| Hemorrhoid is a worldwide common disease among adult men and women with an incidence of 46.3% in China. Prolepses and bleeding are the main symptoms of hemorrhoids. Severe sufferers are complicated with anemia, infarction of incarcerated hemorrhoids, which affect on the patient's quality of life and psycho-psychiatric disorders.The history of hemorrhoids can be traced back to 476 BC. The earlest known description of hemorrhoids can be found in the Shan Hai Jing. But the preliminary investigation on hemorrhoid formation mechanism was in the last hundred years. Pathogenetic studies in hemorrhoid were derived from three main theories, which are varicose vein theory, vascular proliferation theory and anal cushion sloughing theory. The essence of the vascular lesions is an important factor of hemorrhoid formation, but the mechanism is still unclear. It is still unable to explain the hemorrhoidal bleeding and the high rate of postoperative recurrence.Predispositions of internal hemorrhoids contain sedentary, stay-up late, constipation, pregnancy, spicy food, and so on. With aging, the cumulative factors and chronic injuries lead to developing hemorrhoids with a high incidence of human, and the hemorrhoidal lesion is progressive. The pathogenesis is unclear, and effective therapies are still limited on hemorrhoidal prolapse, hemorrhage. Both conservative treatment and surgery can not eradicate hemorrhoids. There is a high recurrence rate on post-operative hemorrhoids. Therefore, if the pathogenesis of internal hemorrhoids can be clarified, the hemorrhoidal patients would revieve better medical intervention, and the complications would be decreased in anemia. Patients' life quality would be better.Several studies have shown that the chronic damages, stay-up late, sedentary, and aging are related to disturbance of anal microcirculation, tissue ischemia and hypoxia. Hypoxia inducible factor (HIF-1 a) combined with hypoxia response binding-elements can mediate transcriptional activation of vascular endothelial growth factor (VEGF), with the production of biological materials such as matrix metalloproteinase (MMPs), transforming growth factor ? (TGF-?). The stimulating molecules promote proliferation of endothelial cells, hyperplasia of vascular smooth muscle, fibrosis of anal tube wall, injuries of anal mucosa and neo-angiogenesis. The hemorrhoids would be resulted from these causative factors, even with the progression and repetitive bleeding.Our previous studies have revealed that chronic progressive hypoxia influenced a boost in various bioactive substances (HIF, VEGF, MMPS, TGF-P) in hemorrhoidal mucosa. Theses stimulation factors influence each other, and to induce angiogenesis, vascular smooth muscle hyperplasia, vascular muscle fibrosis, hemorrhoids mucosa vascular dilation, and eventually lead to generating Dieulafoy's like vascular malformation. Based on the above-referred reasons bring about hemorrhage, prolapse, and progression of the hemorrhoids. In hypoxic environment, HIF-la through multiple downstream target gene and hypoxia bind to and modulate the transcriptional molecules play an important role in the occurrence and development of internal hemorrhoids. This study further applied pathological examination qualitatively and quantitatively to observe the detailed changes in hemorrhoidal tissues, combined with immunohistochemical and molecular biological techniques, electron microscope was used to observe the ultrastructural changes of mucosal injury and vascular anomalies, clear reconstruction neovascularization hemorrhoids dynamic evolution process and the mechanism of bleeding. This thesis will elucidate the pathogenesis of hemorrhoids bleeding from new perspectives, and provides theoretical basis for the prevention and treatment of hemorrhoids.Methods1. Collecting and analyzing the clinical stages of hemorrhoids specimens:After detailed inquiry about the history and records of patients (age, gender, history, history of bleeding, hemoglobin and so on) by excluding tumors, Crohn's disease, the patients were divided into four groups, normal and group ?, ?,? stage hemorrhoids, according to the internal staging system (normal anal cushion tissue; no history of hemorrhoids).2. Obtaining the different clinical staging of hemorrhoids specimens: ?. After hemorrhoidectomy, specimens were immediately trimmed 11 0.2 cm size block, fixed in 10% Formaldehyde Solution, gradient ethanol dehydration, paraffin embedding, slicing, patch and baking sheet (slice using serial sections and the whole section the way).(1). HE Staining:Dewaxing ? Hydration ?hematoxylin semen dyeing ? Hydrochloric acid ethanol acidification ? Back to blue ? Eosin staining ? Dehydrated in graded ethanol ? Xylene transparent ? Mount.(2). MASSON Staining:Dewaxing ? Hydration ? Weiger's iron hematoxylin staining ?Alcoholic hydrochloric differentiation ? Ponceau fuchsin staining ? Phosphomolybdic acid ? aniline blue liquid complex ? Glacial acetic acid ? Dehydrated in graded ethanol ? Xylene transparent ? Mount.(3). Immunohistochemical Staining:Dewaxing ? Hydration ? Citrate repair ? H2O2 oxidation?Anti room temperature incubation ? Two anti room temperature incubation ? DAB coloration ? Hematoxylin staining ? Ethanol hydrochloride acidification ? Water back to blue ? gradient dehydration ? Xylene ? Mount.?. Electron microscope samples making:After the operation, the specimens immediately were divided into 0.5-1mm3 by shear. After giving numbers to the specimens were fixed with 2.5% glutaraldehyde solution 2h, O.lmol/L phosphate buffer 20min 3, osmic acid fixation of 1.5h, distilled water 5min 3. Gradient dehydration, saturated embedding, polymenrization, ultrathin sectioning, and the last step is staining.3. Observing and analyzing the formation mucosal injury and vascular remodeling of internal hemorrhoids:? Observe under the light microscope:(1). Observe the mucosal lining epithelium degree of destruction, erosion and ulcer, gland number, morphology, arrangement, the degree of inflammatory cell clusters in different groups.(2). Observe the specimens with different packet mucosa blood vessel, lumen expansion, the number of thick wall vessels and Dieulafoy's like blood vessels.(3). Observe the specimen of thick wall vessels and Dieulafoy like vascular wall fibrosis in different groups.? Observe under the electron microscope:(1). Observe lining epithelium, the destruction of the capillary endothelial cells of the arrangement, nuclear and chromatin shape of internal hemorrhoids mucosa in different groups.(2). Observe mucosal vascular endothelial surface microvilli, pinocytotic vesicles, the basement membrane is complete, nuclear changes, endothelial cell gap, mitochondrial vacuolization or pyknosis, rod bodies, a circular particle density of cytoplasm on the samples from different groups.4. Signal integration and damage of vascular remodeling formation factor expression:The expression levels of HIF-1 a, VEGF mucosal layer, CD34, CD105, MMP-9, TGF-?, Actin were observed under the light microscope in different groups (all stained sections were observed by two physicians, each patient was randomly were observed in 5 of 20 field).?.The HIF-1 ??MMP-9 judgment standard:Calculate the percentage of positive cells. According to the group of immune Xiaolong Ji provides positive judgement standard:immunohistochemical positive products were brownish yellow or brown finely granular, cells positive degree:<5%(-),5%-20%(+),20%-50%(++), >51%(+++).?.The VEGF, CD34, CD 105 and microvessel density (MVD) determination: Reference Weidner standards and improved the microvessel quantity, microvessel counts the number of 5 visual average as MVD.?.The judgment standard of TGF-?, Actin:The expression of vascular wall as brown as the research object, negative (-), tube wall sparse pale yellow to (+), tube wall is slightly dense brown as (++), tube wall is brown as (+++) density.[Refer in Xiaolong Ji, Zuolin Shi. Diagnosis of immunohistochemistry[M]. Beijing: Military Medical Science Press,1997:25; Weidner N, Semphe J P, Welch W R, et al. Tumor angiogenesis and metastasis correlation in invasive breast carcinoma[J] N Eng J Med,1991,324(1):1-8.]5. Probe the pathological mechanism of hemorrhage of internal hemorrhoids:Comparative observation of ?,?, ? hemorrhoids mucosa injury and hypoxia, microvessel density, vascular smooth muscle proliferation and muscle fibrosis degree, and the number of Dieulafoy's like blood vessels and hemorrhage of internal hemorrhoids and staging of relationship.6. Statistical method:Using statistical analysis using SPSS 13 statistical software package for data analysis and processing, Using multi factor screening analysis, linear regression analysis, chi square test, analysis of variance and other statistical methods. p values< 0.01 were considered statistically significant.RESULTS1.281 cases (male 147 cases, female 134 cases, age 14?78 years old) and clinical stage (? in 91 cases,95 cases of stage ?,91 cases in stage ?,4 cases in stage ?) of internal hemorrhoids tissue samples were divided into three groups (?, ? stage were divided into A, B group, ? and stage IV were divided into C group).2. On the age, gender, history of patients in each group, hemoglobin, defecation time, life habit (stay-up late, sedentariness), dietary habits (like spicy food) analysis and staging for multi-factor screening, three groups of patients with age, sex, history, hemoglobin showed no statistical significance (P> 0.01). Defecation time, living habits of patients (late at night, long wait for), dietary habits (like spicy food) the difference was statistically significant (P< 0.01).3. Observing under electron microscope, we find that the normal and stage I intermediate hemorrhoids anal cushion tissue fibers arranged closely, the connection between the vascular endothelial cell tight, rich mitochondria villi, normal, endothelial cells are arranged on the outer (see photo1-2a,b). Stage II and III internal hemorrhoids tissues of interstitial fiber loose arrangement, endothelial cell proliferation and loose connections, villi reduced, mitochondrial vacuolization, and mitochondrial pyknosis, inner cristae of mitochondria is less, pale stained (see photo1-2c,d).4. Stage II and III hemorrhoids compared with normal mucosa layer, the expression of HIF-1 a increased, covering epithelium damage, increased angiogenesis, and the emergence of the pipe wall focal thickening (tube wall smooth muscle increased and arranged in disorder or decrease of smooth muscle and fibrous tissue replacement), lumen dilatation of the vessel, and the invasion was isolated and mucosal layer Dieulafoy like vascular malformationCONCLUSIONS1. The patient's age, gender, disease history and peripheral blood hemoglobin have no correlation with the hemorrhoids. The life styles, defecation time, life styles (stay-up later, sedentariness), dietary habits (like spicy food) have some correlation with the occurrence of hemorrhoids.2. The disturbance of anal microcirculation may come from unhealthy living habits as stay-up late, over time of defecation, sedentary state, hobbies of spicy food and other adverse factors, which subsequently bring about ischemia and hypoxia of the hemorrhoidal tissue to produce hypoxia inducible factor (HIF-1 a) of harm in the mitochondria of endothelial cells.3. HIF-1 a with hypoxia response element binding mediates vascular endothelial growth factor (VEGF) transcriptional activation, produces various bioactive substances such as matrix metalloproteinase (MMPs) and transforming growth factor-? (TGF-?). The stimulatory factors promote the proliferation of vascular endothelial cells, hyperplasia of smooth muscle, fibrosis of vascular wall. The increased vessels in the mucosa along with focal thickening of the vessels may result from mucosa injuries and vascular reconstruction in the hemorrhoids. Dieulafoy's like malformation of the hemorrhoidal vessels appeared to break down the mucosa muscularis and give rise to repeated bleeding and exacerbation of the hemorrhoids.New findings of this study(1) The published studies cannot well explain bleeding and prolapse of hemorrhoids. This study showed the correlation of the morphological and molecular changes between different clinical stages of internal hemorrhoids, and it also present the data of mechanistic evidences in the progression of hemorrhoids.(2) In this study, using multi level (tissue, cell, molecular) and methods (molecular biology, electron microscope) method, dynamic simulation analysis on the correlation and the progress of mucosal injury and capillary remodeling and internal hemorrhoid hemorrhage.(3) This project provides novel evidences for further understanding the hematochezia of internal hemorrhoids. Mucosal angiogenesis and vascular abnormalities of the hemorrhoids might result from the disturbance of microcirculation due to ischemia and hypoxia in association with prolapse and progression of hemorrhoids. The findings of this study may give a cue to develop new medical intervention of internal hemorrhoids. |
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