知母-黄柏药对抗炎药效部位筛选及作用机制研究
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摘要
目的:筛选知母-黄柏药对的抗炎药效部位,并初步对其抗炎机制进行探讨。方法:制备知母-黄柏药对水煎液,通过D101树脂进行分离,分别用水、30%、60%、95%乙醇-水进行洗脱,制备不同极性部位。采用LPS诱导RAW264.7细胞炎症模型,用不同浓度的药对或各极性部位预处理细胞,MTT法检测细胞凋亡,Griess法检测NO分泌,筛选抗炎活性部位。ELISA法检测炎症因子分泌,Western blot检测炎症相关蛋白COX-2、iNOS蛋白水平及细胞核、细胞质中NF-κB p65含量。结果:知母-黄柏药对及30%醇洗部位能降低LPS诱导RAW264.7细胞分泌NO,其中30%醇洗部位作用更为明显,其他部位无显著作用。30%醇洗部位能显著降低LPS诱导的IL-1β、IL-6、TNF-α分泌及iNOS、COX-2蛋白表达,并促进p65由细胞质进入细胞核。结论:知母-黄柏药对抗炎药效部位为30%醇洗部位,该药效部位可能通过抑制NF-κB信号通路,抑制iNOS、COX-2表达及相关炎症因子的分泌,保护LPS诱导的RAW264.7细胞损伤。
Objective:To screen the anti-inflammatory parts of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair and to study the anti-inflammatory mechanism.Methods:The water-decoction of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair was prepared and separated by D101 resin,the 30%,60%,95% ethanol solution were used to elute,respectively,the different polar sites were prepared.Inflammatory model of RAW264.7 cells was induced by LPS,and cells were pretreated with different concentrations of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair and different polar sites.The cell apoptosis was determined by MTT assay.The NO secretion was detected by Griess method.The anti-inflammatory active sites were screened.Secretion of inflammatory cytokines was determined by ELISA assay.The protein levels of COX-2 and iNOS and the content of NF-κB p65 in nucleus and cytoplasm were determined by Western blot.Results:Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair and 30% ethanol elution site inhibited the NO secretion in RAW264.7 cells induced by LPS.Effection of 30% ethanol elution site was more significant than that of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair.Other parts had no significant effect.30% ethanol elution site inhibited the secretion of cytokines including IL-1β、IL-6 and TNF-α,reduced the protein expressions of iNOS and COX-2,promted p65 into the nucleus.Conclusion:The anti-inflammatory effective fraction of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair was 30% ethanol elution site,the pharmacophore may protect RAW 264.7 cell damage by inhibiting the NF-κB signaling pathway,inhibiting the expressions of iNOS、COX-2 and the secretion of related inflammatory factors.
Objective:To screen the anti-inflammatory parts of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair and to study the anti-inflammatory mechanism.Methods:The water-decoction of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair was prepared and separated by D101 resin,the 30%,60%,95% ethanol solution were used to elute,respectively,the different polar sites were prepared.Inflammatory model of RAW264.7 cells was induced by LPS,and cells were pretreated with different concentrations of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair and different polar sites.The cell apoptosis was determined by MTT assay.The NO secretion was detected by Griess method.The anti-inflammatory active sites were screened.Secretion of inflammatory cytokines was determined by ELISA assay.The protein levels of COX-2 and iNOS and the content of NF-κB p65 in nucleus and cytoplasm were determined by Western blot.Results:Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair and 30% ethanol elution site inhibited the NO secretion in RAW264.7 cells induced by LPS.Effection of 30% ethanol elution site was more significant than that of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair.Other parts had no significant effect.30% ethanol elution site inhibited the secretion of cytokines including IL-1β、IL-6 and TNF-α,reduced the protein expressions of iNOS and COX-2,promted p65 into the nucleus.Conclusion:The anti-inflammatory effective fraction of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair was 30% ethanol elution site,the pharmacophore may protect RAW 264.7 cell damage by inhibiting the NF-κB signaling pathway,inhibiting the expressions of iNOS、COX-2 and the secretion of related inflammatory factors.
引文
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[6] 刘静,党院霞,高英,等.知母-黄柏药对有效部位群改善2型糖尿病大鼠认知功能障碍的药效及机制研究[J].2017,48(19):4037-4043.
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[9] Xie Q,Wu GZ,Yang N,et al.Delavatine A,an unusual isoquinoline alkaloid exerts anti-inflammation on LPS-induced proinflammatory cytokines production by suppressing NF-κB activation in BV-2 microglia[J].Biochem Biophys Res Commun,2018,502(2):202-208.
[10] Cha SH,Hwang Y,Kim KN,et al.Palmitate induces nitric oxide production and inflammatory cytokine expression in zebrafish[J].Fish Shellfish Immunol,2018,79(1):163-167.
[11] Sang W,Zhong Z,Linghu K,et al.Siegesbeckia pubescens Makino inhibits Pam3CSK4-induced inflammation in RAW 264.7 macrophages through suppressing TLR1/TLR2-mediated NF-κB activation[J].Chin Med,2018,13(37):1-10.
[12] 王晓晨,吉爱国.NF-κB信号通路与炎症反应[J].生理科学进展,2014,45(1):68-71.
[13] Natarajan K,Abraham P,Kota R,et al.NF-κB-iNOS-COX2-TNFα inflammatory signaling pathway plays an important role in methotrexate induced small intestinal injury in rats[J].Food Chem Toxicol,2018,118(1):766-783.
[2] Zhao X,Li Y,Huai J,et al.Compatibility effects of herb pair Phellodendri Chinensis Cortex and Anemarrhenae Rhizoma on benign prostatic hyperplasia using targeted metabolomics[J].Biomed Chromatogr,2018,32(10):e4296.
[3] 陈志雄,骆宇戟.知母、黄柏组方治疗解脲脲原体前列腺炎合并精子活力低下症[J].中国临床康复,10(43):76-78.
[4] 黄瑞校.知母黄柏散治疗犬膀胱炎的体会[J].贵州畜牧兽医,2006,30(2):29.
[5] 贺利芳.知母、黄柏、肉桂为主治疗肾阴虚型糖尿病[J].内蒙古中医药,2014,33(31):128.
[6] 刘静,党院霞,高英,等.知母-黄柏药对有效部位群改善2型糖尿病大鼠认知功能障碍的药效及机制研究[J].2017,48(19):4037-4043.
[7] Farzaneh Z,Kalantar K,Iraji A,et al.Inhibition of LPS-induced inflammatory responses by Satureja hortensis extracts in J774.1 macrophages[J].J Immunoassay Immunochem,2018,39(3):274-291.
[8] Sanguigno L,Casamassa A,Funel N,et al.Triticum vulgare extract exerts an anti-inflammatory action in two in vitro models of inflammation in microglial cells[J].PLoS One,2018,13(6):e0197493.
[9] Xie Q,Wu GZ,Yang N,et al.Delavatine A,an unusual isoquinoline alkaloid exerts anti-inflammation on LPS-induced proinflammatory cytokines production by suppressing NF-κB activation in BV-2 microglia[J].Biochem Biophys Res Commun,2018,502(2):202-208.
[10] Cha SH,Hwang Y,Kim KN,et al.Palmitate induces nitric oxide production and inflammatory cytokine expression in zebrafish[J].Fish Shellfish Immunol,2018,79(1):163-167.
[11] Sang W,Zhong Z,Linghu K,et al.Siegesbeckia pubescens Makino inhibits Pam3CSK4-induced inflammation in RAW 264.7 macrophages through suppressing TLR1/TLR2-mediated NF-κB activation[J].Chin Med,2018,13(37):1-10.
[12] 王晓晨,吉爱国.NF-κB信号通路与炎症反应[J].生理科学进展,2014,45(1):68-71.
[13] Natarajan K,Abraham P,Kota R,et al.NF-κB-iNOS-COX2-TNFα inflammatory signaling pathway plays an important role in methotrexate induced small intestinal injury in rats[J].Food Chem Toxicol,2018,118(1):766-783.