Sesamin and vitamin E ameliorate aortic endothelial dysfunction rats induced by D-galactose and aluminum trichloride via regulation of endothelial nitric oxide synthase and NAD(P)H oxidase 4

ZHANG Jun-xiu,YAO Hai,WANG Huan,YANG Jie-ren,ZHENG Shu-guo

(1.Department of Pharmacology,Third-Grade Pharmacology Laboratory of State Administration of Traditional Chinese Medicine,Wannan Medical College,Wuhu 241002,China;2.Jiangsu Hansoh Pharmaceutical Co;Limited.,Nanjing 222000,China)

Abstract:OBJECTlVE To observe the protective effect of sesamin(Ses)and vitamin E(Vit E)against aortic endothelial dysfunction in rats induced by D-galactose(D-gal)and aluminum trichloride(AlCl3),and explore its conceivable mechanisms.METHODS A model of aortic endothelial dysfunction rats was established by D-gal(180 mg·kg-1,ip)combined with AlCl3(15 mg·kg-1,ig)for 84 d.Model rats were randomly divided into model,model+Vit E 10 mg·kg-1,model+Ses 160 mg·kg-1,and model+Ses 160 mg·kg-1+Vit E 10 mg·kg-1groups.After 70 d of treatment with Ses and Vit E,systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean blood pressure(MBP)were measured by tail cuff.The rats were anesthetized by sodium pentobarbital(30 mg·kg-1,ip).Thoracic aortas from the rats were removed and divided into two parts(3 mm in length).The relaxation of the aortic ring induced by acetylcholine(ACh)and sodium nitroprusside was measured.The primary pathologic changes in the aorta were observed by HE staining.Total antioxidant capacity(T-AOC),hydrogen peroxide(H2O2)and nitric oxide(NO)in serum were measured by colorimetric analysis.The expression of endothelial nitric oxide synthase(eNOS)positive cells in the aorta were measured by immunohistochemistry.The expressions of eNOS and NAD(P)H oxidase 4(NOX4)protein in the aortal were detected by Western blotting.RESULTS Compared with the model group,the relaxation response with increase in ACh concentration(1×10-7-1×10-4mol·L-1)was enhanced(P＜0.01)in model+Ses+Vit E,SBP,DBP and MBP decreased(P＜0.01),the serum T-AOC and NO level were increased(P＜0.01),the serum H2O2levels were reduced(P＜0.01),the eNOS expression was increased(P＜0.01)and NOX4 expression was reduced(P＜0.01)in each treatment group.Compared with model+Ses,the SBP,DBP and MBP were lower(P＜0.01 or P＜0.05),the serum H2O2level was lower(P＜0.01),the serum NO level was increased(P＜0.05),the eNOS expression level was higher(P＜0.01)and the NOX4 expression level was reduced(P＜0.05)in model+Ses+Vit E.Compared with the model+Vit E,the serum T-AOC and NO levels were increased(P＜0.05),the serum H2O2level was lower(P＜0.01),eNOS expression was increased(P＜0.01)and NOX4 expression was reduced(P＜0.05)in model+Ses+Vit E group.CONCLUSlON Ses and Vit E can ameliorate aortic endothelial dysfunction of rats induced by D-gal and AlCl3via the regulation of eNOS and NOX4.

Key words:endothelium dysfunction;sesamin;vitamin E;endothelial nitric oxide synthase;NAD(P)H oxidase 4

Sesamin(Ses),a lignan separated from Fagara plants,has health-promoting activities[1].Our previous studies have showed that Ses improves endothelial dysfunction in renovascular hypertensive rats fed with a high-fat,high-sucrose diet[2],Ses exerts renoprotective effects by enhancing nitric oxide(NO)bioactivity in renovascular hypertensive rats fed with a high-fat-sucrose diet[3],Ses ameliorates arterial dysfunction in spontaneously hypertensive rats(SHRs)via down-regulation of NADPH oxidase subunits and up-regulation of endothelial nitric oxide synthase(eNOS)expression[4],Ses can also enhance NO activity in aortas of SHRs[5].But the effect of Ses is weak.In recent years,it has been found that Ses combined with vitamin E(Vit E)can reduce the expressions of transforming growth factor-β1(TGF-β1),connective-tissue growth factor(CTGF),matrix metalloproteinase-9(MMP-9)and tissue inhibitor of metalloproteinases-1(TIMP-1)by inhibiting the activation of protein kinase B/phosphatidylinositol-3-kinase(Akt/PI3K)signaling pathway,thus lowering blood pressure and improving left ventricular fibrosis[6].Therefore,we believe that Ses combined with Vit E has a synergistic effect.

Endothelium dysfunction is believed to play a role in the development of cardiovascular disease[7].D-galactose(D-gal),a reducing sugar normally present in the body,can be oxidized by galactose oxidase to form hydrogen peroxide(H2O2),leading to oxidative stress,inflammation and apoptosis at high levels[8].Aluminum(Al)is a nonessential metal and a significant environmental contaminant and is associated with a number of human diseases including cardiovascular disease[9].Previous studies showed that the chronic administration ofD-gal and aluminum chloride(AlCl3)damages endothelial function,including vascular compliance and reduced blood flow[10],and induces cardiovascular disease[11].Therefore,we used Ses and Vit E in aortic endothelial dysfunction rats induced byD-gal and AlCl3,and explored its possible mechanisms.

1 MATERlALS AND METHODS

1.1 Drugs,reagents and instruments

Ses and Vit E(Guangrun Biological Products,Nanjing,China),HE staining solution(Leagene Biotechnology,China),NO,total antioxidant capacity(T-AOC),hydrogen peroxide(H2O2)detection kits and the Western blotting related chemical reagents(Beyotime Biotechnology,China),the antibodies NAD(P)H oxidase 4(NOX4),eNOS and β-actin(Santa Cruz Biotechnology,USA),sodium nitroprusside(SNP),phenylephrine(Phe),tris,ammonium persulfate(Sigma,USA),Lunimata TM Crescendo luminescent liquid(Millipore Corp,Billerica,MA,USA),ALC-NIBP non-invasive blood pressure measurement,MAP2000 polygraph,jacketed organ chambers and analysis system(Alcott Biotech Co.Ltd.,China),722 episcotister spectrophotometer(Analytical Apparatus Station of Gaomi,China),BX-51 microscope(Olympus,Japan),mini protean electrophoresis system and transfer system(Bio-Rad Laboratories,USA);Image Pro Plus 6.0 software(Media Cybernetics,USA).

1.2 Experimental animals,modeling and grouping

A total of 35 male 14-week-old SPF grade SD rats were seleted[SCXK(Su)2009-0002,body mass 250～290 g](Beijing Weitong Lihua Experimental Animal Technology Co.,Ltd.).Feeding conditions included the temperature of 20-22℃,relative humidity of 50%-70%,and a 12 h day night cycle.Twenty-eight rats were ip givenD-gal 180 mg·kg-1and ig given AlCl315 mg·kg-1for 84 d.Thus,a rat aortic endothelial injury model was prepared.Twenty-eight successfully modeled rats were selected and randomly divided into four groups with seven rats in each group:normal control group that received an equal volume of carboxymethylcellulose sodium,modelgroup,model+Ses 160 mg·kg-1group,model+Vit E 10 mg·kg-1group,and model+Ses 160 mg·kg-1+Vit E 10 mg·kg-1group.Experimental rats were administered once daily for seventy consecutive days.In this study,all rats received humane care in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health(NIH publication№.85-23,revised 1996).The study protocol was approved by the Ethical Committee of Wannan Medical College(Wuhu,China).

1.3 Measurement of blood pressure

After 70 d of administration,systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean blood pressure(MBP)were measured in conscious rats using the tail-cuff method after the animals were maintained in a warm chamber for about 10 min.At least 10 determinations were made on each rat and the mean of 6 readings within a 5-10 mmHg range was taken as the SBP,DBP and MBP[4].

1.4 Thoracic aortas sample collection

At the end of the experiment,the rats were fasted overnight and anesthetized by sodium pentobarbital(30 mg·kg-1,ip).Thoracic aortas from the rats were carefully removed,cleaned of adhering tissue and divided into two parts.One part contained the descending thoracic aorta,which was cut into two transverse rings(3 mm in length),and used for the vascular reactivity experiments.Portions of the arcus aortae were either used for HE and immunohistochemistry or quickly frozen in liquid nitrogen and stored at-80℃for future processing.

1.5 Measurement of aortic relaxation induced by acetylcholine(ACh)and sodium nitroprusside(SNP)

The thoracic aorta was quickly removed and cut into rings 3 mm in length.Two rings were taken from each rat aorta and suspended between two triangular-shaped stainless steel stirrups in two jacketed organ chambers(A and B)containing 10 mL of Krebs solution at 37℃that was continuously oxygenated with 95%O2and 5%CO2.Pre-load(2 g)was applied to the rings,and the vessels were allowed to equilibrate for 60 min(with 4 washes).Changes in isometric tension were detected by force transducers and recorded via a MAP2000 polygraph.The rings were stimulated with norepinephrine(3×10-7mol·L-1)to evaluate their viability and serially washed back to baseline levels and equilibrated once again.In chambers A or B,the concentration-relaxation response curves to ACh(1×10-8-1×10-4mol·L-1)or SNP(1×10-9-1×10-6mol·L-1)were performed respectively in aortic rings,which were precontracted with Phe,1×10-6mol·L-1.

1.6 Measurement of serum NO,T-AOC and H2O2

Griess reagent method was used to detect the content of NO in serum.Serum T-AOC was measured by 2,2′-azino-bis(3-ethylbenzthiazoline)6-sulfonic acid(ABTS)method.H2O2can oxidize Fe2+to produce Fe3+and then form a purple product with xylenol orange in specific solution so as to realize the determination of H2O2concentration in serum.All the experiments were completed according to the instructions.

1.7 HE staining for aortic intima morphological changes

The aortic specimens were fixed in 4% paraformaldehyde solution.After fixation for 24 h,tissues were dehydrated,paraffin-embedded,sectioned at 5 μm,and stained for HE staining[4].The aortic intima morphological changes in the aorta were observed.

1.8 lmmunohistochemistry analysis for the detection of eNOS protein expression in aorta tissue

Slides were incubated in methanol containing 0.3%H2O2for 30 min and blocked with normal goat serum in PBS(containing 0.1%BSA and 0.01%Tween 20)for 30 min.Primary antibodies against eNOS diluted in PBS containing 0.1%BSA and 0.01%Tween 20 were applied to slides for 12 h at 4℃.After counterstaining with hematoxylin,slides were dehydrated and permanently mounted.Sections were digitized using an Olympus BX51 microscope.Digital images were processed using Image Pro Plus 6.0 software,which was used to determine the integral absorbance(IA)of eNOS.

1.9 Western blotting analysis for the detection of eNOS and NOX4 protein expressions in aorta tissue

Total protein from the aorta tissue of each group was extracted,and protein concentration was determined by BCA assay.The isolated protein was separated on a 10% sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE),transferred to a polyvinylidene difluoride(PVDF)membrane and blocked with 5% skim milk powder for 2 h.Then,the primary antibody against eNOS(1∶1000),NOX4(1∶1000),and β-actin(1∶2000)was incubated with the membrane at 4℃overnight.After being washed with PBST,HRP-conjugated secondary antibody(1∶2000)was incubated with the membrane for 30 min at room temperature.The bands were detected using a highly sensitive LunimataTMCrescendo luminescent agent.The gel imaging system was photographed,the appropriate bands were selected,and the band IA values were analyzed using Image Pro Plus 6.0 software.The protein expression was expressed by the ratio of the integral absorbance value of the target protein to that of the internal standard protein.

1.10 Statistical analysis

2 RESULTS

2.1 Effect of Ses and Vit E combination on SBP,DBP and MBP of rats with aortic endothelial dysfunction induced by D-gal and AlCl3

Compared with normal control group,SBP,DBP and MBP in the model group were significantly increased(P＜0.01).Compared with the model group,the SBP,DBP and MBP of each medication group were significantly decreased(P＜0.01).Compared with model+Ses,the SBP,DBP and MBP of model+Ses+VitE group were significantly lower(P＜0.01 orP＜0.05)(Tab.1).

Tab.1Effect of sesamin(Ses)and vitamin E(Vit E)combination on systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean blood pressure(MBP)of rats with aortic endothelial dysfunction induced by D-galactose(D-gal)and AlCl3

2.2 Effect of Ses and Vit E combination on relaxation of aortic rings of rats with aortic endothelial dysfunction induced by D-gal and AlCl3

Compared with the normal control group,with the increase of ACh concentration(1×10-7-1×10-4mol·L-1),the endothelial-dependent relaxation of the aorta in model group decreased gradually(P＜0.01),suggesting that endothelial-dependent relaxation function of the aorta in model group was weakened.Compared with the model group,with the increase in ACh concentration(1×10-7-1×10-4mol·L-1),the endothelium-dependent relaxation response of model+Ses+Vit E group was significantly enhanced(P＜0.01),and was better than that of model+Ses and model+Vit E group(Fig.1A).

Compared with the normal control group,the diastolic response of the aorta to SNP(1×10-9-1×10-6mol·L-1)was not significantly different in the model group,which showed that the nonendothelial diastolic function of rat aorta was not damaged byD-gal and AlCl3.Compared with the model group,there was no significant difference in the aortic diastolic function of each administration group(Fig.1B).

Fig.1 Effect of Ses and Vit E combination on endothelial-dependent（A）and non-endothelial dependent（B）relaxation of aortic rings of rats with aortic endothelial dysfunction induced by D-gal and AlCl3.The concentration-relaxation response curves to ACh(1×10-8-1×10-4mol·L-1)or sodium nitroprusside(SNP)(1×10-9-1×10-6Qmol·L-1)were performed respectively in aortic rings,which were precontracted with phenylephrine(Phe)1×10-6mol·L-1.±s,n=7.＊＊P＜0.01,compared with normal control group;#P＜0.05,##P＜0.01,compared with model group;△△P＜0.01,compared with model+Ses group;††P＜0.01,compared with model+Vit E group.

2.3 Effect of Ses and Vit E combination on serum T-AOC,H2O2and NO level of rats with aortic endothelial dysfunction induced by D-gal and AlCl3

Compared with the normal control group,serum T-AOC and NO were significantly reduced and the serum H2O2was significantly increased in the model group(P＜0.01).Compared with the model group,the serum T-AOC and NO levels were significantly increased(P＜0.01),while the serum H2O2levels were significantly reduced(P＜0.01)in each administration group.Compared with the model+Ses group,the serum H2O2level was significantly lowered(P＜0.01)and the serum NO level was significantly increased(P＜0.05)in the model+Ses+Vit E group.Compared with the model+Vit E group,the serum T-AOC and NO level was significantly increased(P＜0.05),and the serum H2O2level was significantly lower(P＜0.01)in the model+Ses+Vit E group(Tab.2).

Tab.2 Effect of Ses and Vit E combination on serum total antioxidant capacity(T-AOC),H2O2and nitricoxide(NO)level of rats with aortic endothelial dysfunction induced by D-gal and AlCl3

2.4 Effect of Ses and Vit E combination on aortic morphological intima of rats with aortic endothelial dysfunction induced by D-gal and AlCl3

In the normal control group,the aortic intima was smooth,complete and connected,without local defect or thickening,and vascular smooth muscle cells were arranged in good order.The intima of the model group lost the smooth endothelium obviously in the model group.In the model+Ses and model+Vit E group,the pathological damage of the aorta was improved to different extents.In the model+Ses+Vit E group,the intima pathological damage of the aorta was significantly improved,suggesting that Ses combined with Vit E had synergistic protective effect on aortic injury(Fig.2).

Fig.2 Effect of Ses and Vit E combination on the aortic intima morphology of rats with aortic endothelial dysfunction induced by D-gal and AlCl3by HE staining.See Tab.1 for the rat treatment.Arrows show the intima pathological damage of aorta.

2.5 Effect of Ses and Vit E on expression of eNOS in aortas of rats with aortic endothelial dysfunction induced by D-gal and AlCl3

Compared with the normal control group,the eNOS expression level in the model group was significantly lower(P＜0.01).Compared with the model group,the eNOS expression level in each treatment group was increased to varying degrees,and the difference was statistically significant(P＜0.01).Compared with model+Ses group,the eNOS expression level in model+Ses+Vit E group was significantly higher(P＜0.05).Compared with model+Vit E group,the eNOS expression level in model+Ses+Vit E group was significantly increased(P＜0.01).The results suggested that Ses combined with VitE could synergistically upregulate the expression of eNOS in the aorta of rats with aortic endothelial dysfunction induced byD-gal and AlCl3(Fig.3).

Fig.3 Effect of Ses and Vit E on expression of endothelial nitric oxide synthase（eNOS）in aortas of rats with aortic endothelial dysfunction induced by D-gal and AlCl3detected by immunohischemistry.See Tab.1 for the rat treatment.IA：integrated absorbance.± s,n=7.＊＊P＜0.01,compared with normal control group;##P＜0.01 compared with model group;△ P＜0.05,△△ P＜0.01,compared with model+Ses group;††P＜0.01,compared with model+Vit E group.

2.6 Effect of Ses and Vit E combination on eNOS and NOX4 protein expressions in aortas of rats with aortic endothelial dysfunction induced by D-gal and AlCl3

As shown in Fig.4,compared with the normal control group,the eNOS expression level was significantly lower(P＜0.01)and the NOX4 expression level was significantly increased(P＜0.01)in the model group.Compared with the model group,the eNOS expression level was significantly increased(P＜0.01)and the NOX4 expression level was significantly reduced(P＜0.01)in each treatment group.Compared with model+Ses group,the eNOS expression level was significantly higher(P＜0.01)and the NOX4 expression level was significantly reduced(P＜0.05)in model+Ses+Vit E group.Compared with model+Vit E group,the eNOS expression level was significantly increased(P＜0.01)and the NOX4 protein expression level was significantly reduced(P＜0.05)in model+Ses+Vit E group.The results suggested that Ses combined with Vit E could synergistically up-regulate the expression of eNOS and downregulate the expression of NOX4 in the aorta of rats with aortic endothelial dysfunction induced byD-gal and AlCl3.

Fig.4 Effect of Ses and Vit E combination on eNOS and NAD(P)H oxidase 4（NOX4）protein expressions in aortas of rats with aortic endothelial dysfunction induced by D-gal and AlCl3by Western blotting.See Tab.1 for the rat treatment.C was the semi-quantitative result of A and B.± s,n=7.＊＊P＜0.01,compared with normal control group;##P＜0.01,compared with model group;△P＜0.05,△△P＜0.01,compared with model+Ses group;††P＜0.01,compared with model+Vit E group.

3 DlSCUSSlON

After 70 d of treatment with Ses and Vit E,the endothelium-dependent relaxation function of the rat aorta mediated by ACh was significantly enhanced,the content of NO and T-AOC in serum was increased,the expression of eNOS positive cells and protein in the aorta was up-regulated,and the serum H2O2and aortic NOX4 were decreased.The combination of Ses and Vit E group was better than that of Ses and Vit E alone.

Our previous studies showed that Ses reduces hypertension and improves endothelial dysfunction through upregulation of eNOS expression and reduction of NO oxidative inactivation in twokidney,one-clip renovascular hypertensive rats fed with a high-fat,high-sucrose diet(2K1C rats on HFS diet)[2];long-term treatment with Ses had renoprotective effect and improved renal endothelial dysfunction via upregulation of eNOS expression and reduction of NO oxidative inactivation in both clipped and contralateral kidneys of 2K1C rats on HFS diet[3];Ses improved arterial function,which may be attributed to decreased p22phox and p47phox expression and increased eNOS expression[4];chronic treatment with Ses could reduce hypertension and improve endothelial dysfunction through enhancement of NO bioactivity in SHR aortas[5].However,Ses′s effect is relatively weak.In recent years,it was found that Ses combined with Vit E can reduce the expression of TGF-1,CTGF,MMP-9 and TIMP-1 by inhibiting the activation of Akt/PI3K signaling pathway,thus exerting the effect of lowering blood pressure and improving left ventricular fibrosis[6].Therefore,we speculated that combined administration of Ses and Vit E might have synergistic effects.

The endothelium can evoke relaxations of the underlying vascular smooth muscle by releasing vasodilator substances.The best-characterized endothelium-derived relaxing factor(EDRF)is NO which activates soluble guanylyl cyclase in the vascular smooth muscle cells,with the production of cyclic guanosine monophosphate(cGMP)initiating relaxation[12].Endothelium dysfunction is believed to play a role in the development of cardiovascular disease[7].Vascular endothelial dysfunction is manifested in impaired endothelial barrier function,resulting in the muscle layer being damaged by various growth factors or inflammatory factors.On the other hand,it is manifested in impaired vasodilation,vascular remodeling,imbalance of coagulation and anticoagulant mechanism,etc[13].High levels ofD-gal can be converted into aldose and hydroperoxide under the catalysis of galactose oxidase,resulting in the generation of reactive oxygen species(ROS).Increased ROS subsequently cause oxidative stress,inflammation,mitochondrial dysfunction,and apoptosis[8].Al increased SBP,decreased ACh-induced relaxation,increased response to Phe,decreased endothelial modulation of vasoconstrictor responses,the bioavailability of NO[11].Therefore,the chronic administration ofD-gal and AlCl3damages endothelial function,including vascular compliance and reduced blood flow[10],and induces cardiovascular disease[11].

D-gal and Al can increase ROS[8,10]from ADPH oxidase[9,14-15].Excessive superoxide anioncan quickly combine with NO to produce peroxynitrite anion(ONOO-)[16].ONOO-has stronger oxidation ability,which can not only reduce the activity of NO,but also lead to eNOS uncoupling and produce more,and eventually cause the damage of endothelial function[17].NADPH oxidase consists of seven catalytic subunits,including gp91phox,NOX1,NOX3,NOX4,NOX5,dual oxidase 1(DUOX1)and DUOX2[18],while NOX4 is mainly expressed in vascular endothelial cells[19].In this experiment,the serum T-AOC increased significantly in the combination of Ses and Vit E group,and the increase was higher than that in Ses and Vit E alone.The serum H2O2,the aortic NOX4 in Ses and Vit E group decreased significantly,and the decrease was higher than that in Ses and Vit E alone.Subsequently,the reaction between NOX4-mediatedand NO was suppressed,and eNOS expression was increased because of reduced ONOO-.In conclusion,Ses combined with Vit E can improve the aortic endothelial dysfunction of rats induced byD-gal and AlCl3,and the mechanism may be related to down-regulating the expression of NOX4 and up-regulating the expression of eNOS.