飞龙掌血通过调控大鼠M1/M2型小胶质细胞极化减轻脑缺血再灌注损伤

Toddalia asiatica attnuates cerebral ischemia-reperfusion injury in rats through modulating M1/M2 polarization of microglia

目的:观察飞龙掌血(Toddalia asiatica)对脑缺血再灌注损伤(cerebral ischemia-reperfusion injury,CIRI)大鼠的神经功能影响,进一步探讨药物对小胶质细胞M1/M2型极化、Toll样受体4(Toll-like receptor 4,TLR4)/髓样分化因子88(myeloid differentiation factor 88,MyD88)/核因子κB(nuclear factorκB,NF-κB)通路的影响及机制。方法:使用改良线栓法来制备大鼠的CIRI模型,并将大鼠随机分为了模型组、飞龙掌血(1.08 g/kg)组、盐酸多奈哌齐(0.45 mg/kg)组及假手术组,每组各16只,在评估各组大鼠神经功能改变的基础上,采用HE和Nissl染色观察脑组织病理变化,使用TUNEL染色法检测脑神经元的凋亡,免疫组化法检测M1型小胶质细胞标志物离子钙结合衔接分子1(ionized calcium-binding adapter molecule 1,Iba1)和M2型小胶质细胞标志物精氨酸酶1(arginase 1,Arg1)以及TLR4的定位表达。通过Western blot检测海马区小胶质细胞极化蛋白以及与TLR4/MyD88/NF-κB通路相关的蛋白表达。结果:与假手术组比,模型组大鼠神经功能评分较高(P<0.01),同时海马CA1区和皮质区神经元排列松散紊乱,尼氏小体减少,脑神经元凋亡增多。M1型小胶质细胞标志物Iba1、M2型小胶质细胞标志物Arg1及TLR4阳性细胞的数量明显增多。此外,海马组织的TLR4、MyD88、p-NF-κB p65、NF-κB p65、磷酸化NF-κB抑制因子(NF-κB inhibitory factor,IκB)、Iba1、白细胞介素6(interleukin-6,IL-6)和Arg1的蛋白表达升高(P<0.05),而IL-4和IL-10表达降低(P<0.01)。与模型组相比,飞龙掌血组和盐酸多奈哌齐组Arg1、IL-4和IL-10蛋白表达升高(P<0.05),其余指标均降低(P<0.05)。结论:飞龙掌血对CIRI大鼠具有神经保护作用,可能与调控小胶质细胞M1/M2型极化、抑制TLR4/MyD88/NF-κB介导的炎性通路有关。

AIM:Observation of neuroprotective effects of Toddalia asiatica(TA)on cerebral ischemia-reper-fusion injury(CIRI)in rats by investigating the effects and mechanisms of drugs on the polarization of microglia M1/M2 subtype and the Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB)path-way.METHODS:The modified thread occlusion method was used to establish a rat model of CIRI,and the rats were ran-domly divided into the model group,Toddalia asiatica(1.08 g/kg)group,donepezil hydrochloride(0.45 mg/kg)group,and sham group,with 16 rats in each group.Based on the assessment of neurofunctional changes in each group of rats,HE and Nissl staining were used to observe the pathological changes in brain tissue.TUNEL staining was performed to de-tect neuronal apoptosis,immunohistochemistry was used to detect the expression of M1 microglia marker ionized calcium-binding adapter molecule 1(Iba1),M2 microglia marker arginase 1(Arg1),and TLR4 localization.Western blot was used to detect the expression of microglia polarization proteins and proteins related to TLR4/MyD88/NF-κB pathway in the hippocampus region.RESULTS:Compared with sham group,the model group rats had higher neurological function scores(P<0.01),and neuronal arrangement in the hippocampus and cortex was loose and disordered,Nissl bodies de-creased,and neuronal apoptosis increased.The numbers of M1 microglia marker Iba1-,M2 microglia marker Arg1-,and TLR4-positive cells were significantly increased.In addition,the protein levels of TLR4,MyD88,p-NF-κB p65,NF-κB p65,p-NF-κB inhibitory factor(p-IκB),Iba1,interleukin-6(IL-6),and Arg1 in the hippocampus were elevated(P<0.05),while IL-4 and IL-10 expression were decreased(P<0.01).Compared with model group,the Toddalia asiatica group and Donepezil hydrochloride group showed increased protein expression of Arg1,IL-4 and IL-10(P<0.05),while the other indicators were decreased(P<0.05,P<0.01).CONCLUSION:Toddalia asiatica possesses neuroprotective effects on CIRI rats,which may be attributed to its ability to regulate M1/M2 polarization and inhibit the TLR4/MyD88/NF-κB-mediated inflammatory pathway.