| Research background and purpose Crohn’s disease(Crohn’s Disease,CD)is a chronic inflammatory disease of the gastrointestinal tract,the main outcome is intestinal stenosis and intestinal obstruction.The digestive tract is an electrically excited tissue with main mechanical functions,which act as physiological stimuli and feedback signals to maintain normal physiological functions.Intestinal mucosal epithelial cell is a kind of mechanical sensor in the process of intestinal peristalsis,which converts mechanical signal stimulation into intracellular biochemical reaction.Piezo1 is a mechanically sensitive non-selective cation channel with clear biophysical and pharmacological properties,can be directly activated by mechanical forces,and is considered to be the main participant in cell mechanical sensitivity.When intestinal stenosis occurs in patients with CD,it is not clear whether intestinal hypertension affects Piezo1 in intestinal mucosal epithelial cells and whether Piezo1 is involved in the mechanism of inflammation and fibrosis in CD.Therefore,it is of great significance to study the relationship between Piezo1 and intestinal inflammation and fibrosis in patients with CD and its possible mechanism for the prevention and treatment of CD patients.Method1.The clinical data of CD patients with surgical records in the first affiliated Hospital of Anhui Medical University in the past 10 years were collected retrospectively.according to the discharged patients with CD and intestinal surgery records,a total of 154 cases were collected,excluding unclear diagnosis,incomplete medical records,pathology that did not meet the requirements of CD,postoperative follow-up found that did not meet the natural course of CD and loss of follow-up,and finally met the diagnostic criteria of CD in 57 cases.At the same time,the normal intestinal mucosal epithelium of 10 people who underwent enteroscopy in our hospital were collected as the control group.2.According to the clinical data of CD patients,the pathological numbers of surgical specimens were sorted out,and the wax blocks of small intestine samples were obtained from the pathology department of our hospital.The paraffin section machine(specification: RM2255)was used for tissue sections with thickness of 3um,and HE staining and Msson staining were performed.The other two sections of CD patients were stained with immunofluorescence tissue staining(IF)and immunohistochemical staining(IHC).At the same time,the small intestinal mucosal tissues of 10 normal subjects were collected and made into frozen tissue specimens with OCT.The specimens were sliced with a frozen slicer(specification: CM3050S)with a thickness of 6um.they were stained with IF staining,IHC staining,HE staining and Masson staining,respectively.All stained sections of CD group and control group were observed under laser scanning confocal microscope(LSCM,specification: LSM800)to observe the expression and distribution of Piezo1 in intestinal mucosal epithelial cells,inflammation and fiber.The parameters were adjusted uniformly,and fluorescence and bright field pictures were obtained.CZI and TIFF were retained for all images.3.The IF images were semi-quantitatively analyzed by Image J.The HE-stained images were scored by CD mucosal biopsy histological score system(GHAS)and fibrosis score scale,and the inflammation and fibrosis scores were scored by experienced doctors in the pathology department.Result1.57 cases of CD met the diagnostic criteria,including 29 cases of ileal type(50.9%),26 cases of ileal type(45.6%),2 cases of colonic type(3.5%),32 cases of age over 17 years old and less than 40 years old(56.1%),25 cases of older than 40 years old(43.9%),5 cases of smokers(8.8%)and 52 cases of non-smokers(91.2%).12 cases(21.1%)of non-penetrating non-stenotic type,31 cases(54.3%)of stenotic type and 14 cases(24.6%)of penetrating type;the severity of disease activity was assessed by CDAI,mild in 0 cases,moderate in 47 cases(82.5%),severe in 10 cases(17.5%),and 10 cases of intestinal mucosal epithelium in normal control group.2.The pathological sections of CD group and normal control group were stained with IF and IHC,and photographed by LSCM.IHC staining showed the expression of Piezo1,in intestinal mucosal epithelial cells.Semi-quantitative analysis of the fluorescence images taken by Image J software showed that the relative expression level of Piezo1 in intestinal mucosal epithelial cells in CD group was significantly higher than that in normal control group,and the difference was statistically significant(CD group:12.94 ± 4.58 、 Normal control group:8.54 ± 1.10,P=0.012).It is suggested that the increased expression of Piezo1 in intestinal mucosal epithelial cells of patients with CD may be involved in the pathophysiological process of CD patients.3.In CD group,there was no significant difference in the relative expression of Piezo1 in intestinal mucosal epithelial cells in terms of lesion location and disease severity;The expression of Piezo1 in intestinal mucosal epithelial cells was significantly different in different biological behaviors,P < 0.01 was statistically significant,The relative expression of Piezo1 in intestinal mucosal epithelial cells of stenotic and penetrating CD patients was higher than that of non-penetrating and non-stenotic CD patients(P=0.003,P=0.006),There was no significant difference in the relative expression of Piezo1 in intestinal mucosal epithelial cells between stenotic type and penetrating type CD patients(P=0.07).4.The pathological sections of CD group were stained with HE and Masson,read by senior pathologists,and scored by inflammation and fibrosis.finally,it was concluded that the severity of intestinal inflammation and fibrosis was correlated with the relative expression of Piezo1 in intestinal mucosal epithelium.The more serious the intestinal inflammation and fibrosis,the higher the relative expression of Piezo1 in intestinal mucosal epithelium.Conclusion1.The relative expression of Piezo1 in intestinal mucosal epithelial cells of patients with CD was higher than that of normal controls,suggesting that the increased expression of Piezo1 in intestinal mucosal epithelial cells of patients with CD may be involved in the pathophysiological process of CD patients.2.The relative expression of Piezo1 in intestinal mucosal epithelial cells of patients with CD had no statistical significance in the location and severity of the lesions,but had significant differences in different biological behaviors(non-penetration non-stenosis,stenosis,penetration)3.The expression of Piezo1 in intestinal mucosal epithelial cells in patients with CD was correlated with the pathological characteristics of small intestine.According to the correlation analysis of the relative expression of intestinal mucosal epithelial cells and the scores of inflammation and fibrosis,the more serious the intestinal inflammation and fibrosis was,the higher the relative expression of Piezo1 in intestinal mucosal epithelium was.It is speculated that the expression of Piezo1 in intestinal mucosal epithelial cells may be clinically related to the process of intestinal wall fibrosis in CD,but whether it plays an important role in the process of intestinal wall fibrosis in CD and the specific mechanism need to be further studied. |
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