Yang Qingxin, Liu Qi, Lv Hongjun, Wang Feng, Liu Rong, Zeng Nan
Department of Pharmacology, College of Pharmacy, Chengdu University of TCM, Chengdu, Sichuan 611137, P.R. China.
Department of Pharmacology, Sichuan College of Traditional Chinese Medicine, Mianyang, Sichuan 621000, P.R. China.
Exp Ther Med. 2020 Feb;19(2):1304-1312. doi: 10.3892/etm.2019.8327. Epub 2019 Dec 13.
Pulegone is a key active component of Schizonepeta essential oil and has been determined to have anti-inflammatory properties. However, the underlying molecular mechanisms with regard to the NLR family pyrin domain containing 3 (NLRP3) inflammasome, also known as the NALP3 inflammasome, have remained to be elucidated. NLRP3 represents a potential link between inflammation and immunity and may play possible key role in various pathologies. In the present study, the modulatory effects of pulegone on the NLRP3 inflammasome were investigated. THP-1 cells induced with phorbol myristate acetate were divided into various groups, including the Normal (control), lipopolysaccharide (LPS), LPS + ATP/nigericin, LPS + ATP/nigericin + 0.2% DMSO and pulegone (0.2, 0.1 and 0.05 mg/ml) groups. ELISA was used to detect the levels of interleukin (IL)-1β and IL-18 in the cell supernatants and the influence of potassium ions was assessed. PCR was used to determine the expression levels of NLRP3, caspase-1, IL-1β and IL-1α in the cell lysates. Furthermore, NLRP3 and apoptosis-associated speck-like protein (ASC) were detected via immunofluorescence assays and fluorescence microscopy was employed to determine the reactive oxygen species (ROS) levels in the THP-1 cells. The results indicated reduced levels of IL-18 and IL-1β in the supernatant of the cells of the pulegone groups when compared with those in the LPS + ATP/nigericin group. In addition, reduced mRNA production of inflammasome-associated genes was detected in the cell lysates after pulegone treatment. The immunofluorescence analyses indicated significantly reduced protein expression levels of NLRP3 and ASC in the pulegone groups, as well as co-localization of the NLRP3 and ASC proteins. The pulegone groups also exhibited significantly reduced ROS levels. Furthermore, a high concentration of potassium ions significantly reduced the secretion of IL-1β after induction/stimulation. In conclusion, the present study suggested that pulegone exerts its anti-inflammatory effects on LPS + ATP/nigericin-induced THP-1 cells via inhibition of NLRP3 expression, and its regulatory mechanism is associated with potassium channel and ROS pathways. It was hypothesized that pulegone first inhibits ROS signaling, to then inhibit NLRP3 expression as a downstream event. It appeared that NLRP3 may be situated further downstream and represented the link between inflammation and immunity.
胡薄荷酮是荆芥精油的关键活性成分，已被确定具有抗炎特性。然而，关于含NLR家族pyrin结构域3（NLRP3）炎性小体（也称为NALP3炎性小体）的潜在分子机制仍有待阐明。NLRP3是炎症与免疫之间的潜在联系，可能在各种病理过程中发挥关键作用。在本研究中，研究了胡薄荷酮对NLRP3炎性小体的调节作用。用佛波酯诱导的THP-1细胞被分为不同组，包括正常（对照）组、脂多糖（LPS）组、LPS + ATP/尼日利亚菌素组、LPS + ATP/尼日利亚菌素 + 0.2%二甲基亚砜组以及胡薄荷酮（0.2、0.1和0.05 mg/ml）组。采用酶联免疫吸附测定（ELISA）检测细胞上清液中白细胞介素（IL）-1β和IL-18的水平，并评估钾离子的影响。采用聚合酶链反应（PCR）测定细胞裂解物中NLRP3、半胱天冬酶-1、IL-1β和IL-1α的表达水平。此外，通过免疫荧光测定检测NLRP3和凋亡相关斑点样蛋白（ASC），并采用荧光显微镜测定THP-1细胞中的活性氧（ROS）水平。结果表明，与LPS + ATP/尼日利亚菌素组相比，胡薄荷酮组细胞上清液中IL-18和IL-1β水平降低。此外，胡薄荷酮处理后，在细胞裂解物中检测到炎性小体相关基因的mRNA产生减少。免疫荧光分析表明，胡薄荷酮组中NLRP3和ASC的蛋白表达水平显著降低，以及NLRP3和ASC蛋白的共定位。胡薄荷酮组的ROS水平也显著降低。此外，高浓度钾离子在诱导/刺激后显著降低IL-1β的分泌。总之，本研究表明，胡薄荷酮通过抑制NLRP3表达对LPS + ATP/尼日利亚菌素诱导的THP-1细胞发挥抗炎作用，其调节机制与钾通道和ROS途径有关。据推测，胡薄荷酮首先抑制ROS信号传导，进而作为下游事件抑制NLRP3表达。似乎NLRP3可能位于更下游，代表炎症与免疫之间的联系。
