摘要
目的研究趋化因子配体21(CCL21)在小鼠脑出血后继发性脑损伤中的作用。方法将60只C57BL/6J小鼠采用简单随机方法分为假手术组(Sham组)、脑出血组(ICH组)及脑出血CCL21单抗干预组(ICH+anti-CCL21组),每组20只。采用基底节区注射Ⅶ型胶原酶法,建立小鼠脑出血模型。小鼠脑出血后72 h对3组小鼠进行神经损伤功能评分,干湿重法检测患侧半脑脑组织含水量,尼氏染色检测神经细胞损伤,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测神经细胞凋亡,蛋白质印迹法(Western blot)检测Janus激酶2(JAK2)/信号传导与转录激活因子3(STAT3)信号通路蛋白变化情况,实时荧光定量聚合酶链反应(qPCR)检测血肿灶周肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-4、IL-10的基因表达水平。结果与Sham组比较,ICH组小鼠神经功能评分增高[(15.0±1.0)分比(2.4±0.9)分],脑水肿加重,神经细胞损伤增加,神经细胞凋亡增多,差异均有统计学意义(均P<0.05)。小鼠脑血肿灶周围组织的p-JAK2、p-STAT3蛋白表达水平明显增高,促炎因子TNF-α、IL-1β的基因表达水平增加,抗炎因子IL-4、IL-10的基因表达水平降低,差异均有统计学意义(均P<0.05)。与ICH组比较,采用CCL21单抗干预的脑出血小鼠神经功能损伤评分[(9.2±1.3)分]降低,脑水肿程度减轻,神经细胞损伤减少及神经细胞凋亡减少,差异均有统计学意义(均P<0.05)。CCL21单抗干预可明显增加小鼠脑血肿灶周围组织的p-JAK2、p-STAT3蛋白表达水平,减少促炎因子TNF-α、IL-1β的基因表达水平,同时增加抗炎因子IL-4、IL-10的基因表达水平,差异均有统计学意义(均P<0.05)。JAK2、STAT3蛋白的表达水平在各组中均相对稳定,差异均无统计学意义(均P>0.05)。结论CCL21单抗干预显著减少了小鼠脑出血后的神经功能损害,可能是通过激活JAK2/STAT3从而抑制其下游炎性因子TNF-α、IL-1β而引起,提示了脑出血后继发性神经炎性反应的病理生理机制,为脑出血的治疗提供了可能的实验依据和分子靶点。
Objective To investigate the role of the C-C chemokine ligand 21(CCL21)in secondary brain injury after intracerebral hemorrhage in mice.Methods Sixty C57BL/6J mice were randomly divided into sham operation group(Sham group),intracerebral hemorrhage group(ICH group)and intracerebral hemorrhage CCL21 monoclonal antibody intervention group(ICH+anti-CCL21 group),with 20 mice in each group.The mouse intracerebral hemorrhage model was established by injecting typeⅦcollagenase into the basal ganglia.The neurological function of the mice was scored 72 hours after intracerebral hemorrhage.The water content of the injured side brain tissue was measured by dry and wet weight method.The neuronal injury was detected by Nissl staining,and the neuronal apoptosis was detected by terminal deoxynucleotidyl transferase triphosphate Nick end labeling(TUNEL).Western blot was used to detects the changes of Janus kinase 2(JAK2)-signal transducer and activator of transcription 3(STAT3)pathway proteins,and real-time quantitative polymerase chain reaction(qPCR)was used to detects the gene expression levels of tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β),interleukin 4(IL-4),and interleukin 10(IL-10)around the hematoma.Results Compared with the Sham group,mice in the ICH group had higher neurological scores([15.0±1.0]scores vs.[2.4±0.9]scores),increased brain edema,increased neuronal damage,and increased neuronal apoptosis.The differences were statistically significant(all P<0.05).The expression levels of phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)protein molecules in the tissues surrounding the cerebral hematoma lesions in mice were significantly increased,and the gene expression levels of pro-inflammatory factors TNF-αand IL-1βincreased.The gene expression levels of anti-inflammatory factors IL-4 and IL-10 decreased.The differences were statistically significant(all P<0.05).Compared with the ICH group,intracerebral hemorrhage mice treated with anti-CCL21 had lower neurological damage scores[(9.2±1.3)scores],less cerebral edema,decreased neuronal damage and decreased neuronal apoptosis.The differences were statistically significant(all P<0.05).Anti-CCL21 intervention significantly increased the expression levels of p-JAK2 and p-STAT3 protein in tissues surrounding the cerebral hematoma in mice,decreased the gene expression levels of pro-inflammatory factor TNF-αand IL-1β,and increased the gene expression levels of anti-inflammatory factor IL-4 and IL-10.All the differences were statistically significant(all P<0.05).However,the expression levels of JAK2 and STAT3 protein were relatively stable in each group,and the differences were not statistically significant(both P>0.05).Conclusions The intervention of CCL21 monoclonal antibody significantly reduced the neurological damage after intracerebral hemorrhage in mice,which may be caused by JAK2/STAT3 activation to inhibit its downstream inflammatory factors of TNF-αand IL-1β.This reveals the pathophysiological mechanism of secondary neuroinflammation after intracerebral hemorrhage and provides probable experimental evidence and molecular targets for the treatment of intracerebral hemorrhage.
作者
周桂银
赵元丹
高扬
刘潜
张强
曹芳
姚声涛
Zhou Guiyin;Zhao Yuandan;Gao Yang;Liu Qian;Zhang Qiang;Cao Fang;Yao Shengtao(Department of Cerebrovascular Disease,Affiliated Hospital of Zunyi Medical University,Zunyi,Guizhou 563000,China)
出处
《中国脑血管病杂志》
CAS
CSCD
北大核心
2020年第10期602-609,共8页
Chinese Journal of Cerebrovascular Diseases
基金
国家自然科学基金(81960230)
贵州省科技计划项目(黔科合基础[2020]1Y318)。