Expression Chang Of CAT And SOD In Small Intestine Of Hepatic Ischemia Reperfusion Injury Rats

Liver not only is the main organ of the secretion,detoxification and excretion,but also involved in the metabolism of sugar,fat and protein.Therefore,liver has a great demand for energy,which makes it highly dependent on the oxygen supply and very sensitive to ischemia or hypoxia.HIRI?hepatic ischemia reperfusion injury?is a key and common complication in clinical practice.Temporarily blocking of liver blood supply can lead to ischemic injury and rapidly damage tissue with active metabolism.Rerecovery of blood supply after ischemia will activate a series of events,further aggravate the existing damage,this effect is reperfusion injury.Hepatic ischemia reperfusion injury is one of the main reasons of liver dyfunction and liver failure after liver transplantation.HIRI not only increases the morbidity and mortality after surgery,but also lead to recovery delay and poor recovery of patient.A variety of factors have been shown to be involved in the development of HIRI.The major pathological events leading to this kind of injury was the production of a large amount of ROS?the oxygen species?which cause the disturbance of microcirculation.So,oxidative stress is believed to be an important factor which lead to HIRI.There is a balance between the antioxidant system and the reactive oxygen species in the normal body.When this balance is broken,the generation of reactive oxygen species is more than the scavenging ability of the antioxidant system,which leads to the accumulation of a large number of reactive oxygen species in vivo and oxidative stress.Oxidative stress is not only the cause of many diseases,but also aggravating factors.There are many kinds of antioxidation system in the body.Superoxide dismutase?SOD?and catalase?CAT?are the key endogenous antioxidant enzymes of this defense system.They protect the body against oxidative stress by scavenging ROS produced in metabolic reactions.Intestinal ischemia reperfusion injury is a common clinical problem during the development of small bowel transplantation,circulatory shock and strangulated intestinal obstruction and so on.Researches showed that Intestinal ischemia reperfusion injury can cause severe damage to structure and function of liver.Conversely,When hepatic ischemia reperfusion injury happened,Whether the structure and function of small intestine was severely damaged? Whether oxidative stress is an important factor for this damage? How to change of the expression of SOD and CAT,whether SOD and CAT play antioxidation role ? which were not reported.We established HIRI model by clipping hepatic blood vessel to hepatic left,middle lobes and bile duct pedicle with vascular clamp for 30 mins,then restore blood supply.After 6 hours of reperfusion,the H₂O₂ content and MDA content in serum,the H₂O₂ content and MDA content in small intestine,mRNA and protein expression of SOD and CAT in small intestine were detected.To explore the effect of HIRI on the morphology and structure of small intestine and oxidative stress.As well as CAT and SOD play antioxidant role during the process,to provide a new approach for the prevention and treatment of small intestinal injury induced by HIRI.Objective: To observe the morphology changes of small intestinal,oxidative stress state of small intestine and whether small intestinal was subjected to oxidative damage,mRNA and protein expression change of SOD and CAT of HIRI rats.To explore the peroxide damage mechanism of small intestinal injury induced by HIRI,CAT and SOD play antioxidant role during the process,to provide reference data for the prevention and treatment of small intestinal injury induced by HIRI.Methods: 1 The preparation of HIRI models,group and detecting sample12 male Wistar rats weighing 200+10g were randomly divided into control group?Con?and model group?HIRI?.HIRI model was prepared according to the method of Kohli et al.Anesthetized rats with 6% chloral hydrate,first isolate bile duct and hepatic vessels,then established HIRI model by clipping hepatic blood vessel to hepatic left,middle lobes and bile duct pedicle with vascular clamp for 30 mins,then restore blood supply.The hepatic vessels and bile ducts of control group were only isolated,but not clipped.The blood was collected and isolated serum after 6 hours of restore liver blood supply to determine alanine aminotransferase?ALT?activity,hydrogen peroxide?H₂O₂?level and malondialdehyde?MDA?level.The liver and small intestine were collected.one part was fixed with 4% paraformaldehyde to observe morphological changes of liver and small intestine by HE staining.The rest part of small intestine were placed in liquid nitrogen to determe H₂O₂ content,MDA content,mRNA and protein expression change of SOD and CAT.2 The detection index and determination method 2.1 The determination of serum ALT,H₂O₂ content and MDA contentThe collected blood stood for 30 minutes at room temperature was centrifuged for 10 mins?3000rpm?to isolate serum and assay ALT activity,H₂O₂ level and MDA level.The ALT activity was detected by automatic biochemical analyzer.The H₂O₂ content in serum was measured by Molybdate colorimetric method.The MDA content in serum was measured by TBA method.2.2 Observation of morphology and structure of liver and small intestine by HE stainingHE staining was used to observe the morphological changes of liver and small intestine.The liver and small intestine are processed according to the following procedures: fixed,gradient ethanol dehydration,xylene transparent,paraffin embedding,slicing,drying,Hematoxylin eosin staining.The morphology and structure of liver and small intestine were observed by optical microscope.2.3 Preparation of small intestine homogenate and determination of MDA contentThe small intestine tissue is removed from the-80? refrigerator,add 4? homogenate buffer according to 10mg/100?l.Homogenate buffer contains 50mmol/L potassium phosphate buffer,Benzamidine hydrochloride1mmol/L,PH7.4,1mmol/L PMSF,0.5mol/L sodium chloride,0.1% Tween-20,1mmol/L EDTANa3,5mmol/L?-mercaptoethanol.Small intestine tissue was homogenated for 2 minutes in ice bath,then centrifuged for 20 mins at 4? 4000 rpm.The supernatant is the 10% small intestine tissue homogenate.The MDA content in small intestinal tissue homogenate were detected by Nanjing MDA content assay kit.2.4 Preparation of small intestine homogenate and determination of H₂O₂ contentThe small intestine tissue is removed from the-80? refrigerator,add 4? homogenate buffer according to 10mg/100?l.Homogenate buffer contains 50mmol/L potassium phosphate buffer,Benzamidine hydrochloride1mmol/L,PH7.4,1mmol/L PMSF,0.5mol/L sodium chloride,0.1% Tween-20,1mmol/L EDTANa3,5mmol/L?-mercaptoethanol.Small intestine tissue was homogenated for 2 minutes in ice bath,then centrifuged for 20 mins at 4? 4000 rpm.The supernatant is the 10% small intestine tissue homogenate.Molybdate colorimetric method was used to detected the content of hydrogen peroxide in intestinal tissue homogenate,and was expressed with hydrogen peroxide content in each gram of sample protein?pro mmol/g?.2.5 Detection of CAT and SOD gene in small intestine of ratTotal RNA were extracted from small intestine by TRIzol reagent.3?g of total RNA was reverse transcribed into amplified template cDNA for RT-PCR,GAPDH as an internal reference.The relative mRNA expression levels of CAT and SOD were expressed by the ratio of gray value of target gene CAT and SOD amplified bands to GAPDH amplified bands.2.6 Detection of CAT and SOD protein in rat small intestineWestern Blot method was used to detected the CAT and SOD protein levels.small intestine tissue was homogenized to prepare homogenate,then centrifuged and collected supernatant.Total protein was detected by modified Lowry method.The electrophoresis sample was 62 ug.After transfering film and sealing treatment,The anti-rabbit CAT and SOD antibody was added to the PVDF membrane.The PVDF film was overnighting at room temperature.After washing the film the next day,anti-rabbit IgG marked with Fluorescence was added into PVDF film.Dual color infrared imaging system was used to observe strip and analyze gray value.Results:1 Morphological structure changes of liver and small intestineIn control group,the liver cells were cord-like arranged neatly and were distributed around central vein.The average size of hepatic blood sinus was nomal and no dilated congestion.liver cells of HIRI group showed serious compression and congestion.The hepatic blood sinus showed obvious congestion and expansion.Vacuoles could be seen in cytoplasm of liver cells.The colour of HE became shallow.One part of liver cells appeared edema and volume was increased.Compared with control group,the morphology of small intestine of HIRI group was no significant changes under light microscope.2 The ALT level in serumThe ALT level in serum of HIRI group?87.43±9.06 U/L?was significantly higher than that of control group?20.03±5.23U/L?,P<0.01.3 The H₂O₂ content in serumThe H₂O₂ content in serum of HIRI group?23.28±4.19?mol/L?was significantly higher than that of control group?13.52±2.19?mol/L?,P<0.014 The MDA content in serumThe MDA content in serum of HIRI group?17.54±1.96 umol/L?was significantly higher than that of control group?12.69±2.29 umol/L?,P<0.015 Content of MDA in small intestine homogenateThe content of MDA in small intestine homogenate of HIRI group?23.97±3.42 mmol/g?was significantly higher than that of control group?15.94±2.24mmol/g?,P<0.01.6 Content of H₂O₂ in small intestine homogenateThe content of H₂O₂ in small intestine homogenate of HIRI group?36.69±5.98 mmol/g?was significantly higher than that of control group?25.98±4.08 mmol/g?,P<0.01.7 The expression level of CAT and SOD mRNA in small intestineThe expression level of CAT and SOD mRNA in small intestine were determined by RT-PCR.The expression level of CAT?0.55±0.09?and SOD?0.85±0.13?mRNA of HIRI group was significantly higher than CAT?0.38±0.07?and SOD?0.63±0.11?mRNA of control group,P<0.01,which indicated the expression of CAT and SOD gene in small intestine of the HIRI group was increased.8 The protein level of CAT and SOD in small intestineWestern Blot method was used to detected protein level of CAT and SOD.The protein level of CAT?0.59±0.08?and SOD?0.91±0.12?of HIRI group was significantly higher than CAT?0.37±0.06?and SOD?0.67±0.13?of control group,P<0.01,which indicated the protein expression of CAT and SOD in HIRI group were significantly increased.Conclusion:1 The HIRI did not lead to the significant changes of the morphology and structure of small intestine,but the small intestine was in a high oxidative stress state and suffered from peroxide damage.2 The gene and protein expression level of CAT and SOD in the small intestine of the HIRI group were increased,The results showed that CAT and SOD may play antioxidative stress role when small intestine was in highly oxidative stress state.