Oxidative Injury Of Free Radical On The Rat Iecs And Intervention By α-LIPOIC Acid

Objective: Our study improved the method of isolating and purifying the primary culture of small intersitinal epithelial (IEC) in rats, and established two oxidative damage models of IEC by xanthine oxidase (XO) /xanthine (X) and hydrogen peroxid( H2O2) and investigated the protective effects and mechanisms of LA on IEC following the oxidative stress.Methods: Experiment1: Improved the method of isolating and purifying the primary culture of IEC in rats: Enzymatic digestion was used in this study to separate epithelial cells from neonatal rat intestine. The result showed that the intact units(villi and cryp units) and few single cells could be observed by collaborative digestion technique of 300 U/mL collaganase and 100 mg/ml to separate epithelial cells from neonatal rat intestine. IECs could formcell colonies in 3~4days, and fusion in 7~8days which ought to be passaged. IECs with high capacity of proliferation could be obtained by passage with 0.05% Tyspin-EDTA. 80% IECs, which were identified by immunocytochemical characterization, could be got by purifying like scraping and twice digestion and adherence, providing an ideal model of intestianl nutriment effect on IEC.Experiment2: Establishment of two oxidative damage models of IEC by xanthine oxidase (XO) /xanthine (X) and hydrogen peroxid( H2O2) Different concentration of XO (5,10,50,100,200U/L) /10μmol/L X, and H2O2 (0.1μmol/L, 1μmol/L, 2.5μmol/L, 10μmol/L, 30μmol/L, 100μmol/L, 1mmol/L),incubated with IEC for 3, 12, 24, 48h and the cell vitality measured by MTT, from which we could find the optimal damage concentration and exposure time by XO/X and H2O2. Experiment3: Protective effects and intervention mechanisms of LA on IEC following oxidative stress. XO/X Group:Blank Contrast: IECs were normally incubated for 24h; XO/X Injurty Group: Incubated with 50U/L XO, 10μmol/L X for 24 h; XO/X+LA Group:Incubated with 3 different concentration of LA(0.lμg/ml,1μg/ml和10μg/ml)for 3 h,then add 50U/L XO and incubated for 24h. H2O2 Group:Blank Contrast: IECs were normally incubated for 24h; H2O2 Injurty Group: Incubated with 100μmol/L H2O2 for 24 h; H2O2+LA Group: Incubated with 3 different concentration of LA(0.lμg/ml, 1μg/ml, 10μg/ml)for 3 h, then add 100μmol/L H2O2 and incubated for 24h.We measured the protective effects on cell vitality, antioxidative indexs (SOD, GSH-pX, MDA), functional (lipase, amylase), intestinal damage index (DAO, LDH). Results:1. Optimal damage concentration by XO/X and H2O2 were 50U/L XO/10μmol/L X,100μmol/ml H2O2 and optimal damage exposure time of both group was 24h.2. Following oxidative stress by XO/X (50U/L XO/10μmol/L X).and H2O2 (100μmol/ ml), the cell vitality, and the activity of SOD, GSH-pX, lipase, amylase, DAO of IECs were significantly decrease, compared with Blank Contrastgroup (P<0.05), the MDA and LDH in culture was increased significantly (P<0.05). We also found the oxdative injury by50U/L XO was relatively more severe than 100μmol/ml H2O2.3. Following oxidative stress by XO/X (50U/L XO/10μmol/L X).and H2O2 (100μmol/ ml), the cell varity of IEC, and the activity of SOD, GSH-pX, lipase (P>0.05), amylase, DAO of IECs in LA Intervention group were significantly increased (P<0.05). The the MDA and LDH in culture was decreased significantly (P<0.05).