| Objective1.Based on the stomach-brain-related theory,we used N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)for free drinking and combined with irregular fasting on alternate days to establish the Gastric precancerous lesions(GPL)model.Base on it,we combined Chronic unpredictable mild stress(CUMS)to build the GPL rats under emotion intervention,in order to find out whether the emotional disorder would influence the feeding function and the damage of gastric mucosa in GPL rats.Also,we want to select the key indicators contributed to the cancerization and the feeding function in the“inflammatory-cancer”stage of CAG.2.We observed the change of the key indicators contributed to the cancerization and the feeding function in rats with chronic atrophic gastritis(CAG).We also applied electro-acupuncture(EA)at Zusanli(ST36)on them and assessed the effect of EA.We want to explored the mechanism of EA,and hypothesize that it might regulate the feeding function of CAG rats,by mediating the factors and receptors related to feeding in stomach and hypothalamus,repair the damage of gastric mucosa and reduce the inflammation,to produce a protection on the gastric mucosa.Methods1.The study of the establish of the gastric precancerous rats with emotional disorder and the influence of their feeding function(Experiment 1)A total of 60 male Sprague-Dawley(SD)rats were randomly divided into control group,gastric precancerous lesion model group(GPL),and GPL with emotional disorder model group(GPL+CUMS),with 12 rats in each group.GPL group were treated with MNNG(200 ?g/ml)solution for free drinking and irregular fasting for 28 consecutive weeks.GPL+CUMS group was combined with CUMS for 28 consecutive weeks based on GPL group.The general condition and the food intake was dynamically observed.At the end of the 28th week,rats would be sacrificed after the behavior and sucrose preference tests.The expression of IL-6,IL-10,TNF-?,Ghrelin(GHRL),Leptin(LEP),Somatostatin(SS)in serum was measured by ELISA.We observed the general condition of gastric mucosa under naked eyes,and observed the pathological changes of gastric mucosa under light microscope after Hematoxylin and Eosin(HE),Alcian blue/Periodic acid Schiff(AB-PAS),high iron Diamine-Essent Blue(HID-AB)staining.Immunofluorescence was used to observe the expression of Ghrelin and Gastrokine-2(GKN2)in the stomach.2.The study of the mechanism of EA at Zusanli(ST36)on regulating feeding function of rats with CAG(Experiment 2)Sixty male SD rats were randomly divided into control group and CAG group.The rats with CAG was treated with MNNG(200?g/ml)solution for free drinking and irregular fasting for 16 consecutive weeks.The rats with CAG were randomly divided into CAG group,electro-acupuncture at Zusanli(ST36)group(EA group)and electro-acupuncture at Sham-acupoint group(Sham-EA group),with 12 rats in each group.EA group and Sham-EA group both were treated with electro-acupuncture apparatus under the condition of dilatational wave(sparse wave is 2Hz,dense wave is 10Hz)and the intensity range from 0.3 to 0.5mA,for 10min once time,6 times per week.The treatments last for 8weeks,which started from the 17th week.Among this period,all rats were under normal diet.The general condition and the food intake was dynamically observed.At the end of the 28th week,rats would be sacrificed.The expression of IL-6,IL-10,TNF-?,Ghrelin,SS,Leptin,and Prostaglandin E2(PGE2)in serum were measured by ELISA.We observed the general condition of gastric mucosa under naked eyes,and observed the pathological changes of gastric mucosa under light microscope after HE,AB-PAS,HID-AB staining.We observed the expression of Ghrelin,Leptin,Neuropeptide Y(NPY)and GKN2 in gastric tissues by applying immunofluorescence.WB method was used to measure the expression of GKN2,Growth Hormone Secretagogue Receptor(GHSR)and Leptin receptor(LEPR)proteins in gastric tissues.Double immunofluorescence method was used to observe NPY and Agouti-Related protein(AgRP)expression in hypothalamus.WB method was used to measure the expression of GHSR,LEPR,Pro-opiomelanocortin(POMC).Results1.The study of the establish of the gastric precancerous rats with emotional disorder and the influence of their feeding function(Experiment 1)Compared with the control group,the general condition of GPL and GPL+CUMS rats was worse,the weight were reduced and the food intake fluctuated greatly.The results of the open field experiment showed that compared with the control group,the GPL and GPL+CUMS groups had less total distance and lower average speed(P<0.05).Compared with the control group and the GPL group,the GPL+CUMS group significantly increased the ratio of the central area to the total distance(P<0.05),and the time in the central area increased significantly(P<0.05).Compared with the control group,the percentage of sucrose water preference in the GPL+CUMS group was significantly reduced(P<0.05),and the percentage of sucrose water preference in the GPL group did not change(P>0.05).The results showed that GPL+ CUMS can cause tumorigenesis and accelerate GPL progression.Compared with the control group,GPL and GPL+CUMS increased the expression of inflammation-related factors(IL-6,TNF-?)in serum(P<0.05),and decreased the expression of Ghrelin and Leptin(P<0.05),increase the expression of SS(P<0.05).Compared with GPL,GPL+CUMS group significantly increased the expression of TNF-? and SS in serum(P<0.05).Compared with the control group,GPL and GPL+ CUMS groups increased the positive expressions of GKN2 and Ghrelin in gastric tissues and gastric mucosa near the tumor abnormally,while lower expression of GKN2 and Ghrelin were showed in the tumor.The general condition of CAG rats were poor,the weight were reduced and food intake fluctuates greatly.CAG rats increased the expression of inflammation-related factors(IL-6,TNF-?)in serum(P<0.05),the content of IL-10 and PGE2 decreased(P<0.05),decreased the expression of Ghrelin and Leptin(P<0.05),and SS increased the expression of SS(P<0.05).CAG rats increased the positive expression of Ghrelin focally,increased the positive expression of Leptin,and decreased the expression of NPY.CAG rats increased the expression of GKN2 and LEPR proteins(P<0.05),decreased the expression of GHSR protein(P<0.05).The expression of NPY and AgRP in hypothalamus of were increased abnormally,and the expressions of LEPR,GHSR and POMC proteins were increased(P<0.05)in CAG rats.EA can improve the general condition of CAG rats,increase body weight and stabilize food intake,and improve gastric mucosal lesions.Compared with the CAG group,the EA group and Sham-EA group can reduce the serum levels of inf lammation-related factors(IL-6,TNF-?)(P<0.05).EA group increased the expression of IL-10 and PGE2(P<0.05),increased the expression of Ghrelin and Leptin(P<0.05),decrease the expression of SS(P<0.05)in serum.The expression of IL-10,PGE2,Ghrelin,Leptin,and SS in the Sham-EA group were not significantly improved in serum(P>0.05).There was no obvious focal positive expression of Ghrelin in the gastric tissues of EA group and the Sham-EA group,and both groups could increase the expression of NPY in the gastric tissues.EA group could reduce the expression of Leptin in the gastric tissues.EA group could reduce the expression of GKN2 and LEPR(P<0.05),and increase the expression of GHSR(P<0.05)in CAG rats.EA can reduce the high expression of NPY and AgRP in the hypothalamus and reduce the high expression of LEPR,GHSR and POMC in CAG rats(P<0.05).Sham-EA group did not significantly improve the expression of GKN2,LEPR and GHSR in the gastric tissue of CAG rats(P<0.05),and the expression of NPY and AgRP in the hypothalamus.Also the expression of LEPR,GHSR,and POMC protein did not have significant improvement(P<0.05).Conclusion1.Feeding disorders are involved in the "inflammatory-cancer" stage of CAG.2.Emotional disorders are one of the risk factors in the"inflammatory-cancer" stage of CAG.3.Feeding disorders in CAG rats may be related to abnormal NPY signal pathway,or the imbalance between food intake and energy consumption.4.In the "inflammatory-cancer" stage of CAG,severe dysplasia or canceration of gastric mucosal epithelial cells may occur blood metastasis through peripheral blood vessels or neovascularization.The blood metastasis of cancer cells maybe happens before the cancer cells of PLGC infiltrating the basement membrane.5.Ghrelin and GKN2 may be related to early blood metastasis,which is an effective observation indicator for the development and prognosis in the"inflammatory-cancer" stage.6.ST36 showed its specificity on acupoint.Electro-acupuncture at Zusanli(ST36)could stabilize the feeding and improve the gastric mucosal inflammation in CAG rats.EA might mediate the expression of Ghrelin and Leptin and its receptors,to adjust NPY/AgRP neurons and POMC neurons in the hypothalamus to stabilize the feeding. |
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