本文選題：BML-111 + 肺纖維化��； 參考：《華中科技大學(xué)》2013年博士論文

【摘要】：第一部分脂氧素受體激動(dòng)劑BML-111抑制博來霉素誘導(dǎo)的小鼠肺纖維化及其相關(guān)機(jī)制 目的：研究脂氧素受體激動(dòng)劑BML-111對(duì)博來霉素（belymicn, BLM）誘導(dǎo)肺纖維化小鼠生存率、肺組織總膠原蛋白、羥脯氨酸和TGFβ1含量、以及肺組織α-平滑肌肌動(dòng)蛋白（α-SMA）和纖維連接蛋白1（Fn1）表達(dá)的影響，以探討B(tài)ML-111對(duì)肺纖維化的干預(yù)作用及相關(guān)機(jī)制。 方法：通過氣管內(nèi)一次性給予BLM2.5mg或2mg/kg復(fù)制小鼠肺纖維化模型。用于生存率研究的小鼠氣管內(nèi)給予BLM的劑量為2.5mg/kg，動(dòng)物隨機(jī)分為三組：Sham組（生理鹽水（saline, SAL））、BLM組（BLM+SAL）、BML-111組（BLM+BML-111），連續(xù)21天每天記錄動(dòng)物生存情況用于生存率研究。評(píng)估BML-111干預(yù)效果使用的BLM為2mg/kg，動(dòng)物隨機(jī)分為四組：Sham組、BLM組（BLM+SAL）、 BML-111組（BLM+BML-111）、 BOC-2組（BLM+BML-111+BOC-2）。Sham組氣管內(nèi)給予SAL，給予SAL后2h腹腔注射SAL1ml，以后隔天注射。其余三組均氣管內(nèi)給予BLM，BLM組2h后腹腔注射SAL，以后隔天注射。 BML-111組和BOC-2組給予BLM后2h腹腔注射BML-111（1mg/kg），BOC-2組在每次給予BML-111前30min給予B0C-2（50μ g/kg）。而其余三組也于相同時(shí)間給予含0.125%DMSO的SAL1ml作為溶劑對(duì)照。21天后取肺組織標(biāo)本， HE染色及Massion染色用于觀察小鼠肺損傷及纖維化程度；化學(xué)比色法檢測(cè)肺組織羥脯氨酸含量；染料結(jié)合法檢測(cè)肺組織總膠原蛋白濃度；ELISA法檢測(cè)肺組織標(biāo)本中TGFβ1水平；Western blotting檢測(cè)α-SMA, Fn1蛋白表達(dá)。 結(jié)果：BML-111可以顯著改善BLM誘導(dǎo)肺纖維化小鼠的生存情況（P0.01）；HE及massion染色顯示，Sham組肺組織結(jié)構(gòu)無明顯病理改變，BLM組、BML-111組以及BOC-2組都有不同程度的損傷和纖維化，與BML-111組相比，BLM組和BOC-2組損傷和纖維化更加明顯；與Sham組相比，其余三組肺組織總膠原蛋白水平、羥脯氨酸含量、TGFβ1明顯升高(P0.05或P0.01)， BLM組和BOC-2組又高于BML-111組(P0.05或P0.01)。 結(jié)論：BML-111可以減輕BLM誘導(dǎo)肺纖維化小鼠肺組織膠原蛋白沉積，降低α-SMA的表達(dá)，抑制TGFβ1的產(chǎn)生，減輕小鼠肺纖維化程度，而BOC-2可以拮抗BML-111的這種作用。 第二部分脂氧素受體激動(dòng)劑BML-111抑制TGFβ1誘導(dǎo)的胚胎肺成纖維細(xì)胞激活 目的：研究脂氧素受體激動(dòng)劑BML-111對(duì)TGFβ1誘導(dǎo)的小鼠胚胎肺成纖維細(xì)胞α-SMA和Fn1的表達(dá)、總膠原蛋白的合成，以及細(xì)胞增殖能力的影響，以探討B(tài)ML-111是否可以抑制TGFβ1誘導(dǎo)的成纖維細(xì)胞激活。 方法：以5ng/ml的TGFβ1刺激體外培養(yǎng)的NIH3T3細(xì)胞，首先用不同濃度的BML-111(1nM，10nM，100nM，200nM，500nM)進(jìn)行干預(yù)，，選出BML-111作用濃度（200nM）。根據(jù)處理藥物的不同將細(xì)胞分為五組：（1）對(duì)照組：用含0.028%甲醇的無血清培養(yǎng)基處理；（2）TGFβ1組：用含0.028%甲醇的無血清培養(yǎng)基處理后加終濃度為5ng/ml的TGFβ1；(3) BML-111組：用終濃度為200nM的BML-111的無血清培養(yǎng)基處理；(4) BML-111+TGFβ1組：用終濃度為200nM的BML-111的無血清培養(yǎng)基處理30min后加終濃度為5ng/ml的TGFβ1處理；（5）BOC-2+BML-111+TGFβ1組：在用BML-111和TGFβ1處理前加10μM BOC-2處理30min。TGFβ1處理24h后進(jìn)行相應(yīng)檢測(cè)。逆轉(zhuǎn)錄PCR檢測(cè)NIH3T3細(xì)胞脂氧素受體表達(dá)；采用MTT法檢測(cè)細(xì)胞增殖情況；Western blotting和免疫熒光技術(shù)檢測(cè)成纖維細(xì)胞激活標(biāo)記物α-SMA蛋白的表達(dá)；Western blotting檢測(cè)Fn1的表達(dá)；染料結(jié)合法檢測(cè)細(xì)胞培養(yǎng)基中總膠原蛋白的含量。 結(jié)果： NIH3T3內(nèi)檢測(cè)到mRNA水平的脂氧素受體（ALX1/FPR-rs1和ALX2/FPR2）；BML-111可以抑制TGFβ1誘導(dǎo)的NIH3T3細(xì)胞的增殖效應(yīng)(P0.01)；200nM的BML-111預(yù)處理可抑制NIH3T3細(xì)胞Fn1的表達(dá)(P0.05或P0.01)；200nM的BML-111預(yù)處理可以抑制NIH3T3細(xì)胞總膠原蛋白的合成(P0.05或P0.01)；免疫熒光和Western blotting結(jié)果均顯示，200nM的BML-111預(yù)處理可以抑制TGFβ1誘導(dǎo)的α-SMA的表達(dá)(P0.05或P0.01)。脂氧素受體拮抗劑BOC-2可以阻斷BML-111的這種效應(yīng)。 結(jié)論：BML-111可以抑制TGFβ1誘導(dǎo)的NIH3T3細(xì)胞α-SMA的表達(dá)；抑制TGFβ1誘導(dǎo)的Fn1的產(chǎn)生，抑制細(xì)胞合成總膠原蛋白的能力。此外，還可以抑制TGFβ1誘導(dǎo)的細(xì)胞增殖。 第三部分脂氧素受體激動(dòng)劑BML-111抑制TGFβ1誘導(dǎo)的NIH3T3細(xì)胞內(nèi)Smad2/3、Erk、Akt信號(hào)通路的激活 目的：研究BML-111對(duì)TGFβ1介導(dǎo)的Smad依賴的和非Smad依賴的信號(hào)通路激活的影響。 方法：以5ng/ml的TGFβ1刺激體外培養(yǎng)的NIH3T3細(xì)胞作為成纖維激活模型，根據(jù)干預(yù)藥物的不同將細(xì)胞分為4組：⑴對(duì)照組：用含0.028%甲醇的無血清培養(yǎng)基處理；⑵TGFβ1組：用含0.028%甲醇的無血清培養(yǎng)基處理30min后加終濃度為5ng/ml的TGFβ1處理；⑶BML-111組：用含200nM BML-111的無血清培養(yǎng)基處理；⑷BML-111+TGFβ1組：用含200nM BML-111的無血清培養(yǎng)基處理30min后加終濃度為5ng/ml的TGFβ1處理。TGFβ1處理24h后，采用Western blotting檢測(cè)Smad2/3，磷酸化Smad2，磷酸化Smad3，總Erk，磷酸化Erk，總Akt，磷酸化Akt蛋白的表達(dá)。 結(jié)果：5ng/ml TGFβ1刺激體外培養(yǎng)的NIH3T3細(xì)胞,可使細(xì)胞內(nèi)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的這種效應(yīng)。 結(jié)論：BML-111可以抑制TGFβ1誘導(dǎo)的NIH3T3細(xì)胞Smad2/3、Akt、Erk信號(hào)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R563

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