芍药苷对蛛网膜下腔出血早期脑损伤大鼠NLRP3炎性小体表达的影响

Effect of paeoniflorin on the expression of NLRP3 inflammasome in rats with early brain injury after subarachnoid hemorrhage

目的探究芍药苷(Paeoniflorin,PAE)对蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)早期脑损伤大鼠NLR家族Pyrin域蛋白3(NLR family Pyrin domain containing 3,NLRP3)炎性小体表达的影响。方法将60只大鼠随机分为假手术(Sham)组、模型(SAH)组、芍药苷低剂量(PAE-L)组、芍药苷高剂量(PAE-H)组;Sham组不刺破血管,其余各组采用血管内穿孔法构建SAH大鼠模型;PAE-L组、PAE-H组腹腔注射对应剂量的芍药苷(10、20 mg/kg),Sham组、SAH组腹腔注射等量0.9%氯化钠溶液,2次/d,共3 d;采用Garcia评分法评估大鼠神经功能;SAH评分法评估蛛网膜下腔出血情况;脑组织含水量测定评估脑水肿;伊文思蓝(Evans blue,EB)染色评估血脑屏障通透性;苏木精-伊红(Hematoxylin-eosin,HE)染色观察大鼠脑组织病理损伤;TdT介导的dUTP末端标记法(TdT-mediated dUTP nick-end labeling,TUNEL)染色检测神经元凋亡;免疫荧光染色检测NLRP3、离子钙结合衔接分子-1(Ionized calcium binding adapter molecule-1,Iba-1)和髓过氧化物酶(Myeloperoxidase,MPO)表达水平;免疫组化染色检测NLRP3、凋亡相关斑点样蛋白(Apoptosis-associated speck-like protein containing CARD,ASC)、半胱氨酸天冬氨酸酶-1(Cysteinyl aspartate-specific proteinase,Caspase-1)表达水平;Western Blot检测B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)、Bcl-2相关X的蛋白质(Bcl-2-Associated X protein,Bax)、Caspase-3,NLRP3,ASC,Caspase-1、白介素-1β(Interleukin-1β,IL-1β)、IL-18、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、Iba-1和MPO表达水平。采用氧化血红蛋白(Oxygenated hemoglobin,OxyHb)构建原代皮层神经元体外SAH模型,并将其分为对照(Control)组、模型(OxyHb)组、芍药苷低剂量(PAE-L)组和芍药苷高剂量(PAE-H)组;Control组正常培养,OxyHb组、PAE-L组、PAE-H组神经元在含20 uM OxyHb的神经元培养基中培养,PAE-L组、PAE-H组分别加入对应剂量的芍药苷(10、20 uM);各组神经元孵育48 h;采用细胞计数试剂盒8(Cell counting kit 8,CCK8)法和TUNEL染色检测神经元活性及凋亡;Western Blot检测Bcl-2,Bax,Caspase-3的表达水平。结果动物模型显示,Sham组大鼠脑组织未见积血,脑组织细胞结构清晰可辨;与Sham组比较,SAH组脑组织可见大量积血,脑组织细胞排列松散,Garcia评分降低(P<0.05),SAH评分、脑含水量、EB渗出量升高(P<0.05),神经元TUNEL阳性细胞数增加(P<0.05),Bcl-2表达水平降低,Bax,Caspase 3,NLRP3,ASC,Caspase-1,IL-1β,IL-18,TNF-α表达水平升高(P<0.05),Iba-1和MPO阳性细胞数增加(P<0.05);与SAH组比较,PAE-L组、PAE-H组脑组织积血减少,脑组织细胞状态趋于正常,Garcia评分升高(P<0.05),SAH评分、脑含水量、EB渗出量降低(P<0.05),神经元TUNEL阳性细胞数减少(P<0.05),Bcl-2表达水平升高,Bax,Caspase 3,NLRP3,ASC,Caspase-1,IL-1β,IL-18,TNF-α表达水平降低(P<0.05),Iba-1和MPO阳性细胞数减少(P<0.05)。细胞模型显示,Control组神经元形态正常;与Control组比较,OxyHb组神经元肿胀,突触丧失,神经元活性降低,凋亡率增高(P<0.05),Bcl-2表达水平降低,Bax,Caspase 3表达水平升高(P<0.05);与OxyHb组比较,PAE-L组、PAE-H组细胞损伤减轻,神经元活性升高,凋亡率降低(P<0.05),Bcl-2表达水平升高,Bax,Caspase 3表达水平降低(P<0.05)。结论芍药苷能够减轻SAH后大鼠脑水肿、细胞凋亡和神经损伤,其机制可能为通过抑制NLRP3炎性小体的激活,并抑制小胶质细胞和中性粒细胞浸润,改善SAH后早期脑损伤。

Objective To investigate the effect of paeoniflorin(PAE)on the expression of NLR family Pyrin domain containing 3(NLRP3)inflammasome in rats with early brain injury after subarachnoid hemorrhage(SAH).Methods Sixty rats were randomly divided into sham group,model group(SAH group),low-dose paeoniflorin treatment group(PAE-L group)and high-dose paeoniflorin treatment group(PAE-H group).In Sham group,the blood vessels were not punctured,and in the other groups,the SAH model of rats was established by intravascular puncture method.PAE(10 mg/kg,20 mg/kg)was injected intraperitoneally in PAE-L group and PAE-H group respectively,while the same volume of 0.9%sodium chloride solution was injected intraperitoneally in sham group and SAH group twice a day for 3 days.Garcia score was performed to assess the neurologic function of rats;SAH score was used to evaluate SAH grade;the brain water content was measured to assess brain edema;Evans blue staining was used to evaluate the permeability of blood-brain barrier;Hematoxylin-eosin(HE)staining was used to observe the pathological damage of brain tissue;TdT-mediated dUTP nick-end labeling(TUNEL)staining was used to detect neuronal apoptosis;Immunofluorescence staining was used to detect the expression ofNLRP3,ionized calcium binding adapter molecule-1(Iba-1)and myeloperoxidase(MPO);Immunohistochemistry was used to detect the expression of NLRP3,apoptosis-associated speck-like protein containing CARD(ASC)and cysteinyl aspartate-specific proteinase(Caspase)-1;Western blot was used to detect the expression of B cell lymphoma-2(Bcl-2),Bcl-2-Associated X protein(Bax),Caspase-3,NLRP3,ASC,Caspase-1,Interleukin-1β(IL-1β),IL-18,tumor necrosis factor-α(TNF-α),Iba-1 and MPO.The SAH model of cultured primary cortical neurons was established with oxidized hemoglobin(OxyHb)in vitro and divided into control group,model group(OxyHb group),low-dose paeoniflorin treatment group(PAE-L group)and high-dose paeoniflorin treatment group(PAE-H group).Neurons in control group were cultured in normal medium,while neurons were cultured in the medium containing 20μMOxyHb in the OxyHb group,PAE-L group and PAE-H group.And paeoniflorin(10μM and 20μM)was added to the medium of PAE-L group and PAE-H group respectively.Neurons were incubated for 48 h in each group.Cell counting kit 8(CCK8)and TUNEL staining were used to detect the cell activity and apoptosis of neurons;Western blot was used to detect the protein expressions of Bcl-2,Bax and Caspase-3.Results It showed that there was no hematocele in the brain of rats in sham group,and the morphology of cells in brain was clear and recognizable.Compared with the sham group,it showed a large volume of hematocele in the rats'brain,loose arrangement of the cells,decreased Garcia score(P<0.05),increased SAH score,brain water content and EB exudation(P<0.05),increased number of TUNEL positive neurons(P<0.05),decreased the expression of Bcl-2,increased the expressions of Bax,Caspase3,NLRP3,ASC,Caspase-1,IL-1β,IL-18 and TNF-αin the SAH group(P<0.05).Also the number of Iba-1 and MPO positive cells were increased in the SAH group(P<0.05).Compared with SAH group,it showed less hematocele in the brain,normal cellular state,higher Garcia score(P<0.05),lower SAH score,brain water content,and EB exudation(P<0.05),less TUNEL positive neurons(P<0.05),higher expression of Bcl-2,and less expressions of Bax,Caspase 3,NLRP3,ASC,Caspase-1,IL-1β,IL-18,TNF-α(P<0.05)in rats of the PAE-L group and PAE-H group.And the number of Iba-1 and MPO positive cells decreased(P<0.05)in the PAE-L group and PAE-H group.The morphology of neurons in control group was normal in vitro.Compared with the control group,neurons in OxyHb group were swollen,synaptic loss,decreased neuronal activity,increased apoptosis rate(P<0.05),decreased the expression of Bcl-2 and increased the expression of Bax and Caspase3(P<0.05).Compared with OxyHb group,it showed less cell damage,higher neuronal activity,lower apoptosis rate(P<0.05),higher expression of Bcl-2,and lower expression of Bax and Caspase3(P<0.05)in neurons of PAE-L group and PAE-H group.Conclusion Paeoniflorin could reduce brain edema,neuronal apoptosis and neurologic damage in rats after SAH,and improve early brain injury after SAH by inhibiting the activation of NLRP3 inflammasome and inhibiting the infiltration of microglia and neutrophils.