| Background and AimsThe excessive growth and proliferation of vascular smooth muscle cells（VSMCs） induced by the relatively increased insulin results in vessel stenosis diseases in insulin resistance（IR）.Mammalian target of rapamycin（mTOR） is a crucial protein in insulin signal transduction pathway.The changes of mTOR in expression or function plays an important role in abnormal proliferation of VSMCs.Rapamycin（RAP）,a widely used immunosuppressant,is the specific inhibitor of mTOR.The effect of antiproliferation and immunosuppression of RAP have made it become the stent-coated drug for PTCA and antirejection drug for xenograft.Currently,there is no drug else for treating IR-associated vessel-overgrowing diseases by the inhibition of mTOR.Caramonin（CAR）,a flavonoid,is known to have several actions which are benefit for cardiovascular diseases,such as inducing vasorelaxation,inhibiting platelet aggregation,etc.Our previous studies showed that CAR prohibited the proliferation of VSMCs both cultured in normal and mimic IR medium via a mechanism of regulating mTOR signaling.However,the effects of CAR in vivo are still unknown,and its mechanisms are needed to be further clarified.This study was designated to investigate whether CAR could modify IR and prevent vascular pathological changes in fructose-fed model rats,and to evaluate the effect of CAR on the mRNA expression and activity of mTOR.MethodsAfter a week of acclimatization,male Sprague-Dawley rats were randomly divided into two groups.The control group（CG） was fed with regular diet and the fructose-fed group （FrcG） was received fructose-enriched diet.The blood samples were taken from the orbital veins of the rats after end of 4 weeks fed.Free blood glucose（FBG） was detected by glucose-oxidase method and free blood insulin（FINS） was assayed by radioimmunoassay. The homeostasis model assessment for IR（HOMA-IR） and insulin sensitive index（ISI） were used to estimate insulin sensitity.IR model rats were confirmed by their HOMA-IR higher than mean plus a standard deviation.The IR rats were then continuely fed with high fructose diet for 8 weeks,2 rats were sacrificed in every 2 weeks in each group and the morphological changes of aorta thoracalis smooth muscle cells were detected by microscope and electron microscope.If the media shows thickening,VSMCs were rich with organelles and had a plenty of chromatin in aorta of FrcG rats,the model of IR rat with vessel hyperplasia is suggested to be established successfully.Then,the FroG rats were further divided randomly into fructose-diet group（FMG）,solvent group（18%ethanol and 10%tween-80,SG）, rapamycin group(0.8 mg·kg-1,RAPG),high-dose cardamonin group(7.5 mg·kg-1,HCARG), middle-dose cardamonin group(5 mg·kg-1,MCARG) and low-dose cardamonin group(2.5 mg·kg-1,LCARG).Intraperitoneal injection with saline（CG,FMG） or with solvent（SG） or with drugs given before was performed respectively once a day for next 4 weeks.Dosage of RAP and CAR was chosen by literature and our earlier study.FBG,FINS,HOMA-IR,ISI,triacylglycerol（TG）,total serum cholesterol（TC）, carbamide,creatinine,alanine aminotransferase,glutamic oxalacetic transaminase and time of blood coagulation（TBC）,media/lumen area,protein expression of mTOR,p-P70S6K1（by immunohistochemistry） and mRNA level of mTOR,P70S6K1,4E-BP1（by real time RT-PCR） were measured at the end of different treatment.Body weight,daily food and daily water consumption of rats were recorded once a week.Results1.No significant differences were found in body weight,daily food and daily fluid intake in rats with different treatment groups during the whole experiment period.2.IR induced by feeding with high fructose was characterized by an evident increase in FBG, FINS,HOMA-IR（8.09±1.14 vs 6.52±0.95 mmol/L,P<0.01;32.2±9.2 vs 21.7±7.7 mIU/L,P<0.01;11.06±2.70 vs 5.35±0.98,P<0.01,respectively） and by decrease in ISI （-5.49±0.37 vs -4.87±0.38,P<0.01） after 4 weeks of diet.The tendency and statistics difference was similar in week 4 and 8 of FBG;FINS,HOMA-IR and ISI between FrcG and CG rats.FINS was elevated obviously in FrcG rats at week 8 compared with that of week 4（36.8±9.5 vs 32.2±9.2 mIU/L,P<0.01）,all of above facts suggested that hyperinsulinemia was aggravated in rats.Furthermore,cell swelling,cytoplasmic vacuolization,nuclear shrinkage,heterochromation multiplication and more plenty of organelles,indicating an active state of VSMCs proliferation,were observed in aorta media of FroG rats at the end of week 8 by microscope and electron microscope. Considered with the biochemical and stereological data,the model of rat with IR-associated vessel-proliferating disease had been established.3.Continuously medication for 4 weeks,there were no difference in FBG,FINS,HOMA-IR, ISI,TG;TC and TBC between SG and FMG rats,it was revealed that the results were not affected by solvent.FINS,HOMA-IR,TG and TC were significantly elevated whereas ISI and TBC were decreased obviously in FMG rats compared with that of CG rats（P<0.05 or P<0.01）,it suggested that pathological changes of IR,disorder of lipid metabolism and blood coagulation had occurred in model rats.TG and TC were evidently raised（P<0.01, P<0.05,respectively） and other indexes did not alter by treated with RAP.Treating with three dosage of CAR,FINS and HOMA-IR were remarkably decreased and ISI was increased（P<0.01）,meanwhile,the action of middle- and high- dosage of CAR was much stronger than that of low-dose CAR（P<0.01,P<0.05,respectively）,these results indicated that the middle- and high- dose CAR could alleviate IR more effectively;and TG had no marked changes.In addition,the high-dose CAR elevated TC and prolonged thromboxane time in model rats（P<0.01）.There were no significant difference on carbamide,creatinine,alanine aminotransferase,glutamic oxalacetic transaminase among all groups.4.Continuously medication for 4 weeks,MA/LA of aorta was similar in both SG and FMG rats.Remarkably,the MA/LA of aorta was decreased and vascular over-proliferation was inhibited by RAP and CAR of three doses in FMG rats,respectively（P<0.01）.5.Continuously medication for 4 weeks,the protein expression of mTOR and p-P70S6K1 remained unaltered in aorta of SG and FMG rats.Comapred with CG,there was a significant elevation in protein level of both mTOR and p-P70S6K1 in aorta of FMG rats （P<0.01）.The protein amount of mTOR was evidently reduced by treated with RAP and CAR of three doses,respectively,and the effect of low-dose CAR on mTOR protein was weaker than that of RAP and high-dose CAR（P<0.05）.The expression of p-P70S6K1 protein was remarkably inhibited（P<0.01） by treated with RAP and CAR of middle- and high-dose,and there was no effect on that by treated with low-dose CAR.6.Continuously medication for 4 weeks,the mRNA expression of mTOR,P70S6K1 and 4E-BP1 had no marked difference in aorta of SG and FMG rats.The mRNA relative amount of mTOR,P70S6K1 and 4E-BP1 was significantly increased in aorta of FMG rats （P<0.01,respectively）.The mRNA level of mTOR,P70S6K1 and 4E-BP1 were down-regalated by RAP and CAR of three doses（P<0.01）.The action of high-dose CAR on mRNA expression of P70S6K1 and 4E-BP1 was stronger than that of RAP（P<0.01）, and the inhibition on mRNA expression of P70S6K1 was different in three dose of CAR.ConclusionsFed with fructose-enriched diet for 12 weeks,rats displayed IR,hyperinsulinemia, hypertriglyceridemia,hypercholesteremia and hypercoagulable state.Together with excessive activation of mTOR/P70S6K1/4E-BP1 signalling in thoracic aorta,it showed that vessel hyperplasia might associate with proliferation of VSMCs.CAR can improve IR,hypercoagulability and vessel hyperplasia in high-fructose feeding rats,the mechanisms may associate with reducing FINS,prolonging TBC,inhibiting over mRNA expression of mTOR,P70S6K1 and 4E-BP1,protein expression of mTOR and p-P70S6K1,and proliferation of VSMCs.The exact target and mechanism of CAR is needed to be further elucidated. |
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