藏红花素通过抑制JAK2/STAT3信号通路减轻脑缺血再灌注大鼠海马神经元损伤

Crocin Alleviates Hippocampal Neuron Injury in Rats with Cerebral Ischemia-Reperfusion by Inhibiting JAK2/STAT3 Signaling Pathway

目的研究藏红花素(CRO)对脑缺血再灌注(CI/R)大鼠海马神经元损伤的影响并探索其潜在机制。方法将144只雄性SD大鼠按照随机数字表法分为假手术(Sham)组,模型(CI/R)组,CRO低、中、高剂量(CRO-L、CRO-M、CRO-H)组和尼莫地平(NMP)组6组,每组24只。采用线栓法制备CI/R大鼠模型,各组分别于造模前7 d开始1次/d腹腔注射(ip)给药(CRO-L、CRO-M、CRO-H组分别ip给药10、20、40 mg/kg,NMP组ip给药1 mg/kg,Sham组和CI/R组ip给予生理盐水5 mL/kg)。再灌注24 h后,通过Morris水迷宫实验检测大鼠学习记忆能力,TTC染色检测脑梗死率,HE染色法行海马CA1区和CA3区神经元病理学检查,TUNEL染色法行海马CA1区和CA3区神经元凋亡检查,ELISA法检测海马组织白细胞介素-1β(IL-1β)、IL-8、肿瘤坏死因子-α(TNF-α)含量,Western blot法检测海马组织Janus激酶2/信号转导与转录激活子3(JAK2/STAT3)信号通路相关蛋白相对表达量。结果与Sham组比较,CI/R组学习记忆能力明显降低,脑梗死率明显升高(P<0.05);海马CA1区和CA3区神经元呈现数量减少、间隙增大、空泡样变、核膜核仁边界模糊、炎性细胞浸润等病理改变,凋亡率明显升高(P<0.05);海马组织IL-1β、IL-8、TNF-α含量明显升高(P<0.05);p-JAK2、p-STAT3、高迁移率族蛋白B1(HMGB1)、Bcl-2相关X蛋白(Bax)、激活型半胱氨酸蛋白酶-3(cleaved Caspase-3)相对表达量和p-JAK2/JAK2、p-STAT3/STAT3、Bax/Bcl-2表达比值均明显升高,Bcl-2相对表达量明显降低(P<0.05)。与CI/R组比较,CRO-M组、CRO-H组和NMP组大鼠学习记忆能力显著改善、脑梗死率明显降低(P<0.05);海马CA1区和CA3区神经元病理学改变明显改善、凋亡率明显降低(P<0.05);海马组织IL-1β、IL-8、TNF-α含量明显降低(P<0.05);p-JAK2、p-STAT3、HMGB1、Bax、Cleaved Caspase-3相对表达量和p-JAK2/JAK2、p-STAT3/STAT3、Bax/Bcl-2表达比值均明显降低(P<0.05)。CRO上述作用呈现一定的剂量依赖性,且CRO-H组对CI/R大鼠学习记忆能力、海马CA1区和CA3区神经元病理学改变和凋亡率、炎症因子含量、JAK2/STAT3信号通路相关蛋白表达的影响显著优于NMP组(P<0.05)。结论CRO可能通过抑制JAK2/STAT3信号通路活化,减轻炎症和神经元凋亡,从而对CI/R大鼠海马神经元损伤起到保护作用。

Objective To investigate the effect of crocin(CRO)on rats with hippocampal neuron cerebral ischemia reperfu‐sion(CI/R)injury and explore its potential mechanism.Methods A total of 144 male SD rats were selected and randomly divided into sham group,model(CI/R)group,CRO low-,medium-,high-dose(CRO-L,CRO-M,CRO-H)groups and nimodipine(NMP)group,with 24 rats in each group.The CI/R rat models were established by suture method.Rats in each group were administered by intrabitoneal injection(ip)once a day starting 7 days before modeling(the CRO-L,CRO-M,CRO-H groups were given 10,20,40 mg/kg CRO by ip respectively;the NMP group was given 1 mg/kg NMP by ip;and the sham and CI/R groups were given normal saline 5 mL/kg by ip).After 24 hours of reperfusion,the learning and memory ability of rats was detected by Morris water maze test.The cerebral infarction rate was detected using TTC staining.The neuron pathological changes of hippocampal CA1 and CA3 were observed using HE staining,and the neuronal apoptosis of hippocampal CA1 and CA3 was examined by TUNEL staining.The levels of interleukin(IL)-1β,IL-8,tumor necrosis factor-α(TNF-α)in hippocampal tissue were detected by ELISA.The expression of Janus kinase 2/signal transducter and activator of transcription 3(JAK2/STAT3)signaling pathway related proteins in hippocampal tissue were detected by Western blot.Results Compared with the sham group,the learning and memory ability of the rats in the CI/R group was significantly decreased,and the cerebral infarction rate significantly increased(P<0.05);the neurons in hippocampal CA1 and CA3 showed pathological changes,such as reduced neuron number,enlarged gaps,vacuolar degeneration,blurred nucleo‐lar border,and inflammatory cell infiltration,and the apoptosis rate significantly increased(P<0.05);the levels of IL-1β,IL-8,TNF-αin hippocampal tissue significantly increased(P<0.05);the expression of p-JAK2,p-STAT3,high mobility group protein B1,Bcl-2 associated X protein(Bax),cleaved Caspase-3 and the ratio of p-JAK2/JAK2,p-STAT3/STAT3,Bax/Bcl-2 significantly increased,while the expression of Bcl-2 was significantly decreased(P<0.05).Compared with the CI/R group,the learning and memory ability of rats in the CRO-M,CRO-H,and NMP groups significantly improved,and the cerebral infarction rate significantly decreased(P<0.05).The neuron pathological changes of hippocampal CA1 and CA3 significantly improved,and the apoptosis rate significantly decreased(P<0.05).The levels of IL-1β,IL-8,and TNF-αin hippocampal tissue significantly decreased(P<0.05).The expression of p-JAK2,p-STAT3,high mobility group protein B1,Bax,cleaved Caspase-3 and the ratio of p-JAK2/JAK2,p-STAT3/STAT3,and Bax/Bcl-2 significantly decreased(P<0.05).The above effects of CRO were dose-dependent,and the CRO-H group was significant‐ly superior to the NMP group in terms of learning and memory ability,hippocampal CA1,CA3 neuronal pathological changes and apoptosis rate,inflammatory factor level,and the expression of JAK2/STAT3 signaling pathway related proteins(P<0.05).Conclu‐sion CRO may play a protective role against hippocampal neuronal injury in CI/R rats by inhibiting the activation of JAK2/STAT3 signaling pathway,reducing inflammation and neuronal apoptosis.