目的 观察康复新液（美洲大蠊提取物）对脂多糖（LPS）诱导炎症微环境中人食管成纤维细胞（HEFs）损伤 的作用及机制。方法 （1）以不同浓度康复新液（0%、0.5%、1%、2%、4%、8%、16%、32%）及不同浓度 LPS （0、2、4、6、8、10、20、40 μg·mL-1 ）对 HEFs 干预 24 h 后，采用 CCK-8 法检测 HEFs 细胞增殖率，筛选合 适的 LPS 诱导浓度及康复新液干预浓度。（2）将对数生长期 HEFs 细胞分为对照组、LPS 组、0.5% 康复新液组、 1% 康复新液组、2% 康复新液组及醋酸泼尼松龙（PDNN，50 μg·mL-1 ）组；采用 LPS（10 μg·mL-1 ）刺激 1 h 诱导 炎症微环境。采用 ELISA 法检测细胞上清液中白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α、诱导型一 氧化氮合酶（iNOS）的含量；免疫荧光法检测细胞中 IL-6、核因子（NF）-κB p-p65、p-Smad3、α 平滑肌肌动 蛋白（α-SMA）表达水平；qRT-PCR 法检测细胞中 IL-1β、IL-6、TNF-α、NF-κB、转化生长因子（TGF）-β1、 Smad3、α-SMA mRNA 表达水平；Western Blot 法检测细胞中 NF-κB p65、NF-κB p-p65、核因子 κB 抑制蛋 白 α（IκBα）、p-IκBα、TGF-β1、Smad3、p-Smad3、α-SMA 蛋白表达水平。结果 （1）后续实验选择 0.5%、 1%、2% 浓度康复新液进行干预；选择 10 μg·mL-1 LPS 刺激诱导 HEFs 炎症微环境。（2）与对照组比较，LPS 组 HEFs 细胞上清液中的 IL-1β、IL-6、TNF-α、iNOS 含量均显著上升（P＜0.01）；细胞 IL-1β、IL-6、TNF-α、 NF-κB mRNA 表达均显著上调（P＜0.01）；细胞 NF-κB p65、NF-κB p-p65、IκBα、p-IκBα 蛋白表达均显著上 调（P＜0.01）；IL-6 细胞质红色荧光信号及 NF-κB p-p65 细胞核绿色荧光信号均显著增强（P＜0.01）；细胞 TGF-β1、Smad3、α-SMA mRNA 表达均显著上调（P＜0.01）；细胞 TGF-β1、Smad3、p-Smad3、α-SMA 蛋白 表达均显著上调（P＜0.01）；p-Smad3 细胞核绿色荧光信号及 α-SMA 细胞质红色荧光信号均显著增强（P＜ 0.01）。与 LPS 组比较，0.5%、1%、2% 康复新液组及 PDNN 组细胞上清液中的 IL-6、TNF-α、iNOS 含量显著 降低（P＜0.01）；细胞 NF-κB p65、NF-κB p-p65、IκBα、p-IκBα 蛋白表达均显著下调（P＜0.01）；IL-6 细胞 质红色荧光信号及 NF-κB p-p65 细胞核绿色荧光信号均显著减弱（P＜0.01）；细胞 TGF-β1、p-Smad3 蛋白表 达显著下调（P＜0.01）；α-SMA 细胞质红色荧光信号显著减弱（P＜0.01）。与 LPS 组比较，2% 康复新液组及 PDNN 组细胞上清液中的 IL-1β 含量显著降低（P＜0.01）；细胞 IL-1β、IL-6、TNF-α、NF-κB mRNA 表达均 显著下调（P＜0.05，P＜0.01）；细胞 TGF-β1、Smad3、α-SMA mRNA 表达均显著下调（P＜0.01）；细胞 Smad3、α-SMA 蛋白表达显著下调（P＜0.01）；p-Smad3 细胞核绿色荧光信号显著减弱（P＜0.01）。结论 康复 新液可减轻 LPS 诱导炎症微环境中 HEFs 细胞的损伤和肌成纤维细胞表型转化，其作用机制可能与抑制 TGF-β1/Smad3 信号通路过度激活有关。该结果为康复新液治疗内镜黏膜下剥离术后的食管狭窄提供了一定的 实验依据。

Objective To investigate the effects and underlying mechanism of Kangfuxin Liquid [an extract of Periplaneta americana（L.）] on human esophageal fibroblast （HEFs） injury within a lipopolysaccharide （LPS）-induced inflammatory microenvironment. Methods（1） Following intervention with varying concentrations of Kangfuxin Liquid （0%，0.5%，1%，2%，4%，8%，16%，32%） and LPS （0，2，4，6，8，10，20，40 μg·mL-1 ） for 24 hours， the HEFs proliferation rate was assessed via the CCK-8 assay to determine the optimal concentrations for LPS induction and Kangfuxin Liquid intervention.（2） HEFs in the logarithmic growth phase were divided into the following groups： Control， LPS， 0.5% Kangfuxin Liquid， 1% Kangfuxin Liquid， 2% Kangfuxin Liquid， and Prednisolone Acetate （PDNN，50 μg·mL-1 ）. An inflammatory microenvironment was induced by stimulation with LPS （10 μg·mL-1 ） for 1 hour. The levels of interleukin （IL）-1β，IL-6，tumor necrosis factor （TNF）-α，and inducible nitric oxide synthase （iNOS） in the cell supernatant were measured by ELISA. The expression levels of IL-6，nuclear factor （NF）- κB p-p65， p-Smad3， and α -smooth muscle actin （α -SMA） in the cells were detected by immunofluorescence. The mRNA expression levels of IL-1β，IL-6，TNF-α，NF-κB，transforming growth factor （TGF）-β1，Smad3，and α -SMA in the cells were determined using qRT-PCR. The protein expression levels of NF- κB p65，NF- κB p-p65， inhibitor of nuclear factor kappa-B alpha （IκBα），p-IκBα，TGF-β1，Smad3，p-Smad3，and α-SMA in the cells were assessed by Western Blot. Results （1） Subsequent experiments utilized Kangfuxin Liquid at concentrations of 0.5%，1%， and 2%， and an LPS concentration of 10 μg·mL-1 to induce the inflammatory microenvironment in HEFs.（2）Compared with the control group，the LPS group exhibited significantly increased levels of IL-1β，IL-6， TNF-α，and iNOS in the HEF supernatant （P＜0.01）；significantly upregulated mRNA expression of IL-1β，IL-6， TNF-α，and NF-κB （P＜0.01）；significantly increased protein expression of NF-κB p65，NF-κB p-p65，IκBα， and p-IκBα （P＜0.01）； significantly enhanced cytoplasmic red fluorescence signal for IL-6 and nuclear green fluorescence signal for NF- κB p-p65（P＜0.01）；significantly upregulated mRNA expression of TGF- β1，Smad3， and α-SMA （P＜0.01）；significantly increased protein expression of TGF-β1，Smad3，p-Smad3，and α-SMA （P＜ 0.01）；and significantly intensified nuclear green fluorescence signal for p-Smad3 and cytoplasmic red fluorescence signal for α-SMA （P＜0.01）. Compared with the LPS group，the 0.5%，1%，and 2% Kangfuxin Liquid groups and the PDNN group showed significantly reduced levels of IL-6，TNF-α，and iNOS in the cell supernatant （P＜0.01）； significantly downregulated protein expression of NF- κB p65， NF- κB p-p65， IκBα， and p-IκBα（P＜0.01）； significantly attenuated cytoplasmic red fluorescence signal for IL-6 and nuclear green fluorescence signal for NF- κB p-p65（P＜0.01）；significantly decreased protein expression of TGF-β1 and p-Smad3（P＜0.01）；and significantly weakened cytoplasmic red fluorescence signal for α-SMA （P＜0.01）. Compared with the LPS group，the 2% Kangfuxin Liquid group and the PDNN group demonstrated significantly lower IL-1β content in the cell supernatant （P＜0.01）；significantly downregulated mRNA expression of IL-1β， IL-6， TNF- α， and NF- κB （P＜0.05， P＜0.01）； significantly downregulated mRNA expression of TGF-β1，Smad3，and α-SMA （P＜0.01）；significantly decreased protein expression of Smad3 and α-SMA （P＜0.01）；and a significantly weakened nuclear green fluorescence signal for p-Smad3（P＜0.01）. Conclusion Kangfuxin Liquid can alleviate HEF injury and inhibit the phenotypic transition to myofibroblasts in an LPS-induced inflammatory microenvironment. The underlying mechanism may be associated with the suppression of the excessive activation of the TGF-β1/Smad3 signaling pathway. These findings provide experimental evidence supporting the potential use of Kangfuxin Liquid in treating esophageal strictures following endoscopic submucosal dissection.

R285.5

四川省科技厅重点研发项目（2020YFS0376）；西南医科大学科研创新团队项目（2022-CXTD-01）。