基于高通量小RNA测序研究健脾清化汤通过调节iNOS与mir-21治疗慢性萎缩性胃炎异型增生的机制研究

Chronic atrophic gastritis (CAG) with epithelial dysplasia is a kind of precancerous lesions, through the inducible nitric oxide synthase (iNOS),Helicobacter pylori (HP) can be secret of excess nitric oxide(NO) expression. Excess NO promote miRNA-21 expression, inhibit tumor suppressor gene PDCD4 and the cell cycle cyclin dependent kinase inhibitor 1b(P27kipl )expression, which cause gastric mucosal dysplasia. Abnormal proliferation mediated by iNOS-mi-21-PDCD4-p27kipl plays an important role in the pathogenesis of CAG dysplasia but less research. In previous work we have found that Jianpi Qinghua Decoction has exact clinical curative effect, and effectively inhibit HP gastritis mouse gastric mucosa inflammatory injury induced by NO, but its mechanism is still not clear. Therefore, we intend to clarify the intervention of Jianpi Qinghua Decoction on iNOS-mir-21-PDCD4-p27kipl by using the HP combined with MNNG induced CAG with epithelial dysplasia rat model, high-throughput microRNA sequencing technology and other biological method,its treatment targets in CAG with epithelial dysplasia. It will improve therapeutic principle of traditional Chinese medicine(TCM) including as follows: strengthening the spleen and replenishing qi, promoting blood circulation to disperse blood stasis, heat-clearing and detoxifying. Our project also will provide a scientific basis for the development of modern TCM to treat CAG with epithelial dysplasia.

慢性萎缩性胃炎(CAG)伴上皮细胞异型增生属于癌前病变，幽门螺旋杆菌(HP)可通过诱导型NO合酶(iNOS)分泌过量的NO，促进miRNA-21的表达，抑制抑癌基因PDCD4与细胞周期蛋白依赖性激酶抑制剂1B(P27kipl)的表达，导致胃黏膜异型增生。iNOS-mi-21-PDCD4-p27kipl介导的细胞异常增殖在CAG异型增生发病机制中有重要的作用但研究较少。前期工作发现，健脾清化汤临床疗效确切可有效拮抗NO对HP胃炎小鼠胃粘膜的损伤，但其作用机制尚未明确。因此，我们拟以HP感染后联合MNNG诱导的CAG异型增生大鼠模型，采用高通量microRNA测序技术等分子生物学方法，深刻阐明健脾清化汤对iNOS-mir-21-PDCD4-P27kipl的干预，并明确健脾清化汤治疗CAG异型增生的作用靶点，提升中医健脾益气、活血化瘀、清热解毒治则的科学内涵，为研发治疗CAG异型增生现代中药提供

慢性萎缩性胃炎伴异型增生属于胃癌前病变，中医药对本病的治疗有确切的疗效其对异型增生具有一定的逆转作用。因此，深入研究中医药治疗慢性萎缩性胃炎异型增生的机制，有重要的意义。本课题使用SPF 级SD雄性大鼠按随机数字表随机分为正常组、模型组、健脾清化汤低剂量组、健脾清化汤中剂量组、健脾清化汤高剂量组，除正常组外，其余各组使用幽门螺杆菌(Hp)悉尼SSI 菌株悬液灌胃8周，行C13呼气试验后确认感染成功，再按综合造模法(MNNG联合雷尼替丁)连续造模28周。每隔8周随机取2只大鼠观察胃黏膜情况，光镜下确定造模成功后，除正常组和模型组外，健脾清化汤高中低剂量组予不同梯度浓度的健脾清化汤灌胃10周后取材。行HE染色光镜下观察胃黏膜情况，同时应用NO、SOD试剂盒分别测定胃组织中NO含量及SOD活性，运用免疫组化法与实时荧光定量 PCR 法检测 miR-21、P27kip1、PDCD4、iNOS、IL-1β、TNF-α基因表达水平变化。HE病理检测显示：造模组大鼠胃黏膜可见粘膜层变薄，腺体数量减少，腺体排列紊乱，慢性炎性细胞浸润，可达黏膜肌层，可见杯状细胞，细胞形态不一，细胞核出现异常。干预治疗10周后，健脾清化汤高、中剂量组、肉眼及及镜下较模型组有不同程度改善，可见腺体排列相对规则，腺体数目尚可，肠上皮化生及异型增生较少见。健脾清化化瘀汤低剂量组胃黏膜好转不明显。健脾清化汤通过调控 iNOS 表达，下调胃黏膜miR-21表达，解除miR-21对抑癌基因PDCD4与细胞周期蛋白依赖性激酶抑制剂1B(P27kip1)的抑制，促进P27kip1、PDCD4的表达，深刻阐明健脾清化汤对iNOS-mir-21-PDCD4-P27kipl的干预，抑制胃细胞异常增殖。同时健脾清化汤通过调控 iNOS 表达，减少异常 NO生成，从而减少 NO 对大鼠胃黏膜的氧化、促炎、促癌变作用，最终改善大鼠胃黏膜炎症、萎缩、异型增生， 防止癌变。在此基础上，采用高通量小RNA测序技术明确健脾清化汤调控萎缩性胃炎大鼠胃黏膜炎症、细胞周期等生理过程的新型miRNA靶分子，并明确中医健脾益气、活血化瘀、清热解毒治则的科学内涵，为研发治疗CAG异型增生现代中药提供科学依据。