Guo Ya-Jing, Ren Jing, Liu Han, Li Ting-Ting, Zhang Shuai, Wang Hong
Hebei University of Chinese Medicine, Shijiazhuang 050200, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2022 Jan;38(1):11-16. doi: 10.12047/j.cjap.6191.2022.003.
To investigate the effects of the pyrin domain-containing protein 3 (NLRP3) inflammasome inhibitor MCC950 on nerve injury in rats with intracerebral hemorrhage(ICH). Seventy-two SD rats were randomly divided into three groups (=24): Sham group, ICH group and MCC950 group. ICH group and MCC950 group rats were injected with autogenous non-anticoagulant blood to establish ICH model, and then the rats in MCC950 group were intraperitoneally injected with MCC950 at the dose of 10 mg/kg(2 mg/ml) for 3 days after ICH model was established. Seventy-two hours after the establishment of the model, the forelimb placement test, the corner test and mNSS score were performed to observe the neurological function of the rats with ICH. The volume of hematoma was observed in fresh brain tissue sections. HE staining was used to observe the pathological changes of brain tissue. The dry-wet weight ratio was calculated to evaluate the changes of brain tissue edema. The degeneration of neurons was observed by FJC staining. The neuronal apoptosis was observed by TUNEL staining. The protein expression and activation levels of NLRP3, ASC, caspase-1, IL-1β, IL-18 and GSDMD were determined by Western blot. Compared with sham group, the percentage of successful placement of left forelimb and left turn was decreased significantly (＜0.01, ＜0.05), mNSS score was increased significantly (＜0.01) in ICH group. Hematoma volume was increased significantly, the number of microglial cells around the hematoma was increased, the number of neurons was decreased, nerve cell swelled, some cells showed pyknotic necrosis, and the staining was deepened. The water content of the right base was increased significantly (＜0.05). The number of FJC positive and TUNEL positive cells around the hematoma was increased significantly (＜0.05). The levels of NLRP3, ASC, caspase-1, pro-caspase-1, caspase-1/pro-caspase-1 ratio, GSDMD-N, GSDMD, GSDMD-N/GSDMD ratio, IL-1β and IL-18 were increased significantly (＜0.01, ＜ 0.05). Compared with ICH group, the percentage of successful placement of left forelimb and left turn was increased significantly in MCC950 group (＜0.05), while the mNSS score and the volume of hematoma were decreased significantly (＜0.01), the swelling degree of nerve cells around the hematoma was reduced significantly, and the number of pyrotic necrotic cells was decreased. The water content of the right base was decreased significantly (＜0.05), and the number of FJC positive and TUNEL positive cells around the hematoma was decreased significantly (＜0.05). The levels of NLRP3, ASC, caspase-1, pro-caspase-1, caspase-1/pro-caspase-1 ratio, GSDMD-N, GSDMD, GSDMD-N/GSDMD ratio, IL-1β and IL-18 were decreased significantly (＜0.05). MCC950 can ameliorate nerve injury after ICH by inhibiting NLRP3 inflammasome mediated inflammation and pyroptosis.
探讨含吡喃结构域蛋白3（NLRP3）炎性小体抑制剂MCC950对脑出血（ICH）大鼠神经损伤的影响。将72只SD大鼠随机分为三组（每组 = 24只）：假手术组、ICH组和MCC950组。ICH组和MCC950组大鼠注射自体非抗凝血液建立ICH模型，然后MCC950组大鼠在ICH模型建立后以10 mg/kg（2 mg/ml）的剂量腹腔注射MCC950，连续3天。模型建立72小时后，进行前肢放置试验、转角试验和改良神经功能缺损评分（mNSS）以观察ICH大鼠的神经功能。在新鲜脑组织切片上观察血肿体积。采用苏木精-伊红（HE）染色观察脑组织的病理变化。计算干湿重比以评估脑组织水肿的变化。采用荧光金（FJC）染色观察神经元的变性。采用末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色观察神经元凋亡。通过蛋白质印迹法检测NLRP3、凋亡相关斑点样蛋白（ASC）、半胱天冬酶-1（caspase-1）、白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）和Gasdermin D（GSDMD）的蛋白表达和激活水平。与假手术组相比，ICH组左前肢成功放置百分比和左转百分比显著降低（P＜0.01，P＜0.05），mNSS评分显著升高（P＜0.01）。血肿体积显著增大，血肿周围小胶质细胞数量增多，神经元数量减少，神经细胞肿胀，部分细胞呈固缩坏死，染色加深。右侧基底含水量显著增加（P＜0.05）。血肿周围FJC阳性和TUNEL阳性细胞数量显著增加（P＜0.05）。NLRP3、ASC、caspase-1、前半胱天冬酶-1、caspase-1/前半胱天冬酶-1比值、GSDMD-N、GSDMD、GSDMD-N/GSDMD比值、IL-1β和IL-18水平显著升高（P＜0.01，P＜0.05）。与ICH组相比，MCC950组左前肢成功放置百分比显著升高（P＜0.05），而mNSS评分和血肿体积显著降低（P＜0.01），血肿周围神经细胞肿胀程度显著减轻，热坏死细胞数量减少。右侧基底含水量显著降低（P＜0.05），血肿周围FJC阳性和TUNEL阳性细胞数量显著减少（P＜0.05）。NLRP3、ASC、caspase-1、前半胱天冬酶-1、caspase-1/前半胱天冬酶-1比值、GSDMD-N、GSDMD、GSDMD-N/GSDMD比值、IL-1β和IL-18水平显著降低（P＜0.05）。MCC950可通过抑制NLRP3炎性小体介导的炎症和细胞焦亡改善ICH后的神经损伤。
