微小RNAs和长链非编码RNA对胃溃疡肝郁脾虚证线粒体低氧调控机制的研究

Abstract: Gastric ulcer is a common disease, easily complicated by bleeding, perforation, cancer and other serious consequences. As the main drugs used to treat gastric ulcer, proton pump inhibitors and the like have shortages in their effectiveness and safety. Understanding of Traditional Chinese Medicine with the gastric ulcer with syndrome of liver depression and spleen deficiency conforms the modern medical physical and mental illness theory,meanwhile the empirical formula,CHWKN has a fairly good effect on this syndromes clinically.By using two-dimensional gel electrophoresis combined with mass spectrometry technology in the previous study, the differential protein group in the model was found and turned out to be closely related to mitochondrial hypoxia regulatory mechanisms . According to the recent reports that hypoxia associated with ROS、PHD、LDH-A、HIF-1α, and many kinds of microRNAs/LncRNAs were involved in the generation of mitochondrial hypoxia regulatory mechanisms, and these representative proteins were regulated by some microRNAs/LncRNAs.So we assumed there might be microRNAs, LncRNAs losing the control of mitochondrial hypoxia state in the gastric ulcer with syndrome of liver depression and spleen deficiency,losing the control of these representative proteins,leading to the expression of differential protein. By screening the syndrome associated microRNAs first,while from the relevant differential protein group、these representative proteins and known regulatory microRNAs/LncRNAs, then combined with GWAS and other methods,we will explore the syndrome's essence and the therapeutic mechanism of CHWKN on the foundation of our previous study.

胃溃疡是消化系统常见病，易并发出血、癌变等严重后果，西药治疗该病的有效性、安全性仍存在一些问题。中医药对胃溃疡肝郁脾虚证的认识符合现代医学身心疾病理论，验方柴黄胃溃宁对该病证临床疗效良好。前期实验利用2-DE联合质谱技术已找到该模型的差异性蛋白群，这与线粒体中低氧相关，柴黄胃溃宁能改善低氧状态。文献表明线粒体低氧状态与ROS、PHD、LDH-A、HIF-1α等蛋白相关，同时受多种微小RNAs/长链非编码RNAs的调控。本课题组认为胃溃疡肝郁脾虚证中可能存在某些微小RNAs/长链非编码RNAs对ROS、PHD、LDH-A、HIF-1α进行调控，导致差异性蛋白表达。因此，本课题组先筛选病证相关的微小RNAs，同时从差异性蛋白群/低氧相关蛋白/相关微小RNAs、长链非编码RNAs着手，联合GWAS等方法尝试探究胃溃疡肝郁脾虚证的本质和验方柴黄胃溃宁疗效的作用机制。

胃溃疡是消化系统常见病，易并发出血、癌变等严重后果，西药治疗该病的有效性、安全性仍存在一些问题。中医药对胃溃疡肝郁脾虚证的认识符合现代医学身心疾病理论，验方柴黄胃溃宁对该病证临床疗效良好。本课题组据此病因病机建立肝郁脾虚胃溃疡大鼠模型，并检测模型大鼠胃窦组织的低氧诱导因子（HIF-1）、乳酸脱氢酶A（LDH-α）、脯氨酸羟化酶2（PHD2）的蛋白表达水平及ROS含量，揭示该病证的形成与微循环低氧环境有关。以黄芪建中汤为底方的验方柴黄胃溃宁对其有改善作用。课题组从基因（miRNAs）-蛋白调控层面探讨肝郁脾虚胃溃疡在低氧环境中形成的机制，实验表明与低氧相关的miRNA-24、miRNA-122、miRNA-199等表达在肝郁脾虚胃溃疡组与正常组之间有差异，黄芪建中汤、柴黄胃溃宁均可逆转该差异，起到改善低氧环境，达到治疗的作用。通过对筛选的差异miRNA的靶基因预测、GO和KEGG富集性分析显示，验方及其底方还可通过抑制HP、增强机体的免疫力（如影响IL-6趋化等作用）等起到治疗肝郁脾虚胃溃疡的作用。本研究揭示中药验方通过多靶点多通路起效，与现代医学的胃溃疡的病因多样，病机复杂的理论相契合。检测胃溃疡肝郁脾虚证中相关微小RNA的表达是本研究的核心部分，从微小RNA层面去探析胃溃疡肝郁脾虚证发生的机制，为验方柴黄胃溃宁的起效机制进行了更深入的阐释，并为病证结合模型的实验研究提供了更深层次的理论依据。