Role of the cation-chloride-cotransporters in the circadian system

The circadian system plays an immense role in controlling physiological processes in our body.The suprachiasmatic nucleus (SCN) supervises this system,regulating and harmonising the circadian rhythms in our body.Most neurons present in the SCN are GABAergic neurons.Although GABA is considered the main inhibitory neurotransmitter of the CNS,recent studies have shown that excitatory responses were recorded in this area.These responses are enabled by an increase in intracellular chloride ions[Cl;];levels.The chloride (Cl;) levels in GABAergic neurons are controlled by two solute carrier 12 (SLC12)cation-chloride-cotransporters (CCCs):Na^(+)/K^(+)/Cl^(-)co-transporter (NKCC1) and K^(+)/Cl^(-)cotransporter (KCC2),that respectively cause an influx and efflux of Cl^(-).Recent works have found altered expression and/or activity of either of these co-transporters in SCN neurons and have been associated with circadian rhythms.In this review,we summarize and discuss the role of CCCs in circadian rhythms,and highlight these recent advances which attest to CCC’s growing potential as strong research and therapeutic targets.

Anomalous expression of chloride transporters in the sclerosed hippocampus of mesial temporal lobe epilepsy patients

The Na＋-K＋-CI- cotransporter 1 and K＋-CI- cotransporter 2 regulate the levels of intracellular chloride in hippocampal cells. Impaired chloride transport by these proteins is thought to be involved in the pathophysiological mechanisms of mesial temporal lobe epilepsy. Imbalance in the relative expression of these two proteins can lead to a collapse of CI- homeostasis, resulting in a loss of gamma-aminobutyric acid-ergic inhibition and even epileptiform discharges. In this study, we investigated the expression of Na＋-K＋-CI- cotransporter 1 and K＋-CI- cotransporter 2 in the sclerosed hippocampus of patients with mesial temporal lobe epilepsy, using western blot analysis and immunohistochemistry. Compared with the histologically normal hippocampus, the sclerosed hippocampus showed increased Na＋-K＋-Cl- cotransporter 1 expression and decreased K＋-CI- cotransporter 2 expression, especially in CA2 and the dentate gyrus. The change was more prominent for the Na＋-K＋-CI- cotransporter 1 than for the K＋-CI- cotransporter 2. These experimental findings indicate that the balance between intracellular and extracellular chloride may be disturbed in hippocampal sclerosis, contributing to the hyperexcitability underlying epileptic seizures. Changes in Na＋-K＋-CI-cotransporter 1 expression seems to be the main contributor. Our study may shed new light on possible therapies for patients with mesial temporal lobe epilepsy with hippocampal sclerosis.

穿心莲内酯对脑出血大鼠脑水肿及AQP4、NKCC1表达的影响

目的探讨穿心莲内酯(AND)对脑出血大鼠脑水肿及水通道蛋白4(AQP4)、钠-钾-氯协同转运蛋白1(NKCC1)的影响及其机制。方法采用随机数字表法将160只SD大鼠分为假手术组、模型组、穿心莲内酯50 mg/kg组和尼莫地平10 mg/kg组,每组40只。采用自体血注射法制备脑出血大鼠模型,造模完成后,尼莫地平10 mg/kg组给予2 mg/mL尼莫地平溶液(灌胃体积5 mL/kg)灌胃,每日1次;穿心莲内酯50 mg/kg组给予10 mg/mL穿心莲内酯溶液(灌胃体积5 mL/kg)灌胃,每日1次;假手术组和模型组给予生理盐水灌胃5 mL/kg,每日1次。各组疗程7 d。采用改良神经功能缺损评分标准(mNSS)进行神经功能缺失评分,通过多田公式计算脑血肿体积,失重法检测脑含水量,伊文思蓝(EB)法测定血脑屏障通透性;通过透射电子显微镜(TEM)观察血脑屏障超微结构变化;免疫荧光法和蛋白免疫印迹(Western Blot)法检测脑组织AQP4和NKCC1表达。结果模型组大鼠神经功能缺失评分、脑血肿体积、脑含水量、血脑屏障通透性均明显高于假手术组(P<0.01);穿心莲内酯50 mg/kg组和尼莫地平10 mg/kg组神经功能缺失评分、脑血肿体积、血脑屏障通透性均明显低于模型组(P<0.01);且穿心莲内酯50 mg/kg组神经功能缺失评分、脑血肿体积、脑含水量、血脑屏障通透性均明显低于尼莫地平10 mg/kg组(P<0.05或P<0.01)。通过TEM观察发现,模型组大鼠血脑屏障可见基底膜薄厚不均匀、膨大、断裂现象,紧密连接结构(TJPs)疏松,内皮细胞皱缩等超微结构病变;与模型组比较,穿心莲内酯50 mg/kg组和尼莫地平10 mg/kg组血脑屏障超微结构病变不同程度减轻,其中穿心莲内酯50 mg/kg组效果更为明显。通过免疫荧光法和Western Blot法检测发现,模型组大鼠脑组织AQP4、NKCC1蛋白表达量明显高于假手术组(P<0.01);穿心莲内酯50 mg/kg组和尼莫地平10 mg/kg组AQP4、NKCC1蛋白表达量明显低于模型组(P<0.01);且穿心莲内酯50 mg/kg组AQP4、NKCC1蛋白表达量明显低于尼莫地平10 mg/kg组(P<0.01)。结论穿心莲内酯具有减轻脑出血大鼠脑水肿的作用,其机制可能与下调AQP4和NKCC1表达、降低血脑屏障通透性有关。

氯离子协同转运蛋白KCC2和NKCC1在正常成年大鼠背根神经节内的表达

【目的】观察氯离子协同转运蛋白钾离子氯离子共转运体2(potassium-chloride cotransporter-2,KCC2),钠离子钾离子氯共转运体1(sodium potassium chloride cotransporter,NKCC1)在正常成年大鼠背根神经节内的表达。【方法】成年SD大鼠,用4%多聚甲醛固定,取背根神经节,神经肽能物质SP和钠通道蛋白Nav的免疫组织化学用来鉴定背根神经节,KCC2和NKCC1在背根神经节内的免疫组织化学,普通显微镜观察结果拍照。【结果】KCC2在背根神经节内没有表达,NKCC1在背根神经节神经元胞浆有表达。【结论】KCC2在大鼠背根神经节没有表达和NKCC1在背根神经节有表达提示背根神经节内神经元氯离子稳态系统不同于其它神经系统部位,这也可能是背根神经节内神经元对GABA表现为去极化的原因。

神经元细胞KCC2、NKCC1及4E-BP1在外伤性癫痫致痫灶中的表达及其临床意义

目的探讨神经元细胞KCC2、NKCC1及4E-BP1在外伤性癫痫致痫灶中的表达及其临床意义。方法选择2017年1月-2019年6月本院接诊外伤性癫痫患者37例纳入研究组,经手术切除癫痫致癫灶;同期,另选择于单纯脑外伤患者40例纳入对照组,分别于脑外伤后6 h内手术获取脑组织样本。通过蛋白质印迹法测定外伤性癫痫致癫灶中KCC2、NKCC1、4E-BP1表达,并利用RT-PCR法测定外伤性癫痫致癫灶中KCC2 m RNA、NKCC1 m RNA、4E-BP1m RNA相对表达量。结果蛋白质印迹法检测显示:研究组患者的神经元细胞KCC2相对灰度值低于对照组,差异有统计学意义(P <0.05);研究组患者的神经元细胞NKCC1、4E-BP1相对灰度值均高于对照组,差异有统计学意义(P <0.05);RT-PCR检测显示:研究组患者的致癫灶中KCC2 m RNA相对表达量低于对照组,差异有统计学意义(P <0.05);研究组患者的致癫灶中NKCC1 m RNA、4E-BP1 m RNA相对表达量均高于对照组,差异有统计学意义(P <0.05)。结论神经元细胞KCC2、NKCC1及4E-BP1为脑外伤后患者脑组织的组织学改变、癫痫反复发作主要分子机制;KCC2在外伤性癫痫致癫灶中表达异常降低,而神经元细胞NKCC1、4E-BP1的表达异常增高,可以作为外伤性癫痫致痫灶靶向诊断因子。

离子转运／交换蛋白在缺血性脑卒中后脑水肿中的作用机制

脑卒中严重危害着人类生命健康，目前高居世界人类死因排名第二位、中国居民死亡率第二位，且发病率近年来仍呈上升趋势。脑卒中分为缺血性脑卒中及出血性脑卒中，其中缺血性脑卒中的发病率更高，约占脑卒中总数的80％。