荣胃理气方联合守宫对慢性萎缩性胃炎模型大鼠胃黏膜修复作用及Wnt信号通路的影响

发布时间：2018-04-25 00:42

本文选题：荣胃理气方 + 守宫　；参考：《湖北中医药大学》2017年博士论文

【摘要】：研究目的:通过建立慢性萎缩性胃炎(chronic atrophic gastritis,CAG)大鼠模型,从形态学(HE染色,CD34染色切片)、抗氧化损伤(SOD、MDA)、细胞增殖与凋亡(P53,PCNA)、信号通路(Wnt通路)多方面进行研究,观察荣胃理气方联合守宫对CAG模型大鼠胃粘膜的修复作用和机制。研究方法:选用72只健康纯种SPF级SD雄性大鼠,将其饲养在SPF环境中。适应性喂养1周后,给予去氧胆酸钠溶液自由饮用联合酒精灌服及饥饱失常喂养法刺激大鼠诱发CAG模型。将造模成功的大鼠进行随机分组,分别为空白对照组(K)、模型组(M)、中药治疗组(Z)、胃复春组(W)、叶酸组(Y),每组10只。造模成功后于第三天开始灌胃给药,药物干预28天后进行采血以及取材。应用HE染色法观察各组大鼠胃粘膜的病理变化,应用CD34免疫组化染色法检测大鼠胃粘膜的微血管数量及微血管密度;使用ELISA法检测大鼠血清中胃泌素(GAS)、超氧化物歧化酶(S OD)/丙二醛(MDA),血清生长抑素(SS)/血清前列腺素E2(PGE2)的含量;应用免疫组织化学法检测各组大鼠胃粘膜中P53以及PCNA的基因表达水平;同时应用免疫组化法检测大鼠胃粘膜中β-catenin、GSK-3β、cyclin D1以及COX-2蛋白的表达水平。结果:1.对CAG模型大鼠组织形态的影响通过HE染色,结果显示,模型组大鼠的胃粘膜出现了充血、水肿以及腺管排列紊乱等表现,并且黏液层脱落增多,胃小凹可见变浅,而粘膜固有层变薄并且出现了萎缩,腺体腔变小以及数目减少,伴有囊性扩张,粘膜下层水肿明显,炎性细胞散在分布,固有层内出现淋巴细胞浸润等病变。中药治疗组中的各大鼠胃粘膜罕见糜烂和缺损,并未出现胃溃疡病灶,且粘膜层的厚度大致都是正常的,腺体排列也是基本整齐的,数量并未见明显减少,亦无肠上皮化生以及不典型增生等病变。胃复春组的大鼠胃粘膜有少许的糜烂和缺损,偶可见有溃疡病灶,粘膜层稍有增厚,腺体排列略显紊乱,数量减少大致约二分之一左右,偶可见肠上皮化生以及不典型增生等改变。叶酸片组中的大鼠胃炥膜少见糜烂、缺损,偶有溃疡的病灶,粘膜变厚,腺体排列略显紊乱,数量减少大致有1/2左右,偶有肠上皮化生等形态变化。通过CD34染色,然后检测大鼠胃粘膜微血管数量(MVC)以及微血管密度(MVD),结果显示,与空白对照组比较,模型组大鼠MVD值明显降低(P0.01);与模型组比较,中药治疗组数值明显升高(P0.01);免疫组化染色显示空白组胃黏膜有不同程度的充血反应,模型组可见胃黏膜上皮及固有腺体细胞增生,黏膜固有层和黏膜下层间质有血管数量减少。经中药方治疗后胃黏膜微血管密度增多,接近正常,中药治疗组与胃复春组及叶酸组比较亦有统计学意义(P0.05)。2.对慢性萎缩性胃炎大鼠血清GAS、SS、PGE2、SOD、MDA的影响与空白对照组比较,模型组大鼠胃泌素(GAS)水平明显降低(P0.01);与模型组比较,中药治疗组血清胃泌素(GAS)水平均明显升高(P0.01);与胃复春组比较,中药治疗组血清胃泌素(GAS)水平升高具有统计学意义(P0.05);中药治疗组与叶酸组比较,血清胃泌素(GAS)水平无统计学差异(P0.05)。与空白对照组比较,模型组大鼠血清生长抑素(SS)和前列腺素(PGE2)水平显著降低(P0.01);与模型组比较,中药治疗组大鼠血清生长抑素(SS)和前列腺素(PGE2)水平显著升高P0.01),与叶酸组、胃复春组比较,血清生长抑素(SS)和前列腺素(PGE2)变化上无统计学差异(P0.05)。与空白对照组比较,模型组大鼠血清超氧化物歧化酶(SOD)活力降低,丙二醛(MDA)数值升高(P0.05);与模型组比较,中药治疗组SOD活力明显升高,MDA水平明显降低(P0.01),与胃复春组、叶酸组比较,中药治疗组在SOD、MDA变化上无统计学差异。3.对慢性萎缩性胃炎大鼠胃黏膜P53、PCNA基因表达的影响与空白对照组相比,模型组大鼠胃黏膜组织P53水平明显高于正常组(P0.01),胃黏膜组织病变明显。不同治疗组的P53指数明显降低,胃黏膜病理组织学改变明显。中药治疗组与模型组比较差异显著(P0.01);中药治疗组与胃复春组比较差异显著(P0.01),中药治疗组与叶酸组比较无明显差异(P0.05)。与空白对照组相比,模型组大鼠胃黏膜组织PCNA水平明显高于正常组(P0.01),胃黏膜组织病变明显。不同治疗组的PCNA指数明显降低,胃黏膜病理组织学改变明显。中药治疗组与模型组比较差异显著(P0.01);中药治疗组与胃复春组、叶酸组比较差异显著(P0.01)。4.对慢性萎缩性胃炎大鼠胃黏膜Wnt信号通路的影响与空白组相比,模型组GSK-3β的表达显著降低,差异有统计学意义(P0.01)。中药治疗组、叶酸组、胃复春组GSK-3β的表达均升高,中药治疗组GSK-3β蛋白的表达情况要明显优于胃复春组和叶酸组(P0.01)。与空白组相比,模型组β-catenin的表达显著升高,差异有显著统计学意义(P0.01)。中药治疗组、叶酸组、胃复春组GSK-3β的表达均降低,中药治疗组β-catenin蛋白的表达情况要明显优于胃复春组和叶酸组(P0.05)。与空白对照组相比较,模型组cyclin D1的表达增强,差异有显著统计学意义(P0.01)。中药治疗组、叶酸组、胃复春组GSK-3β的表达均降低,与模型对照组相比差异有显著统计学意义(P0.05)。中药治疗组疗效明显优于胃复春组和叶酸组(P0.01)。与空白对照组相比较,模型组COX-2的表达增强,差异有显著统计学意义(P0.01)。中药治疗组、叶酸组、胃复春组GSK-3β的表达均明显降低,与模型对照组相比差异有显著统计学意义(P0.01)。中药治疗组疗效明显优于胃复春组和叶酸组(P0.01)。结论:荣胃理气方联合守宫可以明显改善胃黏膜萎缩状态,增加胃黏膜区新生血管数目,对胃黏膜具有促血管新生作用,其效果优于胃复春和叶酸。荣胃理气方联合守宫可增加胃泌素,生长抑素及前列腺素E2的水平,降低丙二醛含量,抑制炎症反应,修复受损的胃黏膜,并能增加SOD活力。其对胃黏膜的修复作用可能与增加胃泌素、生长抑素、前列腺素E2水平、提升SOD活力,降低MDA含量有关。荣胃理气方联合守宫抑制胃黏膜P53、PCNA基因的过度表达,从而防止慢性萎缩性胃炎向胃癌转化,说明荣胃理气方联合守宫具有一定的优势,且其对胃黏膜修复作用可能与抑制P53、PCNA基因的过度表达有关。荣胃理气方联合守宫能明显提高胃黏膜GSK-3β、cyclin D1以及COX-2的表达,降低胃黏膜β-catenin的表达,其对胃黏膜修复的作用机制可能在于提高胃黏膜中相关生长因子,激活Wnt通路进而逆转胃黏膜萎缩,达到修复胃黏膜的作用。荣胃理气方联合守宫具有较好的胃黏膜修复作用,效果优于胃复春和叶酸。其能有效地抑制胃黏膜的氧化应激反应,提高胃黏膜的修复能力,发挥中医药整体调节、多靶点的作用特点,为荣胃理气方联合守宫临床治疗慢性萎缩性胃炎提供了部分实验依据。
[Abstract]:Objective: to establish a rat model of chronic atrophic gastritis (chronic atrophic gastritis, CAG), from morphologic (HE staining, CD34 stained section), antioxidant injury (SOD, MDA), cell proliferation and apoptosis (P53, PCNA), and signal pathway (Wnt pathway), observe the repair of gastric mucosa in rat model of Rong Wei Qi Fang and Gong Gong on the rat model of CAG model. The study method: 72 healthy pure SPF grade SD male rats were selected to be fed in the SPF environment. After 1 weeks of adaptive feeding, the rats were given free drinking of sodium deoxycholate combined with alcohol irrigation and hunger satiety feeding to induce CAG model in rats. The rats were randomly divided into blank pairs. Group (K), model group (M), Chinese medicine treatment group (Z), gastric Fuchun group (W), folic acid group (Y), 10 in each group. After the success of the model, the medicine was administered at third days. After 28 days of drug intervention, the blood collection and material were collected. The pathological changes of gastric mucosa in each group were observed by HE staining. The microvessels of gastric mucosa of rats were detected by CD34 immunohistochemical staining. Quantity and microvascular density; the content of serum gastrin (GAS), superoxide dismutase (S OD) / malondialdehyde (MDA), serum somatostatin (SS) / serum prostaglandin E2 (PGE2) in rats were detected by ELISA, and the gene expression level of P53 and PCNA in gastric mucosa of each group was detected by immunohistochemical method; and immunohistochemical method was used to detect the gene expression in the gastric mucosa of rats. The expression level of beta -catenin, GSK-3 beta, cyclin D1 and COX-2 protein in the gastric mucosa of rats was measured. Results: the effects of 1. on the tissue morphology of CAG model rats were stained by HE. The results showed that the gastric mucosa of the rat model group showed hyperemia, edema, and the disorder of the gland tube arrangement, and the mucous layer exfoliated and the gastric fovea became shallow and sticky. The lamina propria of the membrane became thinner and atrophied, the cavity of the gland became smaller and the number was reduced, with the cystic dilatation, the edema of the submucosa, the distribution of the inflammatory cells, the infiltration of lymphocytes in the lamina propria. The gastric mucosa of the rats in the Chinese medicine treatment group was rare and the gastric ulcer was rare and there was no gastric ulcer focus and the thickness of the mucosa. The glandular arrangement was basically normal, the glandular arrangement was also basic and neatly. There was no obvious decrease in quantity, no intestinal metaplasia and atypical hyperplasia. The gastric mucosa of the rats of the Fuchun group of the stomach had a little erosion and defect, occasionally the ulcer focus, the thickening of the mucous layer, the glandular arrangement slightly disorder, and the decrease of approximately 1/2. The changes in intestinal metaplasia and atypical hyperplasia were seen occasionally. In the Folic Acid Tablets group, the rat gastric membrane was rarely erosive, defective, occasionally ulcerated, thickened and slightly disordered in the glandular arrangement. The number decreased roughly 1/2, occasionally in intestinal metaplasia and other morphologic changes. The number of microvascular microvessels in the gastric mucosa of rats was detected by CD34 and then the microvascular number of gastric mucosa in rats was detected. The volume (MVC) and microvascular density (MVD) showed that, compared with the blank control group, the MVD value of the model group was significantly lower (P0.01). Compared with the model group, the value of the Chinese medicine treatment group was significantly higher (P0.01). The immunohistochemical staining showed that the gastric mucosa of the blank group had different degree of congestion reaction, and the model group could see the gastric mucosa epithelium and the intrinsic gland cells. The number of blood vessels in the mucous lamina propria and submucosa decreased. The microvascular density of the gastric mucosa increased after the Chinese medicine treatment, which was close to normal. The effect of.2. on GAS, SS, PGE2, SOD and MDA in the serum of rats with chronic atrophic gastritis was compared with that of the blank control group. The level of gastrin (GAS) in the model group was significantly lower (P0.01). Compared with the model group, the level of serum gastrin (GAS) in the Chinese medicine treatment group increased significantly (P0.01). Compared with the stomach Fuchun group, the serum gastrin (GAS) level in the Chinese medicine treatment group was statistically significant (P0.05), and the serum gastrin (GAS) level in the Chinese medicine treatment group was compared with the folic acid group, and the level of serum gastrin (GAS) was no more than that of the Chinese traditional medicine group. Statistical difference (P0.05). Compared with the blank control group, the level of serum somatostatin (SS) and prostaglandin (PGE2) in the model group was significantly lower (P0.01). Compared with the model group, the serum somatostatin (SS) and the prostaglandin (PGE2) level in the Chinese medicine treatment group increased significantly, and the serum somatostatin (SS) and the serum somatostatin (SS) and the folic acid group and the gastric Fuchun group were compared with that of the model group. There was no statistical difference in the changes of prostaglandin (PGE2) (P0.05). Compared with the blank control group, the activity of serum superoxide dismutase (SOD) decreased and the malondialdehyde (MDA) value increased (P0.05) in the model group. Compared with the model group, the SOD activity in the Chinese medicine treatment group was significantly increased, the MDA level was significantly reduced (P0.01), compared with the gastric Fuchun group, folic acid group, and the treatment of traditional Chinese medicine. There was no statistical difference in the changes of SOD and MDA in the treatment group. The effect of.3. on the P53, PCNA gene expression in the gastric mucosa of the rats with chronic atrophic gastritis was significantly higher than that in the blank control group. The level of P53 in the gastric mucosa of the model group was significantly higher than that of the normal group (P0.01), and the pathological changes in the gastric mucosa were obvious. The P53 index in the different treatment groups was significantly reduced and the gastric mucosa was histopathologically histopathological. There was significant difference between the traditional Chinese medicine treatment group and the model group (P0.01), and there was significant difference between the Chinese medicine treatment group and the stomach Fuchun group (P0.01). There was no significant difference between the Chinese medicine treatment group and the folic acid group (P0.05). Compared with the blank control group, the level of PCNA in the gastric mucosa of the model group was significantly higher than that of the normal group (P0.01), and the pathological changes of the gastric mucosa were obvious. The PCNA index of the different treatment groups was obviously decreased and the pathological changes of gastric mucosa were obvious. The difference of the Chinese traditional medicine treatment group and the model group was significant (P0.01); the difference of the folic acid group between the Chinese medicine treatment group and the stomach Fuchun group was significant (P0.01) the effect of.4. on the Wnt signaling pathway in the gastric mucosa of the rats with chronic atrophic gastritis was compared with the blank group, and the model group was GSK-3 beta The expression of GSK-3 beta in the Chinese medicine treatment group, the folic acid group and the stomach Fuchun group increased significantly. The expression of GSK-3 beta protein in the Chinese medicine treatment group was significantly better than that of the gastric Fuchun group and the folic acid group (P0.01). Compared with the blank group, the expression of beta -catenin in the model group was significantly higher than that in the blank group, and the difference was significant statistically significant (P0.01). P0.01). The expression of GSK-3 beta in the Chinese medicine treatment group, the folic acid group and the Wei Fuchun group decreased. The expression of beta -catenin protein in the Chinese medicine treatment group was obviously better than that of the Wei Fuchun group and the folic acid group (P0.05). Compared with the blank control group, the expression of cyclin D1 in the model group was enhanced, the difference was statistically significant (P0.01). Chinese medicine treatment group, folic acid group, gastric Fuchun group were significantly different. The expression of GSK-3 beta in the group was significantly lower than that in the model control group (P0.05). The curative effect of the Chinese medicine treatment group was obviously better than that of the Wei Fuchun group and the folic acid group (P0.01). Compared with the blank control group, the expression of COX-2 in the model group was enhanced, the difference was statistically significant (P0.01). The Chinese medicine treatment group, the folic acid group, the stomach Fuchun group GSK-3 beta group. The expression of the treatment group was significantly lower than that of the model control group (P0.01). The curative effect of the Chinese medicine treatment group was obviously superior to that of the Wei Fuchun group and the folic acid group (P0.01). Conclusion: the combination of the rhono Stomach Qi Fang and the palace of conservation can obviously improve the atrophy of gastric mucosa, increase the number of new blood vessels in the gastric mucosa, and promote the neovascularization of the gastric mucosa. Its effect is superior to gastric Fuchun and folic acid. It can increase the level of gastrin, somatostatin and prostaglandin E2, reduce the content of malondialdehyde, inhibit the inflammatory reaction, repair the damaged gastric mucosa, and increase the activity of SOD. Its effect on gastric mucosa may be associated with increased gastrin, somatostatin, and prostaglandin E2 levels. To improve the activity of SOD and reduce the content of MDA. Rong Wei Qi Qi Fang combined with the palace to inhibit the overexpression of P53 and PCNA genes in the gastric mucosa, thus preventing the transformation of chronic atrophic gastritis to gastric cancer, indicating that the Junwei Qi Fang combined with the conservative palace has some advantages, and its application to the gastric mucosa repair may be related to the inhibition of the overexpression of the P53 and PCNA genes. The expression of GSK-3 beta, cyclin D1 and COX-2 in gastric mucosa and the expression of gastric mucosa beta -catenin can be significantly increased by the combination of stomach qi and Qi Fang, and the mechanism of the effect on gastric mucosa repair may be to improve the growth factors in the gastric mucosa, activate Wnt pathway and reverse the gastric mucosal atrophy, and to repair the gastric mucosa. The palace has a better effect of gastric mucosal repair, the effect is better than the gastric Fuchun and folic acid. It can effectively inhibit the oxidative stress reaction of the gastric mucosa, improve the ability to repair the gastric mucosa, play the role of the overall regulation of traditional Chinese medicine and multi target, and provide some experiments for the clinical treatment of chronic atrophic gastritis by the Junwei Qi Qi Fang and the palace. Basis.

【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5;R-332

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