| Part 1 Correlation research between serum sgp130 and sex hormones in male patients with coronary atherosclerosis diseaseBackgroud and Objective:Coronary atherosclerotic disease(CAD)is a chronic inflammatory disease caused by plenty of factors.C Reactive Protein(CRP)is an important risk factor of CAD.Interlenkin 6(IL-6),the upstream inflammatory factor of CRP,is an independent risk factor of cardiovascular diseases,which can induce the endothelial damage and promote the formation of atherosclerotic plaques.Soluble glycoprotein 130(sgp130),which is the natural antagonist of IL-6 Trans signaling pathway,can inhibt the inflammatory responses induced by IL-6 and then plays important roles in anti-atherosclerosis.In addition,sex hormones(mainly testosterone and estradiol)can inhibit the inflammatory responses in the progression of atherosclerotic plaque formation.Therefore,in this part of research,we aimed to study the relationship among serum sgp130,testosterone,estradiol and the ratio of testosterone to estradiol in male patients of CAD.Methords:1.A total of 376 male patients were recruited from the Department of Cardiovascular of Renmin Hospital of Wuhan University.All subjects were divided into the CAD group(254 cases)and control group(122 cases)according to the results of coronary angiography.2.Circulating IL-6,soluble IL-6 receptor ?(s IL-6R)and sgp130 were measured by enzyme-linked immunosorbent kits.3.Serum testosterone and estradiol were detected by Siemens ADVIA Centaur XP.4.Serum hs-CRP,lipids and other biochemical data were determined by Siemens ADVIA 2400 automatic biochemistry analyzer.5.Effects of testosterone and estradiol on sgp130 were studied in supernatant of primary umbilical vein endothelial cells(HUVECs).Results:1.Serum levels of sgp130,testosterone,estradiol and testosterone/estradiol ratio were decreased in male CAD patients compared with the control group(all P < 0.001).2.Pearson correlation analysis showed that serum sgp130 was positively correlated with testosterone and estradiol(r = 0.295 and 0.338,all P < 0.001).3.Multiple linear regression analysis showed that serum sgp130 was positively correlated with estradiol and testosterone/estradiol ratio(? = 0.450 and 0.257,all P< 0.001).4.The combination of testosterone and estradiol can significantly increase sgp130 concentrations in the culturing supernatant of HUVECs.Conclusions:Low levels of serum sgp130 were positively associated with sex hormones in male patients with CAD,suggesting an important role of sgp130 in the presence of low and imbalanced sex hormones levels.Thus,regulation of sgp130 levels by rebalancing sex hormones has the potential to be a novel therapeutic for the treatment of CAD.Part 2 Mechanistic research of the synergistic effect of estradiol and testosterone on inhibition of IL-6/gp130 signaling pathwayBackgroud and Objective:Atherosclerosis is the potential pathologic basis of coronary atherosclerosis disease(CAD).The vascular endothelial injury induced by inflammation is an important initial factor of atherosclerosis.Interleukin 6(IL-6)is a vital member of the neuro-endocrine-immune regulatory network and plays important roles in inflammatory response.While glycoprotein 130(gp130)is the signal transduction receptor of IL-6and it has been demonstrated that sex hormones can affect the levels and functions of gp130 through their receptors,and then block its downstream signal transduction and biological effects.In addition,sex hormones are closely related to the pathogenesis of CAD.In this section,we study the synergistic effects of testosterone and estradiol on inflammation induced by IL-6 in human umbilical vein endothelial cells(HUVECs).The levels of gp130 and its downstream signaling pathways were detected by western blotting.Furthermore,we explored the possilble mechanism of the synergistic effects of testosterone and estradiol in regulating IL-6/gp130 signaling pathway.Methords:1.IL-6 plus s IL-6R? was used to induce the high expression of gp130 and activate its downstream signaling pathway.In addition,estradiol and(or)testosterone were used for treatments at the same time.2.The expression of gp130 and its downstream signaling pathways including JAK/STAT3,PI3K/AKT and MEK/Erk1/2 were detected by western blotting.3.The HUVECs cultured supernatant levels of tumor necrosis factor(TNF)-? and monocyte chemotactic factor(MCP)-1 were measured by ELISA.4.Immunofluorescence assay was used to detect the levels of intercellular adhesion molecule(ICAM)-1 in HUVECs.5.Gp130-/-HUVECs were established by Crispr-cas9 to determine whether the synergistic effects of testosterone and estradiol were mediated through gp130.6.Immunoprecipitation was used to detect whether there were direct interactions between gp130 and receptors of testosterone and estradiol.7.The inhibitors and agonists of estradiol receptors were used to verify if the inhibition of estradiol on gp130 was through its receptors.Results:1.Testosterone combined with estradiol significantly inhibited the levels of gp130 and the phosphorylation of IL-6/gp130/STAT3 signaling pathway in HUVECs.Otherwise,PI3K/AKT and MEK/Erk1/2 signaling pathways were significantly activated by testosterone plus estradiol.2.Testosterone combined with estradiol significantly inhibited the levels of TNF-?,MCP-1 and ICAM-1 up-regulated by IL-6.3.In gp130-/-HUVECs,IL-6 and s IL-6? no longer inhibited the activation of PI3K/AKT signal pathway,but the combination of testosterone and estrogen still significantly activated the PI3K/AKT signaling pathway.4.In gp130-/-HUVECs,IL-6 and s IL-6? inhibited the phosphorylation of MEK/Erk1/2,but the combination of testosterone and estrogen no longer activated MEK/Erk1/2signaling pathway.5.The results of immunoprecipitation showed that ER? and ER? could direct interact with gp130.What's more,AR could direct interact with AKT and SOCS3.6.The inhibitors of ER? and ER? significantly eliminated the inhibitory effects of estradiol on gp130,while the agonists significantly inhibited gp130 levels.Conclusions:According to the results,synergistic effects of testosterone and estradiol significantly inhibited the expression of gp130 protein and the activation of JAK/STAT3 signaling pathway,simultaneously phosphorylated PI3K/AKT and MEK/Erk1/2signaling pathways,thereby reducing decreased levels of TNF-?,ICAM-1 and MCP-1.Besides,the possible mechanisms are that,through corresponding receptors,testosterone and estradiol inhibit gp130 protein levels and affect the functions of gp130 and its downstream signaling molecules,thus regulating the biological effects of IL-6/gp130 signaling pathway.Part 3 Mechanism Research of the Synergistic Effect of Estradiol and Testosterone on the anti-atherosclerosis through IL-6/gp130 signaling pathway in ApoE-/-miceBackgroud and Objective:Gender and age are independent risk factors of coronary atherosclerosis disease(CAD),which suggest that sex hormones are associated with the occurrence of CAD.However,both estrogen replacement therapy(ERT)and androgen replacement therapy(TRT)are difficult to be used in clinic due to their severe adverse reactions.In recent years,the theories of neuro-endocrine-inflammation network have made new explanations for the pathophysiological process of CAD.Weighing the advantages and disadvantages of androgen and estrogen in the cardiovascular system,the balance of sex hormones plays an important role in maintaining the normal physiological function of the body.Besides,interleukin 6(IL-6)is an important pro-inflammatory factor and activates the downstream signaling pathway through gp130 protein which then promotes the expression of chemokines and adhesion molecules,thus inducing inflammatory responses and endovascular injury.Preliminary reseaches have confirmed that soluble glycoprotein 130(sgp130),the natural inhibitor of IL-6 Trans signal pathway,is closely related to sex hormones in male CAD patients.Besides,we have demonstrated that the combination of testosterone and estradiol inhibited gp130 levels and regulated its downstream signal pathways in human umbilical vein endothelial cells(HUVECs),thus suppressing the inflammatory response induced by IL-6.The purpose of this study was to further investigate the synergistic effect of testosterone and estradiol on IL-6/gp130 signaling pathway and anti-atherosclerosis effect in low-sex-hormones male mice lacking apolipoprotein E(ApoE-/-)model,and to provide new strategies for the prevention and treatment of CAD.Methords:1.Eight-week-old male ApoE-/-mice underwent orchiectomy.Two weeks later,atherogenesis models were established by high-fat diet.At the same time,estradiol and(or)testosterone were used for treatments at the same time.2.Serum testosterone,estradiol,tumor necrosis factor(TNF)-? and IL-6 were measured by enzyme-linked immunosorbent kits.3.The overall oil red O staining was performed to observe the formation of atherosclerotic plaques in the aorta of mice in each group.4.HE staining was performed to observe the injury of endothelial cells and the proliferation of vascular smooth muscle cells.Besides,oil red O staining was used to survey the lipid deposition in the plaques of mice in each group.5.Western blotting was used to detect gp130 levels and the activation status of its downstream signaling pathways in the aorta,as well as the levels of ICAM-1.Results:1.Oil red O staining results of aorta and aortic valve in mice showed that,compared with the model group,the numbers of aorta atheromatous plaques and the lipid in plaques were slightly decreased in testosterone or estradiol treatment groups,while significantly decreased in testosterone combined with estradiol treatment group.2.HE staining results of aorta showed that,compared with the model group,the injury of endothelial cells and the proliferation of vascular smooth muscle cells were slightly improved in estradiol treatment group,while the stability of plaques was improved in testosterone treatment group.However,the injury of endothelial cells,the proliferation of vascular smooth muscle cells and the stability of plaques were significantly improved in testosterone combined with estradiol treatment group.3.The detecting of inflammatory factors showed that,comepared with the model group,the serum levels of TNF-? and IL-6 were significantly deccreaed by testosterone combined with estradiol in ApoE-/-mice(P<0.05),which could not be decreaed by estradiol or testosterone alone.4.The results of western blotting showed that,compared with model group,the levels of gp130,p-gp130,p-STAT3 and ICAM-1 were significantly decreased in aortic of the mice treated with testosterone plus estradiol(All P<0.05).Meanwhile,the PI3K/AKT and MEK/ Erk1/2 signaling pathways were significantly activated(All P<0.05).Conclusions:According to the results,testosterone combined with estradiol inhibited the serum levels of inflammatory cytokines,inhibited the formation of the aorta atheromatous plaque and delayed the development of atherosclerosis in ApoE-/-mice.Furthermore,we confirmed that testosterone combined with estradiol significantly decreased gp130 levels in mice artoa,inhibited the phosphorylation of gp130 and STAT3 phosphorylation,and activated PI3K/AKT and MEK/Erk1/2 signaling pathways.Therefore,we further confirmed that the synergistic effect of estradiol and testosterone played anti-atherosclerosis roles by down-regulating gp130 levels and its downstream signaling pathway which provided target protein and theoretical basis for sex hormones in the treatment of atherosclerosis. |
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