Objectives: To study the protective effect of breviscapine against brain injury induced by intrauterine inflammation in preterm rats and its mechanism.
Methods: A preterm rat model of brain injury caused by intrauterine inflammation was prepared by intraperitoneal injections of lipopolysaccharide in pregnant rats. The pregnant rats and preterm rats were respectively randomly divided into 5 groups: control, model, low-dose breviscapine (45 mg/kg), high-dose breviscapine (90 mg/kg), and high-dose breviscapine (90 mg/kg)+ML385 [a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, 30 mg/kg] (n=10 each). The number and body weight of the live offspring rats were measured for each group. Hematoxylin-eosin staining was used to observe the pathological morphology of the uterus and placenta of pregnant rats and the pathological morphology of the brain tissue of offspring rats. Immunofluorescent staining was used to measure the co-expression of ionized calcium binding adaptor molecule-1 (IBA-1) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex of offspring rats. ELISA was used to measure the levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1β (IL-1β) in the brain tissue of offspring rats. Western blotting was used to measure the expression of Nrf2 pathway-related proteins in the brain tissue of offspring rats.
Results: Pathological injury was found in the uterus, and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, and severe microglia pyroptosis occurred in the cerebral cortex of the offspring rats in the model group. Compared with the control group, the model group had significant reductions in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and heme oxygenase-1 (HO-1) in the brain tissue of the offspring rats (P<0.05), but significant increases in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1β, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). Compared with the model group, the breviscapine administration groups showed alleviated pathological injury of the uterus and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, significant increases in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and HO-1 in the brain tissue of the offspring rats (P<0.05), and significant reductions in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1β, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). The high-dose breviscapine group had a significantly better effect than the low-dose breviscapine (P<0.05). ML385 significantly inhibited the intervention effect of high-dose breviscapine (P<0.05).
Conclusions: Breviscapine can inhibit inflammatory response in brain tissue of preterm rats caused by intrauterine inflammation by activating the Nrf2 pathway, and it can also inhibit microglial pyroptosis and alleviate brain injury.
目的: 探讨灯盏花素对宫内炎症致早产大鼠脑损伤的保护作用及其机制。方法: 向孕鼠腹腔内注射脂多糖制备宫内炎症致早产大鼠脑损伤模型，孕鼠和仔鼠分别随机分为对照组、模型组、灯盏花素低剂量（45 mg/kg）组、灯盏花素高剂量（90 mg/kg）组、灯盏花素高剂量（90 mg/kg）+ML385［核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）抑制剂，30 mg/kg］组（n=10）。测定各组孕鼠产出的仔鼠存活数和体重；苏木精-伊红染色法观察各组孕鼠子宫、胎盘病理形态及仔鼠脑组织病理形态；免疫荧光染色法检测各组仔鼠脑皮质离子钙接头蛋白分子-1（ionized calcium-binding adaptor molecule-1，IBA-1）与核苷酸结合寡聚化结构域样受体蛋白3（nucleotide-binding oligomerization domain-like receptor protein 3，NLRP3）共表达情况；酶联免疫吸附法检测各组仔鼠脑组织中白细胞介素（interleukin，IL）-6、IL-8、IL-1β水平；免疫印迹法检测各组仔鼠脑组织中Nrf2通路相关蛋白表达。结果: 与对照组相比，模型组孕鼠子宫、胎盘组织及仔鼠脑组织产生病理损伤，仔鼠脑皮质产生严重小胶质细胞焦亡，仔鼠存活数和体重、脑组织中Nrf2及血红素加氧酶-1（heme oxygenase-1，HO-1）蛋白表达水平降低（P<0.05），仔鼠脑组织中IBA-1和NLRP3共表达相对荧光强度、炎性因子IL-6、IL-8及IL-1β水平、NLPR3及半胱氨酸天冬氨酸蛋白酶-1（caspase-1）蛋白表达均升高（P<0.05）；与模型组相比，灯盏花素低、高剂量组孕鼠子宫、胎盘组织及仔鼠脑组织病理损伤均减轻，仔鼠存活数和体重、Nrf2及HO-1蛋白表达水平均升高（P<0.05），仔鼠脑组织中IBA-1和NLRP3共表达相对荧光强度、炎性因子IL-6、IL-8及IL-1β水平、脑组织中NLPR3及caspase-1蛋白表达均降低（P<0.05）；且灯盏花素高剂量组效果优于灯盏花素低剂量组，差异有统计学意义（P<0.05）；ML385可显著抑制灯盏花素高剂量组的干预效果（P<0.05）。结论: 灯盏花素可通过激活Nrf2通路而抑制宫内炎症致早产大鼠脑组织炎症反应，抑制小胶质细胞焦亡，减轻早产大鼠脑损伤。.
Keywords: Brain injury; Breviscapine; Intrauterine inflammation; Microglial pyroptosis; Nuclear factor erythroid 2-related factor 2; Preterm rat.