目的 探究厚樸酚抑制瞬時受體電位香草素4（TRPV4）活性緩解脂多糖（LPS）誘導小鼠急性肺水腫的作用。方法 SPF級昆明小鼠隨機分為6組：對照組、模型組、鹽酸多巴酚丁胺(DBT，陽性藥，5 mg·kg^-1)組和厚樸酚低、中、高劑量(3.3、10.0、30.0 mg·kg^-1)組。除對照組外，其他各組建立LPS誘導的小鼠肺水腫模型，LPS滴注前30 min和滴注后8 h分別ip給藥1次，檢測肺濕/干質(zhì)量比、病理損傷（HE染色）；試劑盒法檢測肺泡灌洗液腫瘤壞死因子（TNF）-α、白細胞介素（IL）-6、IL-1β及總蛋白水平；伊文思藍法檢測肺血管通透性；免疫熒光法檢測緊密連接蛋白閉鎖小帶蛋白-1（ZO-1）表達。使用炔基修飾厚樸酚探針在小鼠肺組織切片和肺微血管內(nèi)皮細胞（PMVEC）中驗證厚樸酚與TRPV4共定位。體外培養(yǎng)PMVEC，分為對照組、模型組、GSK219(TRPV4抑制劑，10μmol·L^-1)組和厚樸酚低、中、高濃度(0.1、1.0、10.0μmol·L^-1)組，除對照組外使用LPS (10μmol·L^-1)處理24 h誘導損傷，造模的同時給藥，活細胞鈣成像檢測胞內(nèi)Ca²⁺濃度，鬼筆環(huán)肽染色觀察細胞骨架，Western blotting檢測ZO-1蛋白表達，Transwell實驗檢測跨內(nèi)皮電阻（TEER）。結果 與對照組比較，厚樸酚(30 mg·kg^-1)顯著降低肺濕/干質(zhì)量比（P<0.05）、肺泡灌洗液總蛋白（P<0.05）及炎癥因子水平（P<0.01），減輕肺組織病理損傷、血管滲漏及逆轉(zhuǎn)ZO-1缺失。厚樸酚探針與TRPV4共定位良好。與對照組比較，厚樸酚(1μmol·L^-1)顯著抑制LPS誘導的TRPV4介導Ca²⁺超載（P<0.001），減輕F-actin應力纖維收縮和細胞間連接斷裂，上調(diào)ZO-1表達（P<0.01），恢復TEER（P<0.001）。結論 厚樸酚通過抑制TRPV4活性，調(diào)控肺微血管內(nèi)皮細胞鈣穩(wěn)態(tài)和內(nèi)皮屏障功能，有效緩解LPS誘導的急性肺水腫，為闡釋中藥厚樸“燥濕”功效提供科學依據(jù)。

Objective To investigate the effect and mechanism of magnolol in alleviating lipopolysaccharide（LPS）-induced acute pulmonary edema in mice through inhibition of transient receptor potential vanillin 4（TRPV4） activity. Methods SPF-grade Kunming mice were randomly divided into six groups: control group, model group, dobutamine hydrochloride(DBT, positive drug, 5 mg·kg^-1) group, and the magnolol low-, medium-, and high-dose(3.3, 10.0, 30.0 mg·kg^-1) groups. Except for control group, LPSinduced mouse pulmonary edema models were established in the other groups. Drugs were administered ip once 30 min before LPS injection and 8 h after LPS injection. The lung wet/dry weight ratio, pathological damage（HE staining）, and levels of tumor necrosis factor（TNF）-α, interleukin（IL）-6, IL-1β, and total protein in bronchoalveolar lavage fluid were detected. The lung vascular permeability was measured by Evans blue method, and the expression of tight junction protein zonula occludens-1（ZO-1） was detected by immunofluorescence. The co-localization of magnolol and TRPV4 was verified in mouse lung tissue sections and pulmonary microvascular endothelial cells（PMVEC） using an alkynyl-modified magnolol probe. PMVEC were cultured in vitro and divided into control group, model group, GSK219(TRPV4 inhibitor, 10 μmol·L^-1) group, and magnolol low-, medium-, and high-concentration(0.1, 1.0, 10.0 μmol·L^-1) groups. Except for control group, cells were treated with LPS(10 μmol·L^-1) for 24 h to induce injury, and drugs were administered simultaneously. Intracellular Ca²⁺ concentration was detected by live cell calcium imaging, F-actin stress fiber contraction and intercellular junction disruption were observed by phalloidin staining, and the expression of ZO-1 protein was detected by Western blotting. Transwell assay was used to detect transendothelial electrical resistance（TEER）. Results Compared with the control group, magnolol(30 mg·kg^-1) significantly reduced the lung wet/dry weight ratio（P＜0.05）, total protein in bronchoalveolar lavage fluid（P＜0.05）, and levels of inflammatory factors（P＜0.001）, alleviated lung tissue pathological damage, vascular leakage, and reversed ZO-1 deficiency. The magnolol probe co-localized well with TRPV4. Compared with the control group, magnolol(1 μmol·L^-1) significantly inhibited LPS-induced TRPV4-mediated Ca²⁺ overload（P＜0.001）, alleviated F-actin stress fiber contraction and intercellular junction disruption, upregulated ZO-1 expression（P＜0.01）, and restored TEER（P＜0.001）. Conclusion Magnolol alleviates LPS-induced acute pulmonary edema by inhibiting TRPV4 activity, thereby regulating calcium homeostasis and endothelial barrier function in PMVECs. This study provides scientific evidence elucidating the "drying-dampness" efficacy of the traditional Chinese medicine Magnolia officinalis.

R285.5

廣西“帶土移植”人才引育計劃項目(桂科AA23026008); 天津市大學生創(chuàng)新科研計劃項目(202410055330)