我们报告了Nigella sativa (L.)衍生的草药黑色素（HM）在大鼠模型中具有抗胃溃疡作用。然而，HM胃保护作用的分子机制尚不清楚。环氧合酶-2（COX-2）催化的前列腺素E2（PGE2）和Toll样受体4（TLR4）介导的白细胞介素-6（IL-6）的产生和分泌在胃粘膜保护中起着重要作用。本研究使用人胃癌上皮细胞系AGS作为模型，通过免疫细胞荧光染色、免疫印迹技术和逆转录定量聚合酶链反应（RT-qPCR）研究了HM对TLR4、COX-2、黏蛋白4蛋白和基因表达的影响。通过酶联免疫吸附实验（ELISA）评估了胃保护标志物PGE2和IL-6的产生和分泌。细菌脂多糖（LPS），作为TLR4、COX-2、PGE2和IL-6表达的已知诱导物，被用作阳性对照。我们发现，HM上调了AGS细胞中其主要受体TLR4的基因和蛋白表达水平。HM以剂量和时间依赖方式增加了PGE2的分泌量，同时增加了COX-2 mRNA和蛋白的表达量，检测到它们位于AGS细胞的细胞核、细胞质和主要位于细胞间连接处。此外，HM增强了IL-6的产生和分泌，并上调了黏蛋白4基因的表达，这些是胃保护的标志。为了检查HM是否通过TLR4信号和COX-2生成诱导PGE2和IL-6，我们预先用TLR4信号抑制剂TAK242和COX-2抑制剂NS-398预处理了AGS细胞。观察到在TAK242和NS-398预处理的AGS细胞中HM的COX-2、PGE2和IL-6产生和分泌的刺激效应消失，证实了TLR4信号和COX-2生成在HM胃保护作用中的作用。总之，我们的结果表明HM增强了TLR4/COX-2介导的胃保护标志物PGE2和IL-6的分泌，并上调了人胃上皮细胞系AGS的黏蛋白4基因表达，这可能有助于HM在人类胃预防和治疗中具有有益的胃保护作用。© 2023 BioMed Central Ltd.，Springer Nature的一部分。

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.© 2023. BioMed Central Ltd., part of Springer Nature.