Neuroprotective effects of erythropoietin on neurodegenerative and ischemic brain diseases: the role of erythropoietin receptor

Erythropoietin （Epo） is a fundamental hormone in the regulation of hematopoiesis, and other secondary roles mediated by the binding of the hormone to its specific receptor （EpoR）, which leads to an activation of key signaling pathways that induce an increase in cell differentiation, apoptosis control and neuroprotection. It has been suggested that their ftmction depends on final conformation of glycosylations, related with affinity to the receptor and its half-life. The presence of EpoR has been reported in different tissues including central nervous system, where it has been demonstrated to exert a neuroprotective function against oxidative stress conditions, such as ischemic injury and neurodegenerative diseases. There is also evidence of an increase in EpoR expression in brain cell lysates of Alzheimer＇s patients with respect to healthy patients. These results are related with extensive in vitro experimental data of neuroprotection obtained from cell lines, primary cell cultures and hippocampal slices. Additionally, this data is correlated with in vivo experiments （water maze test） in mouse models of Alzheimer＇s disease where Epo treatment improved cognitive function. These stud- ies support the idea that receptor activation induces a neuroprotective effect in neurodegenerative disorders including dementias, and especially Alzheimer＇s disease. Taken together, available evidence suggests that Epo appears to be a central element for EpoR activation and neuroprotective properties in the central nervous system. In this review, we will describe the mechanisms associated with neuroprotection and its relation with the activation of EpoR in order with identify new targets to develop pharmacological strategies.

Erythropoietin Receptor Gene (EPOR) Polymorphisms are Associated with Sow Litter Sizes

The erythropoietin receptor （EPOR） has shown to play an important role in fetal survival by promoting the maturation of red blood cells in many studies of uterine capacity and litter size in swine. In this study, we screened the porcine EPOR gene for mutations and identified five single nucleotide polymorphisms （SNPs）： g.705G〉T in intron 1, g.2 373C〉T in intron 4, and g.2 882C〉T, g.3 035A〉G, and g.3 132A〉T in intron 6. We then genotyped 247 Beijing Black （BB） sows and compared the polymorphism data with the litter sizes of 1 375 parities among the sows. At first parity, there was no association of g.2 882C〉T and g.3 132A〉T with litter sizes. However, the CT sows in g.2 882C〉T had 2.13 higher total number born （TNB） （P〈0.01） and 1.81 higher number born alive （NBA） （P〈0.01） than the CC sows and the heterozygous sows in g.3 132A〉T had the highest litter size when compared to the two homozygotes for the later parities （P〈0.05）. In the g.3 035A〉G SNP, for the later parities, the TNB of the sows with the GG genotype was 3.81 higher （P〈0.01） and the NBA was 2.75 higher （P〈0.01） than that with the AA genotype but no difference at first parity. The G allele of the EPOR g.705G〉T SNP was associated with a greater litter size at both the first parity （P〈0.05） and later parities （P〈0.01）. Furthermore, we determined the allele frequencies for this SNP among five Chinese indigenous pig breeds （Erhualian, Laiwu Black, Meishan, Min, and Rongchang） and three western commercial pig breeds （Duroc, Landrace, and Large White）. The G allele of the EPOR g.705G〉T SNP was significantly more common in the more prolific Chinese breeds. These results indicated that the EPOR could be an important candidate gene for litter size and g.705G〉T can serve as a useful genetic marker for improving litter size in both first and later parities in swine.

Erythropoietin receptor antibody inhibits oxidative stress induced retinal neovascularization in mice

AIM: To observe the effect of erythropoietin receptor antibody (EpoRA) on oxygen-induced retinal neovascularization. METHODS: C57BL / 63 mice, newly born 7 days, were exposed in high oxygen for 5 days and then placed in normal air for another 5 days, thus the animal models of retinal neovascularization were made. Experimental animals were allocated into 3 groups: normal, experimental and therapeutic. The normal group was fed in the normal environment. Into the vitreous cavity of mice in the therapeutic group were injected 2 mu L of EpoRA for 5 successive days. And the experimental group was injected the same amount of normal saline. Mice were sacrificed 17 days after birth and their eyeballs were removed for detection of malonaldehyde (MDA) content in the retina and by HE staining endothelial cells were counted the breaking through internal limiting membrane. RESULTS: In the experimental group, MDA content in the retina was 25.11 +/- 3.46 mu mol/g, which was obviously less than those in the normal group (5.34 +/- 1.79 mu mol/g, P<0.01) and those in the therapeutic group (12.04 +/- 1.91 mu mol/g). Pathological sections showed the nuclear number of the endothelial cells breaking through internal limiting membrane was 0.7 +/- 0.2 in normal group, and 46.2 +/- 6.5 in high oxygen induced experimental group. In the therapeutic group injected with EpoRA, it was lowered to 24.0 +/- 5.0(P < 0.01). CONCLUSION: EpoRA can effectively inhibit oxygen-induced neovascularization in retina of mouse by reducing oxidative damage.

The Erythropoietin Receptor: Dimerization and Intracellular Signal Transduction

Erythropoietin (EPO), a 34 kD glycopro-tein, is the principal growth factor regulating theproduction of circulating erythrocytes; EPO isessential for committed CFU - E erythroid pro-genitors to divide several times and then to dif-ferentiate into erythrocytes. Like most receptorsfor hematopoietic growth factors, the erythro-poietin receptor (EPO - R) is a type I trans-membrane protein and a member of the cytokinereceptor superfamily. These receptors containfour conserved cysteines and a Trp - Ser - X -

Prognostic significance of erythropoietin and erythropoietin receptor in gastric adenocarcinoma

AIM: To investigate the expression of Erythropoietin (Epo) and its receptor (EpoR) in gastric adenocarcinoma (GAC) and the correlation with angiogenesis and clinicopathological features. METHODS: The expressions of Epo, EpoR and vascular endothelial growth factor (VEGF), as well as mi-crovessel density were evaluated in 172 GAC biopsies by immunohistochemical staining. The correlations between these parameters and patient’s clinicopathological features were analyzed statistically. RESULTS: The proportion of Epo and EpoR alterations in GAC was higher than that in adjacent normal mucosa (P = 0.035 and 0.030). Epo high-expression was associ-ated with EpoR high-expression, Lauren type, extensivelymph node metastasis and advanced stage of GAC (P = 0.018, 0.018, 0.004 and 0), while EpoR expression was linked with older age, World Health Organization type, extensive lymph node metastasis and advanced stage (P = 0.001, 0.013, 0.008 and 0.001). VEGF high expression was significantly correlated with EpoR low-expression, Lauren type, extensive lymph node metastasis and advanced stage (P = 0.001, 0.001, 0.001 and 0.007). The expression of Epo or EpoR was associated with microvessel density (P = 0.004 and 0.046). On multivariate analysis, only lymph node metastasis, abnormal Epo expression and tumor nodes metastases stage were independently associated with survival. In addition, a strong association with the immunohistochemical expression of EpoR and the angiogenic protein, VEGF, was noted. CONCLUSION: Increased expression of Epo and EpoR may play a signif icant role in the carcinogenesis, angiogenesis and progression of GAC. Epo may be an inde-pendent prognostic factor.

Functional significance of erythropoietin receptor on tumor cells

Erythropoietin (Epo) is the regulator of red blood cell formation. Its receptor (EpoR) is now found in many cells and tissues of the body. EpoR is also shown to occur in tumor cells and Epo enhances the proliferation of these cells through cell signaling. EpoR antagonist can reduce the growth of the tumor in vivo. In view of our current knowledge of Epo, its recombinant forms and receptor, use of Epo in cancer patients to enhance the recovery of hematocrit after chemotherapy treatment has to be carefully evaluated.

Constitutively Active Soluble Form of Erythropoietin Receptor Suppresses Growth and Angiogenesis of Xenografts of Transfected Cancer Cell Lines

We examined the effect of blocking the erythropoietin (Epo) signaling using an anti-Epo antibody, soluble form of Epo receptor (sEpoR) capable of binding to Epo or EpoR antagonist, and proved to be effective against xenografts of female reproductive organ malignancies and of cancer cell lines in nude mice. We transfected seven cancer cell lines of various origins to express constitutively active sEpoR, and examined their tumorigenesis in nude mice. Suppression of the tumor growth, decrease in viable and proliferating cells and reduction of vascular density were seen individually in all xenografts of transfected cell lines compared with the controls. Quantitative RT-PCR analyses showed that expression levels of Epo, EpoR, ?1A-adrenaline receptor (?1A-ADR) and muscalinic acetylcholine receptor subunit 3 mRNAs (m3-AchR) were higher in the majority of the wild-type xenografts than in the corresponding cell lines except for A549. In some of the transfected xenografts, EpoR, ?1A-ADR and m3-AchR mRNAs were down-regulated. Western blot analyses revealed that the constitutively activated ERK1/2MAPK was discernible in the majority of non-transfected cell lines and was reduced in the transfected cell lines. However, it was regained after exposure to acetylcholine and/or noradrenaline. These findings suggest that constitutively active sEpoR can effectively destroy the xenografts but signals from the autonomic neurotransmitters of the host produced under stress may interfere with this antitumor activity.

Knockout of the tumor necrosis factor a receptor 1 gene can up-regulate erythropoietin receptor during myocardial ischemia-reperfusion injury in mice

Background Tumor necrosis factor a receptor 1 （TNFaR1） plays an important role in the signal pathway of apoptosis. The objective of this study was to investigate the effects of TNFaR1 knockout on the up-regulation of erythropoietin receptor （Epo-R） and the coordinated anti-apoptosis functions during myocardial ischemia-reperfusion injury in mice. Methods The ischemia-reperfusion injury model for cardiomyocytes was performed by ligating the left circumflex branch artery of TNFaR1 knockout （P55/） C17 B6 mice, as well as wild-type （P55^＋/^＋） C17 B6 mice. Triphenyltetrazolium chloride （TTC） staining was performed to observe the damaged area of the heart. TUNEL staining and DNA fragmentation were used to identify apoptosis. Mitochondrial Bcl-2 and Bax as well as expression of Epo-R and its downstream genes （Jak-2, stat-5, Akt, lkB-a, HIF-1a） were measured by Western blotting. The gene knockout mice were assigned into those undergoing the apoptosis surgical model group （KO group）, and those subjected to sham operation （KOs group）. Similarly, wild-type mice were either exposed to the surgical model （WT group） or subject to a sham operation （WTs group）. Results The myocardial damage ratio of the wild-type group after the operation was significantly higher than that of the knockout group, （50.5±6.4）% vs （36.9±6.9）%, P 〈0.01. Similarly, TUNEL positive ratio of the wild-type group was significantly higher than that of the knockout group, （63.1±5.6）% vs （42.1±4.7）%, P〈0.01. The gray value ratios of Epo-R, Jak-2, stat-5, Akt, IkB-a, HIF-1 and mitochondrial Bcl-2 in the KO group were significantly higher than those of the WT group, P 〈0.05; however, mitochondrial Bax was significantly lower than that of the WT group significantly （P 〈0.05）. Conclusions Using the ischemia-reperfusion injury model in mice, cardiomyocytes of TNFaR1 knockouts exhibited anti-apoptotic characteristics. This information could be used to coordinate the prevention of myocardial apoptosis by up-regulating and activating the Epo-R pathway.

Upregulation of erythropoietin and erythropoietin receptor in castration-resistant progression of prostate cancer

Hypoxia-induced erythropoietin signaling plays an important role in tumor growth and invasion.In the present study,we investigated the contribution of erythropoietin signaling pathway to castration-resistant prostate cancer and the development of a neuroendocrine phenotype.Immunohistochemical staining showed that the erythropoietin and erythropoietin receptor scores in castration-resistant prostate cancer and androgen-dependent prostate cancer were 7.55 versus 4.5 and 7.45 versus 5.9,respectively(P<0.001).Furthermore,a cell proliferation assay was conducted,and the differential expression of erythropoietin and erythropoietin receptor in LNCaP cells and hypoxia-induced LNCaP cells was evaluated using western blot and quantitative real-time PCR.The proliferation capacity of hypoxia-induced LNCaP cells was similar in cultures of both fetal bovine serum and charcoal-stripped fetal bovine serum,suggesting that LNCaP cells acquired hypoxia-induced androgen-independent growth.After 2 weeks of hypoxic culture,LNCaP cells showed a neuroendocrine cell change and increased expression of neuron-specific enolase,erythropoietin,and erythropoietin receptor;knockdown of erythropoietin receptor reversed the hypoxia-induced upregulation of neuron-specific enolase in the LNCaP cells.In conclusion,the concurrent upregulation of erythropoietin and erythropoietin receptor in castration-resistant prostate cancer suggests that the erythropoietin/erythropoietin receptor autocrine loop plays an important role in the progression of castration resistance and is responsible for the development of a neuroendocrine phenotype.

IMMUNOHISTOCHEMICAL DISTRIBUTION OF ERYTHROPOIETIN AND ITS RECEPTOR EXPRESSION IN POSTNATAL RAT RETINA DEVELOPMENT

Objective To investigate the distribution of erythropoietin (EPO) and erythropoietin receptor (EPOR) expression in the postnatal rat retina development. Methods Forty-two male Sprague-Dawley rats were divided into 7 groups according to their various postnatal days: postnatal 1 d (D1 group), 3 d (D3 group), 1 week (W1 group), 2 weeks (W2 group), 3 weeks (W3 group), 4 weeks (W4 group) and 8 weeks (W8 group) (n=6). Single eye was randomly chosen from each rat for the study. The retinal sections were stained with hematoxylin and eosin (HE) and used for the retina development observation. Immunohistochemical staining was used to localize EPO and EPOR expressions in retinas of different stages of development, and the expression intensities were determined by an image plus 4 program. Results The retinal inner nuclear layer (INL) and outer nuclear layer (ONL) were mixed together and had not yet fully differentiated in D1 and D3 groups. The INL and ONL formed their own independent regions and the outer plexiform layer (OPL) appeared between two layers in W1 group. With the postnatal retinal development, the inner plexiform layer (IPL), rods and cones layer (RCL), and OPL were gradually widened and stabilized in W2 to W3 groups. EPO/EPOR expressions located prominently in the inner part of the postnatal rat developing retinas. The expression of EPO in GCL and INL gradually increased from D1 to W4, then the expression decreased in W8. Expression of EPOR in GCL gradually increased from D1 to W1, then decreased in W2; and it gradually increased again from W3 to W8. Expression of EPOR in INL gradually increased from D1 to W1, then decreased in W2; and it continued to decrease from W3 to W8. Expression of EPOR in the external segment of RCL gradually increased from D1 to W8. However, expression in the internal segment of RCL gradually decreased from D1 to W3, then no obvious expression was seen in the internal segment of RCL in W4 and W8. Conclusion EPO/EPOR expressions locate prominently in the inner part of the postnatal rat developing retina. And EPO/EPOR expressions in the rat retinas exist the dynamic changes during the postnatal retina development period.

Expression of EPO Receptor in Pancreatic Cells and Its Effect on Cell Apoptosis

In order to explore the expression of erythropoietin receptor （EPOR） in pancreatic cell line NIT-1 and its effect on cell apoptosis after binding with erythropoietin （EPO）, NIT-1 cells were cultured and expanded. The expression of EPOR was detected using electrophoresis. NIT-1 apoptosis was induced by cytokines and their effects on cell apoptosis and cell insulin secretion were assayed after binding of EPO to EPOR. The results showed that EPOR was expressed in NIT-1 cells. Recombinant human EPO （rHuEPO） had no effect on cell apoptosis but significantly inhibited apoptosis induced by cytokines, rHuEPO had no effect on cell insulin secretion but significantly improved insulin secretion inhibited by cytokines. From these findings, it was concluded that EPOR was expressed in NIT- 1 cells and EPO could protect NIT- 1 cells from apoptosis induced by cytokines.

Bone marrow mesenchymal stem cells transplantation promotes the release of endogenous erythropoietin after ischemic stroke

This study investigated whether bone marrow mesenchymal stem cell（BMSC） transplantation protected ischemic cerebral injury by stimulating endogenous erythropoietin. The model of ischemic stroke was established in rats through transient middle cerebral artery occlusion. Twenty-four hours later, 1 × 106 human BMSCs（h BMSCs） were injected into the tail vein. Fourteen days later, we found that h BMSCs promoted the release of endogenous erythropoietin in the ischemic region of rats. Simultaneously, 3 μg/d soluble erythropoietin receptor（s EPOR） was injected into the lateral ventricle, and on the next 13 consecutive days. s EPOR blocked the release of endogenous erythropoietin. The neurogenesis in the subventricular zone was less in the h BMSCs ＋ s EPOR group than in the h BMSCs ＋ heat-denatured s EPOR group. The adhesive-removal test result and the modified Neurological Severity Scores（m NSS） were lower in the h BMSCs ＋ s EPOR group than in the heat-denatured s EPOR group. The adhesive-removal test result and m NSS were similar between the h BMSCs ＋ heat-denatured s EPOR group and the h BMSCs ＋ s EPOR group. These findings confirm that BMSCs contribute to neurogenesis and improve neurological function by promoting the release of endogenous erythropoietin following ischemic stroke.

Carbamylated Erythropoietin Alleviates Kidney Damage in Diabetic Rats by Suppressing Oxidative Stress

The oxidative stress response plays an important role in the occurrence and development of diabetic kidney disease(DKD).It has become a new treatment target for DKD.In the current study,the effects of carbamylated erythropoietin(CEPO)on renal oxidative stress and damage in diabetic rats were examined.Thirty Sprague Dawley rats were intraperitoneally administered with 60 mg/kg streptozotocin to establish the diabetes model.The diabetic rats were randomly allocated into 4 groups(n=6 each):diabetes model group(DM group),DM+CEPO treatment group(DC group),DM+CEPO+EPO receptor(EPOR)blocking peptide treatment group(DCEB group),and DM+CEPO+CD131 blocking peptide treatment group(DCCB group).Meanwhile,a normal control group(NC group,n=6)was set up.Kidney tissues and blood samples were obtained for evaluation of oxidative stress and renal function.The results showed that diabetic rats exhibited increased oxidative stress in the kidney and early pathological changes associated with DKD.Treatment with CEPO reduced oxidative stress and attenuated renal dysfunction.However,diabetic rats treated with the combination of CEPO and EPOR blocking peptide or CD131 blocking peptide showed increased oxidative stress and reduced renal function when compared with CEPO treatment alone group.These results suggested that CEPO can protect against kidney damage in DKD by inhibiting oxidative stress injury via EPOR-CD131 heterodimers.

ncRNA介导的EphB4上调与低级别胶质瘤不良预后及肿瘤免疫浸润的相关性

目的探讨促红细胞生成素生成人肝细胞受体B4(erythropoietin-producing human hepatocellular receptors B4,EphB4)在低级别胶质瘤(lower-grade glioma,LGG)组织中的表达、其上游靶点以及与患者预后及肿瘤免疫浸润的相关性,分析其作为治疗靶点的潜在作用。方法首先应用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库胶质瘤数据分析EphB4在胶质瘤与正常脑组织中的表达差异,并用基因表达谱交互分析(Gene Expression Profiling Interactive Analysis,GEPIA)数据库分析EphB4对不同癌症生存期的影响。用R语言和starBase数据库分析EphB4可能上游调节非编码RNA(non-coding RNA,ncRNA)。用肿瘤免疫评估资源(Tumor Immune Estimation Resource,TIMER)数据库进行EphB4表达与肿瘤免疫细胞浸润、免疫细胞生物学标志物和免疫检查点相关性分析。结果UBA6-AS1/hsa-miR-346轴是最有潜力影响LGG中EphB4表达的上游ncRNA相关通路。同时,EphB4水平与LGG肿瘤免疫细胞浸润、免疫细胞生物学标志物、免疫检查点表达均呈显著正相关。结论ncRNA介导的EphB4上调与LGG的不良预后和肿瘤免疫浸润相关。

Erythropoietin -induced proliferation of gastric mucosal cells

瞄准：在胃的标本上分析红细胞生成素受体的本地化并且用一个猪的胃的上皮的房间文化模型在正常胃的上皮的增长上描绘红细胞生成素的效果。方法：红细胞生成素受体被 RT-PCR，西方的弄污和 immunohistochermistry 检测。有教养的胃的粘膜房间上的红细胞生成素的生长刺激效果被 ELISA 用 bromodeoxyuridine (BrdU ) 决定。结果：红细胞生成素受体在有教养的猪的胃的粘膜上皮细胞上被检测。红细胞生成素受体也在胃的粘膜上皮的底组织化学地被检测。BrdU 试金由红细胞生成素的管理在有教养的猪的胃的粘膜上皮细胞的生长潜力表明了剂量依赖者增加，以及这些效果被反的管理禁止 -- 红细胞生成素抗体(P【0.01 ) 。结论：这些调查结果显示红细胞生成素有一个潜力增殖经由红细胞生成素的胃的粘膜上皮受体。

Hepc/25(OH)D3、EPO、sTfR-F与慢性肾衰竭MHD患者肾性贫血关系及联合预测并发感染的ROC分析

目的:探讨铁调素与维生素D3比值[Hepc/25(OH)D3]、促红细胞生成素(EPO)、可溶性转铁蛋白受体/铁指数(sTfR-F)与慢性肾衰竭维持性血液透析(MHD)患者肾性贫血的关系及联合预测并发感染的价值。方法:选取2018年1月-2020年12月我院139例慢性肾衰竭MHD患者,根据透析1个月内是否并发感染分为感染组(n=32)、非感染组(n=107),比较两组Hepc/25(OH)D3、EPO、sTfR-F、血红蛋白(Hb)、转铁蛋白饱和度(TSAT)、血细胞比容(HCT),分析Hepc/25(OH)D3、EPO、sTfR-F与肾性贫血相关指标关系、感染相关影响因素及各指标预测感染的价值。结果:感染组Hepc/25(OH)D3、sTfR-F高于非感染组,EPO低于非感染组(P<0.05);感染组Hb、TSAT、HCT低于非感染组(P<0.05);Hepc/25(OH)D3、sTfR-F与Hb、TSAT、HCT呈负相关,EPO与Hb、TSAT、HCT呈正相关(P<0.05);Hepc/25(OH)D3、EPO、sTfR-F均为慢性肾衰竭MHD患者发生感染的影响因素(P<0.05);Hepc/25(OH)D3、EPO、sTfR-F预测慢性肾衰竭MHD患者并发感染的曲线下面积(AUC)分别为0.810、0.784、0.765,各指标联合预测的AUC最大,为0.864。结论:Hepc/25(OH)D3、EPO、sTfR-F与慢性肾衰竭MHD患者肾性贫血密切相关,且是感染发生的影响因素,各指标联合在预测感染发生方面具有较高应用价值。

多瘤病毒增强活化子3和红细胞生成素产生肝细胞受体A2在脑胶质母细胞瘤中的作用

目的探讨多瘤病毒增强活化子3(PEA3)、红细胞生成素产生肝细胞受体A2(EPHA2)在脑胶质母细胞瘤中的作用。方法构建人脑星形胶质母细胞瘤U87细胞基因转染模型,将细胞系分为5组:空白组、PEA3干扰组、PEA3干扰空载组、EPHA2干扰组、EPHA2干扰空载组。通过细胞计数试剂盒8(CCK-8)实验检测细胞增殖率,细胞划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力。结果CCK-8检测细胞增殖结果显示,PEA3干扰组和EPHA2干扰组的细胞增殖率均明显低于空白组[(59.7±1.3)%、(59.5±0.7)%比(67.8±1.3)%](P<0.01或P<0.001),PEA3干扰空载组和EPHA2干扰空载组的细胞增殖率与空白组比较差异均无统计学意义(均P>0.05)。细胞划痕实验结果显示,PEA3干扰组和EPHA2干扰组的划痕愈合率低于空白组[(31.7±1.1)%、(23.0±2.6)%比(42.8±8.8)%],差异均有统计学意义(均P<0.05),PEA3干扰空载组和EPHA2干扰空载组划痕愈合率与空白组比较差异均无统计学意义(均P>0.05)。Transwell检测细胞侵袭能力结果显示,PEA3干扰组和EPHA2干扰组的侵袭细胞数明显少于空白组[(643±20)、(542±165)个比(1225±70)个](均P<0.001),而空白组与PEA3干扰空载组和EPHA2干扰空载组侵袭细胞数比较差异均无统计学意义(均P>0.05)。结论干扰PEA3基因和EPHA2基因能明显降低脑胶质母细胞瘤细胞的增殖、迁移、侵袭能力。

低氧环境下EphrinB2/EphB4调控MC3T3⁃E1细胞成骨分化的实验研究

目的探讨低氧环境下促红细胞生成素肝细胞激酶受体配体B2/促红细胞生成素肝细胞激酶受体B4(erythropoietin producing hepatocyte kinase receptor ligand B2/erythropoietin producing hepatocyte kinase receptor B4,EphrinB2/EphB4)对MC3T3-E1细胞成骨分化的影响,为低氧调控成骨细胞分化提供实验依据。方法设置对照组与氯化钴诱导的低氧组,对MC3T3-E1细胞进行分组培养,应用qRT-PCR检测细胞成骨标志物碱性磷酸酶(alkaline phosphatase,ALP)、runt相关转录因子2(runt related transcription factor 2,RUNX2)、I型胶原(collogen-1,COL I)、骨钙素(osteocalcin,OCN)的mRNA表达变化,ALP染色检测细胞成骨诱导7 d后ALP活性。同时通过qRT-PCR与Western blot检测两组MC3T3-E1细胞缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、EphrinB2、EphB4的mRNA和蛋白表达。然后增设氯化钴+EphB4磷酸化抑制剂组(加入EphB4磷酸化抑制剂NVP-BHG712)阻止EphrinB2与EphB4结合,通过qRT-PCR与Western blot检测三组成骨标志物ALP、RUNX2、COL I、OCN的mRNA与蛋白表达,ALP染色、茜素红染色检测细胞成骨诱导后ALP活性及矿化情况。结果与对照组相比,在氯化钴诱导的体外细胞低氧环境下,MC3T3-E1成骨标志物ALP、RUNX2、COL 1、OCN mRNA表达增加,ALP活性增强,矿化增强(P<0.05)。同时,在氯化钴诱导的低氧环境下,HIF-1α、EphrinB2、EphB4的mRNA和蛋白水平表达增加(P<0.05)。使用NVP-BHG712阻止EphrinB2和EphB4的结合后,细胞的成骨标志物表达下降,ALP活性及矿化能力降低(P<0.05)。结论低氧环境可通过EphrinB2/EphB4信号通路促进MC3T3-E1细胞的成骨分化,增加成骨标志物的表达及组织矿化。

循环EphA2、PGRN与冠状动脉疾病患者内皮炎症及黏附因子的相关性

[目的]探讨促红细胞生成素产生肝细胞受体A2(EphA2)、颗粒蛋白前体(PGRN)与冠状动脉疾病(CAD)患者内皮炎症及黏附因子的相关性。[方法]入选2020年1月—12月复旦大学附属中山医院心内科收治的拟行冠状动脉造影的73例CAD患者,入院次日晨空腹采集肘静脉血5 mL,ELISA测定血清中EphA2、PGRN水平,Premixed Luminex测定肿瘤坏死因子α(TNF-α)、白细胞介素2(IL-2)、白细胞介素6(IL-6)、单核细胞趋化蛋白1(MCP-1)、血管细胞黏附分子1(VCAM-1)及γ干扰素(IFN-γ)水平。[结果]急性冠状动脉综合征(ACS)患者血清EphA2、氨基末端脑钠肽前体(NT-proBNP)、心肌肌钙蛋白T(cTnT)、高敏C反应蛋白(hs-CRP)及肌酸激酶同工酶MB(CK-MB)心肌损伤标志物水平较慢性冠状动脉综合征(CCS)患者升高6.3倍、15倍、161倍、13倍、2.5倍(P<0.001),血清TNF-α、IL-6和VCAM-1较CCS患者升高37.9%、500.0%、196.6%(P<0.01),血清PGRN水平较CCS患者未见明显升高(P=0.051)。血清EphA2、PGRN、IL-6、VCAM-1预测ACS的曲线下面积(AUC)分别为0.932、0.646、0.926和0.861。血清EphA2、VCAM-1、IL-6、TNF-α、MCP-1均与Gensini评分正相关(r=0.533,P<0.001;r=0.549,P<0.001;r=0.621,P<0.001;r=0.263,P=0.027;r=0.264,P=0.026)。血清EphA2与IL-6、VCAM-1呈正相关(r=0.565,P<0.001;r=0.474,P<0.001)。未见PGRN与各炎症因子和黏附因子之间的相关性(P>0.05)。血清PGRN、EphA2与心肌损伤标志物呈正相关(P<0.05)。[结论]本研究从临床的角度支持EphA2参与斑块损伤急性期内皮炎症过程,同时提示EphA2、PGRN有望成为干预内皮炎症与预测动脉粥样硬化病变进展的潜在靶点。

间苯三酚对宫腔内人工授精妊娠结局及患者血清EphA5、SFlt-1表达的影响

目的:探究间苯三酚对宫腔内人工授精妊娠结局及患者血清促红细胞生成素产生肝细胞受体(Eph)A5、可溶性血管内皮细胞生长因子受体-1(SFlt-1)表达的影响。方法:选取2019年5月-2022年2月本院生殖医学中心收治的120例接受宫腔内人工授精术中超声发现子宫内膜蠕动快的患者,按知情同意原则分为对照组(n=60,采用宫腔内人工授精)和观察组(n=60,在对照组基础上给予间苯三酚)。比较两组妊娠结局、血清EphA5、SFlt-1、促黄体生成素(LH)、雌二醇(E_(2))水平、卵泡成熟个数、子宫内膜厚度及不良反应。结果:治疗后,观察组临床妊娠率(23.3%)、异位妊娠率、生化妊娠率(15.0%)和28周以上持续妊娠率(18.3%)均高于对照组(8.3%、3.0%、5.0%),流产率、多胎妊娠率低于对照组(P<0.05)。两组血清SFlt-1、E_(2)水平均较治疗前降低、EphA5、LH水平较治疗前升高,且观察组上述指标变化幅度大于对照组,不良反应总发生率观察组(5.0%)低于对照组(20.0%)(均P<0.05)。结论:间苯三酚可改善宫腔内人工授精妊娠结局,可提高患者血清EphA5、LH水平,降低血清SFlt-1、E_(2)水平,用药安全可靠。