Effect Of Non-steroidal Anti-inflammatory Drugs On Rat Small Intestine

【Objective】Non-steroidal anti-inflammatory drugs(NSAIDs) is a kind of anti-inflammatory drugs which do not contain the steroid structure.These drugs have effect of anti-inflammatory,anti-rheumatism,alleviating pain,pyretolysis and anticoagulated blood and so on.Clinically,NSAIDs are used for short-term is mainly used on alleviating pain,pyretolysis and perioperative analgesia.Long-term using NSAIDs is used to treat rheumatiod arthritis,osteoarthritis and in addition treat gouty arthritis,ankylosing spondylitis,vertebral arthritis and so on.It can be also used for the treatment of acute non-inflammatory diseases such as acute skeletal muscle disease,isthmus and back pain,joint pain,common cold,acute gout and so on. Nowadays NSAIDs is one of the medicine which is used the most in global.Every day about 30 million people use it around the world.In recent years aspirin is used for the prevention of cardiovascular and cerebrovascular diseases,that NSAIDs can reduce the incidence of colon cancer and senile dementia has been initially confirmed.So the using is on the rise.Although the clinical application of NSAIDs is widespread,it's adverse reaction is obvious.One of the most common,but also have the most clinical significance adverse reaction is the gastrointestinal mucosa injury.A study confirmed that in the United States of adverse drug reactions,21 percent for gastrointestinal reaction caused by NSAIDs.In the United Kingdom,25 percent of adverse drug reactions reported was related about NSAIDs.The gastrointestinal damage caused by NSAIDs is common for gastrointestinal bleeding,perforation and pyloric obstruction.It can also cause allergic reactions,liver and kidney damage and blood system,connective tissue of skin,respiratory complications.It is estimated that there is more than one billion prescription of NSAIDs every year in the world and nearly 30 million people taking NSAIDs per day,In the United States,about 6,000 people were killed attributed to taking NSAIDs each year.NSAIDs-related gastrointestinal side effects is a very broad concept,it contains not only the symptoms such as nausea,heartburn,indigestion but also peptic ulcer and the serious complications such as bleeding and perforation,gastric emptying disorder, and can even lead to death of some patients.Studies have shown that the dyspeptic incidence of patients taking NSAIDs is about 10%to 20%,gastric ulcer is about 12 %～30%,duodenal,ulcer is about 2%～19%;Each year,about 2 percent to 4 percent of the NSAIDs users will appear serious gastrointestinal complications. Serious gastrointestinal complications caused by NSAIDs mainly include gastrointestinal bleeding,perforation,gastric emptying disorder,in particular,bleeding and perforation.More importantly,because of the potent analgesic effect of NSAIDs, often"painless",the symptoms were not obvious.About 50%to 80%of patients with complications caused by NSAIDs have no symptom,and the first symptom is usually serious bleeding or perforation can directly lead to death.As these complications often have no warning symptoms,its clinical harm is max.Ever since a long time ago,people think highly of the damage of stomach caused by NSAIDs.And epidemiological investigations revealed that we should also pay attention to adverse reaction of lower digestive tract caused by NSAIDs.But nowadays there are very few reports about lower digestive tract mucosa damage especially intestina parva mucosa damage caused by NSAIDs at home and aboard.In the wake of the wide using of NSAIDs belongs to OTC,intestina parva mucosa damage caused by NSAIDs became excellence day by day.However,due to insidious symtoms,people do not have enough awareness about it.That it's short of effective diagnosis diplomacy leads to misdiagnosis,missed diagnosis and pathogenetic condition delay.This caused the incidence of intestinal damage will be further growth. The incidence of intestinal damage caused by this will be further growth.Therefore, we should strength investigation of clinical features of NSAIDs-related injury of the gastrointestinal mucosa,in particular the lower gastrointestinal mucosa injury research in order to achieve the best clinical effect,and at the same time,gastrointestinal reactions will be reduced to a minimum.This study was designed to observe impaired change of the small intestine mucosal barrier caused by NSAIDs and explore its possible mechanism.We emphasized to pay attention to intestinal tract damage caused by NSAIDs,and raise the consciousness of the protection of patients with intestinal mucosal barrier function to furtherly provide the scientific basis of all aspects of the research,prevention and treatment.【Material and methods】1.Laboratory animals and division32 12-week-old male Sprague-Dawley rats,body mass was 200g-220g,provided by animal experiment center of Zhejiang University of Traditional Chinese Medicine. They were divided into drug injured model group and control group.The two groups were reviewed and determined after administration 1d(24h) and 5d,and the time points were eight.Model group was given diclofenac sodium dual release capsule.2.Methods1)Preparation of intestinal damage in Rats:To dissolve diclofenac sodium(Daifen, Fujisawa German companies,the National Yao Zhunzi J20050064) into 0.9%Saline. Reference to the long-term of human oral dose(150mg/d),then convert into rats oral dose 15 mg/kg·d,lavaged them 2/d(1 ml/100g).The control group used the same dose of saline.2 groups of rats in administration were killed after 1d(24h) and 5d,then took the jejunum and the ileum and observed them.2)The score of anatomical lesion of stomach and small intestine:Rats absoluted diet for 24h,and were put to death by intraperitoneal injection of 1%pentobarbital sodium.Took the stomach,evaginated the mucosa in cavus gastralis,rinsed mucosa 3 times with saline,determined ulcer index with sliding caliper,scored in full refer to Guth.Scored intestine mucosa anatomical lesion refer to anabrosis and accretio,and the total was divided into two.3)Intestinal mucosa treatment and observation:Took jejunum 2 cm for pre-emergency distanced pyloricum 25 cm.Took jejunum to proximate 2 cm for pre-emergency distanced ileocecal junction 5 cm.Fixed the tissue in 10% Formaldehyde.Cut sheet routinely,HE stain,estimated degree of injury of intestinal mucosa.Each sample was counted 20 campus visualis.Quantitative analysed chorionic villi height,film thickness,mucosa section area and so on with Carl Zeiss Imaging Systems.4)Intestinal mucosa ultramicrostructure observation:Took end-piece ileum 1mm×1 mm of each group,fixed in 2.5%glutaraldehyde.After dehydration,replacement and other steps,observed intestinal mucosa with ultramicrostructure transmission electron microscope.5) EGF immunohistochemical detection and image analysis:Used immunohistochemistry(two-step).Automatic staining machine after stained, mounting,microobservation,positive staining took when the cytoplasm and/or membrane was brown.Each section shoule take 3-5 photos for analysis.Calculated the positive area,the optical density and the positive index with single-blind circumstances.3.Statistical treatmentThe data of mormal distribution was demonstrated as X±s,and was analysed using one-factor analysis of variance.The data of nonnormal distribution was demonstrated by median and full range using rank sum test.P＜0.05 showed that difference had statistical significance.【Results】1.change of histological anatomy1)General observation of the stomach,small bowel mucosa:The rats of control group were all alive after intragastric administration.One rat of the model group died at the 4^TM day.The mucous membrane of stomach of model groups was a little oedema,there was no difference between the score of model group and control group. We could see that mucous membrane of small intestine of the first day's model group was presented with erythema,anabrosis and ulcer.The ulcers were distributed along mesenterium.The mucous membrane of small intestine of the fifth day's model group was presented with bleeding,perforation and sinus tract formation,and the score of anatomical lesion was higher than which of control group(P＜0.05).2)Change of histological anatomy under light microscope:The membrane structure of the control group was complete.We could sometimes see chorioepithelium defected and a small quantity of inflammatory cell infiltrate.At the 1st day the rats of the model group showed intestinal villi engorgement,decurtation, thickening,and top part breakage or collapse.At the 5th day they showed mucosa anabrosis,necrosis,diastem augmentation,lamina propria cutaneous dropsy obviously with Inflammatory cell infiltration,cellula epithelialis apomorphosis,cellular necrosis, some villi integration,decurtation,and even cellular necrosis,defluvium,defect leading to exposure of the lamina propria.A great quantity neutrophi granulocytes were infiltrating in the mucosa and underlayer.The scroe of the 1st and 5th day was 3.5 and 5.The difference had statistical significance when prepared with control group(the score was 0)(P＜0.05 ).The height of jejunum's pile was 126.9±32.0μm in model group,and the ileum's was 118.6±22.9μm,it was lower than which of the control group(169.2±25.5μmå’Œ145.8±25.2μm),the difference was significant(P＜0.05).However there was no difference about thickness and section area of them,but thickness and section area showed decreasing tendency.We could also see apomorphosis and sphacelism of cellula epithelialis of the 5th group.Some pile ablated.Lamina propria exposed.The height of jejunum's pile(73.4±25.4μm) and the hight of ileum's(109.3±17.6μm) which were lower than which of the control group(169.2±25.5μmå’Œ145.8±25.2μm,P＜0.05).The thickness(123.8±51.6μm &165.7±37.4μm) became thinningz compared with the control group(123.8±51.6μmå’Œ165.7±37.4μm,P＜0.05).The section area(2.48±1.01 mm2&3.27±0.76 mm²) became small.(P＜0.05 vs control group:4.66±2.11 mm²å’Œ4.54±1.12 mm²).3)ultrastructural changes:microvillus of the mucous membrane in model group A was edematous and ranked disorderly.Cytochondriome swelled,endoplasmic reticulum expanded differently,cellular tight junction became widen partly.The microvilli of 5th day ablated more obviously,cell junction break and destroyed gravely.2.EGF expressionEGF was mainly expressed at cellula epithelialis of villi of small intestine and endochylema of capillary endothelium.At 1st day of making model,the masculine area of EFG of jejunum and ileum(3409.85±1514.32μm² & 3874.09±1313.03μm²),optical density(1203.13±569.90 & 1339.49±467.72) and masculine index (0.017±0.008 & 0.019±0.006) descended significantly compared with the control group(7495.11±4557.88μm² and 7703.04±4165.60μm²;2952.20±2047.92 and 2873.31±1749.19;0.041±0.028 and 0.040±0.024,P＜0.05).There was no significant deviation of the expression of EFG between jejunum and ileum.At 5th day of making model,the masculine area of EFG of jejunum and ileum(3193.45±1695.65μm² & 2919.19±1671.55μm²),optical density(1120.44±639.58 & 1034.45±615.47) and masculine index(0.016±0.009 & 0.014±0.008) descended significantly compared with the control group(6502.51±2364.60μm² & 7001.04±1894.89μm²; 2471.33±1037.67 & 2623.44±796.39;0.034±0.014 & 0.034±0.011,P＜0.05).But there was no statistically significance compared with the 1st group(P＞0.05).【Conclusions】1.The damage in the mucous membrane of small intestine induced by diclofenac was more common and significant than which in the mucous membrane of stomach.The primary mechanisms included detect mucosal toxicity,injury in mitochondria and the damage of the integrity of the intercellular junctions which increased epithelial permeability.2.Diclofenac of routine therapeutic dose could cause damage to function of the barricade of mucous membrane of small intestine.It presented with erythema,anabrosis,ulcer,perforation and may cause sinus tract formation.The ulcers were distributed along mesenterium.Diclofenac could cause damage with cellula epithelialis apomorphosis,cellular necrosis,some villi integration,decurtation, and even cellular necrosis,defluvium,defect leading to exposure of the lamina propria.The height of jejunum's pile and ileum's decreased significantly.The thickness became thinningz and the section area became small in jejuum and ileum.The microvillus of the mucous membrane was edematous and ranked disorderly because of diclofenac.Cytochondriome swelled,endoplasmic reticulum expanded differently,cellular tight junction became widen partly and destroyed gravely.There were proportional relation between administration duration of diclofenac and the lesion classifications.3.The masculine area of EGF of jejunum and ileum descended significantly compared with the control group caused by diclofenac.It suggested that the decrease of EGF could affect the repairing of the small intestine.It may be helpful by using protective agent of intestinal mucosa which promoting endogenous EGF excretion or similar EGF medicine to prevente and cure the damage induced by diclofenac.That were necessary for further research.