目的 基于瞬時(shí)受體電位A1（TRPA1）設(shè)計(jì)開發(fā)一種具有改善特應(yīng)性皮炎（AD）作用的天然活性成分乳膏劑。方法 構(gòu)建TRPA1通道高表達(dá)細(xì)胞，采用細(xì)胞熒光鈣離子成像技術(shù)從31種活性天然產(chǎn)物中篩選TRPA1抑制劑，并篩選最佳組合以及比例；通過單因素實(shí)驗(yàn)和響應(yīng)面分析優(yōu)化乳膏劑中甘油、聚丙烯酸鈉（SP）、辛酸/癸酸甘油三脂（M318）的添加量。構(gòu)建大鼠皮膚紫外線B（UVB）曬傷模型，通過檢測皮膚機(jī)械痛閾評(píng)價(jià)乳膏劑對(duì)痛覺的緩解作用。通過2，4-二硝基氟苯（DNFB）誘導(dǎo)構(gòu)建小鼠AD模型，評(píng)價(jià)乳膏劑對(duì)瘙癢、皮損評(píng)分、皮膚病理損傷以及皮膚中白細(xì)胞介素（IL）-6、IL-4、IL-33、IL-1β、γ干擾素（IFN-γ）、腫瘤壞死因子α（TNF-α）炎癥因子mRNA的表達(dá)水平的改善效果。結(jié)果 篩選出對(duì)TRPA1具有抑制作用的9種活性天然產(chǎn)物，發(fā)現(xiàn)白藜蘆醇與和厚樸酚物質(zhì)的量比值為5∶1時(shí)協(xié)同效果最佳，確定為白藜蘆醇與和厚樸酚雙因組合（BHSY）。經(jīng)配方優(yōu)化BHSY乳膏劑的最佳組成為：甘油5.13%、SP 0.91%、M318 3.01%，其中BHSY質(zhì)量分?jǐn)?shù)為0.17%～0.50%，其粒徑分布均勻且穩(wěn)定性好。與模型組比較，BHSY乳膏劑在UVB照射損傷后的第2、3天可劑量相關(guān)性地提高大鼠皮膚痛閾（P＜0.05、0.01、0.001）；并可明顯降低AD小鼠的搔抓次數(shù)（P＜0.05、0.01），改善皮損評(píng)分（P＜0.05、0.01），抑制背部表皮增厚（P＜0.001）及皮膚中炎癥因子IL-6、IL-4、IL-33、IL-1β、INF-γ、TNF-α mRNA的表達(dá)（P＜0.05、0.01、0.001）。結(jié)論 基于TRPA1通道開發(fā)了具有協(xié)同增效作用的活性天然產(chǎn)物BHSY乳膏劑，對(duì)減輕皮膚的UVB損傷以及舒緩AD癥狀具有顯著效果。

Objective Based on transient receptor potential A1 (TRPA1), a natural active ingredient cream preparation with the effect of improving atopic dermatitis (AD) was developed and optimized. Methods TRPA1 overexpressed cells were constructed to be used in the screening of TRPA1 inhibitors from 31 natural compounds by cell fluorescence calcium ion imaging, and the optimal combination and proportion were screened. The addition amounts of glycerol, sodium polyacrylate (SP), and glyceryl tristearate (M318) in the cream were optimized through single-factor experiments and response surface analysis. A rat skin UVB sunburn model was established to evaluate the analgesic effect of the cream by detecting the mechanical pain threshold of the skin. A mouse AD model was induced by 2, 4-dinitrofluorobenzene (DNFB) to assess the improvement effect of the cream on pruritus, skin lesion score, skin pathological damage, and the mRNA expression levels of inflammatory factors such as interleukin (IL)-6, IL-4, IL-33, IL-1β, interferon-γ (IFN-γ), and tumor necrosis factor α (TNF-α) in the skin. Results Nine active natural products with inhibitory effects on TRPA1 were screened out. It was found that the best synergistic effect was achieved when the molar ratio of resveratrol to magnolol was 5∶1, and this combination was determined as the resveratrol and magnolol dual-component combination (BHSY). After formula optimization, the optimal composition of the BHSY cream was: glycerol 5.13%, SP 0.91%, M318 3.01%, with BHSY content ranging from 0.17% to 0.50%. The particle size distribution was uniform and the stability was good. Compared with the model group, the BHSY cream could dose-dependently increase the mechanical pain threshold of rat skin on the 2nd and 3rd days after UVB irradiation injury (P < 0.05, 0.01, 0.001); it could also significantly reduce the scratching frequency of AD mice (P < 0.05, 0.01), improve the skin lesion score (P < 0.05, 0.01), inhibit the thickening of the epidermis on the back (P < 0.001), and reduce the mRNA expression of inflammatory factors such as IL-6, IL-4, IL-33, IL-1β, INF-γ, and TNF-α in the skin (P < 0.05, 0.01, 0.001). Conclusion Based on the TRPA1 channel, an active natural product BHSY cream with synergistic effects has been developed, which demonstrates significant efficacy in alleviating UVB-induced skin damage and mitigating AD symptoms.

R285.5

廣西科技廳“帶土移植”人才引育計(jì)劃(AA23026008)