| Chronic atrophicgastritis (CAG) with intestinal metaplasia (IM) or dysplasia (Dys) is the most common precancerous lesions of gastric cancer, the possibility of which develop to gastric cancer increases gradually along with the severity of dysplasia. Due to the lack of effective treatment for gastric cancer, it is very important to prevent and control precancerous lesions of gastric cancer, that is presently the focus of research.the treatment of Traditional Chinese Medicine for chronic atrophic gastritis and precancerous lesions has remarkable effect, the study of which pathogenesis has been gradually into the molecular and gene level.This research is mainly divided into two parts:literature research and experimental research.The literature research part following the principle of evidence based medicine, starts from the literature research to explore the syndrome distribution characteristics of precancerous lesions of gastric cancer.The experimental research part studys the possible molecular mechanism of Xiaopi granule based on Yiqi Huayu Jiedu principle to treat the chronic atrophic gastritis precancerous lesion of cancer from the gene level.The first part:literature researchObjective:Through collecting and analyzing the related literatures on TCM Treatment of PLGC, the distribution characteristic of syndrome characteristics and syndrome medicine summary of PLGC, explore and summarize the regularity of the PLGC differentiation treatment from the literature angle to pave the way for the subsequent research.Methods:Through the collection of TCM treatment of PLGC from CBM, CNKI, Wan Fang, VIP database (1994-2014 January), scientifically summarize the type of syndrome and prescription rules of the TCM dialectical treatment to PLGC.Results:A total of 38 literatures,200 dialectical medication data are taken into account. The common syndromes, accounting to the frequency from high to low is weakness of the spleen and stomach,disharmony between liver and stomach, deficiency of stomach yin, dampness-heat of spleen and stomach and blood stasis of stomach.The mechanism characteristics of this disease is deficiency qi and/or yin of spleen and stomach with blood stasis toxin. The drugs accounting to Syndrome differentiation mainly include replenishing spleen qi, regulating qi, eliminating dampness,cooling heat, nourishing Yin and activating blood. Commonly used herbs (the frequency in more than 10%) are white atractylodes rhizome, Radix Paeoniae Alba, Poria cocos, fructus aurantii immaturus, Banxia, Dried tangerine peel, rhizome cyperi, Astragalus, Salvia miltiorrhiza, Codonopsis pilosula, rhizoma coptidis, angelica, Radix Ophiopogonis, Zedoary, Mangnolia officinalis, fructus amomi, semen coicis, radices saussureae, Adenophora stricta, citrus chirocarpus, Dendrobium nobile, Herba Hedyotis, etc, thus the common treatment method of TCM for PLGC is tonifying qi, nourishing Yin, activating blood, detoxification. the weakness of the spleen and stomach syndrome commonly uses Xiangsha six gentleman decoction and according to the needs adds Liqi herbs such as Rhizoma Cyperi, tangerine peel, blood activating herbs such as zedoary, Salvia miltiorrhiza, dispelling internal cold herbs such as ginger etc. the disharmony between liver and stomach syndrome commonly uses ChaiHuShuGan powder, and according to the needs adds blood activating herbs such as Salvia miltiorrhiza, angelica, curcuma aromatic, pseudoginseng, curcuma zedoary, tonifying spleen herbs such as Poria cocos, white atractylodes rhizome, heat-clearing herbs such as rhizome coptidis, radix paeoniae rubrathe, dandelion, Herba Hedyotis. the deficiency of stomach yin syndrome commonly uses Adenophora Ophiopogon soup, and according to the needs adds tonifying spleen herbs such as Poria cocos, white atractylodes rhizome, radix pseudostellariae, Codonopsis pilosula, Rhizoma Dioscoreae, Liqi herbs such as Chinaberry fruit, citrus chirocarpus, Dried tangerine peel, ructus amomi, rhizoma cyperi, resolving food stagnancy herbs such as ventriculi galli mucosa, stir-baked millet sprout, stir-baked FRUCTUS HORDEI GERMINATUS, the dampness-heat of spleen and stomach syndrome commonly uses huopu xialing decoction, and according to the needs adds Liqi herbs such as fructus aurantii immaturus, Mangnolia officinalis, Dried tangerine peel, caulis perllae, radix bupleuri, rhizome cyperi, heat-clearing herbs such as hizoma coptidis,Scutellaria baicalensis, dandelion, Herba Hedyotis, tonifying spleen herbs such as white atractylodes rhizome, rhizoma atractylodis, resolving food stagnancy herbs and blood activating herbs such as ventriculi galli mucosa,curcuma zedoary. The blood stasis of stomach syndrome commonly uses Shixiao Decoction, and according to the needs adds other blood activating herbs such as angelica, curcuma zedoary, pseudoginseng, peach kernel, safflower carthamus, Liqi herbs such as fructus aurantii immaturus, curcuma aro -matic, radices saussureae, Ligusticum wallichii, Bupleurum,tonifying spleen herbs such as Astragalus mongholicus, white atractylodes rhizome, Codonopsis pilosula.Conclusions:The pathogenesis characteristics of the PLGC can be summarized in deficiency qi and/or yin of spleen and stomach with blood stasis toxin. so its treatment method is Yiqi Huayu jiedu principle. the differentiation and treatment of this disease has certain rules, often in the main basis, integrating the syndrome characteristics, interaction pathogenesis beween herbs and physiological and pathological characteristics of the spleen and stomach, accompanied by other classes of herbs, so that both pathogenesis more comprehensive, improving the main berb efficacy.The second part:experimental researchObjective:Study the effects of Yiqi Huayu Jiedu principle on methyltransferasel (DNMT1) and RUNX3 tumor suppressor gene CpG island methylation in the promoter region (CpG Island) on chronic atrophic gastritis with dysplasia rats, and further to explore the influence of Yiqi Huayu Jiedu principle on regulation factor CyclinDl and P21 WAF1/CIP1 of cell cycle.Methods:60 Wistar male rats of SPF level were randomly divided into 10 for blank control group and 50 for molding group. The blank group is provided the ordinary feed and water on clean animal standard, The model group is provided 120ug/ml N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) by gavage, Each 5ml/kg,1 times/24h,0.05% ammonia solution, and feed containing 0.03% ranitidine hydrochloride pellets to establish the CAG with Dys rat model. At the end of the 32 week after the success of modeling, the remained 33 rats of model group were randomly divided into 11 for model group,11 for Weimeisu group,11 for Xiaopi granule group, each group was provided with common feed and water on clean animal standard, the Model group was given physiological saline by gavage,3mL/kg, 1/24h, the Weimeisu group was given Weimeisu suspension by gavage,2ml/kg (including dimension of 0.3g/kg), 1/24h, the Xiaopi granule group was given Xiaopi granule suspension by gavage, 3ml/kg (containing crude drug 9g/kg), 1/24h for 12 weeks. All rats were sacrificed at the end of the forty-fourth week. The pathological changes in rat gastric mucosa were observed with the light microscope, the tumor suppressor gene RUNX3 promoter CpG island methylation and protein expression, the DNMT1 protein expression and the mRNA and protein expression of the cell cycle regulation factor CyclinDl and p21 WAF1/CIP1 were detected with the methods of methylation specific PCR (MSP), immune-histochemistry, RT-PCR and Western-blot techniques.Results:1 In the 26 week, the gastric mucosa of model group rats appeared to atrophy, in the 28 week the atrophy appeared to aggravate, accompanied with intestinal metaplasia, in the 30 and 32 weeks the gastric mucosa of model group rats appeared different degrees of Dys on the basis of atrophy. After intervention, degree of gastric epithelial dysplasia of the model group rats increased; the change of the gastric mucosa of the blank group rats was not obvious. The degree of gastric epithelial dysplasia of Xiaopi granule group and Weimeisu group rats reduced, there were obviously changes of gastric mucosa of the Xiaopi granule group rats, some of which approached normal.2 RUNX3 promoter methylation rates of the Blank group,model group, Weimeisu group and Xiaopi granule group are 14.3%(1/7),100%(7/7),100%(7/7) and 42.9%(3/7), the methylation status of the four groups is overall different or not identical (P=0.000<0.01). Further gray analysis to MSP band appears that RUNX3 CpG island methylation levels of Xiaopi granule group is lower than that of Weimeisu group and model group, there is difference compared with Weimeisu group (P=0.021<0.05), there is significant difference compared with the model group (P=0.001<0.01), there is no significant difference between Weimeisu group and the model group (P=0.067>0.05).3 The positive rates of Runx3 protein expression of the blank group, model group, Weimeisu group and Xiaopi granule group are 100%(10/10),25%(2/8),44.4%(4/9) and 81.8%(9/11), there are significant differences (P=0.001<0.01). Further semi quantitative analysis with the application of IPP6.0 software appears that the expression of RUNX3 protein of Xiaopi granule group is significantly higher than that of Weimeisu group and model group, there is significant difference, respectively P=0.008<0.01 and P=0.004<0.01, there is no significant difference between Xiaopi granule group and the blank group (P=0.324>0.05), and there is no significant difference between Weimeisu group and the model group (P=0.631>0.05).4 The DNMTl protein expression of the model group is the highest,followed by Weimeisu group, The DNMT1 protein expression of Xiaopi granule group is lower than that of the model group,which is close to that of the blank group.the comparison of gray value of each group appears that the DNMT1 protein expression of the model group and Weimeisu group are increased significantly compared with the control group (P=0.001, 0.002<0.01); the DNMT1 protein expression of Xiaopi granule group is lower than that of the model group and Weimeisu group (P=0.000,0.041,P<0.01 and P<0.05); there is no significant difference between Xiaopi granule group and the blank group (P=0.137>0.05), and there is difference between Weimeisu group and the model group decreased (P=0.023<0.05).5 The expression of P21 WAF1/CIP1 mRNA of the model group and Weimeisu group compared with the blank group decreases significantly (P=0.000,0.001,P<0.01); The expression of P21 WAF1/CIP1 mRNA of Xiaopi granule group, model group and Weimeisu group is increased (P=0.001,0.026,P<0.01 and P<0.05); there is no significant difference between the Xiaopi granule group and the blank group (P=0.060>0.05), and there is no significant difference between Weimeisu group and the model group(P=0.088>0.05).6 The expression of P21 WAF1/CIP1 protein is the lowest in the model group, the second is that of Weimeisu group, that of Xiaopi granule group is higher compared with the model group and Weimeisu group, and is close to that of the blank group. the comparison of gray value of each group appears that the expression of P21 WAF1/CIP1 protein of the model group and Weimeisu group decreases significantly compared with the blank group (P=0.000,0.004, P<0.01); the expression of P21 WAF1/CIP1 protein of Xiaopi granule group is increased comopared with the model group and Weimeisu group (P=0.002,0.036,P<0.01 and P<0.05); there is no significant difference between the Xiaopi granule group and the blank group (P=0.260>0.05),there is no significant difference between Weimeisu group and the model group (P=0.123>0.05).7 The expression of CyclinD1mRNA of the model group and Weimeisu group compared with the blank group are increased (P=0.009,0.019,P<0.01 and P<0.05); The expression of CyclinD1mRNA of Xiaopi granule group is lower than that of the model group and Weimeisu group lower (P=0.006,0.013,P<0.01 and P<0.05); there is no significant difference between Xiaopi granule group ahd the blank group (P=0.850>0.05), there is no significant difference between Weimeisu group and the model group (P=0.686>0.05).8 The expression of CyclinDl protein of the model group is the highest, Followed by that of Weimeisu group, the expression of CyclinDl protein of Xiaopi granule group is lower compared with the model group and Weimeisu group, and is close to that of the blank group. the comparison of gray value of each group appears that the expression of CyclinDl protein of the model group and Weimeisu group is higher than that of the blank group (P=0,0.014,P<0.01 and P<0.05); the expression of CyclinDl protein of Xiaopi granule group is lower than that of the model group and Weimeisu group (P=0.000,0.020,P<0.01and P<0.05); there is no significant difference between Xiaopi granule group and the blank control group (P=0.840>0.05),and there is difference between Weimeisu group and the model group (P=0.042<0.05).Conclusions:The possible mechanism of Xiaopi granule based on Yiqi Huayu Jiedu principle to treat PLGC and prevent gastric cancer is inhibiting the DNMT1 expression, so as to improve the tumor suppressor gene RUNX3 promoter CpG island hypermethylation status and promote the expression of RUNX3,and then restore the tumor suppressor function of RUNX3, further enhance the expression of P21 WAF1/CIP1 and inhibit the expression of CyclinD1, then restore the P21-CDK-CyclinDl balance.
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