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Abstract：
OBJECTIVE: This study aimed at exploring the effects of luteolin on psoriasis-like cell model proliferation, apoptosis regulation and the expression of inflammation-related mediators.
METHODS: A Cell Counting Kit-8 (CCK-8) assay was used to determine the survival rate of human immortalized keratinocytes (HaCaT cells) and normal human epidermal keratinocytes (NHEK cells) following stimulation with luteolin and lipopolysaccharide (LPS). Western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis were used to detect the protein and mRNA expressions of nuclear factor (NF)-κB p65 and interleukin (IL)-6 after LPS stimulation. Then a luteolin stimulation protocol (10 μmol/L, 24 h) was determined and a reasonable LPS stimulation concentration (20 μg/mL, 24 h) was chosen to establish the psoriasis cell model. Keratinocytes in luteolin pre-treatment and control groups were stimulated with 20 μg/mL LPS for 24 h, and the expressions of NF-κB p65 and IL-6 were detected by western blot and RT-qPCR. The apoptosis of HaCaT cells was detected by flow cytometry, and the enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of psoriasis-related inflammatory factors.
RESULTS: CCK-8 assay indicated that luteolin inhibited the proliferation of keratinocytes. LPS stimulated the proliferation of keratinocytes and upregulated the expression of NF-κB p65 and IL-6 in a concentration-dependent manner, and induced psoriasis-like changes. Furthermore, the protein and mRNA expression levels of NF-κB p65 and IL-6 were decreased in the luteolin pre-stimulation group (p < 0.05). Treatment with luteolin downregulated the expression of the LPS-induced inflammatory mediators in keratinocytes (p < 0.05). The flow cytometry results showed that luteolin induced HaCaT cells apoptosis. Finally, ELISA results demonstrated that luteolin inhibited the release of the IL-17, IL-23 and tumor necrosis factor α (TNF-α) in the pre-stimulation group (p < 0.05).
CONCLUSIONS: This study confirmed that luteolin can effectively relieve inflammatory mediators in LPS-induced keratinocyte models of psoriasis, which suggested the potential of luteolin in treating psoriasis.
METHODS: A Cell Counting Kit-8 (CCK-8) assay was used to determine the survival rate of human immortalized keratinocytes (HaCaT cells) and normal human epidermal keratinocytes (NHEK cells) following stimulation with luteolin and lipopolysaccharide (LPS). Western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis were used to detect the protein and mRNA expressions of nuclear factor (NF)-κB p65 and interleukin (IL)-6 after LPS stimulation. Then a luteolin stimulation protocol (10 μmol/L, 24 h) was determined and a reasonable LPS stimulation concentration (20 μg/mL, 24 h) was chosen to establish the psoriasis cell model. Keratinocytes in luteolin pre-treatment and control groups were stimulated with 20 μg/mL LPS for 24 h, and the expressions of NF-κB p65 and IL-6 were detected by western blot and RT-qPCR. The apoptosis of HaCaT cells was detected by flow cytometry, and the enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of psoriasis-related inflammatory factors.
RESULTS: CCK-8 assay indicated that luteolin inhibited the proliferation of keratinocytes. LPS stimulated the proliferation of keratinocytes and upregulated the expression of NF-κB p65 and IL-6 in a concentration-dependent manner, and induced psoriasis-like changes. Furthermore, the protein and mRNA expression levels of NF-κB p65 and IL-6 were decreased in the luteolin pre-stimulation group (p < 0.05). Treatment with luteolin downregulated the expression of the LPS-induced inflammatory mediators in keratinocytes (p < 0.05). The flow cytometry results showed that luteolin induced HaCaT cells apoptosis. Finally, ELISA results demonstrated that luteolin inhibited the release of the IL-17, IL-23 and tumor necrosis factor α (TNF-α) in the pre-stimulation group (p < 0.05).
CONCLUSIONS: This study confirmed that luteolin can effectively relieve inflammatory mediators in LPS-induced keratinocyte models of psoriasis, which suggested the potential of luteolin in treating psoriasis.
摘要：
目的：本研究旨在探讨木犀草素对银屑病样细胞模型增殖的影响，细胞凋亡调控和炎症相关介质的表达。
方法:使用细胞计数试剂盒-8(CCK-8)测定法来确定在用木犀草素和脂多糖(LPS)刺激后人永生化角质形成细胞(HaCaT细胞)和正常人表皮角质形成细胞(NHEK细胞)的存活率。采用Westernblot和逆转录-定量聚合酶链反应(RT-qPCR)检测LPS刺激后核因子(NF)-κBp65和白细胞介素(IL)-6蛋白和mRNA的表达。然后采用木犀草素刺激方案(10μmol/L，24h），并确定合理的LPS刺激浓度（20μg/mL，选择24h)树立银屑病细胞模子。木犀草素预处理组和对照组的角质形成细胞用20μg/mLLPS刺激24h，采用Westernblot和RT-qPCR检测NF-κBp65和IL-6的表达。流式细胞术检测HaCaT细胞凋亡,采用酶联免疫吸附试验(ELISA)检测银屑病相关炎症因子的表达。
结果：CCK-8实验表明木犀草素抑制角质形成细胞的增殖。LPS刺激角质形成细胞的增殖，并以浓度依赖的方式上调NF-κBp65和IL-6的表达，并诱导牛皮癣样变化。此外，木犀草素预刺激组NF-κBp65和IL-6的蛋白和mRNA表达水平降低(p<0.05)。用木犀草素处理下调角质形成细胞中LPS诱导的炎症介质的表达(p<0.05)。流式细胞仪检测结果显示木犀草素诱导HaCaT细胞凋亡。最后,酶联免疫吸附试验结果表明,木犀草素抑制预刺激组IL-17、IL-23和肿瘤坏死因子α(TNF-α)的释放(p<0.05)。
结论：本研究证实木犀草素能有效缓解LPS诱导的银屑病角质形成细胞模型的炎症介质，这表明木犀草素治疗银屑病的潜力。
方法:使用细胞计数试剂盒-8(CCK-8)测定法来确定在用木犀草素和脂多糖(LPS)刺激后人永生化角质形成细胞(HaCaT细胞)和正常人表皮角质形成细胞(NHEK细胞)的存活率。采用Westernblot和逆转录-定量聚合酶链反应(RT-qPCR)检测LPS刺激后核因子(NF)-κBp65和白细胞介素(IL)-6蛋白和mRNA的表达。然后采用木犀草素刺激方案(10μmol/L，24h），并确定合理的LPS刺激浓度（20μg/mL，选择24h)树立银屑病细胞模子。木犀草素预处理组和对照组的角质形成细胞用20μg/mLLPS刺激24h，采用Westernblot和RT-qPCR检测NF-κBp65和IL-6的表达。流式细胞术检测HaCaT细胞凋亡,采用酶联免疫吸附试验(ELISA)检测银屑病相关炎症因子的表达。
结果：CCK-8实验表明木犀草素抑制角质形成细胞的增殖。LPS刺激角质形成细胞的增殖，并以浓度依赖的方式上调NF-κBp65和IL-6的表达，并诱导牛皮癣样变化。此外，木犀草素预刺激组NF-κBp65和IL-6的蛋白和mRNA表达水平降低(p<0.05)。用木犀草素处理下调角质形成细胞中LPS诱导的炎症介质的表达(p<0.05)。流式细胞仪检测结果显示木犀草素诱导HaCaT细胞凋亡。最后,酶联免疫吸附试验结果表明,木犀草素抑制预刺激组IL-17、IL-23和肿瘤坏死因子α(TNF-α)的释放(p<0.05)。
结论：本研究证实木犀草素能有效缓解LPS诱导的银屑病角质形成细胞模型的炎症介质，这表明木犀草素治疗银屑病的潜力。
