.01)；200nM的BML-111预处理可抑制NIH3T3细胞Fn1的表达(P0.05或P0.01)；200nM的BML-111预处理可以抑制NIH3T3细胞总胶原蛋白的合成(P0.05或P0.01)；免疫荧光和Western blotting结果均显示，200nM的BML-111预处理可以抑制TGFβ1诱导的α-SMA的表达(P0.05或P0.01)。脂氧素受体拮抗剂BOC-2可以阻断BML-111的这种效应。结论：BML-111可以抑制TGFβ1诱导的NIH3T3细胞α-SMA的表达；抑制TGFβ1诱导的Fn1的产生，抑制细胞合成总胶原蛋白的能力。此外，还可以抑制TGFβ1诱导的细胞增殖。第三部分脂氧素受体激动剂BML-111抑制TGFβ1诱导的NIH3T3细胞内Smad2/3、Erk、Akt信号通路的激活目的：研究BML-111对TGFβ1介导的Smad依赖的和非Smad依赖的信号通路激活的影响。方法：以5ng/ml的TGFβ1刺激体外培养的NIH3T3细胞作为成纤维激活模型，根据干预药物的不同将细胞分为4组：⑴对照组：用含0.028%甲醇的无血清培养基处理；⑵TGFβ1组：用含0.028%甲醇的无血清培养基处理30min后加终浓度为5ng/ml的TGFβ1处理；⑶BML-111组：用含200nM BML-111的无血清培养基处理；⑷BML-111+TGFβ1组：用含200nM BML-111的无血清培养基处理30min后加终浓度为5ng/ml的TGFβ1处理。TGFβ1处理24h后，采用Western blotting检测Smad2/3，磷酸化Smad2，磷酸化Smad3，总Erk，磷酸化Erk，总Akt，磷酸化Akt蛋白的表达。结果：5ng/ml TGFβ1刺激体外培养的NIH3T3细胞,可使细胞内磷酸化Smad2、磷酸化Smad3、磷酸化Erk、磷酸化Akt均升高，而BML-111可以部分抑制TGFβ1的这种效应。结论：BML-111可以抑制TGFβ1诱导的NIH3T3细胞Smad2/3、Akt、Erk信号通路的激活。
[Abstract]:Part 1 lipoxin receptor agonist BML-111 inhibits bleomycin induced pulmonary fibrosis in mice and its related mechanisms
Objective: To study the effect of lipoxygenin receptor agonist BML-111 on the survival rate of pulmonary fibrosis mice induced by belymicn (BLM), the content of total collagen, hydroxyproline and TGF beta 1 in lung tissue, and the expression of alpha smooth muscle actin (alpha -SMA) and fibronectin 1 (Fn1) in lung tissue, in order to explore the intervention effect of BML-111 on pulmonary fibrosis. And related mechanisms.
Methods: the pulmonary fibrosis model of mice was replicated by BLM2.5mg or 2mg/kg in the trachea. The dose of BLM in the trachea of mice used for survival rate was 2.5mg/kg, and the animals were randomly divided into three groups: Sham group (saline, SAL), BLM group (BLM+SAL), BML-111 group (BLM+BML-111), and the animal survival was recorded every day for 21 days. The BLM of BML-111 intervention was 2mg/kg. The animals were randomly divided into four groups: group Sham, BLM group (BLM+SAL), BML-111 group (BLM+BML-111), and BOC-2 group (BLM+BML-111+BOC-2).Sham group. Intraperitoneal injection of SAL was injected every other day. BML-111 (1mg/kg) was intraperitoneally injected with 2h in group BML-111 and BOC-2, BOC-2 group was given B0C-2 (50 mu) before BML-111, while the other three groups were given the same time as a solvent. The lung tissue hydroxyproline content was detected by chemical colorimetry, the concentration of total collagen in lung tissue was detected by dye binding assay, the level of TGF beta 1 in lung tissue samples was detected by ELISA, and the expression of alpha -SMA and Fn1 protein was detected by Western blotting.
Results: BML-111 could significantly improve the survival of pulmonary fibrosis mice induced by BLM (P0.01). HE and massion staining showed that there were no obvious pathological changes in the lung tissue structure of Sham group, and there were different degrees of damage and fibrosis in group BLM, BML-111 and BOC-2 groups, compared with BML-111 group, and more obvious injury and fibrosis in BLM group and BOC-2 group. Compared with group Sham, the total collagen level, hydroxyproline content and TGF beta 1 in the other three groups of lung tissues were significantly increased (P0.05 or P0.01), and in group BLM and BOC-2 were higher than that in group BML-111 (P0.05 or P0.01).
Conclusion: BML-111 can reduce the collagen deposition in lung tissue of pulmonary fibrosis mice induced by BLM, reduce the expression of alpha -SMA, inhibit the production of TGF beta 1 and reduce the degree of pulmonary fibrosis in mice, and BOC-2 can antagonize this effect of BML-111.
The second part of lipoxygenin receptor agonist BML-111 inhibits TGF beta 1 induced activation of embryonic lung fibroblasts.
Objective: To investigate the effect of lipoxygenin receptor agonist BML-111 on the expression of alpha -SMA and Fn1 in TGF beta 1 induced mouse embryonic pulmonary fibroblasts, the synthesis of total collagen and the effect of cell proliferation, so as to explore whether BML-111 can inhibit the activation of fibroblasts induced by TGF beta 1.
Methods: 5ng/ml TGF beta 1 was used to stimulate the NIH3T3 cells in vitro. First, the concentration of BML-111 (1nM, 10nM, 100nM, 200nM, 500nM) was used to select the BML-111 action concentration (200nM). The cells were divided into five groups according to the different treatment drugs: (1) the control group was treated with 0.028% methanol without serum medium; (2) the TGF beta 1 groups: The serum-free medium containing 0.028% methanol was treated with the final concentration of 5ng/ml TGF beta 1; (3) BML-111 group: the serum-free medium treatment with BML-111 of the final concentration of 200nM; (4) BML-111+TGF beta 1: the serum-free medium of BML-111 with the final concentration of 200nM treated 30min after 30min plus the TGF beta 1 after 5ng/ml; (5) BOC-2+BML-111 +TGF beta 1 group: after treating 24h with 30min.TGF beta 1 treated with BML-111 and TGF beta 1, 24h was detected by 30min.TGF beta 1. Reverse transcriptase PCR was used to detect the expression of the NIH3T3 cell lipoxygenin receptor; MTT assay was used to detect cell proliferation; Western blotting and immunofluorescence techniques were used to detect the expression of fibrous cell activation markers. The expression of Fn1 was detected by stern blotting, and the total collagen content in cell culture medium was detected by dye binding assay.
Results: the lipoxygenin receptor (ALX1/FPR-rs1 and ALX2/FPR2) at the level of mRNA was detected in NIH3T3, and BML-111 could inhibit the proliferation effect of TGF beta 1 induced NIH3T3 cells (P0.01), and 200nM BML-111 pretreatment could inhibit the expression of NIH3T3 cell Fn1. Synthesis (P0.05 or P0.01); both immunofluorescence and Western blotting results showed that 200nM BML-111 preconditioning could inhibit the expression of alpha -SMA induced by TGF beta 1 (P0.05 or P0.01). The lipoxygenin receptor antagonist could block this effect.
Conclusion: BML-111 can inhibit the expression of TGF beta 1 induced NIH3T3 cell alpha -SMA, inhibit the production of Fn1 induced by TGF beta 1, and inhibit the ability of cell synthesis of total collagen. In addition, it can inhibit the proliferation of cells induced by TGF beta 1.
The third part of lipoxygenin receptor agonist BML-111 inhibits activation of Smad2/3, Erk and Akt signaling pathways in NIH3T3 cells induced by TGF beta 1.
Objective: To study the effect of BML-111 on TGF beta 1 mediated Smad dependent and non Smad dependent signal pathway activation.
Methods: the NIH3T3 cells cultured in vitro by 5ng/ml TGF beta 1 were used as the fibroblast activation model, and the cells were divided into 4 groups according to the different intervention drugs: (1) the control group was treated with the serum-free medium containing 0.028% methanol; (2) the TGF beta 1 groups were treated with a serum free medium containing 0.028% methanol and TGF beta 1 after 30min and the final concentration of 5ng/ml. Treatment; (3) BML-111 group: serum free culture medium containing 200nM BML-111; (4) BML-111+TGF beta 1 group: after treating 30min with 200nM BML-111 serum-free medium, 30min and 5ng/ml's TGF beta 1 treatment.TGF beta 1 treatment 24h The expression of phosphorylated Akt protein.
Results: 5ng/ml TGF beta 1 stimulated NIH3T3 cells in vitro, which could make the intracellular phosphorylated Smad2, phosphorylated Smad3, phosphorylated Erk, and phosphorylated Akt increased, and BML-111 could partially inhibit the effect of TGF beta 1.
Conclusion: BML-111 can inhibit the activation of Smad2/3, Akt and Erk signaling pathways induced by TGF beta 1 in NIH3T3 cells.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563

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