正丁酸抑制脂多糖诱导肺纤维化的作用及机制

Effect and mechanism of inhibition of lipopolysaccharide-induced pulmonary fibrosis by butyric acid

目的观察组蛋白去乙酰化酶（HDAC）抑制剂正丁酸（BA）抑制脂多糖（LPS）诱导小鼠肺纤维化过程的作用，并探讨其作用机制。方法将30只C57／BL6小鼠按随机数字表法分为LPS刺激组（腹腔注射10mg／kgLPs）、BA预处理＋LPS刺激组（BA10mg／kg灌胃后腹腔注射LPS10mg／kg）和阴性对照组（经口灌注和腹腔注射等量生理盐水），每组10只。制模后2周及4周每组各无痛处死5只小鼠取肺组织，用HDAC活性荧光分析试剂盒检测肺组织HDAC活性；采用蛋白质免疫印迹试验（Western Blot）检测肺组织乙酰化组蛋白H3、H4（Ace—H3、Ace—H4）和胸腺细胞分化抗原-1（Thy-1）蛋白表达；采用实时定量反转录-聚合酶链反应（real—timeRT—PCR）检测肺组织Thy-1mRNA表达；用苏木素-伊红（HE）染色和Masson胶原蛋白染色光镜下观察肺组织炎症和纤维化情况；用羟脯氨酸检测试剂盒测定肺组织羟脯氨酸含量。结果与阴性对照组相比：腹腔注射LPS后肺组织出现明显的炎症反应和纤维化加重；羟脯氨酸含量增加（μg／mg：2周为8．384±0．632比4．388±0．334，4周为8．308±0．244比4．370±0．342，均P〈0．01）；HDAC活化程度增加（1amol／L：2周为7．243±0．384比3．628±0．641，4周为6．479±0．202比3．238±0．524，均P〈0．01）；组蛋白H3、H4去乙酰化程度增加[Ace—H3相对表达量（灰度值）：2周为0．516±0．115比1．005±0．359，4周为0．633±0．143比1．092±0．193，均P〈0．05；Ace—H4相对表达量（灰度值）：2周为0．402±0．164比0．759±0．187，P〉0．05；4周为0．426±0．098比0．858±0．177，P〈0．01]；Thy-1基因及蛋白表达量降低（Thy-1mRNA（2-act）：2周为0．606±0．066比1．005±0．109，P〈0．01，4周为0．824±0．101比1．210±0．400，P〉0．05；Thy-1蛋白相对表达量（灰度值）：2周为0．725±0．284比1．249±0．297，4周为0．589±0．139比1．372±0．343，均P〈0．05]。使用BA后能抑制上述过程，在2周和4周羟脯氨酸含量、HDAC活化，2周Thy-1mRNA表达，4周Ace—H4、Thy-1蛋白表达与LPS刺激组相比，表现出统计学意义[羟脯氨酸含量（μg／mg）：2周为5．943±0．726比8．384±0．632，4周为4．938±0．209比8．308±0．244；HDAC活性（μmol／L）：2周为4．386±0．117比7．243±0．384，4周为4．863±0．096比6．479±0．202；2周Thy-1mRNA（2-act）：0．884±0．216比0．606±0．066；4周Ace—H4相对表达量（灰度值）：0．715±0．145比0．426±0．098；4周Thy-1蛋白相对表达量（灰度值）：0．939±0．098比0．589±0．139；P〈0．05或P〈0．01]。结论LPS诱导小鼠肺组织纤维化过程与肺组织HDAC活化、组蛋白H3和H4去乙酰化以及Thy-1基因表达降低相关。HDAC抑制剂BA预处理能通过抑制HDAC活化以及组蛋白H4去乙酰化过程，从而抑制由LPS诱导的Thy-1基因表达降低及肺纤维化过程。

Objective To evaluate the inhibitory effect of butyric acid （BA） as a histone deacetylase （HDAC） inhibitor on lipopolysaccharide （LPS）-induced pulmonary fibrosis and its mechanism. Methods Thirty C57/BL6 mice were randomly divided into three groups according to the random number method, namely control group （physiological saline was given intraperitoneally and by gavage）, LPS challenge group （LPS-induced murine model of pulmonary fibrosis was reproduced with intraperitoneal injection of 10 mg/kg LPS）, and BA preconditioning ＋ LPS challenge group （10 mg/kg BA was given followed by intraperitoneal injection of 10 mg/kg LPS）, with 10 mice in each group. Mice were sacrificed painlessly, and lung tissue samples were harvested at 2 weeks and 4 weeks respectively （five samples every group each time）. HDAC activity was evaluated with fluorescence analysis kit. Protein expression of acetylated-histone H3 （Ace-H3）, acetylated-histone H4 （Ace-H4） and thymocyte differentiation antigen 1 （Thy-1） were determined by Western Blot. The mRNA expression of Thy-1 was assessed by real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR）. The degree of lung inflammation and fibrosis were microscopic detected after hematoxylin-eosin （HE） staining and Masson collagen staining. The deposition of lung collagen was detected by hydroxyproline content measurement kit. Results Compared to control group, the degree of lung inflammation and fibrosis was aggravated after LPS challenge, as manifested by increased hydroxyproline content （μg/mg, 2 weeks： 8.384±0.632 vs. 4.388±0.334, 4 weeks： 8.308±0.244 vs. 4.370±0.342, both P 〈 0.01）, increased HDAC activity （μmol/L, 2 weeks： 7.243±0.384 vs. 3.628±0.641, 4 weeks： 6.479±0.202 vs. 3.238±0.524, both P 〈 0.01）, increased deacetylation degree of histone H3 and H4 [relative expression of Ace-H3 （gray value）： 0.516±0.115 vs. 1.005 ±0.359 at 2 weeks, 0.633 ±0.143 vs. 1.092±0.193 at 4 weeks, both P 〈 0.05; relative expression of Ace-H4 （gray value）： 0.402±0.164 vs. 0.759±0.187 at 2 weeks, P 〉 0.05; 0.426±0.098 vs. 0.858±0.177 at 4 weeks, P 〈 0.01], and lowered Thy-1 mRNA and protein expression [Thy-1 mRNA （2^-△△ct）： 0.606±0.066 vs. 1.005±0.109 at 2 weeks, P 〈 0.01; 0.824 ± 0.101 vs. 1.210±0.400 at 4 weeks, P 〉 0.05; relative expression of Thy-1 protein （gray value）： 0.725±0.284 vs. 1.249 ± 0.297 at 2 weeks, 0.589±0.139 vs. 1.372 ± 0.343 at 4 weeks, both P 〈 0.05]. Compared with LPS group, BA precondition could inhibit above processes, as manifested by decreased hydroxyproline content （μg/mg： 5.943 ± 0.726 vs. 8.384 ± 0.632 at 2 weeks, 4.938± 0.209 vs. 8.308 ± 0.244 at 4 weeks, both P 〈 0101）, decreased HDAC activity （μmol/L： 4.386 ± 0.117 vs. 7.243 ±0.384 at 2 weeks, 4.863 ± 0.096 vs. 6.479 ± 0.202 at 4 weeks, both P 〈 0.01）, increased Thy-1 mRNA expression at 2 weeks （2^-△△ct： 0.884 ± 0.216 vs. 0.606± 0.066, P 〈 0.05）, increased acetylation degree of histone H4 and Thy-1 protein expression at 4 weeks [relative expression of Ace-H4 （gray value）： 0.715± 0.145 vs. 0.426±0.098, P 〈 0.05; relative protein expression of Thy-1 （gray value）： 0.939±0.098 vs. 0.589 ±0.139, P 〈 0.01]. Conclusions LPS-indueed pulmonary fibrosis was related with activation of HDAC, deacetylation of histone H3 and H4 and Thy-1 gene silencing. HDAC inhibitor BA could inhibit LPS-indueed pulmonary fibrosis and Thy-I gene silencing through inhibiting activation of HDAC and deacetylation of histone H4.