吉林大学学报(医学版) ›› 2026, Vol. 52 ›› Issue (5): 1234-1243.doi: 10.13481/j.1671-587X.20260505

• 基础研究 • 上一篇    

黄芪甲苷与三七总皂苷配伍冰片复方对大鼠脑缺血/再灌注后炎性损伤的抑制作用及其机制

白杨,熊婷婷,李艳玲,胡中吉,兰逍,陈园,黄小平,丁煌(),邓常清()   

  1. 湖南中医药大学中西医结合心脑疾病防治湖南省重点实验室,湖南 长沙 410208
  • 收稿日期:2025-11-28 接受日期:2026-01-05 出版日期:2026-09-28 发布日期:2026-10-15
  • 通讯作者: 丁煌,邓常清 E-mail:004594@hnucm.edu.cn;dchangq@hnucm.edu.cn
  • 作者简介:白 杨(2000-),女,黑龙江省齐齐哈尔市人,在读硕士研究生,主要从事中药成分与脑缺血再灌注后炎症损伤关系方面的研究。
  • 基金资助:
    国家自然科学基金项目(82305031)

Inhibitory effect of combination of Astragaloside Ⅳ, Panax notoginseng saponins, and borneol on inflammatory injury after cerebral ischemia/reperfusion in rats and its mechanism

Yang BAI,Tingting XIONG,Yanling LI,Zhongji HU,Xiao LAN,Yuan CHEN,Xiaoping HUANG,Huang DING(),Changqing DENG()   

  1. Key Laboratory of Hunan Province for Integrated Traditional Chinese and Western Medicine on Prevention and Treatment of Cardio-Cerebral Diseases,Hunan University of Chinese Medicine,Changsha 410208,China
  • Received:2025-11-28 Accepted:2026-01-05 Online:2026-09-28 Published:2026-10-15
  • Contact: Huang DING,Changqing DENG E-mail:004594@hnucm.edu.cn;dchangq@hnucm.edu.cn

摘要:

目的 探讨黄芪甲苷(AST Ⅳ)与三七总皂苷(PNS)配伍冰片复方(APB)调控有氧糖酵解和小胶质细胞极化对大鼠脑缺血/再灌注(CI/R)后神经炎症的抑制作用,并阐明其协同机制。 方法 取25只健康雄性SD大鼠,随机分为假手术组、模型组、低剂量APB组、高剂量APB组和阳性对照药物丁苯酞(NBP)组,每组5只。采用线栓法建立大鼠大脑中动脉阻塞局灶性脑缺血(MCAO)模型,假手术组大鼠未实施血管阻断操作。各组大鼠于缺血2 h后再灌注开始时灌胃给药,每日2次。低剂量APB组大鼠给予60 mg·kg-1冰片+80 mg·kg-1 AST Ⅳ+200 mg·kg-1 PNS,高剂量APB组大鼠给予120 mg·kg-1冰片+160 mg·kg-1 AST Ⅳ+400 mg·kg-1 PNS,NBP组大鼠给予80 mg·kg-1 NBP;假手术组和模型组大鼠给予等体积纯水。处死前2 h,评估各组大鼠改良神经功能缺损评分(mNSS)。再灌注48 h后,各组大鼠麻醉后经腹主动脉取血,处死大鼠并取缺血侧脑组织。采用HE染色和尼氏染色观察各组大鼠脑组织海马区神经细胞损伤情况及尼氏小体数量,酶联免疫吸附试验(ELISA)法检测各组大鼠血清中白细胞介素1β(IL-1β)、白细胞介素10(IL-10)、肿瘤坏死因子α(TNF-α)和白细胞介素4(IL-4)水平,免疫荧光染色检测各组大鼠脑组织中小胶质细胞M1型和M2型极化状态,Western blotting法检测各组大鼠脑组织中糖酵解关键限速酶乳酸脱氢酶A(LDHA)、葡萄糖转运蛋白1(GLUT1)和己糖激酶2(HK2)蛋白表达水平。 结果 mNSS评分法,与假手术组比较,模型组大鼠神经功能缺损评分明显升高(P<0.05);与模型组比较,低剂量APB组、高剂量APB组和NBP组大鼠神经功能缺损评分均明显降低(P<0.05或P<0.01)。HE染色,假手术组大鼠海马区神经细胞排列规则;模型组大鼠海马区神经细胞出现核固缩或空泡样变性;与模型组比较,低剂量APB组、高剂量APB组和NBP组大鼠海马区神经细胞损伤减轻,核固缩和空泡细胞减少。与假手术组比较,模型组大鼠海马区神经细胞存活率明显降低(P<0.05);与模型组比较,低剂量APB、高剂量APB和NBP组大鼠海马区神经细胞存活率均明显升高(P<0.05)。尼氏染色,与假手术组比较,模型组大鼠海马区神经细胞中尼氏小体数量明显减少(P<0.05);与模型组比较,低剂量APB组、高剂量APB组和NBP组大鼠海马区神经细胞中尼氏小体数量均明显增加(P<0.05)。ELISA法,与假手术组比较,模型组大鼠血清中IL-1β和TNF-α水平明显升高(P<0.05),IL-10和IL-4水平明显降低(P<0.05或P<0.01);与模型组比较,低剂量APB组、高剂量APB组和NBP组大鼠血清中IL-1β及TNF-α水平明显降低(P<0.05),IL-10和IL-4水平明显升高(P<0.05或P<0.01)。免疫荧光染色,与假手术组比较,模型组大鼠脑组织中诱导型一氧化氮合酶(iNOS)/离子钙结合接头蛋白分子1(Iba-1)共表达荧光强度明显升高(P<0.01),精氨酸酶1(Arg-1)/Iba-1共表达荧光强度明显降低(P<0.01);与模型组比较,低剂量APB组、高剂量APB组和NBP组大鼠脑组织中iNOS/Iba-1共表达荧光强度均明显降低(P<0.05或P<0.01),Arg-1/Iba-1共表达荧光强度均明显升高(P<0.05或P<0.01)。Western blotting法,与假手术组比较,模型组大鼠脑组织中LDHA、GLUT1和HK2蛋白表达水平均明显升高(P<0.05);与模型组比较,低剂量APB组和高剂量APB组大鼠脑组织中LDHA蛋白表达水平均明显降低(P<0.05),高剂量APB组和NBP组大鼠脑组织中GLUT1及HK2蛋白表达水平均明显降低(P<0.05)。 结论 APB可通过下调糖酵解酶LDHA、GLUT1和HK2蛋白表达抑制小胶质细胞向M1型极化,从而减轻脑组织炎症反应,改善大鼠CI/R后的神经功能。

关键词: 脑缺血/再灌注损伤, 糖代谢重编程, 炎症反应, 黄芪甲苷, 三七总皂苷, 冰片

Abstract:

Objective To investigate the inhibitory effect of the combination of Astragaloside Ⅳ(AST Ⅳ), Panax notoginseng saponins (PNS), and borneol (APB) on neuroinflammation after cerebral ischemia/reperfusion (CI/R) in the rats by modulating aerobic glycolysis and microglial polarization, and to elucidate its synergistic mechanism. Methods Twenty-five healthy male SD rats were selected and randomly divided into sham operation group,model group, low dose of APB group, high dose of APB group, and positive control drug butylphthalide (NBP) group, with 5 rats in each group. The rat models of focal cerebral ischemia were established by middle cerebral artery occlusion (MCAO) using the suture occlusion method, while those in sham operation group did not undergo vascular occlusion.The rats in various groups received intragastric administration twice daily, starting at the onset of reperfusion after 2 h of ischemia. The rats in low dose of APB group were given 60 mg·kg-1 borneol+80 mg·kg-1 AST Ⅳ+200 mg·kg-1 PNS, the rats in high dose of APB group were given 120 mg·kg-1 borneol+160 mg·kg-1 AST Ⅳ+ 400 mg·kg-1 PNS, and the rats in NBP group were given 80 mg·kg-1 NBP. The rats in sham operation group and model group were given equal volumes of pure water. At 2 h before execution, the modified neurological severity score(mNSS) of the rats in various groups were assessed. At 48 h after reperfusion, the rats were anesthetized, and the blood was collected from the abdominal aorta, and then the rats were sacrificed to collect the ischemic side of brain tissue. HE staining and Nissl staining were used to observe the neuronal damage and the number of Nissl bodies in the hippocampal region of brain tissue in the rats in various groups. Enzyme-linked immunosorbent assay (ELISA) method was used to detect the serum levels of interleukin-1β (IL-1β), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interleukin-4(IL-4) in the rats in various groups. Immunofluorescence staining was used to detect the polarization state of microglia towards M1 and M2 phenotypes in the brain tissue of the rats in various groups. Western blotting method was used to detect the expression levels of the key glycolytic rate-limiting enzymes lactate dehydrogenase A (LDHA), glucose transporter 1 (GLUT1), and hexokinase 2 (HK2) proteins in the brain tissue of the rats in various groups. Results ?According to the mNSS score, compared with sham operation group, the neurological deficit score of the rats in model group was significantly increased (P<0.05); compared with model group, the neurological deficit scores of the rats in low dose of APB group, high dose of APB group, and NBP group were significantly decreased (P<0.05 or P<0.01). The HE staining results showed regularly arranged neurons in the hippocampal region of the rats in sham operation group, and pyknosis and vacuolar degeneration of neuronal nuclei were observed in the rats in model group. Compared with model group, the neuronal damage was alleviated in low-dose APB group, high-dose APB group, and NBP group, as indicated by a reduction in pyknotic and vacuolated cells. Compared with sham operation group, the neuronal survival rate in the hippocampal region of the rats in model group was significantly decreased (P<0.05); compared with model group, the neuronal survival rates in the hippocampal regions of the rats in low dose of APB group, high dose of APB group, and NBP group were significantly increased (P<0.05). The Nissl staining results showed that compared with sham operation group, the number of Nissl bodies in neurons in the hippocampal region of the rats in model group was significantly decreased (P<0.05); compared with model group, the numbers of Nissl bodies in the hippocampal neurons of the rats in low dose of APB group, high dose of APB group, and NBP group were significantly increased (P<0.05). The ELISA results showed that compared with sham operation group, the levels of pro-inflammatory factors IL-1β and TNF-α in serum in the rats in model group were significantly increased (P<0.05), while the levels of IL-10 and IL-4 in serum were significantly decreased (P<0.05 or P<0.01); compared with model group, levels of IL-1β and TNF-α in serum in the rats in low dose of APB group, high dose of APB group, and NBP group were significantly decreased (P<0.05), while the levels of IL-10 and IL-4 in serum were significantly increased (P<0.05 or P<0.01). The immunofluorescence staining results showed that compared with sham operation group, the co-expression fluorescence intensity of inducible nitric oxide synthase(iNOS)/ionized calcium-binding adapter molecule 1(Iba-1) in the brain tissue of the rats in model group was significantly increased (P<0.01), while that of arginase-1(Arg-1)/Iba-1 was significantly decreased (P<0.01); compared with model group, the co-expression fluorescence intensities of iNOS/Iba-1 in the brain tissue of the rats in low dose of APB group, high dose of APB group, and NBP group were significantly decreased (P<0.05 or P<0.01), while those of Arg-1/Iba-1 were significantly increased (P<0.05 or P<0.01). The Western blotting results showed that compared with sham operation group, the expression levels of LDHA, GLUT1, and HK2 proteins in the brain tissue of the rats in model group were significantly increased (P<0.05); compared with model group, the expression levels of LDHA protein in the brain tissue of the rats in low dose of APB group and high dose of APB group were significantly decreased (P<0.05), while the expression levels of GLUT1 and HK2 proteins in high dose of APB group and NBP group were significantly decreased(P<0.05). Conclusion ?APB can inhibit the polarization of microglia towards the M1 phenotype by downregulating the expression of glycolytic enzymes LDHA, GLUT1, and HK2 proteins, thereby alleviating inflammatory responses in brain tissue and improving neurological function in the rats after CI/R.

Key words: Cerebral ischemia/reperfusion injury, Glucose metabolism reprogramming, Inflammatory response, Astragaloside Ⅳ, Panax notoginseng saponins, Borneol

中图分类号: 

  • R285.5