Bilirubin encephalopathy (BE) is a neurological disorder caused by the accumulation of unconjugated bilirubin (UCB) in the brain of newborns, resulting in various degrees of neuronal impairment. BE is characterized by cytotoxic edema and neuronal apoptosis. Aquaporin-4 (AQP4), a water channel abundantly expressed in the central nervous system, plays a critical role in maintaining water homeostasis. Dysregulation of AQP4 expression or trafficking is closely associated with brain edema, suggesting that modulation of AQP4 may offer a potential therapeutic approach for BE. Previous studies have indicated that melatonin (MT) possesses neuroprotective and therapeutic potential against BE; however, its precise mechanisms remain unclear. In this study, we optimized rat BE model to investigate the therapeutic effects of melatonin on AQP4 expression, trafficking, and apoptosis in parietal cortical neurons. Furthermore, we explored the molecular mechanisms underlying melatonin’s neuroprotective actions, including the regulation mechanism of AQP4 expression, brain edema formation, and apoptosis induced by UCB accumulation. The results indicate that in the BE model, pathological injury of parietal cortex was significantly aggravated and AQP4’s expression peaked at 24 h after BE modeling. MT activated PI3K/AKT signaling pathway in rat parietal cortex to downregulate AQP4 expression, apoptosis related proteins, and decreased SNX27’s expression to promote the internalization of AQP4, reducing bilirubin induced cytotoxic edema and cortical apoptosis. This data suggest that MT has a neuroprotective role in BE, by potentially delaying its progression.

Retinal ischemia-reperfusion (I/R) injury is a common pathophysiological stress state connected to various diseases, including acute glaucoma, retinal vascular obstruction, and diabetic retinopathy. Recent studies have suggested that geranylgeranylacetone (GGA) could increase heat shock protein70 (HSP70) level and reduce retinal ganglion cells (RGCs) apoptosis in a rat retinal I/R model. However, the underlying mechanism remains unclear. Moreover, the injury caused by retinal I/R includes not only apoptosis but also autophagy and gliosis, and the effects of GGA on autophagy and gliosis have not been reported. Our study established a retinal I/R model by anterior chamber perfusion pressuring to 110 mmHg for 60 min, followed by 4 h of reperfusion. The levels of HSP70, apoptosis-related proteins, GFAP, LC3-II, and PI3K/AKT/mTOR signaling proteins were determined by western blotting and qPCR after treatment with GGA, HSP70 inhibitor quercetin (Q), PI3K in-hibitor LY294002, and mTOR inhibitor rapamycin. Apoptosis was evaluated by TUNEL staining, meanwhile, HSP70 and LC3 were detected by immunofluorescence. Our results demonstrated that GGA-induced HSP70 expression significantly reduced gliosis, autophagosome accumulation, and apoptosis in retinal I/R injury, indicating that GGA exerted protective effects on retinal I/R injury. Moreover, the protective effects of GGA mechanistically relied on the activation of PI3K/AKT/mTOR signaling. In conclusion, GGA-induced HSP70 overexpression has protective effects on retinal I/R injury by activating PI3K/AKT/mTOR signaling.

目的:研究幼年胆红素脑病(BE)模型大鼠海马中水通道蛋白4(AQP4)的表达变化,并且探究其与海马细胞凋亡及认知功能障碍的关系.方法:采用7 d龄SD幼年大鼠经小脑延髓池注射未结合胆红素(UCB)建立BE模型,根据UCB作用时间的不同将其分为12 h、24 h、48 h、72 h和7 d组.采用HE和Nissl染色观察各组海马病理变化;应用TUNEL染色法检测海马细胞凋亡;应用Western Blot检测海马AQP4、LAMP1、caspase-3和胶质纤维酸性蛋白(GFAP)的表达变化;应用免疫荧光染色检测海马AQP4、LAMP1和GFAP的表达变化及AQP4+/GFAP+细胞数量的变化.结果:HE及Nissl染色显示,随着UCB作用时间的延长,海马神经细胞间隙不断变大,空泡逐渐增多,Nissl小体数量减少.TUNEL染色法显示海马凋亡细胞数量在UCB作用24 h组最多(P＜0.05).Western Blot结果显示,随着UCB作用时间的延长,海马细胞中caspase-3和LAMP1均上升至24 h达峰值(P＜0.05).免疫荧光结果显示,AQP4和GFAP均增加至48 h表达最高(P＜0.05),AQP4+/GFAP+细胞数量在48 h组达最多(P＜0.05).结论:UCB的神经毒性作用可导致海马细胞凋亡增加至24 h达峰值,继发星型胶质细胞代偿性增生,并伴随AQP4的表达增加至48 h达峰值,以上变化可能与BE的认知功能障碍具有密切联系.

(AQP4) regulates retinal water homeostasis and participates in retinal oedema pathophys-iology. p-dystroglycan (p-DG) is responsible for AQP4 polarization and can be cleaved by matrix metalloproteinase-9 (MMP9). Retinal oedema induced by ischemia-reperfusion (I/R) injury is an early complica-tion. Bumetanide (BU) has potential efficacy against cytotoxic oedema. Our study investigated the effects of p -DG cleavage on AQP4 and the roles of BU in a rat retinal I/R injury model. The model was induced by applying 110 mm Hg intraocular pressure to the anterior eye chamber. BU and U0126 (a selective ERK inhibitor) were intraperitoneally administered 15 and 30 min, respectively, before I/R induction. Rhodamine isothiocyanate extravasation detection, quantitative real-time PCR, transmission electron microscopy, hematoxylin-eosin stain-ing, immunofluorescence staining, western blotting, and TUNEL staining were performed. AQP4 lost its polariza-tion in the retinal perivascular domain as a result of p-DG cleavage. BU rescued AQP4 depolarization, suppressed AQP4 protein expression, attenuated retinal cytotoxic oedema, and downregulated p-DG and AQP4 mRNA expres-sion. BU suppressed glial responses and mitochondria-mediated apoptotic protein expression, including that of Caspase-3 and Cyto C, raised the Bcl-2/Bax ratio, and lowered the number of apoptotic cells in the retina. Both BU and U0126 downregulated p-ERK and MMP9 expression. Thus, BU treatment suppressed p-DG cleavage, recov-ered AQP4 polarization partially via inhibiting ERK/MMP9 signaling pathway, and possess potential neuroprotec-tive efficacy in the rat retinal ischemia-reperfusion injury model.(c) 2022 IBRO. Published by Elsevier Ltd. All rights reserved.

Bilirubin encephalopathy (BE) is a neurological syndrome in newborns, mainly caused by neuronal injury due to excessive oxidative stress produced by unconjugated bilirubin (UCB). Neuroglobin (NGB) can protect the brain by removing oxidative stress species, but its expression and significance in BE are not clear. To address this question, the neonatal BE model was established by injecting UCB into the cerebellomedullary cistern of 7-day-old SD rats. Rats were divided into a sham and BE 6 hr group, BE 12 hr group, BE 24 hr group, and BE 7 d group according to UCB action times. Hematoxylin/eosin and Nissl staining, and electron microscopy were employed to observe the pathological and ultrastructural changes of nerve cells in each group. Immunofluorescence staining was used to detect NGB expression sites and cell types. Western blotting and quantitative PCR served to detect NGB expression and test the mitochondrial apoptosis signal pathway. The results confirm that UCB can lead to pathological damage and ultrastructural changes in rats' temporal cortex, increasing the expression of apoptosis-related proteins Bax, Bcl-2, Cyt c, Caspase-3, and neuronal NGB. UCB promotes NGB expression with an increase in action time and reach a peak at 12 hr. In summary, brain damage induced by UCB will cause an increase in NGB expression, the increasing NGB can inhibit neuron apoptosis in early BE phases. Therefore, promoting the expression of endogenous NGB, to act as a neuroprotective agent may be a potential treatment strategy for BE.

本研究通过电针刺激介导胆红素脑病(bilirubin encephalopathy,BE)模型鼠大脑颞叶皮质神经珠蛋白(neuroglobin,NGB)及PI3K/AKT通路、凋亡通路相关蛋白质的表达变化,以明确电针(electroacupuncture,EA)对于胆红素脑病的治疗作用,并探讨神经珠蛋白在该过程中的作用机制.将39只7日龄SD乳鼠分为假手术(Sham)组、BE模型组、电针治疗(BE+EA)组.经小脑延髓池注射胆红素溶液(10 μg胆红素/g体重)制备BE模型,假手术组注射等量生理盐水作为对照,BE+EA组选取百会穴与曲池穴以频率2/15 Hz的疏密波,于造模前12h、造模完成时及造模后12 h进行3次电针干预,每次15 min.使用HE、尼氏(Nissl)染色及透射电镜检测各组鼠脑颞叶皮质的病理变化及神经元超微结构的改变,结果显示电针处理可减轻BE乳鼠脑颞叶皮质神经元的损伤及增加尼氏体的数量,透射电镜则证实电针处理可改善神经元线粒体的水肿程度.应用免疫荧光染色法检测各组鼠脑颞叶皮质NGB的表达部位及其表达的细胞种类,结果显示NGB主要表达于颞叶皮质神经元中.进一步通过免疫印迹法检测各组鼠脑颞叶皮质中NGB蛋白及PI3K/AKT通路、线粒体凋亡信号通路相关蛋白质的表达,结果显示电针处理可增加NGB、PI3K p110α和pAKT Ser473的表达(分别为P＜ 0.05、0.05和0.01),上调凋亡相关蛋白Bcl-2/Bax比值(P＜0.001),进而抑制切割胱天蛋白酶3的激活(P＜0.05).通过TUNEL染色法检测各组凋亡细胞的数量,结果证实电针处理使凋亡细胞的数量减少(BE模型组186.00±13.86 vs BE+EA组78.67±11.85,P＜0.01).该研究初步证实,电针刺激可促进胆红素脑病鼠脑颞叶皮质中神经珠蛋白的表达,并进一步激活PI3K/AKT通路,而发挥其神经细胞保护功能,抑制凋亡反应的发生,电针可能成为胆红素脑病治疗潜在的方法.

神经细胞水肿是胆红素脑病(bilirubin encephalopathy,BE)发生发展过程中的重要病理变化.水通道蛋白-4(aquaporin-4,AQP4)的表达及分布异常与多种疾病所致细胞毒性脑水肿的发生发展具有密切联系.但胆红素脑病中AQP4的表达变化规律及其在病理进展中的作用尚不清楚.采用7日龄SD大鼠小脑延髓池注射胆红素溶液的方法,建立新生大鼠胆红素脑病模型.胆红素脑病模型根据胆红素作用时间的不同,分为12h、24 h、48 h、72 h和7d组.采用HE及尼氏染色,检测各新生大鼠脑组织的病理改变;应用透射电镜(TEM),检测胆红素作用24 h后,鼠脑组织超微结构的变化;应用免疫荧光及Western印迹,检测AQP4在脑组织中的表达变化.通过上述实验,以探讨AQP4的表达变化与胆红素所致脑损伤的关系.HE及尼氏染色结果显示,随着胆红素沉积时间的延长,神经细胞逐渐肿胀,细胞间隙增大,尼氏小体数量逐渐减少;电镜结果显示,胆红素脑病24 h后神经细胞线粒体出现肿胀;免疫荧光染色显示,24 h组AQP4的表达范围明显增加,其后表达范围逐渐减少,表达强度也随之减弱;Western印迹结果显示,AQP4表达在不同时间点呈现先增高后降低的趋势,在24 h达到峰值(24 h组1.38±0.11 vs对照组0.87±0.21,P＜0.05),在之后的各时间点上,AQP4的表达呈现下降趋势,而72 h组与7d组AQP4表达均低于48 h组(P＜0.05),基本恢复到对照组的表达水平(P＞0.05).上述结果提示,胆红素脑病中胆红素的毒性作用将引起AQP4表达量的改变,AQP4的表达变化与胆红素脑病中细胞毒性脑水肿的发生相关,并且可能在胆红素脑病脑损伤的进展中发挥作用.