目的:我们旨在解决关于桦木醇是否对蛛网膜下腔出血(SAH)引起的早期脑损伤(EBI)发挥神经保护作用的不确定性。并探讨相关的分子机制。
方法:进行生物信息学分析以预先研究不同表达的基因(DEGs)和可能的信号通路。本研究介绍了SAH的大鼠和细胞模型,还有桦木醇,DJ-1蛋白的激活剂,是为了揭示效果。关于死亡率的总评估,神经功能,SAH等级,脑含水量(BWC)以及体内或/和体外的多种细胞和分子研究,如免疫荧光(IF)染色,蛋白质印迹(WB),活性氧(ROS)测定,在SAH诱导后进行流式细胞术(FCM),以验证DJ-1在不同水平上的保护作用和相关机制。此外,利用MK2206(Akt的选择性抑制剂)和iRNADj-1(干扰Dj-1的RNA)来确认作用机制。
结果:我们研究的数据表明,DJ-1蛋白在神经元中中等表达,小胶质细胞,和星形胶质细胞;SAH诱导后24-72小时,其在脑组织中的水平升高并达到峰值。桦木醇可有效诱导DJ-1的表达,而DJ-1又激活Akt和Bcl-2以及抗氧化酶SOD2和HO-1,从而降低裂解的caspase-3(c-Casp-3)的激活和活性氧(ROS)。诱导的DJ-1可以上调Nrf2的表达。然而,Akt似乎对提高Nrf2的表达没有直接作用。单独的DJ-1也可以通过抑制caspase-8(Casp-8)的激活来激活不依赖Akt的抗凋亡途径。
结论:桦木醇是DJ-1的有效激动剂,具有诱导其在脑组织中表达的能力。DJ-1通过综合机制对EBI具有神经保护作用,包括促进内在和外在抗凋亡途径,并通过上调氧化还原蛋白的表达来减少氧化损伤。桦木醇作为一种廉价的药物显示出治疗SAH的潜力。
OBJECTIVE: We aimed to resolve the uncertainty as to whether betulin exerted neuroprotection on early brain injury (EBI) caused by subarachnoid hemorrhage (SAH), and to investigate the related molecular mechanisms.
METHODS: Bioinformatic analysis was performed to pre-study the differently expressed genes (DEGs) and the possible signaling pathways. Rat and cellular model of SAH were introduced in this study, and betulin, an activator of DJ-1 protein, was administered to reveal the effect. Gross assessment regarding mortality, neurofunctions, SAH grade, brain water content (BWC) along with multiple cellular and molecular studies in vivo or/and in vitro such as immunofluorescence (IF) staining, western blot (WB), reactive oxygen species (ROS) assay, and flow cytometry (FCM) were all conducted after SAH induction to verify the protective effect and the relevant mechanisms of DJ-1 in diverse levels. In addition, MK2206 (selective inhibitor of Akt) and iRNADj-1 (interfering RNA to Dj-1) were utilized to confirm the mechanisms of the effect.
RESULTS: The data from our study showed that DJ-1 protein was moderately expressed in neurons, microglia, and astrocytes; its level in brain tissue elevated and peaked at 24-72 h after SAH induction. Betulin could efficaciously induce the expression of DJ-1 which in turn activated Akt and Bcl-2, and anti-oxidative enzymes SOD2 and HO-1, functioning to reduce the activation of cleaved caspase-3 (c-Casp-3) and reactive oxygen species (ROS). The induced DJ-1 could upregulate the expression of Nrf2. However, Akt seemed no direct effect on elevating the expression of Nrf2. DJ-1 alone could as well activate Akt-independent antiapoptotic pathway via suppressing the activation of caspase-8 (Casp-8).
CONCLUSIONS: Betulin which was a potent agonist of DJ-1 had the ability to induce its expression in brain tissue. DJ-1 had neuroprotective effect on EBI through comprehensive mechanisms, including facilitating intrinsic and extrinsic antiapoptotic pathway, and reducing oxidative injury by upregulating the expression of redox proteins. Betulin as an inexpensive drug showed the potential for SAH treatment.