| Background:Helicobacter pylori(Hp)infection is one of the most common bacterial infections in human beings.According to statistics,half of global population were infected with Hp.Hp,a gram-negative micro-aerobic bacterium,parasitizes human stomach by changing environment and reducing acidity.Hp infection can not only cause benign and malignant gastric diseases such as chronic active gastritis,peptic ulcer and adenocarcinoma,but also cause many diseases outside the stomach.In addition,the articles on the correlation between HP infection and functional gastrointestinal diseases such as gastroesophageal reflux,functional dyspepsia and gastric emptying disorder have been published one after another.The influence of Hp infection on gastric emptying function has been debated for a long time,but no definite conclusion has been obtained.The study of the influence of Hp infection on gastric emptying function and its related mechanism has important guiding significance for predicting and treating delayed gastric emptying(DGE)after operation in patients with Hp infection,and studying Hp-related gastrointestinal motility disorders.Most of the previous studies were clinical trials,which only clarify the influence of Hp infection on gastric emptying,but the related mechanism is rarely studied.And the influence factors of clinical trials are complex,and the subjects may have unpredictable individual differences,so animal experiments may be a better choice.Pancreatoduodenectomy(PD)is the main surgical method to treat benign and malignant diseases around pancreatic head and ampulla in hepatobiliary surgery.DGE is one of the most common complications after PD.Besides postoperative pancreatic fistula and abdominal infection,is Hp infection a new risk factor for DGE after PD?Gastric motility involves interactions among gastrointestinal hormones,smooth muscle,enteric and extrinsic autonomic nerves and interstitial cells of Cajal(ICCs).Changes in gastrointestinal hormone levels,pathological changes in endogenous or exogenous enteric nervous system,changes in the number or function of ICCs,and gastrointestinal smooth muscle diseases all can lead to gastric emptying disorders.The ICCs play an important role in gastrointestinal motility and are acknowledged to act as physiological pacemakers for the gastrointestinal tract.And the smooth muscle is considered as the final effector of gastrointestinal movement.In this study,we establish a model of Hp infection,clarify the influence of Hp infection on gastric emptying function,and explored the related mechanism of Hp infection on gastric emptying function from two aspects of gastrointestinal ICCs and gastrointestinal smooth muscle,and the significance of this study in clinical work by analyzing the clinical cases of patients undergoing PD.Part 1.The effect of Hp infection on gastric emptying functionPurpose:To determine the effect of Hp infection on gastric emptying function.Materials and methods:1.The Helicobacter pylori Sydney strain(HpSS1)was cultured in micro-aerobic environment.2.A total of 70 female specific pathogen-free(SPF)C57BL/6 were purchased.To establish a model of HP infection,40 mice were infected with HP SS1 by oral gavage,and the mice of successful modle were used as the experimental group(Hp+group).Another 30 mice remained uninfected and were used as control(Hp-group).3.After 6 weeks,40 resin beads(diameter,0.4 mm)was administered to each mouse via oral gavage.The stomach was removed 1h after the gavage of beads,and the number of beads in the stomach was counted.Gastric emptying was calculated using the following formula:Gastric emptying(%)=(40-number of beads in the stomach at 1 h)/40 x 100.4.The confirmation of Hp infection:1.rapid urease test of gastric mucosa.2.Bacterial culture of gastric mucosa and identified by biochemical experiments(rapid urease test,catalase test and oxidase test).3.Immunohistochemistry of gastric tissue(Anti HP antibody).5.12 mice were randomly selected in successfully modeled mice without beads in the stomach injection needle as the experimental group(Hp+group),and 12 mice were randomly selected in other 30 mice without beads in the stomach injection needle as the control group(Hp-group),and used to calculate gastric emptying and subsequent experiments.Results:1.Among the 40 mice used for modeling,2 died at the first oral gavage of Helicobacter pylori,and the rest mice survived until the end of the experiment,and 35 mice successfully established the model,with the success rate of about 92%.The other 30 mice in the control group survived until the end of the experiment.2.The gastric emptying rate in the Hp+ group was 65.8±5.6%,which was significantly lower compared with that in the Hp-group(83.8±6.4%)(P<0.05).Summary:It is feasible to establish a mouse model of HP infection by oral gavage.Hp infection affects gastric emptying function and slows down gastric emptying.Part 2.The mechanism of Hp causing DGE by decreasing ICCsPurpose:The mechanism of HP causing DGE by decreasing ICCs,Materials and methods:1.The inflammation of gastric mucosa in two groups of mice was observed by HE staining.2.The expression of miR-222 in gastric tissues of two groups of mice was detected by qPCR.3.The number of ICCs in smooth muscle layer and submucosa in gastric tissue of mice in two groups was determined by immunohistochemistry.4.Western blot was used to measure the protein expression of stem cell factor(SCF)in gastric tissue of mice in two groups,and the results were compared with internal reference GAPDH.Results:1.No visible differences in gastric mucosa between groups were detected by HE staining.2.The expression of miR-222 in gastric tissues of two groups was significantly different.Compared with Hp-group mice,the expression of miR-222 in gastric tissue in Hp+ group mice was significantly up-regulated(P<0.05).3.After Hp infection,the number of ICCs in smooth muscle and submucosa of mouse stomach tissue decreased significantly.The number of ICCs in the intramuscular layer in the Hp+ group was significantly lower compared with that in the Hp-group(3.32±1.60 vs.5.5±2.17,respectively;P<0.05).the number of ICCs in the submucosal layer in the Hp+group was also significantly lower compared with that in the Hp-group(6.29±2.46 vs.14.00±5.18,respectively;P<0.05)4.The SCF/GAPDH ratio was 0.52±0.19 in the Hp+group and 0.69±0.19 in the HP-group.The expression level of SCF in the Hp+ group was significantly lower compared with that in the Hp-group(P<0.05).Summary:Hp reduces the number of ICCs in gastric tissue not only by upregulating miR-222,but also by decreasing the expression of SCF protein,thus cause DGE.Part 3.The mechanism of Hp causing DGE by affecting gastric smooth musclePurpose:The mechanism of HP causing DGE by affecting gastric smooth muscle.Materials and methods:1.Four gastric smooth muscle specimens were randomly selected from the Hp+group and the Hp-group respectively.Proteomics was used to screen out differentially expressed proteins.MicroRNA chips were used to screen out differentially expressed miRNA,and.The genes of proteins related to gastric emptying function were found as target genes among differentially expressed proteins,and the miRNA with negative regulatory relationship with the target genes were selected as target miRNAs by using target gene prediction software(Targetscan).2.qPCR was used to detect the expression level of target miRNAs and target gene mRNA in gastric smooth muscle tissue of two groups of mice.Western blot was used to detect the expression level of target gene’s protein expression in gastric smooth muscle of two groups of mice.3.Using Dual-Luciferase Reporter Assay System to verify that the target miRNAs has a direct regulatory relationship with the target genes.4.Western blot was used to detect the expression level of downstream factors of protein expressed by target gene in gastric tissues of two groups of mice.Results:1.Through proteomics and miRNA chip detection,target gene prediction software(Targetscan)was used to screen out proteins related to gastric emptying function,namely myosin light chain kinase(MLCK/MYLK)as the target gene,and miR-1894-3p,miR-24-3p,miR-365-3p,miR-7018-5p as the target miRNAs regulating MLCK.2.The expression of miR-1894-3p,miR-365-3p in gastric smooth muscle tissue of mice had no significant difference between Hp+group and Hp-group(p>0.05).The expression of miR-24-3p and miR-7018-5p were significantly different in gastric smooth muscle tissue of two groups.The expression of miR-24-3p and miR-7018-5p in gastric smooth muscle tissue of Hp+group mice was significantly up-regulated compared with Hp-group mice(P<0.05).The mRNA expression of MLCK was not significant different in gastric smooth muscle tissue of two groups(P>0.05).The protein expression of MLCK was significantly different in gastric smooth muscle tissue of the two groups mice.Compared with Hp-group mice,the expression of MLCK in gastric smooth muscle tissue of Hp+group mice was significantly down-regulated(P<0.05).3.Dual-Luciferase Reporter Assay showed that miR-7018-5p had no direct regulatory relationship with MLCK,but miR-24-3p had direct regulatory relationship with MLCK.4.The phosphorylation level of myosin light chain(MLC20)was significantly different in gastric tissues between Hp+group and Hp-group mice.Compared with Hp-group mice,the phosphorylation level of MLC20 in gastric tissue of Hp+group mice decreased significantly(P<0.05).Summary:HP infection upregulates miR-24-3p in mice gastric tissue,which leads to the down-regulation of MLCK protein expression,lowers MLC20 phosphorylation level and results in DGE.Part 4.The clinical case analysis of the effect of Hp infection on gastric emptying function after PDPurpose:1.Whether Hp infection is a risk factor for DGE after PD.2.Clinical case verification of the related mechanism of Hp infection affecting gastric emptying.Materials and methods:1.Clinical and pathological data of 134 patients with PD were collected and divided into non-DGE group and DGE group.Gastric immunohistochemistry was used to determine the infection of Hp in patients.Clinical and pathological features of patients both in non-DGE group and DGE group were analyzed.2.Logistic regression analysis was used to analyze the risk factors of DGE after PD.3.Paraffin samples of gastric tissues of 12 patients with positive and negative Hp were randomly selected,and the expression levels of miR-222 and miR-24-3p in gastric tissues of the two groups were detected by qPCR.Immunohistochemistry was used to determine the number of ICCs in smooth muscle layer and submucosa,and expression of MLCK proteinin gastric tissue samples of two groups of patients.Results:1.Among the 134 patients in this study,34(25%)had postoperative DGE.Comparing the clinical data of patients in the DGE group with the non-DGE group,it was found that there were no significant difference in baseline data such as age and gender between the two groups.However,the proportion of Hp infection and abdominal infection in patients with DGE was significantly high.2.The univariate analysis showed that Hp infection and abdominal infection were potential risk factors for DGE after PD.The multivariate analysis showed that Hp infection and abdominal infection were independent risk factors for DGE after PD.3.The expression of miR-222 and miR-24-3p in gastric tissues of Hp+and Hppatients was significantly different.Compared with Hp-group,the expression of miR-222 and miR-24-3p in gastric tissue of Hp+group was significantly up-regulated(P<0.05).Compared with Hp-group,the number of ICCs in gastric smooth muscle layer and submucosa in Hp+group decreased significantly.The number of ICCs in gastric smooth muscle layer of Hp+group and Hp-group was 33.3±14.5 vs 55.0±16.4(P<0.05).The number of gastric submucosa ICCs in Hp+group and Hp-group was 20.9 ± 10.0 vs 30.2±9.2(P<0.05).Compared with Hp-group,the expression of MLCK in gastric tissue of Hp+group decreased significantly.Immunohistochemical staining scores of MLCK in gastric tissues of Hp+group and Hp-group were 5.1±1.9 vs 8.9± 1.7 respectively(P<0.05).Summary:Hp infection is an independent risk factor for DGE after PD.The same as animal experiments,Hp infection upregulates miR-222 in human gastric tissue,which leads to the decrease of ICCs,and increases the risk of DGE after PD.In addition,Hp infection upregulates miR-24-3p in human gastric tissue,which leads to the decrease of MLCK,thus increases the risk of DGE after PD. |
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