背景:包括脂氧素A4(LXA4)在内的Oxylipins通过抑制巨噬细胞浸润和瞬时受体电位(TRP)蛋白表达来促进炎症的消退并具有镇痛特性。玉雪必片(YXB)是一种用于缓解炎症性疼痛的传统中成药。我们之前的研究表明,YXB的镇痛作用与抑制外周炎症和调节巨噬细胞浸润有关,但机制尚不清楚。本研究的目的是从炎症消退的角度探讨YXB对完全弗氏佐剂(CFA)诱导的小鼠炎性疼痛模型的作用机制。
方法:分别使用VonFrey测试和热板测试测量机械异常性疼痛阈值和热超敏反应。采用野外试验和尾部悬吊试验分别测量焦虑和抑郁行为。免疫荧光染色和流式细胞术检测CD68+表达和F4/80+CD11b+细胞比例。免疫荧光染色和免疫印迹法检测瞬时受体电位锚蛋白1(TRPA1)的表达。Oxylipins组学分析提供了爪子中Oxylipins的定量数据,采用酶联免疫吸附试验(ELISA)检测LXA4水平。免疫荧光染色检测白三烯A4羟化酶(LTA4H)在小鼠爪子中的表达。观察到左爪注射甲酰肽受体2(FPR2)拮抗剂WRW4和TRPA1激动剂AITC的影响,专注于机械异常性疼痛阈值的表达,CD68+的表达,TRPA1在爪子,和L5脊髓背角的降钙素基因相关肽(CGRP)。
结果:YXB机械性异常疼痛阈值升高,缓解CFA小鼠的热超敏反应和焦虑抑郁行为。它显著减少了爪子中CD68+的数量和F4/80+CD11b+的比例,从而减少巨噬细胞浸润。此外,它减少了TRPA1在爪子和TRPV1在DRG中的表达,导致外周致敏的抑制。通过定量分析,发现YXB可以调节DHA衍生的氧化脂素和LXA4。ELISA结果表明,YXB提高了LXA4的水平,抑制了LAT4H的表达。此外,服用FPR2拮抗剂后,YXB的促消退和镇痛作用受到阻碍.与AITC组相比,YXB在抗炎和镇痛作用方面没有显着改善。
结论:YXB可以调节CFA小鼠爪子的氧化脂素,促进炎症的消退。LXA4-FPR2-TRPA1通路是决议炎症和镇痛感化的症结机制。
BACKGROUND: Oxylipins including lipoxin A4 (LXA4) facilitate the resolution of inflammation and possess analgesic properties by inhibiting macrophage infiltration and transient receptor potential (TRP) protein expression. Yu-Xue-Bi Tablet (YXB) is a traditional Chinese patent medicine used to relieve inflammatory pain. Our previous research has shown that the analgesic effect of YXB is related to inhibiting peripheral inflammation and regulating macrophage infiltration, but the mechanism is not yet clear. The purpose of this study is to explore the mechanisms of YXB on mice models with Complete Freund\'s Adjuvant (CFA)-induced inflammatory pain from the perspective at the resolution of inflammation.
METHODS: Mechanical allodynia thresholds and heat hypersensitivity were measured using the Von Frey test and the hot plate test respectively. The open field test and the tail suspension test were employed to measure anxiety and depressive behaviors respectively. The expression of CD68+ and the proportion of F4/80+CD11b+ cells were measured by immunofluorescence staining and flow cytometry. The expression of transient receptor potential ankyrin 1(TRPA1) was measured by immunofluorescence staining and western blotting. Oxylipins omics analysis provided quantitative data on oxylipins in the paws, and enzyme linked immunosorbent assay (ELISA) was used to measure the levels of LXA4 there. Immunofluorescence staining was used to perform the expression of Leukotriene A4 hydroxylase (LTA4H) in the paws of mice. The impact of injecting the formyl peptide receptor 2(FPR2) antagonist WRW4 and the TRPA1 agonist AITC into the left paws was observed, focusing on the expression of mechanical allodynia thresholds, the expression of CD68+, TRPA1 in the paws, and Calcitonin gene-related peptide (CGRP) in the L5 spinal dorsal horn.
RESULTS: YXB elevated mechanical allodynia thresholds, alleviated heat hypersensitivity and anxiety and depressive behaviors in CFA mice. It significantly reduced the number of CD68+ and proportion of F4/80+CD11b+ within the paws, thereby decreasing macrophage infiltration. Additionally, it diminished the expression of TRPA1 in the paws and TRPV1 in the DRG, leading to an inhibition of peripheral sensitization. Through quantitative analysis, it was found that YXB could modulate DHA-derived oxylipins and LXA4. ELISA results indicated that YXB elevated the levels of LXA4 and inhibited the expression of LAT4H in the paws. Furthermore, the pro-resolution and analgesic effects of YXB were hindered after administration of the FPR2 antagonist. Compared with the AITC group, YXB showed no significant improvement in anti-inflammatory and analgesic effects.
CONCLUSIONS: YXB can regulate the oxylipins of paws in CFA mice to promote the resolution of inflammation. The LXA4-FPR2-TRPA1 pathway is a key mechanism for the resolution of inflammation and analgesic effects.