| Gastric cancer is a malignant tumor originating from gastric mucosal epithelial cells,and most of the pathological histotype belongs to gastric adenocarcinoma.Globally,gastric cancer has a high incidence and mortality,because there are no obvious specific symptoms in the early stage,it is easy to be ignored,and most gastric cancer patients have progressed to the intermediate and advanced stages when diagnosed.Although the use of various methods has advanced the treatment of gastric cancer,recurrence is still common.Chronic stress is the body in a long-term state of instability,not only will produce depression,restlessness and anxiety,and other negative emotions,but also affect sleep quality,appetite,and gastrointestinal function,more extreme cases will cause damage to the body to relieve stress,escape stressors,in addition to chronic stress will also increase the risk of tumors,promote the malignant progression of tumors.Therefore,based on the fact that chronic stress can promote Epithelial-mesenchymal transition(EMT)in gastric cancer cells at the cellular level in the early stage,this experiment mainly focuses on the relationship between chronic stress and gastric cancer progression at the clinical and animal levels and explores the potential mechanism of its process.The effects of chronic stress on the body are mainly through the activation of the hypothalamic-pituitary-adrenal(HPA)axis and the sympathetic nervous system(SNS),resulting in the release of stress-related hormones,such as epinephrine,glucocorticoids,and norepinephrine,thereby affecting the body’s neuroendocrine system,activating the sustained inflammatory response in the body,and inhibiting the normal function of the immune system.Studies have found that chronic stress stimulates the body to release epinephrine,acting on adrenergic receptors on cell membranes to promote tumor malignant progression,the adrenergic receptors that play a major role are β2-AR.β2-AR is a member of the G protein-coupled receptor family,which plays a role in promoting the malignant progression of tumors in a variety of tumors,such as ovarian cancer,prostate cancer,liver cancer,etc.,and is related to the carcinogenicity,proliferation,immune regulation,invasion,angiogenesis,clinical prognosis,and drug resistance of malignant tumors.At the same time,in the constructed mouse model of anxiety stress,scholars found that anxiety stress is related to the expression of Plexin A1,a member of the transmembrane-mediated SEMA family receptor,which plays a role in axon guidance,invasion growth,and cell migration.At the same time,a large number of studies have found that Plexin A1 is highly expressed in a variety of tumor types and is associated with poor prognosis.EMT the biological process by which epithelial cells acquire mesenchymal cell properties through specific procedures plays an important role in embryonic development,tissue reconstruction,chronic inflammation,a variety of fibrotic diseases,and is related to tumor genesis,invasion,migration,and anti-tumor therapy,which is a key factor determining the malignant progression of tumors.Objective:In this study,clinical medical records,gastric cancer patient samples,and nude mouse gastric orthotopic transplantation tumor models were used as the research subjects,aiming to clarify that chronic stress promotes EMT in gastric cancer cells through the application of epinephrine onβ2-AR/Plexin A1 on cell membranes.Methods:Clinical Part:1.Collect tumor tissue and adjacent normal tissue greater than 5 cm away from cancer in 36 patients with gastric cancer;2.The chronic stress state of gastric cancer patients was evaluated by the Anxiety Self-rated Scale(SAS)and the Depression Self-Rated Scale(SDS),and the patients were divided into stress group and non-stress group;3.The expression of β2-AR and Plexin A1 in gastric cancer tissues and adjacent tissues in stress and non-stress groups was detected by immunohistochemical SP method,and correlation analysis was carried out.Animal Part:1.Construct a model of nude mouse gastric orthotopic transplantation tumor,and detect the success rate,survival rate and tumor homogeneity of nude mouse gastric orthotopic transplantation tumor construction method;2.Construct a chronic stress model of nude mice,according to the experimental method of chronic unpredictable stimulation(CUMS),after a cycle of about 3 w of continuous non-repetitive stimulation methods: fasting,dark,light,tail clamp,water deprivation,cold stimulation,litter moisture,etc.,construct a chronic stress model of nude mice,and detect whether the model is successfully constructed through Tail suspension test(TST)and Light and Dark Box(LDB)animal behavior experiments;3.36 nude mice were randomly divided into 6 groups: control group,chronic stress group(CUMS group),adrenergic agent group(6-OHDA group),chronic stress + adrenergic agent group(CUMS+6-OHDA group),β2-AR inhibitor group(ICI118,551 group),chronic stress + β2-AR inhibitor group(CUMS+ICI118,551 group);4.The expression levels of β2-AR,Plexin A1 and EMT-related markers in gastric cancer tissues of different groups of mice were detected by Western blot and immunohistochemical SP,and the difference was statistically significant with P<0.05.Results:Clinical Part:1.The 36 patients with gastric cancer were evaluated according to the Anxiety Self-rated Scale(SAS)and the Depression Self-Rating Scale(SDS),and found that 12 of them had chronic stress,accounting for about 33%;2.In the tissue samples of 36 gastric cancer patients collected by immunohistochemical staining(IHC)detection,the expression levels ofβ2-AR and Plexin A1 in adjacent tissues were significantly lower than those of gastric cancer tissues;In gastric cancer tissues,the expression levels ofβ2-AR and Plexin A1 in the chronic stress group were significantly higher than those in the non-stressed group,with P<0.05.3.By performing Spearman correlation analysis on β2-AR and IHC scores of Plexin A1 in tissue samples of 12 chronically stressed gastric cancer patients,it was found that β2-AR and Plexin A1 expression were positively correlated,P<0.05.Animal Part:1.Through the results of small animal in vivo imaging system and vivisection,it was found that the success rate of the method of constructing gastric orthotopic transplantation tumors in nude mice was 100%,the survival rate of nude mice was 100%,and the tumor size was uniform.2.The experimental results of the animal behavior of nude mice and the weight changes of mice in chronic stress group and non-stressed group were verified by hanging tail experiment(TST)and light and dark box(LDB),and the successful construction of chronic stress model in nude mice was verified.3.Select 6-OHDA(adrenergic agent)for chemical sympathectomy to block epinephrine,ICI118,551(a specific β2-AR inhibitor)to block β2-AR.Anatomical findings showed that the volume of gastric tumors in situ in nude mice in the CUMS group was significantly increased compared with the control group.Compared with the CUMS group,the volume of gastric orthoplasms in nude mice in the CUMS+6-OHDA group and CUMS+ICI118,551 group decreased significantly,P<0.05.4.Detect the expression of β2-AR and Plexin A1 in nude mouse tumor tissues.Western blot and IHC results showed that β2-AR and Plexin A1 expression in the CUMS group was significantly higher than that in the control group.β2-AR and Plexin A1 were significantly reduced in the CUMS+6-OHDA group and CUMS+ICI118,551 group compared with the CUMS group.β2-AR expression levels in the 6-OHDA group were downregulated compared with the control group;Compared with the control group,the expression level of Plexin A1 in the ICI118,551 group was down-regulated,with P<0.05;5.Detect the expression of EMT-related indexes in nude mouse tumor tissues.The Western blot and IHC results showed that the protein expression of E-cadherin(epithelial marker)in the CUMS group was significantly lower than that in the control group,while the expression of N-cadherin andα-SMA(interstitial marker)in the CUMS group was significantly higher than that in the control group,with a P<0.05.At the same time,it was found that compared with the CUMS group,the protein expression levels of E-cadherin in the CUMS+6-OHDA group and CUMS+ICI118,551 group were increased,and the protein expression levels of N-cadherin and α-SMA were significantly reduced,with P<0.05.Conclusions:1.β2-AR and Plexin A1 were highly expressed in chronic stressed gastric cancer tissues,and the expression of the two was positively correlated.2.The promoting effect of chronic stress on gastric cancer is related to the activation of the epinephrine-β2-AR/Plexin A1 pathway;3.Chronic stress promotes EMT in gastric cancer cells through the epinephrine-β2-AR/Plexin A1 pathway. |
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