Intramuscular Injection Of SRAGE Gene Prevents The Development Of STZ-induced Diabetes In Rats On High Fat Diet

BackgroundHigh fat diet is characteristic of current foodstyle, which is accompanied with high incidence of diabetes mellitus. Hight temper- ature-cooked high fat diet results in high level of advanced glycation end products (AGEs or glycotoxin). Interaction of AGEs with their receptor RAGE can induce oxidative stress, which can lead to insulin resistance (IR). IR is a key component in the pathogenesis of type 2 diabetes. The increased intake of AGEs exceeds body's clearance, thereafter food-borne AGEs will interact with RAGE, an vircious cycle of oxidative stress will incur, and IR, even diabetes develops. Suppression the formation of AGEs, degradation of AGEs and interruption the binding of AGEs with RAGE can decrease the state of oxidative stress. Therefore, intervention AGEs-RAGE pathway might stop the progression to diabetes mellitus in the current state of AGEs surplus.ObjectivesTo construct a rat sRAGE gene expression plasmid and to study if the expression of sRAGE could prevent the development of type 2 diabetes in rats on high fat diet.Methods1. Construction of sRAGE gene expression plasmid Total RNA was extracted from rat lung tissue, Primers were designed according to the published sequence and restriction enzyme cutting sites of SalI and ClaI were designed in primers. The fragement from SalI and ClaI digestion of the RT-PCR product was cloned to the plasmid pMD18-T simple Vector (designated as pMD18-sRAGE). The sRAGE fragment from SalI and ClaIdigestion of plasmid pMD18-sRAGE was cloned to the SalI and ClaI digested plasmid pLNCX2 under the promoter of CMV (designated as pLNCX2-sRAGE). Direct sequencing was undertaken to check the nucleotide sequence and the alignment of the inserted sRAGE fragement.2. Plasmid pLNCX2-sRAGE and pLNCX2 were amplified by E.Coli. Plasmid was extracted by alkaline lysis method and purified by polyethylene glycol method.3. Expession of pLNCX2-sRAGE Plasmids were injected into quadriceps muscle of thigh bilaterally (300μg each) after surgical exposure. In five minutes after injection electric pulses (voltage 200V/cm,pulse 9 and frequency 1Hz) were applied by two stainless steel needle electrodes placed about 5 mm apart in parallel of the injection area. After that the exposures were closed. For the study of injected muscle sRAGE mRNA expression six Sprague-Dawley (SD) rats (12 weeks old, weighted 300g±10g,male) were divided into two groups. Group pLNCX2-sRAGE received plasmid pLNCX2-sRAGE; Group pLNCX2 received the parent plasmid pLNCX2. One week after injection, all rats were executed and total RNA was extracted from musle of injected sites. sRAGE mRNA was amplified by RT-PCR and separated on 1% agarose gel.4. Effects of muscular injection of pLNCX2-sRAGE on the state of oxidative stress in rats on high fat diet4.1 For preliminary experiment 8 of 12 SD rats (6 weeks old, weighted 150g～160g,male) were fed on high fat diet for 8 weeks and divided into three groups (4 rats in each group) with free access to water and 12-hour light and dark cycle: (1)Group one was injected with plasmid pLNCX2-sRAGE (designed as sRAGE group); (2)Group two was injected with plasmid pLNCX2 (designed as high fat control). (3)Group three was fed with regular forage and no plasmid was injected (designed as normal chew control). 500-1000μl of blood sample was collected from tail bleeding before and every week after transfection, serum malondialdehyde (MDA) levels and superoxide dismutatse (SOD) activities were detected with commercial methods.4.2 For formal experiment 20 SD rats (6 weeks old, weighted 150g-160g,male) were fed on high fat diet for 8 weeks and divided into twogroups (10 rats in each group): (1)One group was injected with plasmid pLNCX2-sRAGE (sRAGE group). (2)One group was injected with plasmid pLNCX2 (high fat control). Rats were given a second injection of plasmid every 3 weeks after the first injection. SOD and MDA before and 7th, 14th, 21th day after each injection of the plasmid were analyzed. For study of the effects of plasmid pLNCX2-sRAGE injection on blood glucose and plasma insulin in rats fed on high fat diet, blood glucose (glucose dehydrogenase method, tip strips from Roche Diagnostics) and plasma insulin (ELISA from Mercodia Diagnostics) in rats were analyzed.5. For study of the effect of intramuscular injection of pLNCX2-sRAGE on the streptozotocin (STZ) induced incidence of diabetes after high fat diet rats in method 4.3 were injected with STZ (30mg/kg)intraperitoneally 3 weeks after the second transfection. 10 rats (20 weeks old, same age to the rat on high fat diet, weight 350～380g) that fed with regular forage were divided into two groups: one group (n=5) was given the same dosage of STZ as in the high fat groups; another group (n=5) was injected normal saline. One week after injection, fasting plasma glucose(FPG) were detected , diabetes was made when fasting tail blood glucose (FPG)≥7.0mmol/L. Pancreas was fixed and sliced after rat executed. The islet was studied immunohistochemistically with first rabbit anti-rat antibody and horseradish peroxidase labelled second goat anti- rabbit antibody.Results1. Direct sequencing of the final plasmid showed that the direction of insertion and nucleotide sequence of the inserted rat sRAGE cDNA was correct.2. sRAGE mRNA was detected at mRNA level after intramuscular injection of plasmid pLNCX2-sRAGE, and not detected in muscle of plamid pLNCX2 injection.3. The weights were similar between groups of the pLNCX2-sRAGE and pLNCX2 (382g vs 380g,P>0.05).4. The level of MDA decreased after one week of injection,and lasted for 3 weeks in group treated with pLNCX2-sRAGE, and the difference was signigicant compared with the high fat controls (P<0.01). The level ofSOD was noted to increase after injection of pLNCX2-sRAGE, but only significant after the third week (P< 0.05) of injection. For the second injection the level of MDA decreases similarly to the first, and the level of SOD continued to increase until a platform was reached.5. There was no difference in blood sugar among the rats of the two treatment group on high fat diet and on regular forage before injected STZ. The serum insulin level in rats of high fat control was higher than in sRAGE rats and rats on normal chew.There was no significant difference between sRAGE rats and normal chew rats.6. In the pLNCX2-sRAGE group there were two rats with FPG≥7.0mmol/L, while in the PLNCX2 group eight rats with FPG≥7.0mmol/L. No rats on regular forage were with FPG≥7.0mmol/L.7. Immunohistochemistry study showed that the pancreas islets were more intact and insulin staining was higher in density in sRAGE rats than in high fat controls, and both were worse than in normal chew controls.Conclusions1. Rat sRAGE was successfully cloned and a sRAGE expression plasmid which could expressed in the skeletal muscle was constructed.2. Intramuscular injection of pLNCX2-sRAGE could prevent rats on high fat diet from STZ-induced diabetes. This effect might due to the decreased state of oxidative stress and increased antioxidative activity from pLNCX2-sRAGE treatment.