摘要
目的:探讨大黄酸对急性脑出血大鼠小胶质细胞焦亡的抑制作用及机制。方法:采用国际公认的注射Ⅶ型胶原酶来诱导脑出血(ICH)大鼠模型;依据核苷酸结合寡聚化结构域样受体3(NLRP3)基因序列,构建核苷酸结合寡聚化结构域样受体3(NLRP3)沉默腺病毒,建立沉默NLRP3的ICH大鼠模型;尾静脉注射半胱氨酸天冬氨酸蛋白酶1抑制剂(VX-765),建立半胱氨酸天冬氨酸蛋白酶(Caspase-1)抑制的ICH大鼠模型。将50只SD大鼠随机分为假手术组(Sham组,n=5)、脑出血组(ICH组,n=5)、假手术+大黄酸治疗组(Sham+Rhein组,n=5)、脑出血+大黄酸治疗组(ICH+Rhein组,n=5)、假手术+大黄酸+NLRP3沉默组(Sham+Rhein+shNLRP3组,n=5)、脑出血+大黄酸+NLRP3沉默组(ICH+Rhein+shNLRP3组,n=5)、假手术+大黄酸+腺病毒阴性对照组(Sham+Rhein+Ad-NC组,n=5)、脑出血+大黄酸+腺病毒阴性对照组(ICH+Rhein+Ad-NC组,n=5)、脑出血+Caspase-1抑制剂组(ICH+Caspase-1 inhibitor组,n=5)、脑出血+大黄酸+Caspase-1抑制剂组(ICH+Rhein+Caspase-1 inhibitor组,n=5);大黄酸治疗第7天后,ELISA检测白细胞介素-1β、-18、-6(IL-1β、IL-18、IL-6)、肿瘤坏死因子α(TNF-α)、诱导性一氧化氮合酶(iNOS)水平,HE检测脑组织炎症水平,Western blot及RT-qPCR检测NLRP3、抗体分泌细胞(ASC)、Caspase-1、消皮素D(GSDMD)表达。结果:(1)中剂量(Adv-M:5×10^(9)pfu/只)及高剂量(Adv-H:1×10^(10)pfu/只)的腺病毒能明显降低NLRP3的表达,二者之间比较没有统计学差异(P>0.05);(2)中剂量(MDR,1.73 mg/kg)为大黄酸治疗的最佳剂量;(3)与Sham组比较,ICH组中炎症因子(IL-1β、IL-18、TNF-α、IL-6、iNOS)及炎症水平显著升高(均P<0.05);与ICH组比较,ICH+Rhein组大鼠的炎症水平显著降低(P<0.05);与ICH+Rhein组比较,ICH+Rhein+shNLRP3组和ICH+Rhein+Caspase-1 inhibitor组明显使得炎症因子的水平更低(均P<0.05);(4)与Sham组比较,ICH组中NLRP3、ASC、Caspase-1、GSDMD的水平显著升高(均P<0.05);与ICH组比较,ICH+Rhein组大鼠脑组织中NLRP3、ASC、Caspase-1、GSDMD的水平有所降低(均P<0.05),ICH+Rhein+shNLRP3组以及ICH+Rhein+Caspase-1 inhibitor组均显著降低了中NLRP3、ASC、Caspase-1、GSDMD的水平(均P<0.05)。结论:大黄酸可通过调控NLRP3炎症小体活化,抑制ICH后小胶质细胞焦亡,从而减少炎症因子的释放,减轻脑组织炎症水平,从而发挥抑制炎症反应、减轻脑组织继发性损伤的作用。
Objective:To explore the inhibitory effect and mechanism of rhubarbic acid on microglial cell focal death in rats with acute cerebral hemorrhage.Methods:The internationally recognized rat model of intracerebral hemorrhage(ICH)was induced by injection of typeⅦcollagenase;based on the sequence of NLRP3 gene,NLRP3-silencing adenovirus was constructed to establish an NLRP3-silencing rat model of ICH;VX-765 was injected into the tail vein to establish a Caspase-1-inhibited rat model of ICH.Fifty SD rats were randomly divided into a sham-operated group(Sham group,n=5),a cerebral hemorrhage group(ICH group,n=5),a sham-operated+rhubarbic acid-treated group(Sham+Rhein group,n=5),a cerebral hemorrhage+rhubarbic acid-treated group(ICH+Rhein group,n=5)a sham-operated+rhubarbic acid+shNLRP3-silenced group(Sham+Rhein+shNLRP3 group,n=5),cerebral hemorrhage+rhubarbic acid+NLRP3 silencing group(ICH+Rhein+shNLRP3 group,n=5),sham-operated+rhubarbic acid+adenovirus-negative control group(Sham+Rhein+Ad-NC group,n=5),cerebral hemorrhage+rhubarbic acid+adenovirus-negative control group(ICH+Rhein+Ad-NC group,n=5),cerebral hemorrhage+Caspase-1 inhibitor group(ICH+Caspase-1 inhibitor group,n=5),and cerebral hemorrhage+Rhein+Caspase-1 inhibitor group(ICH+Rhein+Caspase-1 inhibitor group,n=5);After the 7th day of rhubarbic acid treatment,the levels of IL-1β,IL-18,TNF-α,IL-6,and iNOS were detected by ELISA,the inflammation level of brain tissue was detected by HE,and the expression of NLRP3,ASC,Caspase-1,and GSDMD were detected by Western blot and RT-qPCR.Results:①Medium-dose(Adv-M:5×10^(9) pfu/pupil)and high-dose(Adv-H:1×10^(10) pfu/pupil)adenoviruses significantly reduced the expression of NLRP3,and there was no statistically significant difference between them(P>0.05);②Medium-dose(MDR,1.73 mg/kg)was the optimal dose for rhubarbic acid treatment;③The levels of inflammatory factors(IL-1β,IL-18,TNF-α,IL-6,iNOS)and inflammation were significantly higher in the ICH group compared with the Sham group(all P<0.05);the levels of inflammation in rats in the ICH+Rhein group were significantly lower in the ICH group compared with the ICH group(P<0.05);the levels of inflammatory factors in rats in the ICH+Rhein group were significantly lower in the ICH+Rhein+shNLRP3 group and ICH+Rhein+Caspase-1 inhibitor group significantly made the level of inflammatory factors lower(all P<0.05);④The levels of NLRP3,ASC,Caspase-1,and GSDMD were significantly higher in the ICH group compared with the Sham group(all P<0.05);the levels of NLRP3,ASC,Caspase-1,and GSDMD were decreased in the brain tissues of the rats in the ICH+Rhein group compared with the ICH group(all P<0.05),and the ICH+Rhein+shNLRP3 group as well as ICH+Rhein+Caspase-1 inhibitor group significantly reduced the levels of NLRP3,ASC,Caspase-1,and GSDMD in the middle(all P<0.05).Conclusion:Rhubarbic acid can inhibit microglial cell pyroptosis after ICH by regulating the activation of NLRP3 inflammatory vesicles,thereby reducing the release of inflammatory factors and attenuating the level of inflammation in brain tissues,thus exerting the effects of inhibiting inflammatory responses and attenuating the secondary damage in brain tissues.
作者
陈臣
李倩
刘涛
阿达来提·艾麦提
CHEN Chen;LI Qian;LIU Tao;ADALAITI·Aimaiti(The Fourth Clinical Medical College,Xinjiang Medical University,Urumqi 830000,China;The Affiliated Hospital of Traditional Chinese Medicine,Xinjiang Medical University,Urumqi 830000,China)
出处
《陕西医学杂志》
2025年第10期1316-1323,共8页
Shaanxi Medical Journal
基金
新疆维吾尔自治区自然科学基金杰出青年科学基金资助项目(2022D01E79)。
关键词
大黄酸
脑出血
NLRP3炎症小体
小胶质细胞
细胞焦亡
机制
Rhubarbic acid
Intracerebral hemorrhage
NLRP3 inflammatory vesicles
Microglia
Cellular pyroptosis
Mechanism
