| ObjectiveGastric cancer is characterized as a high incidence,high metastasis rate and high mortality rate malignant digestive system cancer,which is possessing low rate of early diagnosis in the developing countries(area).The early diagnosis rate of gastric cancer in China is about 10%,while over 90%of confirmed patients were with advanced gastric cancer.Most patients are already in the advanced stage after gastric cancer screening,and the prognosis is poor,which seriously threatens patients’ survival.Pathological changes before gastric carcinoma(defined as the precancerous lesions of gastric cancer,GPL)include the chronic atrophic gastritis(chronic atrophic gastritis,CAG),intestinal metaplasia(IM),and severe hyperplasia.Up to date,there is still in the absence of specific effective Western medicine for GPL.Our group previous studies suggested that Traditional Chinese Medicine can be considered to provide promising strategy for the prevention and treatment of GPL.In the present study,GPL-related bio-medical database was used to screen candidate targets and involved pathways in each key-stage of GPL by the bioinformatics network pharmacology approach,which subsequently predicted and screened the candidate targets and their underlying mechanisms of Jianpihuayujiedu decoction on treating GPL.In addition,studies related to gastric cancer in Taiwan indicated that chewing areca can produce a large number of reactive oxygen species and may activate oncogenes to promote the occurrence and development of gastric cancer.Therefore,this study the free drinking N-methyl-N ’-Nitro-NNitrosoguanidine(MNNG)plus intermittent fasting method was used to construct a GPL mice model,oral administration of Jianpihuayujiedu decoction,pathological histology observation detection were performed to valuate morphology and pathological situation in the vehicle or treated GPL mice,then qPCR,Western blot and ELISA method were performed to investigate the effect mechanism of jianpi Huayu Jiedu prescription on GPL by antioxidative stress.Methods1.The DisGeNET online database was used to screen the GPL-related candidate genes,we then constructed protein to protein network of the candidate genes by using STRING online tool and Cytoscape software.The candidate KEGG pathways of GPL were enriched and analyzed by using the DAVID online tool,we then picked up the candidate hub genes to perform Biological process,Cellular component,and Molecular function analysis.The overall survival and disease-free survival of candidate genes were accessed by using the GEPIA online tool.2.Balb/c pure mice were sensitive to alkylating agents,they were applied to feed by free drinking MNNG plus intermittent fasting method to construct the GPL animal model.The H&E staining was then performed to confirm the GPL animal model.3.Fifty SPF male Balb/c mice were randomly divided into blank group,model group,high-dose Jianpihuayujiedu decoction group,low-dose Jianpihuayujiedu decoction group and positive drug group,with 10 mice in each group.Corresponding intervention methods were given after successful modeling,and samples were taken after 8 weeks of continuous administration.Gastric mucosa specimens and splenic specimens of GPL mice were prepared for pathological histology observation and immunohistochemical detection by HE staining and HID-AB-PAS staining.The protein expression of Nrf2,p38,p-p38,Akt,p-Akt,HO-1,NQO1,SOD2,and GAPDH were detected by Western blot.The SOD serum level was detected by SOD ELISA kit.Real time PCR was performed to evaluated Nrf2,HO-1,and NQOl transcriptional levels.Results(1)The PI3K/AKT pathway was aberrant and significant activated in the GPL disease,CCND1 was observed co-expression with EGFR,ERBB2,and MYC.MYC was also observed co-expression with TP53,ERBB2 was also observed co-expression with EGFR in GPL patients.Besides,aberrant EGF,HGF,and IL-6 upregulation significantly correlated with poor prognosis in gastric cancer patients.Jianpihuayujiedu decoction attenuated inflammatory damage of CAG and inhibited inflammatory cytokine induced precancerous transformation of gastric cancer via targeting PI3K signaling.(2)MNNG free drinking and alternating-day fasting can cause gastric mucosa damage in mice,which gradually develops from chronic non-atrophic gastritis to intestinal metaplasia and gradually develops into dysplasia.The lesion degree increases over time,and the selection of Balb/c mice sensitive to gastric cancer significantly reduces the modeling time(3)As for the appearance,the hair of the blank group were luxuriant,soft smooth.shiny;the hair of MNNG-treated group was sparse,dull,and coarse.The fur density of mice in the low/high dose of the Jianpihuayujiedu decoction group and positive drug group were similar to that of the blank group,which were soft,shiny and smooth.(4)The gained weight of the blank group grew significantly fastest when compared to the lest of groups,though all groups body weight were significantly increased after 14 weeks feeding(P<0.05).The spleen index of the blank group was significantly higher than that of the MNNG-treated group(P<0.05),and the spleen index of the high dose group of the Jianpihuayujiedu decoction and positive drug group were significantly higher than that of MNNG-treated group(P<0.05).There was no significant difference in spleen index between the low dose group of the Jianpihuayujiedu decoction and the MNNG-treated group.The thymus index of the MNNG-treated group and the low dose group of the Jianpihuayujiedu decoction were lower than that of the blank group,and the thymus index of the high dose group of the Jianpihuayujiedu decoction and the positive drug group was higher than that of the MNNG-treated group,but the individual difference was large.(5)For pathological morphology observation,the gastric mucosa epithelium of the blank group mice was orderly and intact,with normal cell morphology,normal mucosal thickness and uniform muscle thickness.The gastric mucosa gland of the MNNG-treated group mice were atrophy,disrupt epithelial barrier structure,mucous membrane and lamina propria appeared intestinal metaplasia,the cell morphology were abnormal,cellular slime secretion of lamina propria and mucus were increased,appeared vacuolated changes,adjacent gland blended with each other to form hollow mucilage lake;the near the nuclei of the basal layer mucosa were stained by purple or black,while their cytoplasm were violet.The gastric mucosa glands of the low dose group were slightly disrupted epithelial barrier structure,there was slightly erosion or ulcer in the epithelial layer,intestinal metaplasia appeared in lamina propria and the epithelial layer,and the nuclei of the mucosal basal layer were stained by dark purple,but the color and intestinal metaplasia were slighter than that in the MNNG-treated group.In the high dose group,the gastric mucosa glands appeared normal epithelial barrier structure,and there were new normal glands in the muscularis and lamina propria of the mucosa.Intestinal metaplasia cells had been transferred to the upper cortex.The mucosa of positive drug group was intact,with smooth arrangement of glands,normal thickness,without any hyperplasia or atrophy,except for a few vacuolar changes.After HIDAB-PAS staining,The gastric mucosa epithelial cells of the blank group were stained as bright red,while it was widely blue,even locally blue-purple in the gastric mucosa ofMNNG-treated group.The gastric mucosa of the low dose group was stained as violet,the blue foci were less than that in the MNNG-treated group.The gastric mucosa of the high dose group was pink,and the degree of red staining was higher than that in the low dose group while less than that the blank group.The color of gastric mucosa in positive drug group was almost as bright red as that in the blank group,but less evenly staining that which were still mixed with slight blue.(6)Compared with the blank group,the serum SOD level of the MNNG-treated group was significantly lower than that in the blank group(P<0.01);the SOD level of the low dose group was close to that of the MNNG-treated group(P>0.05),the serum SOD level of the Jianpihuayujiedu decoction high dose group and positive drug group was apparently higher than that of the MNNG-treated group(P<0.01),and the positive drug group was slightly higher than that of high dose group.(7)Compared with the blank group,the phosphorylation level of p38 and AKT in the MNNG-treated group were upregulated,Jianpihuayujiedu decoction significantly decreased the phosphorylation level of p38 and AKT in dose dependent manner,the positive drug also significantly decreased the phosphorylation level of p38 and AKT.Compared with the blank group,the protein level of NQOl in MNNG-treated group was significantly decreased;compared with the MNNG-treated group,the low dose group was significantly increased,while the positive drug group and high dose group was decreased.Compared with the blank group,the protein level of SOD2 and HO-1 in the MNNG-treated group were decreased,and Jianpihuayujiedu decoction significantly rescued the protein level of SOD2 and HO-1 in dose dependent manner,the positive drug also significantly rescued the protein level of SOD2 and HO-1.Compared with the blank group,the protein level of Nrf2 in MNNG-treated group were decreased,and Jianpihuayujiedu decoction significantly rescued the protein level of Nrf2 in dose dependent manner,the positive drug had no effect on the protein level of Nrf2 while compared with the MNNG-treated treatment.Compared with the blank group,the mRNA level of Nrf2,NOQ1,and HO-1 in the MNNG-treated group were decreased,and Jianpihuayujiedu decoction significantly rescued the mRNA level of Nrf2,NOQ1,and HO1 in dose dependent manner.The positive drug also significantly rescued the mRNA level of Nrf2 and HO-1,but it had no effect on the mRNA level of NOQ1.ConclusionWe successfully constructed GPL animal model,our results suggested that Jianpihuayujiedu decoction would inactivate PI3K/AKT pathway,further rescued the expression of NQO1,SOD2,HO-1 and Nrf2 to suppress oxidative stress,which in turn to ameliorate gastric mucosa dysplasia and intestinal metaplasia,maintain the physiological structure and function of spleen and stomach,and significantly improve the symptoms of GPL such as emaciation,which in all provided solid evidence to promote clinic application of Jianpihuayujiedu decoction.And it would provide theoretical guidance for the improvement of GPL treatment and gastric cancer prevention in Taiwan region by giving full play to the advantages of TCM treatment. |
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