Objective The present study aimed to elucidate the exact phase of chronic atrophic gastritis(CAG)that Weierning(WEN)protects and to illuminate its potential mechanisms.Methods 1.Establishment of CAG model:(1)MNNG free drinking and combined with multiple factors was used to construct CAG model mice:The 8-week-old ICR mice were randomLy divided into the normal group(6 mice)and the model group(34 mice)after adaptive feeding.The model group was given 180 mg·mL-1 MNNG solution every day,combined with irregular fasting(two days full diet,one day fast)and gavage with 30%ethanol solution 0.4 mL/mouse on the fasting day.At the 9th,10th,and 11th week of modeling,two mice were randomLy sacrificed,and gastric mucosal tissues were taken for pathological detection to determine whether the model was successful.Morphological indexes(color of hair,auricle and paw)and behavioral indexes(autonomic activity and grasping power)were observed after successful modeling.Caudal and stomach microcirculation blood flow.The entire molding cycle lasts 11 weeks.(2)CAG rats were induced by gavage of sodium ethosalicylate combined with multiple factors:After 7 days of adaptive feeding,70 SPF-grade male SD rats with body weight of(200±20)g were randomLy divided into normal group(10 animals)and model group(60 animals)according to body weight.Rats in normal group were given free water and intragastrically deionized water.The CAG model was established by gavage rats with a modeling solution(consisting of 2%sodium salicylate and 30%alcohol)with irregular diets(two days full feeding,one day fasting,and so repeatedly)and free access to 0.1%ammonia solution.At 19 weeks of modeling,gastric tissue was taken for biopsy and pathological diagnosis.2.The overall protective effect of WEN on CAG:The rats were randomLy divided into model group,after 70 days of modeling,the model rats were randomLy divided into model group,positive control group(Vitacoenzyme 240 mg·kg-1)and WEN low-dose,medium-dose and high-dose groups(180,360,720 mg·kg-1)according to body weight,with 9 rats in each group.During administration,except normal group and model group were given 0.01 mL·g-1 deionized water by intragastric gavage(ig)according to body mass,each administration group was given corresponding drugs by ig,once a day,for consecutive 9 weeks.Blood was collected to detect inflammatory and anemic indicators in peripheral blood(WBC,NEUT,NEUT%,LYMPH,and LYMPH%).Enzyme-linked immunosorbent assay was applied to measure serum levels of GAS,GT-17,PGI,and PGII and IL-1β.qRT-PCR was applied to measure mRNA expressions of TNF-α,IL-6,IL-8,IL-10,and γ-IFN.HE staining and transmission electron microscopy was applied to observe the pathological and ultrastructural changes of gastric mucosa.Tunel staining was used to detect the apoptosis of gastric mucosa cells.3.Preliminary study on the mechanism of WEN on CAG rats:Immunohistochemistry was used to observe the expression of PCNA,Bax,and Bcl2 in gastric mucosa,and western blotting were applied to detect the expression levels of Bcl2,Bax,Caspase3,Cleave-caspase9,and Cytochrome c.AB-PAS staining was applied to observe the intestinal metaplasia of gastric mucosa.The expressions of Cdx2 and Muc2 proteins were detected by immunofluorescence staining,and precancerous lesion related proteins(Glil,Smo,and Shh)was detected by Western blot.Results 1.Model evaluation results:Compared with the normal group,the model mice induced by MNNG combined with multiple factors had hard and yellow hair,fluffy and dull hair,lazy clumps,tired posture,slow weight gain,and granular feces.Under light microscope,atrophy and glandular reduction of intrinsic glands in gastric mucosa were observed,infiltration of a large number of inflammatory cells and lymphocytes,reduction of main and parietal cells,thickening of mucosal myometria,and intestinal metaplasia were observed.Compared with the normal group,the model rats with sodium ethanolsalicylate combined with multi-factor showed hard and yellow hair,fluffy and dull hair,slow weight gain,some rats have loose stools.Under light microscope,the intrinsic glands of the gastric mucosa were atrophic,the glands were reduced,the muscularis mucosa was thickened,and intestinal metaplasia was observed.2.WEN has a good therapeutic effect on CAG and can improve intestinal metaplasia(IM)of gastric mucosa:① Comparison of body weight:Compared with normal group,body weight increment of model group was significantly increased(P<0.01).Compared with model group,the increment of body mass in Vatacoenayme group and WEN groups were significantly increased(P<0.05).② Comparison of the pathological morphology of gastric mucosa tissues:The normal group of glands and cells arranged neatly and intact structure,no inflammatory infiltration.In the model group,the number of adenoid cells decreased,the gland atrophy increased,the volume of adenoid cavity increased,the main and parietal cells decreased substantially,and neutrophil and plasma cell infiltration was observed,accompanied by IM.After treatment,the cell arrangement of WEN groups were more orderly,and the atrophy of glands and IM were improved.③ Anemic and inflammatory indexs:Compared with normal group,the level of WBC,LYMPH,and LYMPH%of model group was significantly increased(P<0.01),after WEN treated the levels of WBC,LYMPH,and LYMPH%were decreased.Compared with normal group,the level of NEUT and NEUT%in model group was significantly decreased(P<0.01),WEN increased CAG-reduced neutrophils(NEUT)and percentage of neutrophils(NEUT%).④ Inflammatory markers:Compared with normal group,the level of TNF-α,IL-6,IL-8,IL-10,γ-IFN,and IL-1β in model group was significantly increased,after WEN treated the levels of TNF-α,IL-6,IL-8,IL-10,γ-IFN,and IL-1β were decreased.⑤Gastrointestinal hormone levels:Compared with normal group,the level of GAS,GT-17,and PGI decreased,after WEN treated the levels of GAS,GT-17,and PGI increased.Compared with normal group,the level of PGII increased,after WEN treated the levels of PGII decreased.⑥ Apoptosis index:Compared with the normal group,the apoptosis of gastric mucosa epithelial cells increased in the model group,but decreased significantly after administration of WEN with different dosage.3.Potential mechanisms of WEN on anti CAG:①Mitochondrial apoptosis-related proteins:Compared with the normal group,the expression level of Bcl2 protein in the model group was significantly decreased,and the expression level of Bcl2 protein was increased after administration of WEN.Compared with the normal group,the expression levels of PCNA,Bax,Caspase3,Cleaved-caspase9,and Cytochrome c were increased in the model group.The expression levels of PCNA,Bax,Cleaved-caspase9,and Cytochrome c proteins were decreased after administration of WEN.② Indexes of IM:Compared with the normal group,the expression levels of Muc2 and Cdx2 were increased in the model group.The expression levels of PCNA,Bax,Cleaved-caspase9,and Cytochrome c proteins were decreased after administration of WEN.③Related indexes of gastric precancerous lesions:Compared with the normal group,the expression levels of Gli 1,Shh,and Smo were increased in the model group.After WEN treated the expression levels of Gli1,Shh,and Smo proteins were decreased.Conclusion 1.WEN has a good therapeutic effect on CAG and can improve IM of gastric mucosa.2.WEN could improve inflammation,regulate gastrointestinal hormone secretion,inhibit the apoptosis of gastric mucosa cells,and improve IM of CAG.3.The mechanism of WEN in the treatment of CAG may be related to the regulation of mitochondrial apoptosis pathway and Hedgehog pathway. |