减味连梅汤诱导脓毒症小鼠粪便上清保护RAW264.7细胞炎症与Caco-2细胞氧化 损伤的作用研究

Protective Effects of Fecal Supernatant from Jianwei Lianmei Decoction-Treated Septic Mice on Inflammation in RAW264.7 Cells and Oxidative Injury in Caco-2 Cells

摘要: 目的：探讨经减味连梅汤干预后不同组别脓毒症小鼠粪便上清(fecal supernatant, FS)对RAW264.7巨噬细胞炎症模型及Caco-2肠上皮细胞损伤模型的影响，并初步分析其经肠道微环境介导的体外生物学效应。方法：收集正常组、脓毒症模型组及减味连梅汤干预组小鼠新鲜粪便，制备FS。采用CCK-8法筛选FS对RAW264.7和Caco-2细胞的安全作用浓度，并分别建立LPS诱导的RAW264.7炎症模型和H₂O₂诱导的Caco-2氧化损伤模型。以安全浓度FS预处理细胞后进行造模，检测细胞活力、RAW264.7细胞NO分泌，并采用RT-qPCR检测炎症因子及肠屏障相关蛋白表达水平。结果：各组FS在筛选浓度范围内对RAW264.7和Caco-2细胞均未见明显细胞毒性。LPS刺激后，RAW264.7细胞炎症反应被成功激活；与模型组比较，不同组FS处理后能一定程度降低NO水平，但iNOS及IL-6等炎症相关指标未见明显逆转，提示其对巨噬细胞炎症表型的直接调控作用有限。H₂O₂刺激后，Caco-2细胞活力下降并伴随肠屏障相关分子表达受损；与模型组比较，减味连梅汤干预组来源FS可在一定程度上改善Caco-2细胞紧密连接蛋白ZO-1等屏障相关指标的表达，但改善程度有限，部分指标差异未达到统计学显著。结论：减味连梅汤干预后小鼠FS对LPS诱导的RAW264.7细胞炎症反应调控作用较弱，但对H₂O₂诱导的Caco-2细胞屏障损伤具有一定改善趋势。

Abstract: Objective: To investigate the effects of fecal supernatant (FS) derived from septic mice in different groups after intervention with Jianwei Lianmei Decoction on an inflammatory model of RAW264.7 macrophages and an injury model of Caco-2 intestinal epithelial cells, and to preliminarily explore its in vitro biological effects mediated through the intestinal microenvironment. Methods: Fresh feces were collected from mice in the normal group, the DSS model group, and the Jianwei Lianmei Decoction intervention group to prepare fecal supernatant (FS). The safe concentration range of FS for RAW264.7 and Caco-2 cells was screened using the CCK-8 assay. An inflammatory response model was constructed in RAW264.7 macrophages using lipopolysaccharide (LPS), while an oxidative damage model in Caco-2 cells was established through stimulation with hydrogen peroxide (H₂O₂). Prior to the induction of these models, the cells were pretreated with FS at concentrations verified to be non-cytotoxic. Following treatment, the viability of RAW264.7 cells and the production of nitric oxide (NO) were determined. In addition, the mRNA expression levels of inflammation-associated cytokines as well as proteins related to the intestinal barrier were further evaluated using RT-qPCR analysis. Results: Within the tested concentration range, FS from all groups showed no obvious cytotoxicity toward RAW264.7 and Caco-2 cells. After LPS stimulation, inflammatory responses in RAW264.7 cells were successfully induced. Compared with the model group, FS treatment from different groups reduced NO levels to a certain extent; however, inflammatory indicators such as iNOS and IL-6 were not significantly reversed, suggesting that the direct regulatory effect of FS on the macrophage inflammatory phenotype may be limited. Following H₂O₂ stimulation, the viability of Caco-2 cells decreased and the expression of intestinal barrier-related molecules was impaired. Compared with the model group, FS derived from the Jianwei Lianmei Decoction intervention group slightly improved the expression of tight junction proteins such as ZO-1 in Caco-2 cells; however, the improvement was modest, and some indicators did not reach statistical significance. Conclusion: FS derived from mice after Jianwei Lianmei Decoction intervention showed a relatively weak regulatory effect on the inflammatory response in LPS-induced RAW264.7 cells, but exhibited a potential trend toward improving barrier injury in H₂O₂-induced Caco-2 cells.