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熊夷, 易琼, 王壮雄, 邓丽敏, 杨璐琪, 彭筱平.祛风化痰稳斑汤抑制NLRP3/Caspase-1/GSDMD信号通路调控VSMC焦亡的机制研究[J].湖南中医药大学学报,2026,46(7):1365-1378[点击复制] |
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| 祛风化痰稳斑汤抑制NLRP3/Caspase-1/GSDMD信号通路调控VSMC焦亡的机制研究 |
| 熊夷,易琼,王壮雄,邓丽敏,杨璐琪,彭筱平 |
| (湖南中医药大学, 湖南 长沙 410208;湖南中医药大学第一附属医院, 湖南 长沙 410007;湖南中医药高等专科学校第一附属医院, 湖南 株洲 412000;丰城市中医院, 江西 丰城 331100) |
| 摘要: |
| 目的 在“从风论治”的中医治疗原则指导下，探讨祛风化痰稳斑汤能否通过抑制NOD样受体热蛋白结构域相关蛋白3（NLRP3）半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）/消皮素D（GSDMD）信号通路调控血管平滑肌细胞（VSMC）焦亡。方法 将56只新西兰兔随机分为空白组（8只）和造模组（48只），造模组采用高脂饮食联合牛血清白蛋白（125 mg·kg-1，每天1次，共3次）、卵清白蛋白（每次2.5 mg·kg-1，每2天1次，共5次）注射建立动脉粥样硬化（AS）模型。造模成功后，将48只造模动物均分为6组（每组8只）：模型组、祛风化痰稳斑汤低剂量组（5.8 g·kg-1·d-1灌胃）、祛风化痰稳斑汤中剂量组（11.6 g·kg-1·d-1灌胃）、祛风化痰稳斑汤高剂量组（23.2 g·kg-1·d-1灌胃）、阿托伐他汀钙组（1.1 mg·kg-1·d-1灌胃）、NLRP3抑制剂组（MCC950首剂31.2 mg·kg-1，后续每2天1次、每次1.5 mg·kg-1腹腔注射）。空白组与模型组灌服等体积0.9%氯化钠注射液，共干预8周。体外培养兔颈动脉VSMC，设置5组干预：空白组以正常DMEM完全培养基培养24 h；其余4组均采用200 μg·mL-1氧化型低密度脂蛋白（OX-LDL）处理24 h，构建AS斑块VSMC损伤模型，在此基础上分别施加对应干预：模型组不额外给药，祛风化痰稳斑汤含药血清组加入20%浓度的祛风化痰稳斑汤高剂量含药血清，NLRP3激动剂组加入1 μmol/L BMS-986299，NLRP3抑制剂组加入1 μmol/L MCC950。HE染色、天狼星红染色观察主动脉组织病理形态学变化；免疫组织化学、油红O染色及天狼星红染色计算斑块易损指数（VI）；透射电镜观察VSMC焦亡相关形态改变；ELISA测定主动脉组织和VSMC中Ⅰ型胶原蛋白（COLⅠ）、Ⅲ型胶原蛋白（COLⅢ）表达水平及COLⅠ/COLⅢ的比值；Western Blot检测主动脉组织和VSMC中NLRP3、Caspase-1、GSDMD-N、白细胞介素（IL）-1β、IL-18、凋亡相关斑点样蛋白（ASC）蛋白表达水平；qRT-PCR检测主动脉组织和VSMC中Caspase-1、IL-1β、IL-18的mRNA表达水平。结果 体内实验结果显示，与空白组比较，模型组VI和NLRP3、Caspase-1、GSDMD-N、IL-1β、IL-18、ASC蛋白表达水平以及Caspase-1、IL-1β、IL-18 mRNA表达水平均升高（P<0.05），COLⅠ表达水平、COLⅠ/COLⅢ比值均降低（P<0.05）。与模型组比较，祛风化痰稳斑汤低、中、高剂量组和阿托伐他汀钙组、NLRP3抑制剂组VI和NLRP3、Caspase-1、IL-1β、IL-18及ASC蛋白表达水平以及IL-1β、IL-18 mRNA表达水平均降低（P<0.05），祛风化痰稳斑汤中、高剂量组及NLRP3抑制剂组GSDMD-N蛋白表达水平均降低（P<0.05）；祛风化痰稳斑汤中、高剂量组和阿托伐他汀钙组、NLRP3抑制剂组Caspase-1 mRNA表达水平均降低（P<0.05）；祛风化痰稳斑汤低、中、高剂量组和阿托伐他汀钙组、NLRP3抑制剂组COLⅠ表达水平、COLⅠ/COLⅢ比值均升高（P<0.05）；祛风化痰委稳斑汤中剂量组COLⅢ表达水平降低（P<0.05）。与祛风化痰稳斑汤低剂量组比较，祛风化痰稳斑汤中、高剂量组和阿托伐他汀钙组、NLRP3抑制剂组VI和NLRP3、Caspase-1、IL-1β、ASC蛋白表达水平以及Caspase-1、IL-1β、IL-18 mRNA表达水平均降低（P<0.05）；祛风化痰稳斑汤中、高剂量组、NLRP3抑制剂组GSDMD-N蛋白表达水平均降低（P<0.05）；祛风化痰稳斑汤中、高剂量组和NLRP3抑制剂组COLⅠ表达水平、COLⅠ/COLⅢ均升高（P<0.05）；祛风化痰稳斑汤中剂量组COLⅢ表达水平降低（P<0.05）。与祛风化痰稳斑汤中剂量组比较，祛风化痰稳斑汤高剂量组、阿托伐他汀钙组、NLRP3抑制剂组VI和Caspase-1、IL-18 mRNA表达水平均降低（P<0.05）；NLRP3抑制剂组Caspase-1、GSDMD-N、IL-1β蛋白表达水平和IL-1β mRNA表达水平均降低（P<0.05），COLⅠ表达水平升高（P<0.05）。与祛风化痰稳斑汤高剂量组比较，阿托伐他汀钙组、NLRP3抑制剂组VI、IL-18 mRNA表达水平均降低（P<0.05）；NLRP3抑制剂组Caspase-1、GSDMD-N、ASC蛋白表达水平和IL-1β mRNA表达水平均降低（P<0.05），COLⅠ表达水平升高（P<0.05）。与阿托伐他汀钙组比较，NLRP3抑制剂组VI和NLRP3、Caspase-1、GSDMD-N、IL-1β、ASC蛋白表达水平以及IL-1β、Caspase-1 mRNA表达水平均降低（P<0.05），COLⅠ表达水平、COLⅠ/COLⅢ比值均升高（P<0.05）。体外结果显示，与空白组比较，模型组COLⅠ表达水平、COLⅠ/COLⅢ比值均降低（P<0.05），COLⅢ表达水平和NLRP3、Caspase-1、GSDMD-N、IL-1β、IL-18、ASC蛋白表达水平以及Caspase-1、IL-1β及IL-18 mRNA表达水平均升高（P<0.05）。与模型组比较，祛风化痰稳斑汤含药血清组、NLRP3抑制剂组COLⅠ表达水平及COLⅠ/COLⅢ比值均升高（P<0.05），COLⅢ表达水平和NLRP3、Caspase-1、GSDMD-N、IL-1β、IL-18、ASC 蛋白表达水平以及Caspase-1、IL-1β、IL-18 mRNA表达水平均降低（P<0.05）；NLRP3激动剂组COLⅠ表达水平及COLⅠ/COLⅢ比值均降低（P<0.05），COLⅢ表达水平和NLRP3、Caspase-1、GSDMD-N、IL-1β、IL-18、ASC蛋白表达水平以及Caspase-1、IL-1β及IL-18 mRNA表达水平均升高（P<0.05）。与祛风化痰稳斑汤含药血清组比较，NLRP3激动剂组COLⅠ表达水平及COLⅠ/COLⅢ比值均降低（P<0.05），COLⅢ表达水平和NLRP3、Caspase-1、GSDMD-N、IL-1β、IL-18、ASC蛋白表达水平以及Caspase-1、IL-1β及IL-18 mRNA表达水平均升高（P<0.05）。与NLRP3激动剂组比较，NLRP3抑制剂组COLⅠ表达水平、COLⅠ/COLⅢ比值均升高（P<0.05），COLⅢ表达水平和NLRP3、Caspase-1、GSDMD-N、IL-1β、IL-18、ASC 蛋白表达水平以及Caspase-1、IL-1β、IL-18 mRNA表达水平均降低（P<0.05）。结论 祛风化痰稳斑汤可能通过调控NLRP3/Caspase-1/GSDMD信号通路抑制VSMC焦亡，为“从风论治”干预AS斑块形成提供了实验依据，其具体活性成分及直接靶点有待进一步阐明。 |
| 关键词: 祛风化痰稳斑汤|动脉粥样硬化|NLRP3/Caspase-1/GSDMD信号通路|细胞焦亡|从风论治 |
| DOI：10.3969/j.issn.1674-070X.2026.07.007 |
| 投稿时间：2026-03-16 |
| 基金项目:湖南省中医药科研计划项目（A2023028）；湖南省自然科学基金项目（2023JJ60045）；湖南省“十四五”第一批中医药学科带头人项目（湘中医药函〔2022〕4号）。 |
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| Mechanism investigation of Qufeng Huatan Wenban Decoction in regulating VSMC pyroptosis by inhibiting the NLRP3/Caspase-1/ GSDMD signaling pathway |
| XIONG Yi, YI Qiong, WANG Zhuangxiong, DENG Limin, YANG Luqi, PENG Xiaoping |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, 410007 Hunan, China;The First Affiliated Hospital of Hunan Traditional Chinese Medical College, Zhuzhou, Hunan 412000, China;Fengcheng Hospital of Traditional Chinese Medicine, Fengcheng, Jiangxi 331100, China) |
| Abstract: |
| Objective Guided by the Chinese medicine (TCM) therapeutic principle of "treating from wind", to investigate whether Qufeng Huatan Wenban Decoction (QHWD) could regulate vascular smooth muscle cell (VSMC) pyroptosis by suppressing the NOD-like receptor family pyrin domain-containing 3 (NLRP3)/Cysteine-aspartic acid protease-1 (Caspase-1)/gasdermin D (GSDMD) signaling pathway. Methods Fifty-six New Zealand rabbits were randomly divided into blank group (n=8) and model group (n=48). In the model group, an atherosclerosis (AS) model was established by a high-fat diet combined with injection of bovine serum albumin (125 mg·kg-1, once daily, 3 doses in total) and ovalbumin (2.5 mg·kg-1 per dose, once every 2 days, 5 doses in total). After successful modeling, the 48 modeled rabbits were equally divided into six groups (n=8): the model group; the low-, medium-, and high-dose QHWD groups (5.8, 11.6, and 23.2 g·kg-1·d-1 by gavage, respectively); the atorvastatin calcium group (1.1 mg·kg-1·d-1 by gavage); and the NLRP3 inhibitor group [MCC950, an initial dose of 31.2 mg·kg-1 followed by 1.5 mg·kg-1 per dose, injected intraperitoneally once every 2 days]. The blank group and the model group were given an equal volume of 0.9% sodium chloride injection by gavage, and the intervention lasted for 8 weeks. Rabbit carotid artery VSMCs were cultured in vitro with five intervention settings: the blank group was cultured in normal DMEM complete medium for 24 h; the other four groups were all treated with 200 μg·mL-1 oxidized low-density lipoprotein (OX-LDL) for 24 h to establish an AS plaque VSMC injury model, on the basis of which the corresponding interventions were applied: the model group received no additional drug treatment, the QHWD drug-containing serum groups were supplemented with high-dose QHWD-containing serum at a concentration of 20%, the NLRP3 agonist group was supplemented with 1 μmol/L BMS-986299, and the NLRP3 inhibitor group was supplemented with 1 μmol/L MCC950. HE staining and Sirius red staining were performed to observe the histopathological and morphological changes of the aortic tissues; immunohistochemistry, Oil Red O staining, and Sirius red staining were used to calculate the plaque vulnerability index (VI); transmission electron microscopy was used to observe the pyroptosis-related morphological changes of VSMCs; ELISA was used to determine the expression levels of collagen types I and Ⅲ (COLⅠ and COLⅢ) and the COLⅠ/COLⅢ ratio in the aortic tissues and VSMCs; Western blot was used to test the protein expression levels of NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-18, and apoptosis-associated speck-like protein (ASC) in the aortic tissues and VSMCs; and qRT-PCR was used to measure the mRNA expression levels of Caspase-1, IL-1β, and IL-18 in the aortic tissues and VSMCs. Results The in vivo results showed that, compared with the blank group, the model group showed elevated VI, protein expression levels of NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18, and ASC, as well as mRNA expression levels of Caspase-1, IL-1β, and IL-18 (P<0.05); conversely, expression level of COLⅠ and the COLⅠ/COLⅢ ratio were reduced (P<0.05). Compared with the model group, the low-, medium-, and high-dose QHWD groups, the atorvastatin calcium group, and the NLRP3 inhibitor group showed attenuated VI, protein expression levels of NLRP3, Caspase-1, IL-1β, IL-18, and ASC, as well as mRNA expression levels of IL-1β and IL-18 (P<0.05); the GSDMD-N protein expression level was downregulated in the medium- and high-dose QHWD groups and the NLRP3 inhibitor group (P<0.05); the Caspase-1 mRNA expression level was decreased in the medium- and high-dose QHWD groups, atorvastatin calcium group, and NLRP3 inhibitor group (P<0.05); expression level of COLⅠ and the COLⅠ/COLⅢ ratio were upregulated in the low-, medium-, and high-dose QHWD groups, atorvastatin calcium group, and NLRP3 inhibitor group (P<0.05); the expression level of COL III in the medium-dose QHWD group was reduced (P<0.05). Compared with the low-dose QHWD group, the medium- and high-dose QHWD groups, atorvastatin calcium group, and NLRP3 inhibitor group exhibited lower VI, protein expression levels of NLRP3, Caspase-1, IL-1β, and ASC as well as mRNA expression levels of Caspase-1, IL-1β, and IL-18 (P<0.05); the GSDMD-N protein expression level was decreased in the medium- and high-dose QHWD groups and the NLRP3 inhibitor group (P<0.05); expression level of COLⅠ and the COLⅠ/COLⅢ ratio were elevated in the medium- and high-dose QHWD groups and the NLRP3 inhibitor group (P<0.05), while expression level of COLⅢ was dropped in the medium-dose QHWD group (P<0.05). Compared with the medium-dose QHWD group, the high-dose QHWD group, the atorvastatin calcium group, and the NLRP3 inhibitor group showed decreased VI and mRNA expression levels of Caspase-1 and IL-18 (P<0.05); the NLRP3 inhibitor group showed reduced protein expression levels of Caspase-1, GSDMD-N, and IL-1β and mRNA expression level of IL-1β (P<0.05), as well as increased expression level of COLⅠ (P<0.05). Compared with the high-dose QHWD group, the atorvastatin calcium group and the NLRP3 inhibitor group showed downregulated VI and mRNA expression level of IL-18 (P<0.05); the NLRP3 inhibitor group demonstrated decreased protein expression levels of Caspase-1, GSDMD-N, and ASC and mRNA expression level of IL-1β (P<0.05); the expression level of COLⅠ was improved (P<0.05). Compared with the atorvastatin calcium group, the NLRP3 inhibitor group showed reduced VI, protein expression levels of NLRP3, Caspase-1, GSDMD-N, IL-1β, and ASC, as well as mRNA expression levels of IL-1β and Caspase-1 (P<0.05), together with high erexpression level of COLⅠ and COLⅠ/COLⅢ ratio (P<0.05). The in vitro results showed that, compared with the blank group, the model group showed decreased expression of COLⅠ and COLⅠ/COLⅢ ratio (P<0.05), together with increased expression of COLⅢ, protein expression levels of NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18, and ASC as well as mRNA expression levels of Caspase-1, IL-1β, and IL-18 (P<0.05). Compared with the model group, the QHWD drug-containing serum group and the NLRP3 inhibitor group exhibited elevated expression of COLⅠ and COLⅠ/COLⅢ ratio (P<0.05), and decreased expression of COLⅢ, protein expression levels of NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18, and ASC, as well as mRNA expression levels of Caspase-1, IL-1β, and IL-18 (P<0.05); the NLRP3 agonist group showed decreased expression of COLⅠ and COLⅠ/COLⅢ ratio (P<0.05), and enhanced expression of COLⅢ, protein expression levels of NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18, and ASC, as well as mRNA expression levels of Caspase-1, IL-1β, and IL-18 (P<0.05). Compared with the QHWD drug-containing serum group, the NLRP3 agonist group showed decreased expression of COLⅠ and COLⅠ/COLⅢ ratio (P<0.05), together with increased expression of COLⅢ, protein expression levels of NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18, and ASC, as well as mRNA expression levels of Caspase-1, IL-1β, and IL-18 (P<0.05). Compared with the NLRP3 agonist group, the NLRP3 inhibitor group showed increased expression of COLⅠ and COL I/COLⅢ ratio (P<0.05), and decreased expression of COLⅢ, protein expression levels of NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18, and ASC, as well as mRNA expression levels of Caspase-1, IL-1β, and IL-18 (P<0.05). Conclusion QHWD may inhibit VSMC pyroptosis by regulating the NLRP3/Caspase-1/GSDMD signaling pathway, which provides an experimental basis for the intervention of AS plaque formation based on the principle of "treating from wind"; its specific active components and direct targets remain to be further elucidated. |
| Key words: atherosclerosis|Qufeng Huatan Wenban Decoction|NLRP3/Caspase-1/GSDMD signaling pathway|pyroptosis|treating from wind |
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