| Background and purpose:Previous studies suggest that arachidonic acid (AA) has a plenty of containing in vivo and is the precursor of various important biological active substance. AA exists mainly in the form of combined etherified fatty acids in the membrane on the inside. When stimulated by various physiological or pathological, actived phospholidase A2 release arachidonic acid from cell membrane. The lipoxygenases and cyclooxygenases metabolize pathways have been well known. Recently, the researchers have found the third way to metabolize AA, the cytochrome P450 pathway. This pathway can convert AA to 5,6-EET,8,9-EET,11,12-EET and 14,15-EET. Soluble epoxide hydrolase is the main enzyme which metabolizes EETs to their corresponding dihydroxyeicosatrienoic acids (DHET) and DHET has less biological active than EETs.Accumulating evidence suggests that EETs play an important role in regulating cardiovascular system functions. It is well established that EETs possess potent vasodilatory effects, which are mediated by smooth muscle cell hyperpolarization via the activation of calcium-sensitive potassium channels (BKCa2+), the release of nitric oxide (NO) from L-arginine metabolized by endothelial nitric oxide synthase (3) also partly contribute to the vasodilatory effects. EETs can promote the proliferation and angiogenesis of endothelial cells through the activation of MAPK and PI3K/AKT signaling pathways, and to some extent, the eNOS pathway. CYP epoxygenase overexpression, which is known to increase EETs biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-alpha. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/AKT signaling. CYP-derived EETs possess potent anti-inflammatory properties in the vasculature, and improved endothelial dysfunction. Recent studies show that inhibiter of sEH has a significant protective effect in the cardiovascular system, as well as in other systems. sEH gene knock out can significantly increase EETs level, lower gene knock out animal systolic pressure; sEH gene mutation is tightly related to the coronary heart disease. In the myocardial ischemia-reperfusion test, the sEH gene knock out animals immediately resume heart function, shrink infarct size and delay the heart failure and arrhythmia caused by myocardial ischemia. The mechanism under the phenomena is the activation of PI3K pathway and myocardial K+ channel. sEH inhibitor can prevent myocardial hypertrophy and ventricular remodeling. sEH gene mutation is closely related to the coronary atherosclerosis. sEH inhibitor can also improve vascular remodeling, inhibit vascular imflammatio.It is estimated that 225 million people are affected worldwide. Moreover, the diabetic population is subject to a high incidence of cardiovascular and renal diseases Diabetes is characterized by hyperglycemia and beta cell dysfunction. Beta cell destruction and dysfunction are the central events in the development and progress of diabetes, the prevention of beta-cell destruction and the improvement of beta-cell function could be important strategies for controlling the advance of diabetesThese findings indicated that rAAV-CYP2J2 gene delivery and the co-treatment of sEHI can improve glucose homeostasis, prevent pancreas cell apoptosis and delaying the progression of DM. So, the effects of CYP2J2 gene delivery on DM and islet apoptosis and the possible molecular mechanisms were investigated in KKAy type 2 diabetic mice models.Experimental methods and resultsExperimental methods:1. Type 8 rAAV vectors containing human CYP2J2 or GFP were prepared by triple plasmid co-transfection in HEK293 cells as described previously. The sEH inhibitor 1-adamantyl-3-cycloexylurea (Winkler and Arenhovel-Pacula), trans-4-[4-(3- adamantan-1-ylureido) cyclohexyloxy]-benzoic acid (sEHI-1471) was kindly granted by Dr. Hammurg.2. Male non-diabetic C57BL/6J mice (Experimental Animal Center, Shanghai, China) and obese diabetic KKAy mice (HFK Bio-technology, Beijing, China) were used experimentally when they reached the age of 10-11 weeks. Forty male KKAy mice were randomized into 4 groups as follow:saline control group, rAAV-GFP group, rAAV-CYP2J2 group and rAAV-CYP2J2+sEHI group. All mice received a single injection of either 0.9% NaCl or rAAV (1×1012 vector genomes per mouse) via tail vein respectively. Additionally, rAAV-CYP2J2+sEHI group mice received sEHI-1471 (8mg/L) in the drinking water for 12 weeks;3.At 0,1,2,4,8,12 weeks of experiment, the following parameters were measured in all five groups as described previously:fasting plasma glucose, triglycerides, cholesterol, LDL cholesterol and HDL cholesterol levels. Body weight and food consumption were monitored weekly. A day before the end of treatment,24 hours urine samples were collected in metabolic cages with triphenylphosphine to prevent the decay of urine and the oxidation of EETs. At the end of experiment, animals were anesthetized with pentobarbital (40 mg/kg). Blood was collected through cardiac puncture, centrifuged and serum was used. Serum insulin levels were determined by using the Rat/Mouse insulin ELISA Kit (Linco Reseach, Inc., USA); serum FFA levels were determined by using enzymatic colorimetric method (Wako Chemicals, Inc., USA). The mouse pancreas were rapidly dissected out and cut into two part, with one half snap frozen in lipid nitrogen and stored at -80℃while another fixed in 4% paraformaldehyde and embedded in paraffin for immunohisto-chemical study;4.Five groups of mice were fasted for 16h and intraperitoneally injected with D-glucose (20% solution; 2g/kg body wt). At 0,30,60 and 120 min after glucose administration, blood samples were taken from the tail vain;5.Urine 14,15-DHET was quantified by an ELISA kit; 6.Fourμm thick sections of pancreas were stained with hematoxylin and eosin (H&E) for morphological analysis and immunolabeled by a combination of mouse anti-insulin and rabbit anti- glucagon antibodies.7.Western blot was carried out to detect CYP2J2 expression in pancreas, liver and kidney of KKAy type 2 diabetic mice and C57BL/6J normal mice; the expression of Bax, Bcl-xL,Bcl-2,pBad,Bim,Bid,p-Y473-AKT,p-Y989-IRS-1 and p-S307-IRS-1 in KKAy type 2 diabetic mice and C57BL/6J normal mice pancreas were also detected.Research results:1. Results showed that compared with the normal mice and mice injected with saline and rAAV-GFP, the CYP2J2 expression in pancreas, liver and kidney of mice injected with single rAAV-2J2 or co-treatment of sEHI increased significantly (P<0.05). Results of ELISA indicated that 14,15-DHET content (24.33±1.85) ng/mL of rAAV-CYP2J2 group in urine was significantly higher than that (6.13±0.74) ng/mL, (6.76±0.93) ng/mL and (5.7±0.31) ng/mL of saline, rAAV-GFP and normal group. Content in rAAV-CYP2J2+sEHI group (17.18±1.52) ng/mL was higher than rAAV-CYP2J2 single treatment.2. Daily food consumptions of all mice were measured and remained similar throughout the study (6.5 g/day for KKAy mice vs.5.8 g/day for C57BL/6J mice). Body weight in saline and rAAV-GFP group mice increased significantly with time, rAAV-2J2 significantly prevented the weight gain after 8 weeks of treatment (p<0.05), after this period, body weight increased slightly. The adding of sEHI earlier the prevention to 4 weeks and the effect kept to the end of experiment. Fasting blood glucose (FBG) increased significantly with time in both saline and rAAV-GFP groups. rAAV-2J2 reduced FBG by～4 mm/1 compared to pre-injection levels 4 weeks after gene delivery. and the hypoglycemia effect continued throughout the experiment compared with saline and rAAV-GFP groups. The co-treatment with sEHI spent 1 week to significantly reduce the FBG compared to saline and rAAV-GFP groups. Then Levels of triglycerides and total cholesterol increased significantly in KKAy mice than the C57BL/6J. The HDL cholesterol and LDL cholesterol had the same levels neither in KKAy nor in C57BL/6J mice. rAAV-2J2 or rAAV-CYP2J2+sEHI treatment had no effect on the four targets above. At the end of the experiment, serum insulin and FFA levels were also detected, compared with values in C57BL/6J group both serum insulin and FFA levels were significantly increased in saline and rAAV-GFP treated groups. Injection of rAAV-2J2 can significantly decrease the high levels of them and the co-treatment of sEHI enhances the decreasing effect of rAAV-2J2.3. A glucose tolerance test was performed and fasting plasma glucose levels before and after glucose loading was significantly increased in all KKAy groups. Compared with C57BL/6 mice 0,30,60,120 min glucose level (4.68±2.17) mmol/L,(14.17±1.56) mmol/L,(8.89±1.63) mmol/L,(5.19±1.74) mmol/L, four groups of KKAy mice glucose level significantly increased before and after glucose loading (P<0.05); However, the result showed that in rAAV-2J2 treated group,0,30,60,120 min glucose level were (10.56±2.2) mmol/L,(28.54±1.8) mmol/L,(21.88±2.3) mmol/L,(15.32±1.25) mmol/L, rAAV-2J2+sEHI treated mice glucose level in the corresponding time point were (7.71±23.84) mmol/L,(23.84±1.81) mmol/L,(15.33±2.05) mmol/L,(12.08±1.87) mmol/L significantly lower than the saline treated group (13.22±2.1) mmol/L (39.54±2.39) mmol/L,(32.46±1.71) mmol/L,(25.62±2.16) mmol/L (P<0.05)和rAAV-GFP group (12.56±2.31) mmol/L,(38.07±2.51) mmol/L,(31.24±1.86) mmol/L,(25.13±2.28) mmol/L (P<0.05). Area under the curve (AUC) analysis revealed significantly lower glucose levels in rAAV-2J2 treated KKAy mice and the co-treatment of sEHI enhance the glucose lowing ability of rAAV-2J2. Insulin secretion was assessed at the same time point of glucose tolerance test and a similar profile for fasting plasma insulin levels and plasma insulin levels after glucose loading was observed. Treatment of rAAV-CYP2J2 significantly lower insulin levels at all time points assessed and rAAV-CYP2J2+sEHI treatment greater the lowing effect but they all higher than those of the C57BL/6J mice.4. The morphology and cell apoptosis of pancreas:H&E staining revealed severely decreasedβ-cell mass and disrupted islet organization, the clear round islet boundary was lost, and islet shrinkage was observed in saline and rAAV-GFP groups compared with normal control. In contrast, rAAV-2J2 delivery significantly attenuated these pathological changes and co- treatment of sEHI restored them to nearly normal. Furthermore, in saline and rAAV-GFP groups insulin-positive area (red staining) was significantly decreased (p<0.05) whereas glucagon-positive area (green staining) was significantly increased (p<0.05) compared with the normal control. rAAV-2J2 can significantly increase insulin-positive area (p<0.01) and co-treatment of sEHI greater this increase. TUNEL staining showed that islet cell apoptosis was significantly increased in saline and rAAV-GFP groups compared with normal (p<0.05). rAAV-2J2 treatment dramatically reduced apoptosis and sEHI co-treatment elevated the protective effect of rAAV-2J2.5. Western blot analysis showed that, compared with C57BL/6J mice, pancreas in saline and rAAV-GFP group had significantly decreased p-Y989-IRS-1, p-Y473 Akt, Bcl-2, Bcl-xL and pBad expression levels whereas p-S307-IRS-1,Bax, Bim and Bid expression increased significantly. The rAAV-CYP2J2 gene delivery obviously inhibited p-Y989-IRS-1, p-Y473 Akt, Bcl-2,Bcl-xL and pBad expression levels decreasing and p-S307-IRS-1, Bax, Bim and Bid expression increasing.Statistical AnalysisContinuous data were expressed as means+S.E.M. Comparisons between groups were performed by a one-way analysis of variance. Two-way analysis of variance was used to examine differences in response to treatments and between groups, with post hoc analysis performed using the Student-Newman-Keuls method. Statistical significance was defined as P<0.05Concusions1. CYP2J2 was successfully expressed in various organs of C57BL/6J and KKAy type 2 diabetic mice, both CYP2J2 and sEHI were functionalyy active in the metabolism of arachidonic acid into EETs.2. CYP2J2 protein overexpression and the co-treatment with sEHI significantly reduced KKAy mice body weight gain, fasting blood glucose, insulin and FFA.3. CYP2J2 protein overexpression and the co-treatment with sEHI improve KKAy mice glucose homeostasis and insulin release.4. CYP2J2 protein overexpression and the co-treatment with sEHI improve KKAy mice islet morphology, increase insulin-positive cell numbers and decrease islet cell apoptosis.5. CYP2J2 protein overexpression and the co-treatment with sEHI protect islet cell apoptosis in KKAy type 2 diabetic mice and its possible molecular mechanism are that EETs activated IRS-1/Akt signaling pathways and the Bcl-2 family related apoptosis proteins.6. The finding of this study provide a new research direction, ideas and theoretical basis for future studies of prevention and treatment of insulin resistance and diabetes, as well as a new strategy to find new drug targes. |
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