Study On Biological Basis Of Chaishao Liujun Decoction In Regulating Proliferation And Apoptosis Of Gastric Mucosa Cells In Rats With Chronic Atrophic Gastritis (CAG) Of Liver Depression And Spleen Deficiency Syndrome

ObjectiveIn this study,a rat model of chronic atrophic gastritis(CAG)with liver depression and spleen deficiency syndrome was established by compound factors.The therapeutic effect of Chaishao Liujun Decoction was observed from the perspective of "corresponding prescriptions and syndromes",and the molecular biological basis of Chaishao Liujun Decoction in regulating the proliferation and apoptosis of gastric mucosal cells in CAG with liver depression and spleen deficiency syndrome was preliminarily explored.Methods8 week old clean grade Sprague Dawley rats,half male and half female,were randomly divided into two groups: NG group(n=6)and MG group(n=20).NG group were not given any treatment.MG group were given 20 mmol/L sodium deoxycholate solution and 0.1%ammonia solution alternately for chemical induction every day.In the meantime,MG group were given tail clipping stimulation for 1 hour every day for 10 weeks.2 model rats were randomly selected to observe the morphology of gastric mucosa.The changes of general conditions,the results of behavioral experiments and the pathological morphology of gastric mucosa were used as the evaluation criteria.The model rats were randomly divided into CAG group,VI group and CS group,with 6 rats in each group.CS group was given Chaishao Liujun Decoction with concentration of 0.51g/mL by gavage,VI group was given vitamin suspension with concentration of 24mg/mL by gavage,NG group and CAG group were given[10mL/(kg·d)] sterilized drinking water by gavage for 4 weeks.The morphology of gastric mucosa was observed by hematoxylin eosin(HE)staining.The levels of serum EGF and TGF-α were detected by enzyme-linked immunosorbent assay(ELISA).The levels of p53 and c-myc in gastric mucosa were detected by quantitative real-time PCR(q PCR)Immunohistochemistry(IHC)was used to detect the expression of VEGF and PCNA in gastric mucosa.TUNEL was used to detect the apoptosis index of gastric mucosa.Results1.Model evaluation results:After modeling,compared with NG group,the hair of MG group was hard,light yellow,fluffy and lusterless,less activity,quiet and clumping,slow reaction,tired look,less food and water intake,slow weight growth,smelly stool and loose knot.The total distance and the number of upright explorations in open field experiment were significantly reduced,and the rest time of forced swimming experiment was significantly prolonged(P<0.01 or P<0.001).There were atrophy and decrease in the number of proper glands in gastric mucosa,infiltration of a large number of plasma cells and lymphocytes,hyperemia and edema in submucosa,deep staining of nuclei,reduction of main cells and parietal cells,thickening of mucosal muscle layer,no intestinal metaplasia or intraepithelial neoplasia.2.Changes of indexes after intervention of traditional Chinese medicine(1)Comparison of biological characterization: In NG group,the hair was white and glossy,active and sensitive,with good mental state,obvious resistance by gavage,moderate stool hardness and light smell;in CAG group,the hair was fluffy,light yellow,hard and dull,slow reaction,quiet and less movement,still tired,mild and not resistant by gavage,with slightly soft stool texture,yellowish and smelly;in VI group,the hair was fluffy and less glossy In CS group,the hair of the rats was light yellow,hard,slightly glossy,lively and active,and resisted by gavage,the stool was moderate soft and hard,the color turned brown,and the smell was light.There was no significant difference in body weight among the three groups after intervention(P>0.05);the body weight increment of NG group,CAG group and VI group was basically similar,and the body weight increment of CS group had an upward trend compared with the three groups,and the difference was not statistically significant(P>0.05).(2)Comparison of the appearance and pathological morphology of gastric mucosa: By gross observation,the color of gastric mucosa in NG group was bright red,the shape was normal,and the mucosal folds were rich and full;the color of gastric mucosa in CAG group was gray,the thickness became thinner,and the mucosal folds decreased or even disappeared;the color of gastric mucosa in the VI group was light red,slightly pale,and the mucosal thickness was slightly thinner than that in CAG group.In CS group,the color of gastric mucosa was light red,the shape tended to NG group,the thickness was significantly thickened,and the mucosal folds were significantly increased.Under light microscope,the structure of gastric mucosa in NG group was complete,the layers were clear,the cells were closely arranged,the main cells and parietal cells were obvious,and only a few plasma cells were infiltrated;in CAG group,the inherent glands of gastric mucosa atrophied,the cells arranged disorderly,the main and parietal cells lost,the gland cavity volume widened,a large number of macrophages,plasma cells and lymphocytes infiltrated,some cells fell off and necrotized,and the cells disappeared the results showed that obvious congestion and edema could be seen in the gap;compared with CAG group,the atrophy degree of gastric mucosa proper gland in VI group was slightly improved,the arrangement of cells was more orderly,the number of main cells and parietal cells increased,the volume of gland cavity was still larger,the infiltration of inflammatory cells decreased,and the congestion and edema were still seen in the intercellular space;the atrophy of inherent glands in gastric mucosa tissue in CS group was improved to a certain extent,the arrangement of cells was more orderly,the number of main and parietal cells increased the volume of glandular cavity decreased,a small amount of eosinophils and plasma cells infiltrated,and the congestion and edema of intercellular space decreased slightly.(3)Comparison of serum EGF and TGF-α contents: Compared with NG group,the serum EGF and TGF-α contents of CAG group were significantly increased(P<0.01).Compared with CAG group,the levels of serum EGF and TGF-α in VI group decreased,and the difference was not statistically significant(P>0.05);the levels of serum EGF and TGF-αin CS group decreased(P<0.05).There was no significant difference in serum EGF and TGF-α between VI group and CS group(P>0.05).(4)Comparison of p53 and c-myc mRNA expression in gastric mucosa: Compared with NG group,the expression levels of p53 and c-myc mRNA in gastric mucosa of CAG group were significantly increased(P<0.01);the expression levels of p53 mRNA in gastric mucosa of VI group and CS group were significantly increased(P<0.05 or P<0.01),and the expression of c-myc mRNA had an upward trend,but the difference was not statistically significant(P>0.05).Compared with CAG group,the expression levels of p53 and c-myc mRNA in gastric mucosa of rats in VI group and CS group were decreased(P<0.05 or P<0.01).Compared with VI group,the expression levels of p53 and c-myc mRNA in gastric mucosa of CS group were increased,and the difference was not statistically significant(P>0.05).(5)Comparison of VEGF and PCNA protein expressionin gastric mucosa: Compar ed with NG group,the expression levels of VEGF and PCNA in CAG group were in creased(P<0.05 or P<0.01),and the expression level of PCNA in VI group was significantly increased(P<0.01).Compared with CAG group,the expression levels of VEGF and PCNA protein in VI group were decreased,and the difference was not stat istically significant(P>0.05).The expression level of PCNA protein in CS group was decreased(P<0.05),and the expression level of VEGF protein was decreased,and the difference was not statistically significant(P>0.05).There was no significant differenc e in the expression levels of VEGF and PCNA protein between VI group and CS gro up(P>0.05).(6)Comparison of apoptosis index: Compared with NG group,the apoptosis index of gastric mucosa in CAG group increased(P<0.05).Compared with CAG group,the apoptotic index of gastric mucosa of rats in VI group decreased,and the difference was not statistically significant(P>0.05).The apoptotic index of gastric mucosa of rats in CS group decreased(P<0.05).There was no significant difference in the apoptosis index of gastric mucosa between VI group and CS group(P>0.05).Conclusions1.There is abnormal expression of cell proliferation and apoptosis factor in gastric mucosa of rats with chronic atrophic gastritis of liver stagnation and spleen deficiency syndrome.Imbalance of cell proliferation and apoptosis in gastric mucosa may be one of the biological basis of chronic atrophic gastritis of liver stagnation and spleen deficiency syndrome.2.The rat model of chronic atrophic gastritis with liver depression and spleen deficiency syndrome can be successfully established by the compound factor model method mainly induced by sodium deoxycholate and ammonia.Chaishao Liujun Decoction can improve the general condition of the CAG rats,reduce the inflammatory reaction of gastric mucosa and reverse the atrophy of gastric mucosa glands.3.Chaishao Liujun Decoction plays an important role in regulating the excessive proliferation and apoptosis of gastric mucosal tissue cells,which may regulate the proliferation and apoptosis factors EGF and TGF-α of gastric mucosal tissue cells in rats,The expression of p53,c-myc and PCNA can promote the repair of gastric mucosal tissue damage and improve the syndrome of liver stagnation and spleen deficiency in CAG.