| BackgroundCardiovascular disease?CVD?has caused the death rate of urban and rural residents in China to increase year by year.It has become a major public health problem in China.Therefore,the prevention and treatment of cardiovascular diseases cannot be delayed.In 2015,epidemiological studies in China showed that the prevalence of myocardial infarction and angina pectoris in premenopausal women was lower than that of men of the same age,and the incidence of cardiovascular disease in women after menopause increased gradually,revealing that the reduction of estrogen was inseparable from the occurrence of cardiovascular disease.Estrogen replacement therapy?ERT?can protect cardiovascular disease in postmenopausal women.Although early results of the Women's Health Initiative?WHI?dismissed the cardiovascular benefits of ERT,its follow-up analysis found that ERT can reduce postmenopausal women?age<60years?in atherosclerosis?AS?who are in the"opportunity window".Incidence rate;and the results of the 2012 DOPS study in Denmark showed that women received ERT at the early stage of menopause,and their overall mortality,heart failure,and myocardial infarction rates decreased significantly after 10 years compared with the control group.Therefore,further understanding of the mechanism of action of estrogen in cardiovascular and finding more effective molecular targets and their mechanisms of action provide a reliable theoretical basis for the rational use of ERT for postmenopausal women to delay cardiovascular disease.The biological effects of estrogens are divided into gene effects and non-gene effects.The gene effect is that estrogen enters the cytoplasm and binds to cytoplasmic receptors.It enters the nucleus and binds to nuclear receptors and regulates gene expression.The non-genetic effect is essentially the membrane estrogen receptor?mER?on the cell membrane.After binding to estrogen,a series of signal transduction pathways are recruited and activated in the cell to produce various rapid effects.In recent years,the non-genomic effects of estrogens have gradually become a research hotspot.This is because studies have found that non-genomic effects can have important long-term effects on their function in multiple tissue systems.For example,estrogen can inhibit neonatal death after vascular injury.Endometrial formation inhibits bone loss;but also can avoid some of the side effects of estrogen,such as not stimulating mouse endometrial formation,and has no stimulatory effect on the growth and metastasis of breast cancer in nude mice.H2S is the third kind of gas signal molecule following NO and CO.In the body,the substrate L-cysteine is catalyzed by cystathionine synthase?CBS?,cystathionine lyase?CSE?and 3-thiopyruvate thiotransferase?3-MST?produce.This molecule can freely cross the cell membrane and reach the intracellular target site,thus affecting the downstream signaling pathway.More and more experiments show that H2S is involved in in vivo immune modification,vasodilatation,oxidative stress attenuation and angiogenesis.It has been reported in the literature that endogenous H2S in vivo can protect vascular tissues from atherosclerotic lesions by reducing the intimal proliferation and inhibiting the expression of adhesion molecules.Our previous study found that estrogen stimulated the rapid release of hydrogen sulfide from endothelial cells and down-regulated the aortic atherosclerosis in mice.Based on this,we propose the scientific hypothesis that estrogen stimulation of endothelial cells to release hydrogen sulfide is mediated through non-genetic effects.To understand the non-genetic effects of estrogen-stimulated endothelial cell release of hydrogen sulfide,we have done the following research.ObjectivesTo explore the mechanism of E2 release of H2S from vascular endothelial cells through non-genetic effects and its effect on vascular function.Methods and Results1.E2 activation of mER-stimulated HUVECs releases H2S as non-gene-effect transductionSelect 10nM,1nM,100uM,10uM,1uM E2 to process the HUVECs for 15 min,collect the supernatant of the culture medium,use the sulphur sensitive electrode to measure the sulphur ion level in the supernatant to represent the level of hydrogen sulfide released by the endothelium,and find that 10uM is the optimal treatment concentration The HUVECs were treated with 10uM E2 for 5,10,15,30,and 60 min.The culture supernatant was collected.The sulphur sensitive electrode was used to measure the supernatant sulphur ion level representing the level of hydrogen sulfide released from the endothelium.The results showed that:In contrast,E2time-dependently up-regulated H2S levels,with 15 minutes being the most effective?P<0.05,n=3?.The transcription inhibitor actinomycin D?ActD?and the protein synthesis inhibitor cycloheximide?CHX?pretreated HUVECs with ActD?10uM?and CHX?200uM?for30 min by inhibiting the transcription and synthesis of the protein and inhibiting the gene effect.After treatment with E2?10uM?for 15 min,the results showed that compared with the CON group,the E2 treatment alone could increase the release of H2S?P<0.001,n=3?,and ActD?10uM?and CHX?200uM?were added after pretreatment.E2 treatment increased H2S release compared with CON?P<0.001,P<0.001,n=3?.E2-BSA blocked the E2 gene effect by connecting a macromolecule BSA that could not pass through the cell membrane on E2,and then selected 10nM,1nM,100uM,10uM,and 1uM E2-BSA to treat HUVECs for 15 min,and then collected the culture supernatant.The sulphur sensing electrode was used to measure the supernatant sulphur ion level representing the level of hydrogen sulfide released from the endothelium.It was found that 10 uM was the optimal concentration and 10 uM E2was used to treat HUVECs for 5,10,15,30,and 60 min,and the culture supernatant was collected.The sulphur sensitive electrode was used to measure the supernatant sulphur ion level representing the level of hydrogen sulfide released by the endothelium.The results showed that E2 time-dependently up-regulated H2S levels and was highest at 15 min H2S level?P<0.05,n=3?.There are three types of ER on the endothelial cell membrane:mER?,mER?,and GPR30.To understand which receptor on the E2 activation membrane exerts a non-genetic effect,GPR30 agonist G1(10-6M)treats HUVECs separately for 5 min,and ER inhibition occurs.After ICI(10-7M)and GPR30 blocker G15(10-6M)were pretreated for 30 min,E2?10uM?was used for 15 min to observe H2S release.The results showed that compared with CON group,E2 treatment alone could increase the release of H2S.?P<0.001,n=3?,the effect of E2 on H2S release was inhibited after ER inhibition?P<0.001,n=3?.After G15 pretreatment of HUVECs for 30 min,E2stimulation was added.Compared with CON group,E2 remained Can increase the level of H2S?P<0.01,n=3?.2.E2 up-regulates the phosphorylation level of CSE protein in HUVECs to promote the release of H2SE2 treatment of HUVECs 5,10,15,30,60 min,co-immunoprecipitation CSE protein phosphorylation levels,the results show that:Compared with the CON group,E2time-dependently upregulate CSE protein tyrosine and silk-threonine in endothelial cells The phosphorylation level of the acid site was the highest,and the level of tyrosine and threonine upregulated at 15 min.3.E2 activates PKGI?to up-regulate phosphorylation of threonine in CSE protein Our previous experiments showed that E2 increased H2S release by upregulating PKG activity.To prove that PKG participates in E2 release of H2S signaling pathway,PKG inhibitor?KT5823,10 mM?was used to pretreat HUVECs for 30 min,and co-precipitation was used to detect CSE protein phosphoric acid The level of H2S in the cell supernatant was measured by a free radical electrode.The results showed that compared with CON group,E2 up-regulated the phosphorylation level of tyrosine and silk-threonine sites of CSE protein,but KT5823 pretreated with E2 treatment for 15min,compared with E2 treatment group,CSE tyrosine Acid,threonine phosphorylation was significantly inhibited.Compared with CON group,E2up-regulated H2S levels?P<0.05,n=3?,but KT5823 pretreated with E2 for 15 min,compared with E2 alone,H2S release was inhibited?P<0.05,n=3?.After pretreatment of HUVECs with PKGI-?siRNA?20 nM?and PKGI-?siRNA?40 nM?for 24 h,the cells were treated with E2?10 uM?for 15 min,and HUVECs were treated with E2 for15 min.The results showed that E2 was up-regulated compared with CON group.After the release of H2S,E2 was treated with PKGI-?siRNA.Compared with the E2alone group,the phosphorylation level of CSE protein did not change significantly.E2treatment after transfection with PKGI-?siRNA and E2 treatment alone In contrast,the level of threonine phosphorylation of the CSE protein is reduced.The HUVECs were pretreated with PKGI-??2ug?and PKGI-??2ug?overexpression plasmids for 24h,and then treated with E2?10uM?for 15 min.The results showed that compared with CON group,E2 up-regulated H2S.After the release of PKGI-?Plasmid,there was no statistically significant increase in the threonine phosphorylation level of CSE protein.The phosphorylation of CSE protein was increased after transfection of PKGI-?Plasmid.The co-immunoprecipitation method used CSE antibody as IP antibody.Western blot analysis of PKGI-?and PKGI-?protein expression in CON group and E2?10?M?treatment group for 15 min showed that both PKGI-?and PKGI-?could interact with CSE.Cross-linking occurred,compared with the CON group,E2 treatment group can increase the cross-linking between PKGI-?and CSE,at the same time,compared with the CON group,the E2 treatment group reduced the cross-linking of PKG1?and CSE protein.4.E2 upregulates cGMP levels mediating PKG/CSE pathway upregulates H2S releasecGMP is the upstream signal molecule of PKG.In order to clarify whether cGMP is involved in the process of E2 promoting H2S release,we first detected the cyclic guanosine monophosphate after 5 min,10 min,15 min,30 min,and 1 h of HUVECs after E2?10uM?treatment by ELISA.The level of guanosine monophosphate?cGMP?showed that E2 could up-regulate cGMP levels in a time-dependent manner,with the strongest effect at 15 min?P<0.001,n=3?.10-12,10-13,10-14,10-15,and 10-16M cGMP-treated HUVECs were used for 15 min.Western blot was used to deter mine the level of p-VASP in the cell lysate.Immunoprecipitation was used to measure CSE as an IP antibody.The expression level of threonine was measured.The cell supernatant after dosing was used to measure the level of H2S.The results suggested that compared with the CON group,cGMP concentration-dependently increased the activity of PKG,of which the effect of 10-15M was the strongest?P<0.001,n=3?,compared with the CON group,cGMP increased the serine threonine phosphorylation level of the CSE protein in a concentration-dependent manner for 15 min,of which the 10-14 M effect was the strongest.Compared with CON group,cGMP treatment increased concentration of H2S in a concentration-dependent manner for 15 min,of which 10-15 M had the strongest effect?P<0.05,n=3?.We chose 10-15 M as the treatment concentration.HUVECs were treated with cGMP(10-15M)for 5 min,10 min,15 min,30 min,and 1h respectively.Western blot was used to deter mine the level of p-VASP in the cell lysate,and CSE was used as an IP antibody by immunoprecipitation.Western blot was used to measure the expression of serinethreonine.The cell supernatant after dosing was used to measure H2S levels.The results showed that cGMP(10-15M)increased PKG activity in a time-dependent manner compared with CON group,with the strongest effect at 10 min?P<0.01,n=3?.Compared with CON group,cGMP(10-15M)increased the phosphorylation level of CSE protein in a time-dependent manner,with the strongest effect at 15 min.Compared with the CON group,cGMP(10-15M)enhanced the release of H2S in a time-dependent manner,with the strongest effect at 10 min?P<0.01,n=3?.5.E2 mediates the level of cGMP that up-regulates PKG's upstream signaling molecules by GCGC-catalyzed GTP production in intracellular cGMP by GC,there are two types of intracellular GC:pGC-And sGC,in order to understand which kind of GC participates in the process of E2 to promote H2S release,add sGC inhibitor?NS2028,10nM?and agonist?BAY41-2272,3nM?,respectively.Pretreated HUVECs for 30 min,E2?10uM?treated cells for 15 min,the results showed that compared with CON group,E2increased the level of cGMP in cells?P<0.001,n=3?,and NS2028 pretreated 30 min after E2 After treatment for 15 min,there was a significant difference in cGMP levels compared with the E2 alone group?P<0.05,n=3?.The level of p-VASP was detected by Western blot.Compared with CON group,the expression of p-VASP was increased in E2 group?P<0.05,n=3?.The sGC agonist BAY41-2272 group also increased the expression of p-VASP?P<0.05,n=3?,E2 treatment for 15 min after NS2028 pretreatment for 30 min,the expression level of p-VASP decreased compared with E2 alone treatment group?P<0.05,n=3?.The H2S level in the cell supernatant was detected by free radical electrode:Compared with the CON group,the H2S level in the E2 group was increased?P<0.01,n=3?,and E2 was treated for 15min after the NS2028 pretreatment for 30 min,compared with the E2 alone group.The level of H2S decreased?P<0.001,n=3?.sGC is a cytoplasmic receptor of NO.After adding the NO donor?SNP,100 nM?and NO inhibitor?LNNA,1 mM?for 1 h,the cells were treated with E2?10 uM?for 15min.The results showed that:Compared with the E2 alone treatment group,the PKG activity was increased?P<0.05,n=3?.The SNP treatment group could increase the expression of p-VASP?P<0.01,n=3?;LNNA pretreatment HUVECs 1h after E2treatment At 15 min,there was no significant difference in PKG activity compared with E2 alone?P>0.05,n=3?.The HUVECs were pretreated with soluble guanylyl cyclase?pGC?siRNA?20 nM?for 24 h,and then added E2?10 uM?for 15 min.The results showed that E2 increased the H2S level compared with the CON group?P<0.05,n=3?,pGC siRNA pretreatment with E2 for 15 min,compared with E2 group,H2S levels decreased?P<0.01,n=3?.pGC ligand ANP?1 mM?was used to treat HUVECs for 5 min,10 min,15 min,30min,and 1 h.Western blot was used to deter mine the expression of p-VASP in the cell lysates.CSE was used as IP antibody and phosphorylated serthrine was detected by Western blot.The results showed that ANP increased the PKG activity in a time-dependent manner compared with the CON group.Among them,the effect of 15min was the strongest?P<0.001,n=3?.Compared with CON group,ANP time-dependently promoted the threonine phosphorylation of CSE protein,and the effect was most significant at 15 min.6.ERa/G?i2/pGC-A mediated E2 promote H2S releaseTo elucidate how E2 activates pGC-A in endothelial cells to promote H2S release effect,E2?10uM?acts on HUVECs 15 min later,the two subtypes of ER,ER?and ER?,are used as IP antibodies for co-immunoprecipitation,Western blot detection of pGC-A The level of expression.The results showed that both pGC-A and ER?and ER?were cross-linked.Compared with the CON group,cross-linking between ER?and pGC-A in the E2 treatment group increased,and cross-linking between ER?and pGC-A decreased.It has been reported that mER can bind to the non-gene effect of Gi protein's functional subunit Gi?.To understand whether G?i participates in the process of E2-mediated ER?/pGC-A pathway and increase H2S release,which G?i subtype is One process played a leading role.After E2?10uM?exposure to HUVECs for 15 min,G?i1,2,3 were used as IP antibodies respectively.After co-immunoprecipitation experiments,Western blot was used to detect the expression of ER?and pGC-A.The results showed that G?i-1 could not cross-link with ER?and pGC-A,and G?i-2 and 3could cross-link with ER?and pGC-A.Compared with CON group,E2 could increase G?i-2,3 and ER?and pGC-A Cross-linking.In order to confirm which G?i subtypes play a major role,HUVECs were transfected with G?i-2 and G?i-3 siRNA for 24 h and E2?10 uM?for HUVECs for 15 min.Western blot was used to detect the expression of p-VASP in cell lysates and cell supernatants were harvested.Liquid detection of H2S levels.The results showed that compared with the CON group,the expression level of p-VASP in E2 group increased?P<0.05,n=3?and the H2S level increased?P<0.05,n=3?.E2 was pre-transfected with G?i-2 siRNA after 24h.After 15 min of treatment,the activity of PKG was inhibited?P<0.05,n=3?and the level of H2S was reduced?P<0.01,n=3?compared with the E2alone group.Pre-transfected G?i-3 siRNA for 24 h,compared with E2 alone,E2 was treated for 15 min after pre-transfection with G?i-3 siRNA.Compared with E2 alone,there was no significant difference in PKG activity?P>0.05,n=3?.),but there was a difference in H2S levels?P<0.05,n=3?.7.E2 mediates the release of H2S through non-gene effectsThe endothelial cells were transfected with the estrogen receptor mutant ERa?D258A?plasmid,which blocked the non-gene effects of E2 by cleaving the cross-linking of membrane receptors ER?and Gi?.After transfection of ER??D258A?mutant plasmids into HUVECs for 48 h,E2?10?M?-treated cells were treated with ERa as an IP antibody and Western blot was used to detect the expression of G?i protein.Western blot analysis of cell lysates8.E2 stimulates the release of H2S from vascular endothelium and influences the overall function of blood vesselsIn order to study the effect of E2 promoting H2S release on the overall function of blood vessels,we used ApoE–/–mice as a model and were randomly divided into three groups:OVX,OVX+E2,and OVX+PPG+E2.The vascular tone was measured systematically,and blood was taken from the eyeballs to measure H2S levels in plasma using H2S radical electrodes.The mouse heart and aorta were harvested for cryosectioning,hematoxylin and oil red O staining,and aortic valve plaque area was quantified using Leica Qwin software.The results showed that compared with OVX group,the vasodilatation increased in OVX+E2 group.Compared with OVX+E2group,OVX+PPG+E2 group attenuated the effect of Ach on the vasorelaxation effect of Ach?P<0.05,n=4?;Compared with OVX group,the area of aortic valve plaque in OVX+E2 group decreased?P<0.05,n=7?,but the level of H2S increased?P<0.05,n=7?;and OVX Compared with+E2 group,the area of aortic valve plaque in OVX+PPG+E2 group increased?P<0.05,n=7?,and H2S level decreased?P<0.05,n=7?.Conclusions1.E2 binds to membrane receptor ER?and recruits Gi?as an intermediate signal,which binds to the intracellular catalytic region of guanylate cyclase?GC?and exerts a non-genetic effect.2.E2 activates PKG to cause extensive phosphorylation of downstream target proteins.3.E-stimulation of phosphorylation of the Ser-a mino acid site on CSE protein in HUVECs stimulates H2S release. |
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