| BackgroundGastric Cancer is one of the most common types of cancer and it is the second leading cause of cancer-related death worldwide.The present studies show that patients with chronic atrophic gastritis follow-up atypical hyperplasia and intestinal metaplasia,the rate of cancer are 2.27% and 4.26%,the rate of severe epithelial dysplasia and intestinal metaplasia is 25% to 9.09%.And the occurrence of gastric carcinoma is an evolutionary histopathological stage,starting with chronic gastritis,followed by GPL,including chronic atrophic gastritis(CAG),intestinal metaplasia,dysplasia,even carcinoma.As a result,further research of GPL,which is a borderline of gastric cancer,is indispensable for preventing the formation and development of gastric carcinoma.p62,also known as SQSTM1,a tumor-associated autoantigen,is importa nt for cellular events that control proliferation and apoptosis,particul arly during tumorigenic conditions.The autophagy-independent role for p62 is reported in the digestive system tumors including esophageal cancer,large intestine cancer and gastric cancer.Recent advances in the unders tanding of p62 function suggest that it increases the activity of the nuc lear factor kappa B(NF-?B)pathway in many cell systems.Hyperactivatio n of NF-?B contributes to tumorigenesis by regulating cell cycle progres sion,promoting cancer cell proliferation,preventing apoptosis,and gene rating chemotherapeutic resistance.However,the mechanism of over-expres sed p62 in GPL has not been fully demonstrated.Based on our previous stu dy,we employed GPL model to explore whether the formlua could improve the protective function for gastric mucosa through p62.Objective1.To model GPL rats,and observe the effect of formlua on general conditions and macroscopic characters of GPL rats.Meanwhile,we would explore the effect of the formlua on gastric intestinal metaplasia and epithelial dysplasia.2.Based on our previous study,we employed GPL model to explore whether the formlua could improve the protective function for gastric mucosa through p62/RIP1-NF-?B signaling pathway.MethodBriefly,70 SD rats were randomly divided into normal group(n=13),the rest were model group(n=57).The model group free drinking MNNG solution(200 ?g/mL)everyday,and were subjected to feed every other day,while the rats in the control group were fed normally.At the sixteenth week,the model group were randomly divided into four groups: model group,WFC group(0.2g/kg),the formlua high dose group(19.8g/kg)and the formlua low dose group(9.9g/kg).After treatment for 10 weeks,all rats were sacrificed.All procedures proceeded for 26 consecutive weeks.(?)Effects of Weipixiao formula on histopathological changes of the Gastric Mucosa.1.The degree of gastric mucosal lesion was judged by H&E histopathological examination.2.HID-Alcian Blue-Periodic acid-Schiff(HID-AB-PAS)staining was used to accurately evaluate the types of the intestinal metaplasia.(?)Effects of Weipixiao formula on protection of gastric mucosa through autophagy pathway.1.Immunohistochemistry(IHC)was used to observed the protein expression of CDX2 and MUC2 in GPL.2.Western Blot analysis of autophagy expression in the GPL rats after treatment of the Weipixiao formula.(?)Effects of Weipixiao formula on protection of gastric mucosa through inflammation pathway.1.ELISA analysis of the TNF-? and IL-1? expression in the GPL rats after treatment of the Weipixiao formula.2.Western Blot analysis of the NF-?B signaling pathway expression in the GPL rats after treatment of the Weipixiao formula.(?)Effects of Weipixiao formula on protection of gastric mucosa through p62/RIP1-NF-?B signaling pathway.1.The mRNA levels of p62 and RIP1 in GPL rats were detected by real-time fluorescent quantitative PCR.2.Western Blot analysis of p62 and RIP1 expression in the GPL rats after treatment of the Weipixiao formula.Result(?)Histopathological Changes of the Gastric Mucosa.The degree of gastric mucosal lesion was judged by H&E histopathological examination.Compared to control group,the dysplastic glands were significantly increased,irregularly arranged,and weakly stained,which indicated that the abnormality of gastric epithelial dysplasia was diffusely augmented in the model group,moreover lined by atypical cells with overlapping,hyperchromatic and pleomorphic nuclei.HID-Alcian Blue-Periodic acid-Schiff(HID-AB-PAS)staining was used to accurately evaluate the types of the intestinal metaplasia.As the model group showed,the tissue of mucous metaplasia was positive for HID-AB-PAS staining,which elucidated the replacement of fundic parietal and chief cells by foamy cells containing neutral and acid mucins.Combined the results of both HE and HID-AB-PAS,the MNNG-exposed rats appeared intestinal metaplasia and epithelial dysplasia,namely precancerous lesions.Interestingly,after treatment of formula,the lesions of GIM and GED were significantly ameliorated compared with the model rats,especially crowded tubular glandular and back-to-back tubular structure,which were the dangerous borderline between GPL and GC.(?)Effects of Weipixiao formula on protection of gastric mucosa through autophagy pathway.1.Immunohistochemistry(IHC)was used to observed the protein expression of CDX2 and MUC2 in GPL.Immunohistochemistry analysis showed that the levels of CDX2 were decreased in the group of Weipixiao formula compared with the model group(P<0.05);The levels of MUC2 were decreased in the group of Weipixiao formula compared with the model group(P<0.01).2.Western Blot analysis of autophagy expression in the GPL rats after treatment of the Weipixiao formula.Western Blot analysis showed that the ratio of Bcl-2/Bax were decreased in the group of Weipixiao formula compared with the model group(P<0.01).The level of Bcl-XL were decreased in the group of Weipixiao formula compared with the model group(P<0.01,P<0.05).(?)Effects of Weipixiao formula on protectoion of gastric mucosa through inflammation pathway.1.ELISA analysis of the TNF-? and IL-1? expression in the GPL rats after treatment of the Weipixiao formula.The ELISA results showed that compared with the model group,the expression of TNF-? in the group of Weipixiao High and Lose dose were significantly lower than the model group(P<0.05,P<0.05).The ELISA results showed that compared with the model group,the expression of IL-1? in the group of Weipixiao High dose and Low dose were significantly lower than the model group(P<0.05,P<0.05).2.Western Blot analysis of the NF-?B signaling pathway expression in the GPL rats after treatment of the Weipixiao formula.The Western Blot results showed that compared with the model group,the expression of p-IKK?p-IKB and p-NF-?B in the group of Weipixiao Lose dose was significantly lower than the model group(P<0.05).The Western Blot results showed that compared with the model group,the expression of p-IKK and p-NF-?B in the group of Weipixiao High dose was significantly lower than the model group(P<0.05).(?)Effects of Weipixiao formula on protection of gastric mucosa through p62/RIP1-NF-?B signaling pathway.1.The mRNA levels of p62 and RIP1 in GPL rats were detected by real-time fluorescent quantitative PCR.The results of Fluorescence quantitative PCR showed that compared with the model group,the expression of p62 in the group of Weipixiao High dose and Low dose were significantly lower than the model group(P<0.05,P<0.05).The results of Fluorescence quantitative PCR showed that compared with the model group,the expression of RIP1 in the group of Weipixiao Low dose was significantly lower than the model group(P<0.05).2.Western Blot analysis of p62 and RIP1 expression in the GPL rats after treatment of the Weipixiao formula.The Western Blot results showed that compared with the model group,the expression of p62 in the group of Weipixiao Lose dose was significantly lower than the model group(P<0.05).The Western Blot results showed that compared with the model group,the expression of RIP1 in the group of Weipixiao formula was significantly lower than the model group(P<0.01,P<0.05).ConclusionThis research manifested that the occurrence of gastric cancer was a pathological process of chronic progression,was preceded by a prolonged precancerous stage,which could be ameliorated by the formlua.Taken these into together,the p62 elevating the cascade of apoptosis and inhibiting the pathway of autophagy in the gastric mucosa,which would be critical for preventing and curing GC.Further,the formlua could regulate NF-?B signaling pathway regulated by the p62 in GPL.It provided a potential therapeutic strategy in reversing intestinal metaplasia and dysplasia of gastric precancerous lesions. |
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