| Background In insulin resistance states, the overproduction of reactive oxygenspecies (ROS) and proinflammatory cytokines play a major role in the pathogenesis ofendothelial dysfunction, hypertension and atherosclerosis. Heme oxygenase-1 (HO-1) isa stress response protein involved in vascular disease, transplantation, andinflammatory disorders. Recently, increasing attention has been drawn to the beneficialeffects of heme oxygenase-1 (HO-1) in the cardiovascular system. HO is a microsomalenzyme that catalyzes the degradation of heme into biliverdin, which is subsequentlyreduced to bilirubin, plus one iron atom and one carbon monoxide (CO) molecule. Thesebreakdown products play many vital physiological and pathological roles in anti-oxidantprocesses and regulation of vascular tension, which implies that the induction of HO-1might function as an alternative defense mechanism against the vascular injury in insulinresistance states.Objective This study aimed at investigating the regulatory effects of HO-1 on vascularfunction and morphology in vascular dysfunction in the aortas of insulin resistance ratsand demonstrating the probable mechanisms. These would provide experimentalevidences for potential therapeutic option for vascular dysfunction in insulin resistancestates.Methods1. Established insulin resistance (IR) model by feeding with high fat diet (HFD). IRmodel was successfully established by feeding with high fat diet (HFD) for 6 weeks.Sprague-Dawley rats fed with high fat diet (HFD) for 6 weeks and the IR models wereconfirmed to be established successfully through hyperinsulinemic- euglycemic clamp. 2. The model rats were randomly divided into 3 subgroups, namely, IR group, hemingroup and zinc protoporphyrin-Ⅸ(ZnPP-Ⅸ) group. Then the model rats and the normalcontrol group were treated as described below for 4 weeks.①control group Controlrats continued the standard chow diet and treated with intraperitoneal injection of normalsaline every other day;②IR group IR rats continued a high-fat diet and treated withintraperitoneal injection of normal saline every other day;③hemin group IR ratscontinued a high-fat diet and treated with intraperitoneal injection of hemin (30pmol/kg)every other day;④ZnPP group IR rats continued a high-fat diet and treated withintraperitoneal injection of ZnPP (10μmol/kg) every other day. Each week, all the ratswere determined for body weight and blood glucose, as well as systolic blood pressure(SBP) and heartbeats by tail-cuff plethysmographic method. The levels of free fatty acid(FFA), total antioxidant capacity (TAOC), superoxide dismutase (SOD), malondialdehyde(MDA), tumor necrosis factor a (TNF-α), nitric oxide(NO), inducible nitric oxide synthase(iNOS) and endothelial nitric oxide synthase(eNOS)in serum or in aorta were measured.The blood carbon monoxide (CO) was measured by blood gas analysis. Theexpressions of mRNA of HO-1, TNF-α, iNOSand eNOS were detected by reversetranscription-polymerase chain reaction (RT-PCR) and theexpression of HO-1 protein bywestern blot. The response of thoracic aortic rings (TARs) was studied with isolatedartery ring technique. The ultrastructure of thoracic aorta was observed under scanningelectron microscope (SEM).Results1. Results of IR models1.1. Compared with normal control (NC) group (on standard chow diet), the body weight, blood pressure , blood glucose and the level of insulin were significantlyincreased after 6 weeks of high fat diet (HFD) (P＜0.05), and the glucose infusion rates(GIR) of IR group as shown by hyperinsulinemic-euglycemic clamp was lower than thatof NC group, suggesting decreasedinsulin sensitivity and development of insulinresistance in HFD rats.1.2. The IR group demonstrated abnormal metabolism of blood fat, with increased levelsof TC, TG and FFA as compared with NC group. Compared with NC group, the higherlevel of MDA and the lower levels of TAOC and SOD in IR group outlined the presenceof oxidative damage and impaired antioxidant capacity in IR rats. The level of TNF-αinIR group was increased compared with NC group.2. Results of intervention with hemin and ZnPP2.1. Changes of Blood glucose, body weight, and SBP IR group rats progressivelydeveloped hypertension as evidenced by elevated SBP with no change in the heart rate.The 4-week treatment with hemin had no significant effect on the blood glucose andbody weights compared with 4wk IR group (P＞0.05), however, it significantly decreasedSBP to a level comparable with that in control group (P＞0.05). The 4-week treatmentwith ZnPP significantly elevated SBP compared to 4wk IR group (P＜0.05).2.2. Change of oxidative damage markers in aorta Changes of serum TAOC and MDAcontents, TAOC and SOD in aorta of 0wk and 4wk IR groups were decreased in atime-dependent manner (both P＜0.01), and the content of MDA was progressivelyincreased (P＜0.01). After pretreatment with hemin for 4 weeks in IR rats, the levels ofTAOC and SOD were significantly increased compared with IR group (P＜0.05).Alternatively, after pretreatment with ZnPP in IR rats, lower contents of TAOC and SOD and a higher level of MDA in aorta were measured.2.3. Changes of blood CO contents and serum NO contents Compared with controlgroup, the CO content in the blood was decreased in 0wk and 4wk IR rats (P＜0.05), andthe NO level in aorta of IR rats markedly increased (P＜0.05). After pretreatment withhemin in IR rats, a high level of CO in serum and decreased of NO in aorta wasmeasured (P＜0.05). Alternatively, after pretreatment with ZnPP in 4wk IR rats, thecontents of serum CO further decreased (P＜0.01), and the level of NO significantlyincreased (P＜0.01).2.4. Changes of iNOS and eNOS activities and mRNA expression Compared withcontrol group, the iNOS activity was significantly enhanced, while eNOS activity wassignificantly inhibited. Upregulated iNOS mRNA expression and eNOS mRNAdown-regulation were observed in aorta of 4wk IR group. Pretreatment with hemin for 4weeks in IR rats could inhibit the mRNA expression and activity of iNOS and promote theexpression and activity of eNOS (both P＜0.01) compared with those of 4-week IR rats.ZnPP treatment in IR rats could enhance iNOS activity (P＜0.05) but had no effect oneNOS activity and mRNA expression.2.5. Changes of TNF-αlevel and TNF-αmRNA expression Compared with controlgroup, the level of TNF-αwas increased and the expression of TNF-αmRNA wasenhanced in aorta of IR rats (P＜0.05). And there was no significant difference with thelevel of TNF-αand the expression of TNF-αmRNA between ZnPP group and hemingroup (P＞0.05).2.6. Vascular reactivity As detected at each time points, the endothelium-dependentrelaxation responses to Ach (10-6mol/L) in IR rats were diminished compared with control group (P＜0.05). Administration of hemin in IR rats could improve vasorelaxationdisorder and cause the curve of cumulative dose responses to ACh (10-8～10-5mol/L) ofhemin group to shift upward compared with that of 4wk IR group (P＜0.05), whiletreatment with ZnPP in IR rats aggravated the vasorelaxation disturbance. Contractiveresponses to PE (10-6mol/L) were significantly higher in TARs from IR rats than thosefrom control group, while the contractile responses to PE were significantly reduced inTARs from hemin group. ZnPP treatment did not significantly alter the contractions to PEin aortas of IR rats. No changes were detected in endothelium-independent relaxationresponses to SNP (10-6mol/L) among the four groups.2.7. Ultrastucture changes Scanning electron micrographs of rat thoracic aorticendothelium from control group showed intact endothelial cell layer; while IR groupexhibited structural damages of endothelial cell and cell gap; compared with IR group,ZnPP induced serious structural damages and hemin attenuated such morphologicchanges of endothelial cells.Conclusion1. Insulin resistance (IR) model could be successfully established by feeding with high fatdiet (HFD) for 6 weeks. In insulin resistance states, the oxidative damage andoverproduction of proinflammatory cytokines play a major role in the pathogenesis ofvascular dysfunction and endothelial dysfunction.2. Upregulation of HO-1 gene expression in IR rats could partly reverse vasculardysfunction and hypertension; while downregulation of HO-1 could worsen vasculardysfunction.3. It was further demonstrated that upregulation of HO-1 might increase the anti-oxidant capacities, decrease lipid peroxidation powerfully and inhibit the generation ofpro-inflammatory cytokines in IR rats. The protective effect of HO-1 against vascularinjury in insulin resistance states was due to its property as an antioxidants.4. The interaction of HO /CO and NOS/NO syesterm could play the protective role.Upregulation of HO-1 could promote CO content and eNOS expression, inhibit iNOSactivity and decrease NO production, thus improving the vasodilatation dysfunction ofartery.5. Since nitrogen monoxide is a free radical, endogenous carbon monoxide produced byHO-1 is more potently protective against vascular tissue lesion than nitrogen monoxidein insulin resistance states.
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