| Objective: The therapy of HuaZhuoJieDu formula in treating the rats with Chronic Atrophic Gastritis(CAG)was observed,by observing the changes of the general condition of CAG rats,histopathological changes of gastric mucosa,and the expression levels of MOB1,LATS1,P-YAP protein and mRNA of Hippo/YAP signaling pathway in gastric mucosal tissue,in order to provide experimental basis for the Zhuodu theory in treating CAG and explore the mechanism and efficacy of HuaZhuoJieDu formula in treating CAG.Methods:1 A total of 70 healthy clean-grade male SD rats were selected with weighing 250-300 g,and fed for 7 days.Grouping: Using random number table method,3 groups of each with 4 rats were selected as normal group(Normal Group,N).The remaining 58 rats were randomized into 14 groups as model groups(Model Group,M).Subsequently,a combined modelling method of free drinking N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)solution,ranitidine solution administered,combined with irregular diet were given to rats in M groups,while the rats in N group only received regular feeding.At the 20 th week,2 rats of each group N and M were randomly selected to examine the pathological state of gastric mucosa,and the successful set up of models were confirmed.Regrouping: The models were divided into 5 groups according to the principle of randomization: Spontaneous Recovery Group(SR),Vitacoenzyme Group(VG),high-dose HuaZhuoJieDu formula treatment group(High-doze Group,HG),moderate-dose HuaZhuoJieDu formula treatment group(Moderate-doze Group,MG),low-dose HuaZhuoJieDu formula treatment group(Low-doze Group,LG),and eachgroup had 10 rats(Note: 6 rats were deaths during the modelling stage).Treatment: The VG were administered 10 ml/kg/d of Vitacoenzyme solution,and the dosage of crude drug is 0.006g/ml.The HG,MG,and LG groups were administered 10 ml/kg/d of HuaZhuoJieDu formula solution.The crude drug dosages were 2.8g/ml,1.4g/ml,and 0.7g/ml respectively.The N and SR groups were administered 10ml/kg/d distilled water,and all groups were treated with lavage for 12 weeks.Without eating and drinking for 48 hours after the last administration.After that,the materials of rats were taken after anesthesia,finally to put rats to death.2 To observe changes in general conditions such as: body weight,coat color,tooth and claw color,mental status,appetite,stool condition,and tongue color during(including before and after)the modeling and treatment stage,and to observe the pathological changes of gastric mucosa by HE staining.3 Immunohistochemistry(IHC)and Western Blot were used to detect the protein levels of MOB1,LATS1 and P-YAP in gastric mucosa of 6 groups after treatment.4 Real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect mRNA levels of MOB1 and LATS1 in gastric mucosa of 6groups after treatment.5 All data are analyzed with SPSS 21.0 software.Results:1 General situation: Modeling stage: In group N,there were no significant difference in coat color,mental status,intake of food and water,stool,and flexibility compared with former stage,but body weight increased significantly.In group M,the coat color was dull,messy,and had hair loss phenomenon,and the quantity of intaking water and food was decreased,mental status was poor and violent injury behaviors were appeared,the stool was soft,water,smelly and odious,unresponsive,and body weight decreased significantly compared with before.Treatment stage: The general condition of rats in HG and VG improved significantly compared with that before treatment,the MG improved better than before treatment,and the LG and SRgroups did not improve significantly.2 Observation of gastric mucosaVisual observation: Modeling stage: In group N,the gastric mucosa was pink,the tissue surface was smooth,and the mucosal folds were neatly arranged;in group M,the gastric mucosa was grayish,the surface of the tissue was rough,and there were some grainy bulges,mucosal folds are sparse,flat or disorganized,and even partially disappear.Treatment stage: Gastric mucosal lesions of rats in HG,MG,and VG were significantly improved;gastric mucosal lesions in LG and SR were partially improved.Pathological observation of gastric mucosa: Modeling stage: In group N,the thickness of mucosal layer was normal,and the epithelial cells were intact.The intrinsic glands were arranged neatly and moderately in quantity,without inflammatory cells were infiltrated.In group M,the mucous layer became thinner,the epithelial cells were degeneration,necrosis and shedding,and the intrinsic glands were arranged disorderly,and the number was reduced.Treatment stage:The HG,MG and VG had obvious improvement,the thickness of gastric mucosa was moderately,and epithelial cells without degeneration and necrosis,the intrinsic glands were arranged properly and in proper quantity,the quantity of intrinsic glands were as same as N,and more than M,and a small amount of inflammatory cells in the interstitium;The LG and SR had a certain improvement,but the quantity of intrinsic glands were low,obviously less than N,HG,VG,MG,and as same as M.3 Effect of HuaZhuoJieDu formula on MOB1 expression in gastric mucosa of CAG rats: The average optical density,the grey value and the mRNA of high-doze groups were higher than Vitacoenzyme groups,moderate-doze groups and low-doze groups,the difference was statistically significant(P<0.01),moderate-doze groups and low-doze groups were lower than Vitacoenzyme groups,the difference was statistically significant(P<0.01),and moderate-doze groups were higher than low-doze groups,the difference was statistically significant(P<0.01).4 Effect of HuaZhuoJieDu formula on LATS1 expression in gastricmucosa of CAG rats: The average optical density,the grey value and the mRNA of high-doze groups were higher than Vitacoenzyme groups,moderate-doze groups and low-doze groups,the difference was statistically significant(P<0.01),moderate-doze groups and low-doze groups were lower than Vitacoenzyme groups,the difference was statistically significant(P<0.01),and moderate-doze groups were higher than low-doze groups,the difference was statistically significant(P<0.01).5 Effect of HuaZhuoJieDu formula on P-YAP expression in gastric mucosa of CAG rats: The average optical density and the grey value of high-doze groups were higher than Vitacoenzyme groups,moderate-doze groups and low-doze groups,the difference was statistically significant(P<0.01),moderate-doze groups and low-doze groups were lower than Vitacoenzyme groups,the difference was statistically significant(P<0.01),and moderate-doze groups were higher than low-doze groups,the difference was statistically significant(P<0.01).Conclusions:HuaZhuoJie Du formula can relieve the general condition,significantly improve the pathological state of gastric mucosa of CAG rats,and up-regulate the expression of MOB1,LATS1 and P-YAP in gastric mucosa of CAG rats,the high-dose HuaZhuoJieDu formula is better than Vitacoenzyme in the aspects of improving pathological condition of gastric mucosa and up-regulating the expression of MOB1,LATS1 and P-YAP. |
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