目的 评估基于卟啉结构的新型声敏剂5,10,15,20-四-{4-[(S)-2,6-二氨基己酰胺]苯基}卟啉（4i）介导的声动力疗法（SDT）在小鼠急性肺炎模型中的治疗效果。方法 将4i与2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）溶液混合后超声，观察活性氧（ROS）的生成；体外培养肺癌人类肺泡基底上皮细胞A549和人正常支气管上皮细胞系BEAS-2B，经脂多糖（LPS，500 ng·mL^-1）诱导炎症后，给予4i（16、32、64 mg·mL^-1）联合超声（1.0 MHz、1 W·cm^-2）作用，通过MTT实验和碘化丙啶（PI）染色法检测细胞活性，实时荧光定量PCR（qRT-PCR）法检测p38、细胞外信号调节激酶（ERK） 1/2、c-Jun氨基端激酶（JNK） 1/2 mRNA水平。小鼠通过气管插管吸入50 μL的4i（60、120、180 mg·kg_-1） 2 h后，处死动物并提取其内脏器官，观察4i的分布。建立小鼠急性肺炎（50 μL 0.2 μmol·L^-1 LPS溶液通过鼻腔吸入）模型后3 d，4i通过气管滴注给药，每隔1天给药1次，剂量分别为60、90、120 mg·kg_-1，4i吸入2 h后进行超声照射（1.0 MHz、10 min），共进行4次治疗；动态监测各组小鼠体质量变化；检测小鼠肺功能；采用苏木素-伊红（HE）染色观察肺组织病理学改变，通过Wright-Giemsa染色分析支气管肺泡灌洗液（BALF）中炎症细胞浸润情况，使用ELISA法检测BALF中促炎因子肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6及总蛋白水平。结果 随着4i浓度和声强的增加，生成的ROS也呈增加趋势。细胞实验结果表明，与模型组相比，4i联合SDT处理后，死亡细胞的红色荧光明显增强；细胞存活率显著降低（P＜0.01、0.001），JNK2、p38、ERK1/2 mRNA表达显著降低（P＜0.01、0.001）。动物实验结果表明，4i沿气管给药确保了其在肺部的有效分散和分布，吸入2 h后，肺部4i浓度与荧光强度呈正相关。与对照组比较，模型小鼠从第3天开始体质量下降，随着4i介导的SDT进行逐渐改善；与模型组比较，接受4i介导SDT的小鼠的用力肺活量（FVC）、第一秒用力呼气容积与用力肺活量的比值（FEV₁/FVC）、峰值呼气流速（PEF）显著增加（P＜0.05、0.01、0.001），肺组织病理变化均有不同程度的逆转，炎症评分显著降低（P＜0.001），BALF中炎症细胞数量显著减少（P＜0.001）、TNF-α、IL-6和总蛋白浓度显著降低（P＜0.01、0.001）。结论 新型卟啉衍生物4i可通过SDT显著减轻肺部炎症反应，对急性肺炎具有显著的治疗效果，可能通过影响丝裂原活化蛋白激酶（MAPK）通路发挥作用。

Objective To evaluate the therapeutic effect of a novel porphyrin-based photosensitizer, 5,10,15,20-tetra-{4-[(S)-2,6- diaminohexanoylamino]phenyl}porphyrin (4i), mediated sonodynamic therapy (SDT) in a mouse model of acute pneumonia. Methods 4i was mixed with 2', 7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) solution and then subjected to ultrasound to observe the generation of reactive oxygen species (ROS). Human lung cancer alveolar basal epithelial cells A549 and human normal bronchial epithelial cell line BEAS-2B were cultured in vitro. After induction of inflammation with lipopolysaccharide (LPS, 500 ng·mL^-1), 4i (16, 32, 64 mg·mL^-1) was combined with ultrasound (1.0 MHz, 1 W·cm^-2) treatment. Cell viability was detected by MTT assay and propidium iodide (PI) staining, and the mRNA levels of p38, extracellular signal-regulated kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK) 1/2 were detected by real-time fluorescence quantitative PCR (qRT-PCR). Mice were intratracheally instilled with 50 μL of 4i (60, 120, 180 mg·kg^-1) for 2 h, and then the animals were sacrificed and their internal organs were extracted to observe the distribution of 4i. A mouse model of acute pneumonia was established by intranasal inhalation of 50 μL of 0.2 μmol·L^-1 LPS solution for 3 d. 4i was administered via intratracheal instillation at doses of 60, 90, and 120 mg·kg^-1 every other day, and ultrasound irradiation (1.0 MHz, 10 min) was performed 2 h after 4i inhalation for a total of four treatments. Body weight changes were dynamically monitored in each group. Pulmonary function was detected. Hematoxylin-eosin (HE) staining was used to observe the pathological changes in lung tissue, and Wright-Giemsa staining was used to analyze the infiltration of inflammatory cells in bronchoalveolar lavage fluid (BALF). The levels of pro-inflammatory factors tumor necrosis factor (TNF)-α, interleukin (IL)-6, and total protein in BALF were detected by ELISA. Results With the increase of 4i concentration and sound intensity, the generation of ROS also showed an increasing trend. The results of cell experiments indicated that compared with the model group, the red fluorescence of dead cells was significantly enhanced after 4i combined with SDT treatment; the cell survival rate was significantly decreased (P < 0.01, 0.001), and the mRNA expression of JNK2, p38, and ERK1/2 was significantly decreased (P < 0.01, 0.001). The results of animal experiments showed that intratracheal administration of 4i ensured its effective dispersion and distribution in the lungs. Two hours after inhalation, the concentration of 4i in the lungs was positively correlated with the fluorescence intensity. Compared with the control group, the body weight of model mice decreased from the third day and gradually improved with the administration of 4i-mediated SDT. Compared with the model group, the forced vital capacity (FVC), the ratio of forced expiratory volume in one second to forced vital capacity (FEV₁/FVC), and peak expiratory flow rate (PEF) of mice treated with 4i-mediated SDT were significantly increased (P < 0.05, 0.01, 0.001), and the pathological changes in lung tissue were reversed to varying degrees, the inflammatory score was significantly decreased (P < 0.001), the number of inflammatory cells in BALF was significantly reduced (P < 0.001), and the concentrations of TNF-α, IL-6, and total protein in BALF were significantly decreased (P < 0.01, 0.001). Conclusion The novel porphyrin derivative 4i can significantly mitigate pulmonary inflammatory responses through sonodynamic therapy (SDT), exhibiting notable therapeutic effects on acute pneumonia by alleviating acute lung injury via modulation of the MAPK pathway.

R965

中国医学科学院医学创新基金资助项目（2021-I2M-1-052/2021-I2M-1-015）