Experimental Study Of The Regulatory Effect Of TXNIP On Vascular Endothelial Dysfunction In Hypertension

PART Ⅰ HYPERTENSION INDUCES THE UPREGULATION OF TXNIP EXPRESSION IN VASCULAR ENDOTHELIAL CELLObjective:An obvious imbalance of“oxidation-antioxidation”often occurs in the course of hypertension,and oxidative stress is one of the major causes which induce the vascular endothelial dysfunction.As an important pro-oxidant protein in organism,thioredoxin interacting protein(TXNIP)has found to promote cellular oxidative damage under many pathological conditions.In this part,the main object is to examine the changes in TXNIP expression in vascular endothelial cell under hypertension condition,and to explore the mechanism involved.Methods:Two-kidney and one-clip(2K1C)was used to establish a rat model of experimental hypertension.The 30 male SD rats were randomly divided into 3 groups:control,sham and hypertension,respectively,and 10rats in each group.A noninvasive tail-cuff system was used to regularly monitor blood pressure and heart rate of rats in each group,and some rats were eliminated for unsuccessful surgery.After 4 weeks,the heart,aorta and carotid artery were harvested for HE,immunohistochemistry and Western blot analysis.Enzyme-linked immunosorbent assay was used to detect the angiotensin(Ang)Ⅱ concentration in plasma of rats in each group.In vitro,human umbilical vein endothelial cell(HUVEC)was cultured and exogenously treated with different concentration and duration of Ang Ⅱ to mimic the endothelial cell injury in the course of hypertension;and the expression of TXNIP was then detected.HUVEC was pretreated with Losartan(an AT-1R blocker)and Tempol(a membrane-permeable antioxidant),Western blot and immunofluorescence were performed to detected the change in TXNIP expression in HUVEC under Ang Ⅱ stimulation.Results:(1)After surgery,hypertensive rats exhibited a consistent increase in blood pressure.At 4 weeks after surgery,both the systolic pressure(181±6 vs.120±5 mmHg;P<0.01)and diastolic blood pressure(156±8 vs.105±3 mmHg;P<0.01)were higher than those in the sham group;while,no difference in heart rate was observed among the groups.(2)Compared with sham-operated rats,hypertensive rats also exhibited increases in heart mass index(4.38±0.16 vs.3.67±0.19;P<0.05),cardiomyocyte area(586±44vs.353±27μm~2;P<0.01)and medial thickness of carotid artery(38.28±2.37 vs.23.64±1.68μm,P<0.05)and aorta(82.04±2.52 vs.54.68±1.43μm;P<0.05).In addition,the plasma concentration of Ang Ⅱ was elevated in hypertensive rats when compared with sham-operated rats(556.18±9.14vs.501.78±7.16 pg/mL,P<0.01).(3)Compared with sham-operated rats,the expression of TXNIP in the carotid artery and aorta was obviously increased,and such increase was predominantly presented in the vascular endothelial cells,as detected by immunohistochemistry.(4)In vitro,we found that Ang Ⅱ could upregulate TXNIP expression in HUVEC in a time-dependent manner.(5)The upregulation of TXNIP in Ang Ⅱ-treated HUVEC could be reversed by Losartan and Tempol.Conclusion:In hypertension,the expression of TXNIP is upregulated in the vascular endothelial cell,which may be related to oxidative stress induced by Ang Ⅱ.PART Ⅱ EFFECTS OF TXNIP ON VASCULAR ENDOTHELIAL FUNCTION IN HYPERTENSIVE RATSObjective: As a barrier between blood and vessel wall,vascular endothelial cell can secrete multiple vasoactive substances,and is essential for maintaining the normal function of blood vessels and hemodynamic instability.Hypertension leads to endothelial dysfunction;however,the concrete mechanism involved is still not fully elucidated.This part is mainly designed to explore the effects of TXNIP on endothelial function in hypertensive rats.Methods: A total of 32 male SD rats were randomly divided into 4 groups(8 for each group): sham,sham + TXNIP inhibitor(Resveratrol),hypertension,and hypertension + TXNIP inhibitor(Resveratrol).2K1 C was used to establish the hypertensive rat model;Resveratrol(2mg/kg/d)or equal volume of solvent was administrated to rats via oral gavage on the next day after surgery.After consistently administered for 4 weeks,carotid artery,thoracic aorta,and plasma were harvested.A Wire Myograph System was used to detect the endothelium-dependent vascular responses of thoracic aorta,and the protein levels of TXNIP,e NOS,p-e NOS(ser 1177)and several representative redox proteins(NOX2、NOX4、SOD2 and NRF2)in aorta were detected by Western blot.Then the malondialdehyde(MDA)concentration and SOD activity in plasma,associated with dihydroethidium(DHE)staining of aorta were performed to evaluate the systemic and vascular oxidative stress,respectively.Results:(1)The expression of TXNIP in the vascular tissues was significantly inhibited by Resveratrol.(2)Compared with sham-operated rats,hypertensive rats exhibited an impairment of acetylcholine-induced endothelium-dependent vasodilation,in parallel with a decrease in e NOS protein in the vascular tissues.Inhibition of TXNIP significantly improved the endothelium-dependent vasodilation and restored e NOS expression and activation.(3)Compared with hypertensive rats,inhibition of TXNIP significantly decreased plasma MDA concentration(9.39 ± 1.11 vs.16.61 ± 0.67μM;P < 0.01),while increased SOD activity(68.65 ± 6.37 vs.48.18 ± 4.61 U/m L;P < 0.05)of hypertensive rats.DHE staining showed that ROS generation of aorta was effectively suppressed after TXNIP inhibition.Western blot further indicated that inhibition of TXNIP could upregulate antioxidant proteins(SOD2 and NRF2),but downregulate pro-oxidants(NOX4 and NOX2)in aorta of hypertensive rats.Conclusion: The vascular endothelium-dependent relaxation function was impaired in hypertensive rats;inhibition of TXNIP can improve endothelial function and suppress systematic and vascular oxidative stress in hypertensive rats.PART Ⅲ REGULATORY EFFECTS OF TXNIP ON ENDOTHELIAL FUNCTION IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLObjective: The increased Ang Ⅱ in circulation is a curial pathological stimulus contributing to endothelial dysfunction in hypertension.Ang Ⅱ can lead to vascular endothelial cell injury through promoting oxidative stress and apoptosis.This part is aimed to investigate the regulatory effects of TXNIP on endothelial function in human umbilical vein endothelial cell(HUVEC)upon Ang Ⅱ stimulation.Methods: Specific small interfering RNA(si RNA)targeting TXNIP and negative control(NC)RNA were constructed,and were transiently transfected into HUVEC prior to Ang Ⅱ treatment(10-6M).According to different treatments,cells were dived into the following 5 groups: control,control + TXNIP-si RNA,Ang Ⅱ,Ang Ⅱ+ NC-si RNA,and Ang Ⅱ+ TXNIP-si RNA.DCFH solution was used to detect intracellular ROS generation.Annexin V/PI staining associated with flow cytometry were used to evaluate the apoptosis rates of HUVEC.The intracellular nitric oxide(NO)production was stained by a DAF-FM fluorescent probe.The protein levels of AKT、p-AKT(ser 473)、e NOS、p-e NOS(ser 1177)in associated with several redox proteins(NOX2、NOX4、SOD2、NRF2)and apoptosis-related proteins(ASK1、BCL2、BAX、cleaved-Caspase 3、Caspase 9)were determined by Western blot.Results:(1)Using TXNIP-si RNA successfully approached an obvious reduction of the m RNA and protein levels of TXNIP in HUVEC(both P < 0.01);while,NC-si RNA didn’t affect TXNIP expression(P > 0.05).(2)Compared with the control group,the intracellular ROS and apoptosis rates were significantly increased in the Ang Ⅱ group,and administration of Ang Ⅱ obviously suppressed e NOS activation,resulting in an impairment of NO production in HUVEC(all P < 0.05).(3)Compared with the Ang Ⅱ+ NC-si RNA group,TXNIP knockdown diminished Ang Ⅱ-induced intracellular ROS generation,and downregulated NOX2 and NOX4 expression,but upregulated SOD2 and NRF2 expression in HUVEC(all P < 0.05).(4)Compared with the Ang Ⅱ+ NC-si RNA group,TXNIP knockdown alleviated Ang Ⅱ-induced cell apoptosis as evidenced by a reduction of apoptosis rate [(8.75 ± 0.23)% vs.(14.23 ± 1.60)%,P < 0.05],and decreased the protein levels of ASK1,cleaved-Caspase 3 and Caspase 9,but enhanced the BCL2/BAX ratio in Ang Ⅱ-treated HUVEC(all P < 0.05).(5)TXNIP knockdown significantly upregulated the ratios of p-AKT(ser 473)/AKT and p-e NOS(ser 1177)/e NOS in Ang Ⅱ-treated HUVEC(both P < 0.05).Importantly,compared with the Ang Ⅱ+ NC-si RNA group,the impaired NO production of HUVEC induced by Ang Ⅱ was partly rescued in the Ang Ⅱ+ TXNIP-si RNA group(P < 0.05).Conclusion: TXNIP is closely related to Ang Ⅱ-induced vascular endothelial dysfunction,and inhibition of TXNIP can effectively improve endothelial function through suppressing oxidative stress and cell apoptosis.PART Ⅳ REGULATORY MECHANISMS OF TXNIP ON VASCULAR ENDOTHELIAL CELL FUNCTIONObjective: As a major endogenous inhibitor of thioredoxin(TRX)system in organism;in hypertension,whether TXNIP promotes endothelial dysfunction through negatively regulating TRX system is currently unknown.This part is designed to explore the underlying molecular mechanisms of TXNIP on endothelial function.Methods: After transfected with TXNIP-si RNA and NC-si RNA,HUVEC was stimulated with Ang Ⅱ;the grouping mode was referenced to PART ⅡI.The expression of TRX was determined by RT-q PCR,Western blot,and cell immunofluorescence,respectively.The nuclear extracts of HUVEC were isolated for TRX,AP-1,and REF-1 detection.In addition,the activity of thioredoxin reductase(TRXR)in each group was detected by a TRXR assay kit.Subsequently,HUVEC was pretreated with PX-12(an inhibitor of TRX),and then dived into the following 4 groups: Control,Ang Ⅱ,Ang Ⅱ+TXNIP-si RNA,and Ang Ⅱ+TXNIP-si RNA+PX-12.The intracellular ROS,apoptosis rate,and the protein levels of e NOS and p-e NOS(ser 1177)in each group were detected.A TRX-loaded GV230 plasmid(overexpressing TRX protein)and NC plasmid were constructed and transfected into HUVEC;and the expression of e NOS and p-e NOS(ser 1177)in paralleled with intracellular NO production were evaluated in the presence or absence of Ang Ⅱ stimulation.Results:(1)The TRX system could be activated by Ang Ⅱ,as a compensatory response to external stimulus;compared with the control group,the m RNA and protein levels of TRX were relatively increased in the Ang Ⅱ group(both P < 0.05).(2)As compared with the Ang Ⅱ+ NC-si RNA group,the expression of TRX was further increased associated with higher activity of TRXR in the Ang Ⅱ+ TXNIP-si RNA group(both P < 0.05).(3)TXNIP knockdown promoted the nuclear translocation of TRX,and further upregulated the expression of transcription factors(AP-1 and REF-1)in the nucleus of Ang Ⅱ-treated HUVEC(both P < 0.05).(4)The regulatory effects of TXNIP on oxidative stress,cell apoptosis,and e NOS activation could be completely reversed by PX-12.(5)Upon Ang Ⅱ stimulation,overexpression of TRX could upregulate p-e NOS(ser 1177)/e NOS ratio and promote intracellular NO production in HUVEC(all P < 0.05).Conclusion: TXNIP is involved in mediating endothelial dysfunction through negatively regulating TRX system in hypertension;maintaining TXNIP/TRX system homeostasis may be a favorable target for alleviating vascular endothelial injury induced by hypertension.