The Phenomenon Of Insulin Resistance Of Receptors And Post Receptors In L02Cells Induced By Peroxidative Damage

Background:Insulin resistance is the common mechanism and the risk factor of the chronic diseases such as type2diabetes, coronary heart disease and high blood pressure. Previous studies have found that the body showed insulin resistance to a certain degree under traumatic stress condition, directly affecting the prognosis of the diseases. In recent years, the research between insulin resistance and ischemia-reperfusion gradually became one of the new and hot spots. The studies showed that there was high blood sugar phenomenon after hepatic ischemia reperfusion[1-2]; however, the mechanism was not well revealed and discussed in different opinions. Zanaro NL[3] thought that the secretion of the hormone such as endogenous catecholamine, cortisol and glucagon resulted in the high blood sugar, which were induced by the formation of inflammatory cytokines （such as TNF-a） in the process of hepatic ischemia reperfusion; Choi CS[4] thout that it was caused by the large dose of steroid hormones used for restoring blood flow; Nowsk G[5] thout that it was due to the decomposition and concentrated release of glycogen stored in liver. The insulin resistance （IR） can be divided into three levels of before receptor, receptor and after receptor. The IR level before receptor is related to the release of all kinds of hormone resistance to insulin in vivo. The other two levels are mainly related to the signal system of IR-IRSs--PI3k-PKB/AKT. Studies displayed that the decreased expression of any target protein molecules of this signal system can cause insulin resistance. The expression of insulin receptor anchoring on the cell membrane will be affected by the destruction of cell membrane fluidity and the structure.therefore,to detect the residual glucose content in culture medium, the expression of Annexin V, the changes of cell proliferation inhibition rate and other indicators, to a certain extent, may reflects the question whether there were insulin resistance of receptors and post receptors. Insulin receptor is constituted of two subunits of alpha and beta respectively, in which beta subunit is the function unit of cell membrane and intracellular effect,we can observe the insunlin resistance of receptors level by test the expression of InsR β. As the key molecule in the signaling pathway, the phosphorylation of AKT can activate the glucose transporter and further activate the transportation of glucose from the outside of membrane to the inside of membrane, we can observe the insunlin resistance of post receptors level by test the expression of P-AKT. The insulin resistance phenomenon of before insulin receptor in vivo experiment has been confirmed. It is required to be confirmed whether there was insulin resistance in vitro liver parenchyma cells during ischemia reperfusion. The ischemia-reperfusion injury in nature is mainly the peroxidation damage of liver parenchyma cells because of ischemia hypoxia%lipid superoxide and a large number of free radicals. In order to verify whether the peroxidation damage could cause appearance of insulin resistance phenomenon in liver cells and its mechanism, herein on the basis of peroxidation damage model via hydrogen peroxide （H2O2） processing of human normal liver cells （L02cells） we study in vitro MDA, residual glucose in culture medium, annexin5, cell survival rate,cell morphology change,and the expression of InsRβ and AKT to confirm if there is the phenomenon of insulin resistance of receptors level and post receptors level in L02cells with peroxidation damage, and is possible mechanisms, providing new way of thinking for clinical drug prevention and treatment of ischemia-reperfusion injury.Materials and Methods:1. Materials:L02cells were bought from Chinese classic culture preservation center （wuhan）. Hydrogen peroxide was bought from national medicine group chemical reagent co., LTD. DMEM/High Glucose medium and altered RPMI-1640medium were bought from Hyclone co., LTD. Fetal bovine serum was bought from hangzhou sijiqing biological engineering materials co., LTD. Pancreatic enzymes was bought from Amrescoco., LTD. Glucose determination kit was purchased from Shanghai classical biological engineeringco.,LTD. Determination of malondialdehyde （MDA） kits were bought from nanjing to build science and technology co., LTD.2.Cell cultureâ‘ Abdicate after the coverage of cells in the culture bottle （25cm） is reaching80%-90%â‘¡Abandon the original medium, add2ml of PBS to clean cells, then remove PBSâ‘¢Add appropriate trypsin to cover cell and digest for1to2minutes.â‘£Suck out pancreatic enzyme to terminate digestion after cells become round⑤Added3ml of complete medium, beat cells with pipetting gun until the cells are suspending.â‘¥The cells are spread to three in the same specifications of the culture bottle and continued to be developed at37℃and5%CO2incubator.⑦prepare cell suspension using3-5generation cells with stable growth and seed in6orifice with about5×105cells in each hole.3. experimental groups:the experiment includes3groups:â‘ Group A: normal cultured L02cells without H2O2treatment for24h;â‘¡Group B:Cultured L02cells with160μmol/LH2O2for24h;â‘¢Group C:Incuba tion of normal L02cells after24h with517μmol/LH2O2. In0,12,24h, collect the cells and supernatant, and test respectively.4. The oxidative injury:Cells were inoculated in6well plates. After24h of culture, different concentrations of H2O2were added to make cellular oxidative damage models with different degrees.5. Glucose oxidase peroxidase method （GOD-POD） for detecting residual glucose content in cell supernatant:Mixture of reagent1and reagent2was prepared by mixing liquid according to the proportion of4:1. After balancing to the determination temperature, each supernatant of cultured cell was added in10μl automatic biochemical analyzer. The residual glucose in culture medium was determined with the unit of mmol/L.6Detection of malondialdehyde thiobarbituric acid （MDA） content:The collected cells in the4°C refrigerator was determined according to kit instructions by thiobarbituric acid assay malondialdehyde. By UV spectrophotometer, at532nm, with1cm optical path and distilled water zero, all tube absorbance values are determined. The blank tube was instead for standard blank pipe. The content of MDA=determined tube absorbance-Determination of blank tube absorbance/standard tube absorbance-standard blank tube absorbance*standards/protein content.7. Annexin5expression by RT-PCR method:According to the kit instruction for total RNA extraction and purification and the expression of Annexin5of L02cell was detected using RT-PCR kit. Primers were synthesized by Invitrogen Biotechnology Co., LTD China Company. Annexin5mRNA serial number XM₀01154, the upstream primer:5TTATCACCATCTTTGGAACACG-31, downstream primer:5’-CTTCCTAAACTCCTTCCTGATGT-3’. Beta-actin mRNA serial number XM₀01101, the upstream primer:5’-GTCCACCGCAAATGCTTCTA-31,5’-TGCTGTCACCTTCACCGTTC-3’ downstream primer5. First in total RNA reverse transcription reaction, then use cDNA as template using RT-PCR Kit （TOYOBO THUNDERBIRD SYBR qPCR Mix QPS-201） by real-time fluorescence quantitative PCR reaction in SLAN fluorescence quantitative PCR detection system. The reaction conditions were:95℃denaturing1min, denaturation15s at95℃,58℃for20s,72℃for20s, a total of40cycles,72℃terminal extension5min. Melting curve analysis was completed by relative quantitative method using actin as the reference. The CT value was used to calculate the relative expression of Annexin5mRNA:expression ofâ–³=2-â–³CT （△ CT=CT gene-CT internal standard gene,â–³CT=â–³CTâ–³sample-â–³CT control sample）.8. Western blot （Western Blot） for detection of insulin receptor beta （insulin β Receptor β, InsR） and phosphorylation of protein kinase B （phospho-Protein Kinase B, PKB/AKT） expression:Tissue was washed using cold TBS for2-3times to remove blood and cut into small pieces. Then place in the ehomogenizer for homogenate completely on ice. Add10times volume extraction reagent （add the phosphorylation of cooktail and protease inhibitors before use）. The homogenate is transferred into the1.5ml centrifuge tube for oscillation and ice bath of30min. Repeat pipetting for complete lysis of cells. The collected supernatant after12000r/min centrifugal5min is the total protein solution. Protein concentration was determined by Bradford method. The calculated volume of solution containing40μ G protein is the sample quantity. The used separation gel concentration was10%. The stacking gel is twelve alkyl sulfide-5%polyacrylamide gel electrophoresis （SDS-PAGE）, separation, transfer film （PVDF film before using methanol activation, to film in accordance with200mA,1H condition）,5%skim milk at room temperature after closed30min diluted500times PERK, p-EIF2a and Caspase12antibodies were added, room temperature incubation of2hours with TBST at room temperature, the decolorization table and wash three times, each time5min, two anti diluted3000times with TBST, incubate at room temperature for45min, the use of0.5‰TBST decolorization at room temperature table and wash three times, each time5min, membrane cleaning after adding two anti incubation. Chemiluminescence reagents, X-ray film exposure, observations by developing fixing process. Gel imaging and analysis system of scanning image, using Alpha software processing system analysis of optical density value of the target zone, ratio of sample and reference for comparative analysis. 9. Determined cell inhibition rate by MTT experiment test:â‘ The composite material was cut into96well culture plate hole size, with75%ethanol and ultraviolet light disinfection, drying. Placed in a96well culture plate holes in1rows in2-6.â‘¡To adjust the density of5×104/mL cells which were seeded on the material placed in holes, each hole0.2ml vaccination, i.e.104per cell.1and7hole with no cell culture fluid with wet environment. The culture plates into the CO2incubator, at37℃,5%CO2and saturated humidity conditions after incubation of24h by adherent.â‘¢Until the cells attached to the wall after overnight, adding different concentrations of H2O2（0μmol/LH2O2,160μmol/LH2O2,570μ mol/LH202）, cultured for24h, and no drug control group and only with medium of zero hole, each sample concentration of three replicates. The corresponding training time, take a culture plate, each hole by adding MTT solution （5mg/ml） of50μl, an incubator to incubation with2H, the end of culture supernatant discarded, careful suction hole. Each hole plus200μl DMSO （two,10min dimethyl sulfoxide） shocks, the surrounding cells and cells of MTT-α Cuan particles completely dissolve. The570nm wavelength was selected, the hole was measured in the ELISA meter on the light absorption value （A）, record the results, calculating the average value, and compared with the control group.10. Morphological observation:Cells were inoculated in6well plates. After24h of culture with different concentration of H2O2treatment, cells morphology was observed with inverted microscope.11. Statistical methods:Mean±standard deviation for all data （+s）, using SPSS13.0statistical software for data analysis, several groups of samples were compared using analysis of variance （compared with LSD method:the homogeneity of variance, variance not neat by Games-Howell method）, with P<0.05as criterion for significant differences.Results:1.The residual glucose content:The residual glucose content of A group was a linear downward trend from0hour （3.24+0.08） to24h （0.98+0.03）. There was significant differences（P<0.01） compared with0hour. Group B and C were also falling, but the falling range in group B was lower than that in group A at the time of12hour （2.71±0.08,2.90±0.09）,24h （2.18±0.03,2.34±0.06）. Group C was lower than group B.2.The MDA contents:The MDA content was no obvious difference at0hour. Two values in group B at12h （0.63±0.02） significantly increased, and continued to rise to24hour （0.72±0.04）. Compared with group A（0.49±0.05） there was significant differences（P<0.01）. Two values in group C at12hour （0.71±0.03） were obvious increased, which were not only higher than that of group A （P<0.01） but also significantly higher than that of group B. The values were declining until24hour （0.62±0.02）, which were slightly lower than that of group B. But they were still higher than that of group A.3. The Annexin5mRNA levels:The Annexin5mRNA expression level was no obvious difference at0hour. Two values in group B at12h （2.51±0.07） significantly increased, and continued to rise to24hour （2.83±0.04）, Compared with group A（1.00±0.00） there is significant differences（P<0.01）.Two values in group C at12hour（2.81±0.07） obvious increased. Not only higher than that of group A （P<0.01）, but also significantly higher than that of group B; But the values were on the decline to24hour（1.67±0.04）, which were slightly lower than that of group B, but still higher than that of group A.4.The expression of InsRβ、p-AKT by WesternBlot:The expression of InsRβ, p-AKT were no obvious difference at0hour. Two values in group B at12h （0.33±0.02,0.25±0.02） were significantly decreased, and continued to be declined to24hour （0.22±0.03,0.16±0.02）.Compared with group A （0.47±0.02、0.41±0.04） there were significant differences （P<0.01）. Two values in group C at12hour （0.26±0.02,0.14±0.01） were obvious increased, which were not only lower than group B （0.33±0.02,0.25±0.02）（P<0.01）, but also significantly lower than that of group A. The values were on the declining to24hour （0.13±0.02,0.06±0.02）, which were slightly lower than that of group B and significantly lower than that of group A.5. L02cells living conditions:The inhibition rate between the three groups was not in difference at0hour. The inhibition rate of A group was0.00%in the0to24hour. Under the microscope the growth momentum of cells was good, adherently growing, closely packing. The contour was clear and the form was normal. Group B and C were on the rise, the inhibition rate of both B and C group were higher than that of group A （P<0.05） at12hour （10.7%,49.3%） and24hour （31.1%,59.5%）. Group C was higher than group B. At24hour, Under the microscope the cells were ambiguous, the gap between cells was increased and a small float cells were seen in group B. At the same time, the cells were sparse, there were a large number of floating cells, its refraction became weak and particles deposited in the cytoplasm.Conclusions:The experimental results that the MDA, cell inhibition rate and Annexin5were increased,The cell uptake and utilization of glucose were decreased and residual glucose in culture medium was increased demonstrated that there is the phenomenon of insulin resistance when L02cells suffering from oxidative damage. At the same time, cell morphology was changed and the expression of InsR β was decreased, indicating that the phenomenon was associated with the receptors. In addition, the phosphorylation of AKT played very important role on uptaking glucose after receptors. It was found p-AKT expression was decreased, showing that the peroxidation damage can cause not only L02cells insulin resistance on receptor, but also insulin resistance phenomenon after receptors. Together, these results can provide new way of thinking for treatment of ischemia-reperfusion injury clinically for us by improving the insulin resistance on and after receptors.