| ObjectiveIn this study,a rat model of liver-depression and spleen-deeficiency chronic atrophic gastritis（CAG）was established by compound factors and chronic stress.The purpose of this research was to explore the biological basis of liver-depression and spleen-deficiency CAG from hedgehog signaling pathway.The mechanism of Chaishao Liujun Decoction in treating liver-depression and spleen-deficiency CAG was preliminarily determined.MethodsA total of 52 SPF Wistar male rats aged 5-6 weeks（110±10g）were divided into 2 groups by random number table method,including 10 normal group（NG）and 42 model group（CAG）.NG were not given any treatment.CAG were give 0.1%ammonia solution and 20 mmol/L sodium deoxycholate solution alternately for chemical induction every day with hunger and satiety disorders（2 days of adequate food,1 day of fasting）.In the meantime,CAG were given chronic binding for 3h and tail clamping for 1h every day,continuous modeling for 16weeks.Two rats in the NG and two rats in the CAG were randomly selected to observe the pathological morphology of gastric mucosa.The success of modeling was determined by general morphology,behavioral experiments and pathological morphology of gastric mucosa of rats.The successful modeling rats were randomly divided into model group,Vitacoenzyme group(2.4g·kg-1·d-1),Chaishao Liujun Decoction（CLD）group(5.1g·kg-1·d-1),Hedgehog blocker（GDC-0449）group(50mg·kg-1·d-1),and CLD plus GDC-0449 group(5.1g·kg-1·d-1+50mg·kg-1·d-1),and the NG was used as the control to receive corresponding dose of drug intervention.The model group and the NG were given equal volume of sterilized drinking water for continuous intervention for 4 weeks.The histomorphology of gastric mucosa was observed by htoxylin eosin（HE）staining,and the contents of interleukin-1β（IL-1β）,interleukin-6（IL-6）and tumor necrosis factor（TNF-α）in serum were detected by enzyme-linked immunosorbent assay（ELISA）,the mRNA and protein expression levels of Shh,Ptch1 and Gli1 in gastric mucosa of rats were detected by q-PCR and Western Blot.Results1.Model evaluation results:After 16 weeks of modeling,compared with the NG,rats in the CAG had hard,yellow,fluffy,hair,dull and lumpy hair,lazy and weary,tired expression,slow weight gain,thin stool and smelly smell.In the open field experiment,the number of crossing squares and upright times decreased obviously,and the rest time of forced swimming experiment increased obviously.Under the light microscope,the inherent glands in gastric mucosa were atrophied and reduced,and a large number of inflammatory cells and lymphocytes were infiltrated,the nuclei were hyperchromatic,the main and parietal cells were reduced,the mucosal muscle layer was thickened,and intestinal metaplasia was observed.2.Changes of indexes in each group after drug intervention（1）Comparison of physiological characterizations:Rats in NG were active and alert,with shiny white hair,normal stool shape and light odor.Rats in the model group were tired in expression,obviously clustered,unresponsive,with dry,yellow and dull hair,soft stool,yellowish and smelly.Compared with the model group,the mental state of the rats in the Vitacoenzyme group was improved with fluffy hair and a a little luster and stools become hard in texture and slightly smelly.The rats of the CLD group were lively and active,with light yellow and slightly shiny hair and the stools are slightly normal.The rats in GDC-0449group and CLD plus GDC-0449 group showed no improvement.（2）Comparison of body mass:Compared with NG,the body weight of model group increased significantly（P<0.01）.Compared with model group,the body weight of Vitacoenzyme group and CLD group increased significantly（P<0.01）,while there was no statistical significance between GDC-0449 group and CLD plus GDC-0449 group（P>0.05）.Compared with CLD group,there was no significant difference in body weight increment in Vitacoenzyme group（P>0.05）,while body weight increment in GDC-0449group and CLD plus GDC-0449 group was significantly decreased（P<0.01）.（3）Comparison of behavioral experiments:Compared with the NG,the number of square crossing and upright times in the model group were significantly reduced（P<0.01）,but there was no significant difference in the number of washing face modification（P>0.05）.Compared with model group,the number of square crossing and upright times in Vitacoenzyme group and CLD group were significantly increased（P<0.01）,but there was no significant difference in the number of washing face modification（P>0.05）.The number of square crossing,upright times and the number of washing face modification in GDC-0449group and CLD plus GDC-0449 group were no significant differences（P>0.05）.Compared with CLD group,there was no significant difference in the number of crossing squares and the number of washing face modification in Vitacoenzyme group（P>0.05）,but the number of standing upright was significantly decreased（P<0.01）.The number of square crossing and upright times in GDC-0449 group and CLD plus GDC-0449 group were significantly decreased（P<0.01）,but there was no significant difference in the number of washing face modification（P>0.05）.Compared with the NG,the rest time of forcced swimming experiment in the model group was significantly increased（P<0.01）.Compared with model group,the rest time of forced swimming experiment in Vitacoenzyme group and CLD group was significantly reduced（P<0.01）,while there were no statistical difference in the rest time of forced swimming experiment in GDC-0449 group and CLD plus GDC-0449 group（P>0.05）.Compared with CLD group,there was no significant difference in the rest time of Vitacoenzyme group（P>0.05）,while the rest time of GDC-0449 group and CLD plus GDC-0449 group was significantly increased（P<0.01）.（4）Comparison of pathological morphology of gastric mucosa:In the NG,the glands and cells were arranged neatly and the structure was intact without inflammatory infiltration,which was the normal pathological manifestation of gastric mucosa.In model group,adenosine cells decreased,glands atrophied,lumen volume increased,main and parietal cells decreased significantly,neutrophils and plasma cells infiltrated,accompanied by intestinal metaplasia,indicating that CAG model was successfully constructed in rats.After treatment,the cells were arranged more orderly,the atrophy of glands was improved,inflammatory infiltration was slightly improved,and the main and parietal cells were increased in the Vitacoenzyme group and the CLD group,while GDC-0499 group and CLD plus GDC-0449group were not improved.（5）Comparison of inflammatory factors in serum:Compared with NG,the contents of IL-6,IL-1βand TNF-αin model group were significantly increased（P<0.01）.Compared with model group,the contents of IL-6 and TNF-αin Vitacoenzyme group were significantly decreased（P<0.01）.The content of IL-1βin Vitacoenzyme group showed a downward trend,but there was no statistical significance（P>0.05）.The contents of IL-6,IL-1βand TNF-αin CLD group were significantly decreased（P<0.05,P<0.01）.There were no significant differences in the contents of IL-6,IL-1βand TNF-αin GDC-0449 group and CLD plus GDC-0449 group（P>0.05）.Compared with CLD group,the contents of IL-6,IL-1βand TNF-αin Vitacoenzyme group had no significant difference（P>0.05）.The contents of IL-6,IL-1βand TNF-αin GDC-0449 group and CLD plus GDC-0449 group were significantly increased（P<0.01,P<0.05）.（6）Comparison of mRNA expression levels of Shh,Ptch1 and Gli1:Compared with NG,the mRNA expression levels of Shh,Ptch1 and Gli1 in model group were significantly decreased（P<0.01）.Compared with model group,the mRNA expression levels of Shh,Ptch1and Gli1 in Vitacoenzyme group and CLD group were significantly increased（P<0.01）,while there was no significant difference in Shh and Gli1 mRNA in GDC-0449 group（P>0.05）.The Ptch1 mRNA expression was increased（P<0.05）,but not as high as that in the treatment group.The mRNA expression levels of Shh,Ptch1 and Gli1 in GDC-0449 group of CLD group were not significantly different（P>0.05）.Compared with CLD group,the expression level of Shh mRNA in vitrase group was increased（P<0.01）,the expression level of Ptch1mRNA in Vitacoenzyme group was decreased（P<0.01）,and there was no statistical significance in the expression level of Gli1mRNA in vitrase group（P>0.05）.The mRNA expression levels of Shh,Ptch1 and Gli1 in GDC-0449 group and CLDplus GDC-0449 group were significantly decreased（P<0.01）.（7）Comparison of protein expression levels of Shh,Ptch1 and Gli1:Compared with NG,the protein expression levels of Shh,Ptch1 and Gli1 in model group were significantly decreased（P<0.01）.Compared with model group,the protein expression levels of Shh,Ptch1and Gli1 in Vitacoenzymegroup and CLD group were significantly increased（P<0.01）,while there was no significant difference in the protein expression levels of Shh,Ptch1 and Gli1between GDC-0449 group and CLD plus GDC-0449 group（P>0.05）.Compared with CLD group,there was no significant difference in the protein expression levels of Shh,Ptch1 and Gli1 in the vitase group（P>0.05）,and the protein expression levels of Shh,Ptch1 and Gli1 in GDC-0449 group and CLD plus GDC-0449 group were significantly decreased（P<0.01）.Conclusions1.The compound modeling method based on chemical induction of ammonia water and sodium deoxycholic acid combined with chronic stress can successfully establish the rat model of chronic atrophic gastritis with liver depression and spleen deeficiency,and some rats can develop intestinal metaplasia.2.Excessive expression of inflammatory factors in serum and abnormal expression of hedgehog signaling pathway in gastric mucosa of chronic atrophic gastritis rats with liver depression and spleen deficiency are considered to be one of the biological bases of chronic atrophic gastritis with liver depression and spleen deficiency.3.Chaishao Liujun Decoction can reduce the expression of inflammatory factors and improve the pathological morphology of gastric mucosa by regulating the mRNA and protein expressions of Shh,Ptch1 and Gli1. |
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