基于HIF-1α-SIRT1-beclin1介导自噬失衡研究从瘀毒论治胃癌前病变的分子机制

Our research team has undertaken the treatment of precancerous lesions of gastric cancer(PLGC) from blood stasis, toxin in traditional Chinese medicine(TCM), especially we have achieved the nobly reversing effect on the stage of abnormal hyperplasia, a critical pathology of PLGC, and make a breakthrough in prevention of this malady. According to basic researches, the alteration of gastric mucosal micro - environment and unbalance of autophagy have been observed during the development of PLGC. However, a TCM formula(JPHY), which is able to strengthen spleen and dissolve stasis, can inhibit formation of autophagosome in gastric mucosa as well as micro-environmental hypoxia induced by HIF-1α, and selectively regulated associated-autophagy protein expression of beclin1. Hence, this project hypothesizes this formula can reverse the development of PLGC by regulating autophagy via HIF-1α-SIRT1-beclin1 pathway. Additionally, the research will make a GES-1 cell model, which is combined with siRNA and plasmid transfection, as well as differential stages of chemical inducements and Kras transgenic PLGC animal models. Based on these designs, we are allowed to detect the regulations of the formula on signal network of HIF-1α-SIRT1-beclin1 pathway and expression of miRNA. Briefly, we are able to systematically elucidate the role of this signaling transduction within the development of PLCG, and the reversing mechanism as well as cure traits of JPHY, which will provide powerful experimental evidences in its clinical utilization and applications.

课题组从中医瘀毒论治胃癌前病变(PLGC)，尤其在逆转胃癌“临界性病变”PLGC的异形增生阶段取得显著临床疗效，突破防治PLGC的瓶颈。前期研究发现，PLGC的发展过程中出现胃粘膜微环境的改变和自噬失衡；而瘀毒论治方剂-健脾化瘀解毒方及有效成分可显著抑制PLGC胃黏膜自噬小体和HIF-1α介导缺氧微环境的形成，且选择性调控自噬相关蛋白beclin1。故，本课题提出健脾化瘀解毒方通过调控HIF-1α-SIRT1-beclin1介导自噬而逆转PLGC发展的假说。为此，拟采用siRNA和质粒转染GES-1细胞模型，并于化学诱变及Kras转基因PLGC动物模型的各阶段，检测该方及有效成分对HIF-1α-SIRT1-beclin1信号网络相关基因及miRNA的调控，从而系统阐述该信号转导网络在PLGC发展中的角色，及瘀毒论治方剂逆转PLGC作用机理和治疗特点，为该方的临床应用提供有力实验依据。

根据经典的Correa假说，胃癌往往经历胃癌前病变(PLGC)（正常胃黏膜→慢性非萎缩性胃炎→肠化生→异型增生→侵袭性胃腺癌）逐步演化的过程。可见，PLGC的早诊断和早干预对于阻断其向胃癌不可逆发展具有重要意义。前期发现PLGC胃粘膜细胞呈现自噬的异常变化，且有低氧应激，能量代谢异常。故提出健脾化瘀解毒方通过调控HIF-1α-SIRT1-beclin1介导自噬而逆转PLGC发展的假说。. 本课题采用MNNG诱变PLGC动物模型，动态观察PLGC发展中不同病理阶段出现胃粘膜损伤与修复、微环境变化和自噬失衡的变化，发现在早中晚期胃黏膜细胞均处于缺氧微环境状态。受缺氧应激的微环境变化所影响，PLGC模型大鼠在三个时间节点均出现胃黏膜细胞自噬凋亡失衡的表现，即自噬激活和凋亡抑制状态。同时，所构建的Atp4a-/-自发小鼠模型，证实在PLGC发展过程中（肠化阶段），HIF-1α-SIRT1-Beclin1调控轴被激活。通过MNU诱导和质粒转染GES-1构建的MTCs细胞模型，在胃粘膜细胞处于缺氧微环境改变时，微环境自噬是细胞调控内稳态应对微环境改变的重要方式。其中HIF-1α与SIRT1发生相互作用，激活自噬关键蛋白Beclin1，从而发生自噬，与此同时，自噬蛋白通过抑制caspase介导的凋亡蛋白抑制细胞凋亡，发生自噬凋亡失衡，出现“选择性细胞增殖”现象，即正常细胞低增殖以及恶化细胞的高增殖可逃避凋亡，逐渐形成肿瘤细胞。. JPHY对PLGC不同阶段的黏膜损伤均有保护作用，初期以抑制胃黏膜炎性反应，减少病变细胞为主，后期以改善腺腔结构、修复受损胃黏膜上皮细胞为主。可能通过调控PI3K/Akt/mTOR改善PLGC胃黏膜病理情况，并激活HIF-1α-SIRT1-Beclin1通路介导自噬，改善缺氧微环境，调节细胞的能量代谢方式，从而逆转胃黏膜萎缩、肠上皮化生和异型增生。. 此研究针对“微环境”改变对胃黏膜的损伤，与胃癌前病变“瘀毒”致病理论相符合，同时JPHY可能通过改善动物模型主、壁细胞超微结构和组织学病变，缓解胃黏膜萎缩、肠上皮化生和异型增生性病变，减轻微环境改变对胃黏膜的损伤并延缓胃黏膜恶性进展，防止其恶变为侵袭性胃腺癌，从而丰富了健脾化瘀解毒法修复胃黏膜损伤的科学内涵。