2023 年第 3 期 第 18 卷

吡咯烷二硫代甲酸铵通过抑制Toll样受体4/核因子κB信号通路改善脂多糖诱导的脓毒症大鼠心肌纤维化

Ammonium pyrrolidine dithiocarbamate inhibiting Toll-like receptor 4/nuclear factor-κB signaling pathway to improve myocardial fibrosis induced by lipopolysaccharide in sepsis rats

作者：许哲敏杨凯杨梦黎雪琴李鑫鹏缪莎莎彭鹏

英文作者：Xu Zhemin Yang Kai Yang Meng Li Xueqin Li Xinpeng Miu Shasha Peng Peng

单位：新疆医科大学第一附属医院急救创伤中心·急救重症监护室，乌鲁木齐830011

英文单位：Emergency Intensive Care Unit Emergency Trauma Center First Affiliated Hospital of Xinjiang Medical University Urumqi 830011 China

关键词：脓毒症；心肌纤维化；心肌成纤维细胞；Toll样受体4/核因子κB信号通路

英文关键词：Sepsis;Myocardialfibrosis;Myocardialfibroblasts;Toll-likereceptor4/nuclearfactor-κBsignalingpathway

• 摘要：

• 目的 探讨脓毒症大鼠心肌纤维化和Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路的关系及吡咯烷二硫代甲酸铵（PDTC）对脂多糖刺激的大鼠心肌成纤维细胞的干预作用。方法 将大鼠原代心肌成纤维细胞复苏后，等待细胞贴壁生长至镜下视野内贴壁细胞大于90%后，按1∶3的比例传代培养。采用细胞计数试剂盒8(CCK-8)法检测第3代细胞在不同浓度脂多糖和PDTC干预下的细胞增殖活力。传代培养并取第3代细胞分为对照组、脂多糖组、PDTC组，对照组细胞培养瓶内加入50 μl的二甲基亚砜(DMSO)和4 950 μl的完全培养基, 脂多糖组细胞培养瓶内加入50 μl的DMSO和4 900 μl的完全培养基以及1 mg/L的脂多糖原液50 μl，PDTC组加入10 mmol/L的PDTC溶液50 μl和4 900 μl的完全培养基以及1 mg/L的脂多糖原液50 μl。比较各组细胞增殖活力和炎症介质水平。采用蛋白质印迹法检测大鼠心肌成纤维细胞中TLR4、NF-κB-p65和磷酸化NF-κB-p65（p-NF-κB-p65）、转化生长因子β1（TGF-β1）蛋白表达水平。采用实时荧光定量聚合酶链反应(RT-PCR)法检测Ⅰ型胶原和TGF-β1的mRNA相对表达量。结果 以脂多糖浓度为0 mg/L的对照组细胞增殖活力为参照，其他不同浓度脂多糖组的细胞增殖活力均高于对照组（均P＜0.001）。以含1 mg/L脂多糖的完全培养基为基础再使用不同浓度的PDTC干预24 h，结果显示各浓度PDTC组细胞增殖活力均明显低于脂多糖组（均P＜0.001）。脂多糖组IL-6和TNF-α蛋白表达水平均明显高于对照组［（2.02±0.37）比（1.00±0.00）、（2.00±0.38）比（1.00±0.00）］(均P＜0.05)，PDTC组IL-6和TNF-α蛋白表达水平［（1.05±0.19）、（0.72±0.40）］均明显低于脂多糖组（均P＜0.05）。脂多糖组TLR4/NF-κB信号通路TLR4蛋白表达水平及p-NF-κB-p65/NF-κB-p65比例均明显高于对照组［（1.51±0.06）比（1.00±0.00）、（1.96±0.38）比（1.00±0.00）］（均P＜0.05）。PDTC组TLR4蛋白表达水平及p-NF-κB-p65/NF-κB-p65比例［（0.88±0.24）、（0.72±0.40）］均明显低于脂多糖组（均P＜0.05）。脂多糖组TGF-β1蛋白和mRNA表达水平及Ⅰ型胶原 mRNA表达水平均明显高于对照组［（2.18±0.24）比（1.00±0.00）、（1.24±0.25）比（1.00±0.00）、（4.70±0.52）比（1.00±0.00）］（均P＜0.05）。PDTC组TGF-β1蛋白和mRNA表达水平及Ⅰ型胶原 mRNA表达水平［（1.52±0.29）、（1.12±0.02）、（2.28±0.66）］均明显低于脂多糖组（均P＜0.05）。结论 PDTC可抑制TLR4/NF-κB信号通路，降低脂多糖刺激的大鼠心肌成纤维细胞的增殖以及炎症介质和纤维化标志物的表达，进而抑制纤维化进程。

• Objective  To explore the relationship between myocardial fibrosis and Toll-like receptor 4 (TLR4)/nuclear factor-κB(NF-κB) signaling pathway in sepsis rats and the intervention of ammonium pyrrolidine dithiocarbamate (PDTC) on lipopolysaccharide (LPS) -stimulated rat myocardial fibroblasts. Methods  After resuscitation, rat primary myocardial fibroblasts were cultured in a ratio of 1∶3 after they grown to more than 90% of the adherent cells in the microscopic field. Cell counting kit-8 (CCK-8) method was used to detect the cell proliferation activity of the third generation rat myocardial fibroblasts under the intervention of LPS and PDTC. After subculture, the third generation cells were divided into control group, LPS group and PDTC group. Control group added dimethyl sulfoxide (DMSO) 50 μl and complete medium 4 950 μl. LPS group added DMSO 50 μl, complete medium 4 900 μl and 1 mg/L LPS stock solution 50 μl. PDTC group added 10 mmol/L PDTC solution 50 μl, complete medium 4 900 μl and 1 mg/L LPS stock solution 50 μl. The cell proliferation activity and inflammatory mediator levels in each group were compared. Levels of TLR4, NF-κB-p65, phosphate NF-κB-p65 (p-NF-κB-p65) and transforming growth factor-β1(TGF-β1) in rat myocardial fibroblasts were detected by western blotting. Expressions of type Ⅰ collagen mRNA and TGF-β1 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). Results  Taking the cell proliferation activity of control group with 0 mg/L LPS as reference, the cell proliferation activity of other LPS concentration groups was higher than that of the control group (all P＜0.001). Based on the complete medium containing 1 mg/L LPS, PDTC with different concentrations was used to intervene for 24 h, and the Results   showed that the cell proliferation activity of each PDTC concentration group was significantly lower than that of LPS group (all P＜0.001). Compared with control group, levels of IL-6 and TNF-α in LPS group significantly increased［（2.02±0.37） vs （1.00±0.00）, （2.00±0.38） vs （1.00±0.00）］ (both P＜0.05). Compared with LPS group, levels of IL-6 and TNF-α in PDTC group ［（1.05±0.19）,（0.72±0.40）］ decreased significantly (both P＜0.05). Compared with control group, the level of TLR4 and the proportion of p-NF-κB-p65/NF-κB-p65 of TLR4/NF-κB signaling pathway in LPS group increased significantly ［（1.51±0.06） vs （1.00±0.00）,（1.96±0.38） vs （1.00±0.00）］ (both P＜0.05). Compared with LPS group, the level of TLR4 and the proportion of p-NF-κB-p65/NF-κB-p65 in PDTC group ［（0.88±0.24）,（0.72±0.40）］ decreased significantly (both P＜0.05). Compared with control group, levels of TGF-β1 protein and mRNA and type Ⅰ collagen mRNA in LPS group significantly increased［（2.18±0.24） vs （1.00±0.00）, （1.24±0.25） vs （1.00±0.00）, （4.70±0.52） vs （1.00±0.00）］ (all P＜0.05). Compared with LPS group, levels of TGF-β1 protein and mRNA and type Ⅰ collagen mRNA in PDTC group［（1.52±0.29）, （1.12±0.02）, （2.28±0.66）］ significantly decreased (all P＜0.05). Conclusion  PDTC can inhibit TLR4/NF-κ B signaling pathway, reducing the proliferation of LPS-stimulated rat myocardial fibroblasts and the expression of inflammatory mediators and fibrosis markers, thereby inhibiting the fibrosis process.