Standardized ethanol extract of Tinospora crispa upregulates pro-inflammatory mediators release in LPS-primed U937 human macrophages through stimulation of MAPK, NF-κB and PI3K-Akt signaling networks
Standardized ethanol extract of Tinospora crispa upregulates pro-inflammatory mediators release in LPS-primed U937 human macrophages through stimulation of MAPK, NF-κB and PI3K-Akt signaling networks
复制标题
DOI：
10.1186/s12906-020-03039-7
复制
发表时间：
2020-08-06
影响因子：
3.9
通讯作者：
Ahmad, Waqas
中科院分区：
文献类型：
--
作者：
Haque, Md Areeful;Jantan, Ibrahim;Ahmad, Waqas
背景资料：由于其传统用途是治疗与免疫系统相关的几种炎症性疾病，因此研究了青牛胆的免疫调节作用。本研究报道了雷公藤80%乙醇提取物刺激神经元的作用机制。方法：采用酶联免疫吸附试验（ELISA）检测脂多糖（LPS）致敏的U937人巨噬细胞释放白细胞介素（IL）-1 β、肿瘤坏死因子（TNF）-α和前列腺素E-2（PGE（2））的情况。免疫印迹法检测MAPK、NF-κ B、PI 3 K-Akt和考克斯-2蛋白的活化。采用实时定量逆转录聚合酶链反应（qRT-PCR）测定促炎细胞因子和考克斯-2相关基因表达水平。反相高效液相色谱法被开发和验证，以标准化T。结果：色谱数据的定性和定量分析表明，卷柏提取物中主要成分为卷柏苷和木兰花碱。Crispa处理通过增强IKK α/β和NF-κ B（p65）磷酸化以及I κ B α的降解促进NF-κ B的活化。该提取物上调考克斯-2蛋白表达、促炎介质释放和MAPK（ERK、p38和JNK）磷酸化以及Akt的量效依赖性。香葱提取物还上调上游信号衔接分子Toll样受体4（TLR 4）和MyD 88。Crispa处理还上调了促炎标志物mRNA的表达。香葱提取物通过上调各种免疫炎症相关参数刺激MyD 88依赖性信号通路。
Background: Immunomodulatory effects ofTinospora crispahave been investigated due to its traditional use to treat several inflammatory disorders associated to the immune system. The present study reports the underlying mechanisms involved in the stimulation of 80% ethanol extract ofT. crispastems on pro-inflammatory mediators release in lipopolysaccharide (LPS)-primed U937 human macrophages via MyD88-dependent pathways.Methods: Release of interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha, and production of prostaglandin E-2(PGE(2)) were determined by using enzyme-linked immunosorbent assay (ELISA). Immunoblot technique was executed to determine the activation of MAPKs molecules, NF-kappa B, PI3K-Akt and cyclooxygenase-2 (COX-2) protein. Determination of pro-inflammatory cytokines and COX-2 relative gene expression levels was by performing the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). A reversed-phase HPLC method was developed and validated to standardize theT. crispaextract and chemical profiling of its secondary metabolites was performed by LC-MS/MS.Results: Qualitative and quantitative analyses of chromatographic data indicated that syringin and magnoflorine were found as the major components of the extract.T. crispa-treatment prompted activation of NF-kappa B by enhancing IKK alpha/beta and NF-kappa B (p65) phosphorylation, and degradation of I kappa B alpha. The extract upregulated COX-2 protein expression, release of pro-inflammatory mediators and MAPKs (ERK, p38 and JNK) phosphorylation as well as Akt dose-dependently.T. crispaextract also upregulated the upstream signaling adaptor molecules, toll-like receptor 4 (TLR4) and MyD88.T. crispa-treatment also upregulated the pro-inflammatory markers mRNA expression.Conclusion: The results suggested thatT. crispaextract stimulated the MyD88-dependent signaling pathways by upregulating the various immune inflammatory related parameters.