Zhongguo cuzhong zazhi (Nov 2022)
基于高通量RNA-Seq技术探讨补阳还五汤治疗小鼠脑梗死的作用机制 Mechanism of Buyanghuanwu Decoction in Treatment of Cerebral Infarction Mice Based on High-Throughput RNA Sequencing Technology
Abstract
目的 采用高通量RNA-Seq技术探讨补阳还五汤治疗小鼠脑梗死的作用机制。 方法 通过电凝法制作左侧大脑中动脉闭塞（middle cerebral artery occlusion，MCAO）的脑梗死小鼠模型，随机分为治疗组和模型组（每组6只），分别从MCAO术后1 d起每天灌胃18.59 g/kg补阳还五汤生药或等容积蒸馏水。另设假手术组（6只）。在治疗前和MCAO术后7 d采用改良神经损伤严重程度评分（modified neurological severity scores，mNSS）行神经功能评分。通过高通量RNA-Seq技术分析MCAO术后7 d三组小鼠脑组织差异基因的表达谱，随机挑选6个差异基因采用定量反转录PCR（quantitative reverse transcriptase-mediated PCR，qRT-PCR）法进行验证。对小鼠脑梗死后表达失调且补阳还五汤治疗后逆转的差异基因进行基因本体论（gene ontology，GO）和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路分析，预测补阳还五汤治疗脑梗死的潜在生物学过程及相关信号通路。 结果 治疗前，治疗组与模型组mNSS差异无统计学意义[5.00（4.00～5.25） vs. 5.00（4.00～6.00），P＞0.017]；MCAO术后7 d，治疗组mNSS较模型组下降[3.00（2.75～4.00）vs. 5.00（4.00～6.00），P＜0.017]。模型组与假手术组相比上调且补阳还五汤治疗后下调的差异基因有134个，模型组与假手术组相比下调且补阳还五汤治疗后上调的差异基因有19个。Myd88、Map3k8、Il1r1、Lamc2和Tlr9的mRNA表达水平在模型组升高，在治疗组降低；而Cxcl10则相反，均与高通量测序结果一致。GO分析和KEGG分析显示，小鼠脑梗死后上调且补阳还五汤治疗后逆转的差异基因主要富集于“急性炎症反应、免疫调节、细胞死亡、自噬”和“IL-17信号通路、Tod样受体信号通路、趋化因子信号通路及NF-kB信号通路”等与炎症反应及细胞死亡相关的生物学过程和信号通路；小鼠脑梗死后下调且补阳还五汤治疗后上调的差异基因则主要富集于“锥体神经元分化、突触可塑性的正向调节、突触囊泡循环、神经递质运输”和“逆行内源性大麻素信号转导、钙信号转导、长寿调节通路、长时程增强”等与神经可塑性相关的生物学过程和信号通路。 结论 小鼠脑梗死后缺血组织的基因表达水平发生了明显变化。补阳还五汤可能作用于与炎症反应和神经可塑性密切相关的多个分子靶点，减轻炎症导致的脑组织损伤，促进神经重塑，从而改善急性脑梗死后神经功能。 Abstract: Objective To explore the mechanism of buyanghuanwu decoction (BYHWD) in treating cerebral infarction mice based on high-throughput RNA sequencing technology. Methods The mouse cerebral infarction model of middle cerebral artery occlusion (MCAO) was established by electrocoagulation. The MCAO mice were randomly divided into BYHWD treatment group and control group (n=6, each group). From 1 day after MCAO, 18.59 g/kg BYHWD or equivalent volume distilled water were administered by gavage for consecutive 7 days. Another 6 mice were selected as sham operation group. The modified neurological severity scores (mNSS) was used to evaluate the neurological function of mice in the three groups before treatment and at 7 days after MCAO. High-throughput RNA-sequencing technology was used to analyze the expression profile of differential genes of mice brain tissues in the three groups at 7 days after MCAO operation. Six differential genes were randomly selected and verified by quantitative reverse transcriptase-mediated PCR (qRT-PCR). The differential genes were analyzed by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway methods to identify the potential biological process and related signaling pathways of genes dysregulation after MCAO and reversed by BYHWD. Results Before treatment, there was no statistical difference in the mNSS score between the BYHWD and control groups [5.00 (4.00-5.25) vs. 5.00 (4.00-6.00), P>0.017]. At 7 day after MCAO, the mNSS score in BYHWD group decreased compared with the control group [3.00 (2.75-4.00) vs. 5.00 (4.00-6.00), P<0.017]. Compared with the sham operation group, 134 genes were up-regulated after MCAO and down-regulated by BYHWD treatment, while 19 genes were down-regulated after MCAO and up-regulated by BYHWD treatment. The mRNA expression of Myd88, Map3k8, Il1r1, Lamc2 and Tlr9 increased in control group and decreased in BYHWD group, while Cxcl10 was on the contrary, which were consistent with the results of high-throughput sequencing. GO and KEGG analysis showed that the up-regulated genes after MCAO and reversed by BYHWD treatment were mainly enriched in the biological processes and signaling pathways related to inflammation and cell death, such as “acute inflammatory response, immune regulation, cell death, autophagy” and “IL-17 signaling pathway, Tod-like receptor signaling pathway, chemokine signaling pathway and NF-Kappa B signaling pathway”, etc. The down-regulated genes after MCAO and reversed by BYHWD treatment were mainly enriched in the biological processes and signaling pathways related to neural plasticity, such as “pyramidal neuron differentiation, positive regulation of synaptic plasticity, synaptic vesicle circulation, neurotransmitter transport” and “retrograde endocannabinoid signal transduction, calcium signal transduction, longevity regulation pathway, long-term enhancement”, etc. Conclusions The level of gene expression in ischemic brain tissue of mice changed significantly after acute cerebral infarction. BYHWD can alleviate brain damage and promote neuroplasticity by acting on multiple molecular targets closely related to inflammatory response and neural plasticity, and thus improve neurological function of acute cerebral infarction mice.
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