| Objective: Chronic Atrophic Gastritis(CAG)is a precancerous lesion of gastric cancer,particularly with Intestinal metaplasia and dysplasia that have the more possibility of cancer.The signal pathway of Hippo/YAP as a new target for the onset and treatment of gastric cancer is more and more popular.HuaZhuoJieDu formula has achieved remarkable curative effect in treatment of CAG.Observing the changes of CAG rats treating with HuaZhuoJieDu formula,such as general condition,gastric mucosa pathology,the level of YAP and TEAD,the main composition of the signal pathway of Hippo/YAP,and the protein of Merlin and Survivin,this experiment investigates the mechanism of HuaZhuoJieDu formula action,which provides the theory basis of modern research for the clinical application of Zhuodu theory.Methods: 70 clean SD rats in 250-300 g weight,feeding one week adaptively,were divided into two groups by random number table method.The one was Normal Group with 12 rats.The other was Model Group with the remaining 58 rats which were fed in model method with drinking N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)freely and lavaging ranitidine in combination with the hunger disorder.Modelling after 20 weeks,two rats were killed from each Group at random,observing gastric mucosa to confirm the model in the success of the copy.Then in accordance with the principle of random,rats in this group were divided into 5 goups: Spontaneous Recovery Group,Vitacoenzyme Group,High-doze Group,Moderate-doze Group and Low-doze Group,each group of 10(6 rats died in the process of modelling).In the second day after the success of modeling,Normal Group and Spontaneous Recovery Group were lavaged with 10 ml/kg/d distilled water.Vitacoenzyme Group was given 10 ml/kg/d Vitacoenzyme suspension solution in which had 0.006g/ml Vitacoenzyme.The others group ranging from high-doze to moderate-doze to low-doze of HuaZhuoJieDu Formula were given 10 ml/kg/d herb suspension solution in which have 2.8g/ml,1.4g/ml and 0.7g/ml HuaZhuoJieDu Formula.The therpy remained 12 weeks.During the period of therpy,the general situation of rats was observed such as weight,hair color,tooth claw,mental state,appetite,stool color and so on.The fasting time with banning water continued 48 h after the last treatment.After that,the gastric mucosae with significantly pathological change were moved and rats were executed.The pathological change of gastric mucosae was observed by HE.The expression of YAP,Merlin,TEAD and Survivin were detected by RT-PCR,Western Blot and immunohistochemical method(SP).The data was analyzed by SPSS 21.0 statistical software.Results:1 The general situationThere were no different with Normal Group between before test and after.Model group had changes such as dark and lackluster hair,hair removal,depression,slow response,less activity,compulsive nuzzles,poor appetite,decresscing weiht,dung soft or deformed and so on.After treatment,all group had improved,especially general situation of High-doze Group improved markedly.Vitacoenzyme Group and Moderate-doze Group also had improved.But the improvement of Low-doze Group and Spontaneous Recovery Group were not obvious.2 Observation of gastric mucosa tissue pathological changeMacroscopic observation: the gastric mucosa tissue of Normal Group is smooth,the pink and mucosal fold neatly;the gastric mucosa tissue of Model Group with rough surface,part of pale granular uplift,Numerous point flake erythema and haemorrhage and a few erosion appeared mucosal fold flat,scarce,disorder or even disappear;the gastric mucosa tissue of Vitacoenzyme Group,High-doze Group and Moderate-doze Group had improved obviously.The gastric mucosa tissue of Low-doze Group and Spontaneous Recovery Group had little improvement.Histopathological observation: the mucosal thickness,epithelial cells and inherent glands of Normal Group were normal with moderate quantity and no inflammatory cell.The epithelial cells of Model Group were thinned,degeneration,necrosis and shedding.The inherent glands were decreased with disordered arrangement.The mucosal muscularis has thickened with interstitial inflammatory cell infiltration;The gastric mucosa tissue of Vitacoenzyme Group,High-doze Group and Moderate-doze Group had moderated thickness of mucosa,on obvious degeneration and necrosis of epithelial cells,moderate quantity and arrangement of inherent glands with small interstitial inflammatory cell infiltration;The gastric mucosa tissue of Low-doze Group and Spontaneous Recovery Group had little improvement.3 Comparison of the signal pathway of Hippo/YAP and related protein expression level in gastric mucosa3.1 Comparison of the signal pathway of Hippo/YAP expression level in gastric mucosa3.1.1 Comparison of the YAP expression level in gastric mucosaThe YAP expression level of MOD,mRNA and protein in Spontaneous Recovery Group were higher than Normal Group(P<0.01).The YAP expression level of MOD,mRNA and protein in Vitacoenzyme Group,High-doze Group,Moderate-doze Group and Low-doze Group were lower than Spontaneous Recovery Group(P<0.01).The YAP expression level of MOD,mRNA and protein in High-doze Group were lower than Moderate-doze Group,and Low-doze Group(P<0.01).The YAP expression level of MOD,mRNA and protein in Moderate-doze Group were lower than Low-doze Group(P<0.01);The YAP expression level of MOD,mRNA and protein in High-doze Group were lower than Vitacoenzyme Group(P<0.01).The YAP expression level of MOD,mRNA and protein in Normal Group were lowest.The YAP expression level of MOD,mRNA and protein in Normal Group were lower than Vitacoenzyme Group,Moderate-doze Group and Low-doze Group(P<0.01).The YAP expression level of MOD in Normal Group is lower than High-doze Group(P<0.01).The YAP expression level of mRNA and protein in Normal Group is similar with High-doze Group(P>0.05).3.1.2 Comparison of the TEAD expression level in gastric mucosaThe TEAD expression level of MOD,mRNA and protein in Spontaneous Recovery Group were higher than Normal Group(P<0.01).The TEAD expression level of MOD,mRNA and protein in Vitacoenzyme Group,High-doze Group and Moderate-doze Group were lower than Spontaneous Recovery Group(P<0.01).The TEAD expression level of MOD,mRNA and protein in High-doze Group were lower than Moderate-doze Group,and Low-doze Group(P<0.01).The TEAD expression level of MOD,mRNA and protein in Moderate-doze Group were lower than Low-doze Group(P<0.01);The TEAD expression level of MOD,mRNA and protein in Vitacoenzyme Group were higher than High-doze Group(P<0.01).The TEAD expression level of MOD,mRNA and protein in Vitacoenzyme Group were higher than Moderate-doze Group(P<0.01).The TEAD expression level of MOD,mRNA and protein in Normal Group were lowest.The TEAD expression level of MOD,mRNA and protein in Normal Group were lower than Vitacoenzyme Group,Moderate-doze Group and Low-doze Group(P<0.01).The TEAD expression level of MOD in Normal Group is lower than High-doze Group(P<0.01).The TEAD expression level of mRNA and protein in Normal Group is similar with High-doze Group(P>0.05).3.2 Comparison of the Merlin expression level in gastric mucosaThe Merlin expression level of MOD,mRNA and protein in Spontaneous Recovery Group were lower than Normal Group(P<0.01).The Merlin expression level of MOD,mRNA and protein in Vitacoenzyme Group,High-doze Group,Moderate-doze Group and Low-doze Group were higher than Spontaneous Recovery Group(P<0.05).The Merlin expression level of MOD,mRNA and protein in High-doze Group were higher than Moderate-doze Group and Low-doze Group(P<0.01).The Merlin expression level of MOD,mRNA and protein in Moderate-doze Group is similar with Low-doze Group(P>0.05).The Merlin expression level of MOD,mRNA and protein in Normal Group were higher than High-doze Group,Vitacoenzyme Group,Moderate-doze Group and Low-doze Group(P<0.01).3.3 Comparison of the Survivin expression level in gastric mucosaThe Survivin expression level of MOD,mRNA and protein in Spontaneous Recovery Group were higher than Normal Group(P<0.01).The Survivin expression level of MOD,mRNA and protein in Vitacoenzyme Group and High-doze Group were lower than Spontaneous Recovery Group(P<0.01).There were no difference among Moderate-doze Group,Low-doze Group and Spontaneous Recovery Group in MOD,mRNA and protein(P>0.05).The Survivin expression level of MOD,mRNA and protein in High-doze Group were lower than Moderate-doze Group,Low-doze Group(P<0.01).The Survivin expression level of MOD,mRNA and protein in High-doze Group were higher than Vitacoenzyme Group(P<0.05).The Survivin expression level of MOD,mRNA and protein in Normal Group were lowest.The Survivin expression level of MOD,mRNA and protein in Normal Group were lower than Vitacoenzyme Group,High-doze Group,Moderate-doze Group and Low-doze Group(P<0.01).Conclusions:1 HuaZhuoJie Du Formula can improve the general situation and repair the damaged gastric mucosa.2 Gastric mucosa of CAG rats have the high expression level of YAP,TEAD and Survivin and the loss of Merlin.3 HuaZhuoJieDu Formula has obvious effect on repairing the damaged gastric mucosa,decreasing the expression level of YAP,TEAD and Survivin and increasing the expression level of Merlin.The mechanism of action of HuaZhuoJieDu Formula is that play a role through regulating the Hippo/YAP signaling pathways and the expression of Survivin protein level.4 High-doze Group is better than Vitacoenzyme Group in the treatment of CAG in suppressing the abnormal expression of YAP and TEAD,and increasing the level of Merlin. |
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