【作者】 黄亮；

【导师】 黄恺；

【作者基本信息】 华中科技大学 ， 内科学（心血管病学）， 2019， 硕士

【摘要】 目的:地黄根是一种在东亚国家广泛应用的中草药,具有抗炎、抗过敏的功效。然而地黄根的重要成分梓醇在RAW264.7细胞中的作用及其机制尚不清楚。因此,本研究旨在探究梓醇是否对脂多糖诱导的RAW264.7细胞炎症反应有抑制作用及其可能的机制,其意义在于为全身炎症反应或者代谢综合征慢性炎症等炎症相关疾病的治疗提供理论基础与思路。方法:培养小鼠单核巨噬细胞系RAW264.7细胞。（1）将RAW264.7细胞分为6组,即正常对照组、LPS组（10ng/ml）、LPS+梓醇（25μmol/L）组、LPS+梓醇（50μmol/L）组、LPS+梓醇（100μmol/L）组、单用梓醇（100μmol/L）组。（2）MMT试验检测不同药物浓度处理时的细胞活性。（3）实时荧光定量PCR（qRT-PCR）检测各组细胞炎症因子TNFα、IL-1β、IL-6 mRNA的表达量。（4）用ELISE试剂盒检测各组细胞上清中IL-1β、IL-6、TNFα、PGE₂表达量。格里斯试剂检测各组细胞培养基中NO的含量。（5）蛋白印迹法（western-blot）检测各组iNOS、COX-2、ERK、p-ERK、JNK、p-JNK、p38、p-p38、p65、p-p65、IκBα、p-IκBα的蛋白表达量。（6）细胞免疫荧光检测各组RAW264.7细胞中p65的表达和分布情况。结果:（1）MTT试验发现梓醇对RAW264.7细胞的活性无不良影响。（2）qRT-PCR结果表明LPS刺激可以明显增加RAW264.7细胞炎症因子IL-1β、IL-6、TNFαmRNA的表达,梓醇处理可明显下调IL-1β、IL-6 mRNA的表达且浓度愈高下降趋势愈加明显;只有当梓醇浓度达到100μM时才能抑制TNFαmRNA的表达。（3）IL-1β、IL-6、TNFα因子ELISE检测表明,在LPS刺激下RAW264.7细胞上清中IL-1β、IL-6、TNFα、PGE₂分泌量明显增多,梓醇可以改善这一作用,使IL-1β、IL-6、PGE₂表达量降低,同mRNA的表达一致,只有当梓醇浓度达到100μM上清中TNFα的表达才明显下降。格里斯试剂检测细胞上清中NO的表达发现LPS刺激时明显升高,药物处理组则呈现下降趋势且呈现浓度依赖性。（4）Western-blot结果表明在LPS刺激下与空白对照组比较iNOS、COX-2蛋白表达量升高,梓醇处理组与LPS对照其表达量明显下调。LPS作用下与对照组比较,p-p65、p-IкBα蛋白表达量明显升高,IкBα的表达量则明显下降,梓醇处理组与LPS组比较,p-p65、p-IкBα蛋白表达量显着下降,而IкBα的表达量增加。此外MAPK通路蛋白的检测发现LPS刺激后与对照组比较p-ERK、p-JNK、p-p38的表达增加,药物处理组与LPS组比较p-ERK、p-p38的表达随着给药浓度的增加呈梯度下降,而当给药浓度为25μM、50μM时p-JNK的表达无显著变化,当给药浓度达到100μM时出现明显下降。（5）细胞免疫荧光检测显示在LPS作用下与对照组比较,RAW264.7细胞p65的表达增多且进入细胞核中增多,药物处理组与LPS组比较发现p65的表达和入核均减少。结论:以上结果表明梓醇可以通过抑制NF-κB通路的活化来缓解脂多糖诱导的RAW264.7细胞炎症反应。此外MAPK信号通路中的p38、ERK在梓醇的抗炎反应中也起作用,而JNK的作用不甚明显。更多还原

【Abstract】 Objective: Rehmanniae Radix is a traditional herbal medicine widely used in East Asia that has anti-inflammatory and anti-allergic effects.Catalpol is the primary active component of Rehmanniae Radix.However,it′s effect on RAW264.7 cells and the underlying molecular mechanisms remained poorly understood.This study aimed to assess the anti-inflammation effect of catalpol on lipopolysaccharide-induced RAW264.7 cells and explore the potential mechanisms.Its significance is to provide theoretical basis and ideas for the treatment of inflammatory diseases such as systemic inflammatory response or chronic inflammation of metabolic syndrome.Methods: Mouse mononuclear macrophage RAW264.7 cells were cultured.(1)RAW264.7 cells were divided into six groups: Control,LPS(10ng/ml),LPS + catapol(25μmol/L),LPS + catapol(50μmol/L),LPS + catapol(100μmol/L),catapol(100μmol/L).(2)MTT assay was used to detect cell viability.(3)The messenger RNA(m RNA)expression of IL-1β、IL-6、TNFα was measured by real-time PCR.(4)The expression of IL-1β、IL-6、TNFα、PGE2 in cell supernatant was detected by ELISE kits.(5)The protein expression of i NOS、COX-2、ERK、p-ERK、JNK、p-JNK、p38、p-p38、p65、p-p65、IκBα、p-IкBα was detected by western blot.(6)The expression and distribution of p65 in RAW264.7 cells were detected by cell immunofluorescence.Result:(1)The MTT assay showed that catalpol had no adverse effect on the viability of RAW264.7 cells.(2)LPS significantly stimulated the m RNA expression of IL-1β、IL-6、TNFα in RAW264.7.Catalpol significantly inhibited the LPS-induced m RNA expression of IL-1β、IL-6 but inhibited TNFα m RNA expression only when the drug concentration reached 100 μM.(3)The results of ELISE showed that LPS evidently induced secretion of IL-1β、IL-6、PGE2,which was significantly inhibited by catalpol.However,the inhibitory effect on TNFα appeared when the drug concentration reached 100 μM.The expression of NO in cell supernatant detected by griss reagent was significantly increased in LPS group while decreased in drug group in a concentration dependence.(4)Catalpol significantly decreased LPS-induced protein expression of i NOS 、 COX-2.Furthermore,catalpol pretreatment restored LPS-induced increase of p-p65、p-IκBα、p-p38 、p-ERK dose-dependently,but influenced the JNK activation when the drug concentration reached 100 μM.(5)Cell immunofluorescence assay showed that the expression and nuclear translocation of p65 increased in LPS group compared with control group in RAW264.7 cells.However,the expression and nuclear translocation of p65 decreased in drug group compared with LPS group.Conclusions: Results above indicated that catalpol alleviated LPS-induced inflammatory response by inhibiting the activation of NF-κB pathways and partial MAPK pathways.更多还原