Fatty Acid Synthase and Hormone-sensitive Lipase Expression in Liver Are Involved in Zinc-α2-glycoprotein-induced Body Fat Loss in Obese Mice

Objective To explore the effects of zinc-0t2-glycoprotein （ZAG） on body weight and body fat in high-fat-diet （HF1））-induced obesity in mice and the possible mechanism. Methods Thirty-six male mice were fed with standard food （SF） （n=9） and HFD （n=27）, respectively. Five weeks later, 9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method, and another 9 mice to negative control plasmid transfection. Two weeks later, serum ZAG level in the mice was assayed by Western blot, and the effects of ZAG over-expression on body weight, body fat, serum biochemical indexes, and adipose tissue of obese mice were evaluated. The mRNA expressions of fatty acid synthase （FAS） and hormone sensitive lipase （HSL） in liver tissue were deterlnined by reverse transcription-polymerase chain reaction. Results Serum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice （0.51±0.10 AU vs. 0.75±0.07 AU, P〈0.01）. Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight （r =-0.56, P〈0.001）, epididymal fat mass （r=-0.67, P〈O. 001）, percentage of epididymal fat （r= 0.65, P〈0.001）, and increased weight （r= 0.57, P〈0.001） in simple SF- and HFD fed mice. ZAG over-expression in obese mice reduced body weight and the percentage of epididyreal fat. Furthermore, FAS mRNA expression decreased （P〈0.01） and HSL mRNA expression increased （P〈0.001） in the liver in ZAG over-expressing mice. Conclusions ZAG is closely related to obesity. Serum ZAG level is inversely correlated with body weight and percentage of body fat. The action of ZAG is associated with reduced FAS expression and increased HSL expression in the liver of obese mice.

Zinc-α2-glycoprotein 1 attenuates non-alcoholic fatty liver disease by negatively regulating tumour necrosis factor-α

BACKGROUND Zinc-α2-glycoprotein 1 (AZGP1) plays important roles in metabolism-related diseases. The underlying molecular mechanisms and therapeutic effects of AZGP1 remain unknown in non-alcoholic fatty liver disease (NAFLD). AIM To explore the effects and potential mechanism of AZGP1 on NAFLD in vivo and in vitro. METHODS The expression of AZGP1 and its effects on hepatocytes were examined in NAFLD patients, CCl4-treated mice fed a high fat diet (HFD), and human LO2 cells. RESULTS AZGP1 levels were significantly decreased in liver tissues of NAFLD patients and mice. AZGP1 knockdown was found to activate inflammation;enhance steatogenesis, including promoting lipogenesis [sterol regulatory elementbinding protein (SREBP)-1c, liver X receptor (LXR), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl CoA desaturase 1 (SCD)-1], increasing lipid transport and accumulation [fatty acid transport protein (FATP), carnitine palmitoyl transferase (CPT)-1A, and adiponectin], and reducing fatty acid β-oxidation [farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR)-α];accelerate proliferation;and reverse apoptosis in LO2 cells. AZGP1 overexpression (OV-AZGP1) had the opposite effects. Furthermore, AZGP1 alleviated NAFLD by blocking TNF-α-mediated inflammation and intracellular lipid deposition, promoting proliferation, and inhibiting apoptosis in LO2 cells. Finally, treatment with OV-AZGP1 plasmid dramatically improved liver injury and eliminated liver fat in NAFLD mice. CONCLUSION AZGP1 attenuates NAFLD with regard to ameliorating inflammation, accelerating lipolysis, promoting proliferation, and reducing apoptosis by negatively regulating TNF-α. AZGP1 is suggested to be a novel promising therapeutic target for NAFLD.

Thrombophilia Caused by Beta2-Glycoprotein I Deficiency： In Vitro Study of a Rare Mutation in APOH Gene

This study aimed to explore the mechanism of a novel mutation （p.Lys38Glu） in apolipoprotein H （APOH） gene causing hereditary beta2-glycoprotein I （β2GPI） deficiency and thrombosis in a proband with thrombophilia. The plasma level of β2GPI was measured by ELISA and Western blotting, and anti-β2GPI antibody by ELISA. Lupus anticoagulant （LA） was assayed using the dilute Russell viper venom time. Deficiency of the major natural anticoagulants including protein C （PC）, protein S （PS）, antithrombin （AT） and thrombomodulin （TM） was excluded from the proband. A mutation analysis was performed by amplification and sequencing of the APOH gene. Wild type and mutant （c.112A〉G） APOH expression plasmids were constructed and transfected into HEK293T cells. The results showed that the thrornbin generation capacity of the proband was higher than that of the other family members. Missense mutation p.Lys38Glu in APOH gene and LA coexisted in the proband. The mutation led to β2GPI deficiency and thrombosis by impairing the protein production and inhibiting the platelet aggregation. It was concluded that the recurrent thrombosis of the proband is associated with the coexistence ofp.Lys38Glu mutation in APOH gene and LA in plasma.

Relationship and significance between anti-b2-glycoproteinⅠantibodies and platelet activation state in patients with ulcerative colitis

AIM： To study the relationship between anti-β2- glycoprotein Ⅰ （aβ2GPⅠ） antibodies and platelet activation state in patients with ulcerative colitis （UC） and its significance. METHODS： Peripheral blood samples were collected from 56 UC patients （34 males and 22 females, aged 43.5 years, range 21-66 years）, including 36 at active stage and 20 at remission stage, and 25 sex-and age-matched controls. The level of aβ2GP Ⅰ was measured by ELISA. The platelet activation markers, platelet activation complex- Ⅰ （PAC- Ⅰ ） and P-selectin （CD62P） were detected by flow cytometry. RESULTS： The A value for IgG aβ2GP Ⅰ in the active UC group was 0.61 ± 0.13, significantly higher than that in the remittent UC and control groups （0.50 ± 0.13 and 0.22 ± 0.14, P 〈 0.01）. There was a significant difference between the two groups （P 〈 0.01）. The A value for IgM aβ2GP Ⅰ in the active and remittent UC groups was 0.43 ± 0.13 and 0.38 ± 0.12, significantly higher than that in the control group （0.20 ± 0.12, P 〈 0.01）. However, there was no significant difference between the two groups （P 〉 0.05）. The PAC- Ⅰ positive rate for the active and remittent UC groups was 30.6% ± 7.6% and 19.6% ± 7.8% respectively, significantly higher than that for the control group （6.3% ± 1.7%,P 〈 0.01）. There was a significant difference between the two groups （P 〈 0.01）. The CD62P positive rate for the active and remittent UC groups was 45.0% ± 8.8% and 31.9% ± 7.8% respectively, significantly higher than that for the control group （9.2% ± 2.7%, P 〈 0.01）. There was a significant difference between the two groups （P 〈 0.01）. In the active UC group, the more severe the state of illness was, the higher the A value for IgG aβ2GP Ⅰ was, and the positive rate for PAC-Ⅰ and CD62P was positively correlated with the state of illness （Faβ2GP Ⅰ = 3.679, P 〈 0.05; FPAC-Ⅰ （%） = 5.346, P 〈 0.01; and FCD62P （%） = 5. 418, P 〈 0.01）. Meanwhile, in the same state of illness, the A value for IgG aβ2GP Ⅰ was positively correlated to the positive rates for PAC-Ⅰ and CD62P. CONCLUSION： aβ2GP Ⅰ level, platelet activation state and their relationship of them are closely correlated with the pathogenesis and development of UC.

High levels of Zinc-α-2-Glycoprotein among Omani AIDS patients on combined antiretroviral therapy

Objective:To investigate the levels of zinc-α-2-glycoprotein(ZAG) among Omani AIDS patients receiving combined antiretroviral therapy(cART).Methods:A total of 80 Omani AIDS patients(45 males and 33 females),average age of 36 vears.who were receiving cART at the Saltan Qaboos University Hospital(SQUH).Muscat,Oman,were tested for the levels of ZAG.In addition,SO healthy blood donors(46 males and 34 females),average age of 26 years,attending the SOUH Blood Bank,were tested in parallel as a control group.Measurement of the ZAG levels was performed using a competitive enzyme—linked immunosorbent assay and in accordance with the manufacturer's instructions.Results:The ZAG levels were found to he significantly higher among AIDS patients compared to the healthy individuals(P=0.033).A total of 56(70%) of the AIDS patients were found to have higher levels of ZAG and 16(20%) AIDS patients were found to have high ZAG levels,which are significantly(P>0.031) associated with weight loss.Conclusions:ZAG levels are high among Omani AIDS patients on cART and this necessitales the measurement of ZAG on routine basis,as it is associated with weight loss.

锌-α2-糖蛋白在食管鳞癌组织中的表达及预后意义

目的研究锌-α2-糖蛋白（Zn-alpha2-glycoprotein,ZGP1）在食管鳞状细胞癌（esophageal squamous cell carcinoma,ESCC）组织中的表达及AZGP1蛋白表达水平对患者预后的影响。方法采用免疫组织化学方法检测112例食管鳞癌组织及25例癌旁正常食管黏膜组织中AZGP1蛋白的表达。分析食管鳞癌组织中AZGP1蛋白表达与临床病理特征的关系及对患者预后的影响。结果 ESCC组织中AZGP1蛋白高表达率低于癌旁正常食管黏膜组织（47.3%vs 76.0%,χ2=6.741,P=0.008）;在高-中分化（60.9%）、T1~3分期（61.7%）及临床分期Ⅰ期＋Ⅱ期（58.3%）食管鳞癌组织中AZGP1蛋白高表达多（P〈0.05）;生存分析表明,AZGP1蛋白高表达组较低表达组具有生存优势,无进展生存期（progression-free survival,PFS）及总生存期（overall surviva L,OS）均延长,差异有统计学意义（P〈0.05）。结论 AZGP1蛋白高表达可能对ESCC有抑制作用,AZGP1蛋白表达水平可预测ESCC患者的预后。

锌-α2-糖蛋白(ZAG)在SD大鼠附睾管中表达的研究

目的:探讨锌-α2-糖蛋白(ZAG)在成年雄性SD大鼠附睾管中的表达情况。方法:分别抽提附睾头、体、尾部精子和附睾液中的蛋白质,利用免疫印迹的方法研究ZAG在SD大鼠附睾管各段中的表达情况。结果:附睾头、体、尾部中的精子和附睾液中均有ZAG蛋白的存在,表达有一定的差异,附睾头部至尾部中的精子和附睾液表达逐渐增加。结论:ZAG主要分布在附睾尾部的精子和进一步的附睾液中,为后续的功能研究奠定了必要的基础。

大鼠肾小管上皮细胞转分化程度与锌-α2-糖蛋白的关系

目的研究白蛋白诱导大鼠肾小管上皮细胞(NRK-52E)转分化程度与锌-α2-糖蛋白(ZAG)表达程度的关系。方法将去脂牛血清白蛋白(d-BSA)稀释成0.5、1、5、10、30 mg/m L,分别用以上不同浓度的dBSA刺激体外培养的NRK-52E细胞48 h。将d-BSA稀释成10 mg/m L,分别刺激NRK-52E细胞0、2、6、12、24、48 h。提取总蛋白,用Western Blot检测α-SMA与ZAG表达。结果用0.5、1、5、10、30 mg/m L d-BSA刺激NRK-52E细胞48 h,随d-BSA浓度增加,ZAG表达增加。用10 mg/m L d-BSA刺激NRK-52E细胞,在0、2、6、12 h ZAG无表达;在24、48 h ZAG表达,且48 h ZAG表达比24 h增加。在白蛋白诱导的NRK-52E细胞中,α-SMA表达量升高后,ZAG的表达量亦升高。结论 ZAG的表达程度与d-BSA呈浓度和时间依赖性,且α-SMA表达量升高后,ZAG的表达量亦升高。ZAG可能成为肾小管上皮细胞转分化程度的生物标志物。

The receptor for β2GPⅠon membrane of hepatocellular carcinoma cell line SMMC-7721 is annexin Ⅱ

AIM: To evaluate the receptor protein which can specifically bind to β2GPⅠon the membrane of hepatocellular carcinoma (HCC) cell line SMMC-7721, and to study the biological function of the receptor.METHODS: Through β2GPⅠ-affinity chromatography column, the peptid-polysome-mRNA complex, which can specially bind to β2GPⅠ, stayed with the column and was separated from the whole polysome of liver cells, and then eluted and collected. Using cDNA synthesis kit and cDNA PCR kit, the corresponding cDNA was obtained and sequenced. RT-PCR was used to amplify annexinⅡ, and flow cytometry was used to study the competitive binding of annexinⅡ with β2GPⅠto SMMC-7721.RESULTS: A total of 1.1 kb of the cDNA fragment of the specific binding protein of β2GPⅠon liver cell membrane was obtained. The sequence of cDNA shared high homology with human annexinⅡ (98%). AnnexinⅡ was expressed on the membrane of SMMC-7721, and could compete with β2GPⅠfor combining with SMMC-7721.CONCLUSION: The receptor for β2GPⅠon membrane of SMMC-7721 cells is annexinⅡ, which might bridge HBV to infect hepatocytes.

Expression and Function of Zinc-α2-Glycoprotein

Zinc-α2-glycoprotein(ZAG), encoded by the AZGP1 gene, is a major histocompatibility complex I molecule and a lipid-mobilizing factor. ZAG has been demonstrated to promote lipid metabolism and glucose utilization, and to regulate insulin sensitivity. Apart from adipose tissue, skeletal muscle, liver, and kidney, ZAG also occurs in brain tissue, but its distribution in brain is debatable. Only a few studies have investigated ZAG in the brain. It has been found in the brains of patients with Krabbe disease and epilepsy, and in the cerebrospinal fluid of patients with Alzheimer disease, frontotemporal lobe dementia, and amyotrophic lateral sclerosis. Both ZAG protein and AZGP1 m RNA are decreased in epilepsy patients and animal models, while overexpression of ZAG suppresses seizure and epileptic discharges in animal models of epilepsy, but knowledge of the specific mechanism of ZAG in epilepsy is limited. In this review, we summarize the known roles and molecular mechanisms of ZAG in lipid metabolism and glucose metabolism, and in the regulation of insulin sensitivity, and discuss the possible mechanisms by which it suppresses epilepsy.

Proteomic analysis of human serum from diabetic retinopathy

AIM: To establish and compare serum proteomic of diabetic retinopathy (DR) patients in various phases and discuss pathogenesis of DR so as to find out possible serum specific molecular markers for early diagnosis of DR. METHODS: Thirty-two subjects were divided into four groups: one group of eight type 2 diabetes mellitus (T2DM) patients without apparent DR (No-DR, NDR), one group of eight T2DM patients with non-proliferative diabetic retinopathy (NPDR), one group of eight T2DM patients with proliferative diabetic retinopathy(PDR) and one group of eight healthy volunteer participants. Two dimensional fluorescence difference gel electrophoresis (2D-DIGE) was applied to establish differential protein expression profiles in four groups. Matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI-TOF-TOF MS) was applied to identify mass spectrometry of differential proteins and analyze follow-up bioinformatics. RESULTS: 2D-DIGE maps of serum protein were satisfactory obtained from NDR, NPDR, PDR and normal control groups. Twenty-six different proteins spots were screened(the volume ratio was > 1.5 based on DeCyder software analysis). Twenty-four of them were verified and two of them were not. Fifteen proteins were verified. Most of them were high-abundant proteins in serum. The four relatively low-abundant ones were beta 2-glycoprotein I (beta (2)-GPI), alpha2-HS-glycoprotein (AHSG), alpha1-acid glycoprotein (alpha(1)-AGP) and apolipoprotein A-1 (apo A-1). beta (2)-GPI expression was gradually increased in the development of DR but unrelated to the severity of DR. The volume ratio of beta (2)-GPI is 1.54, 2.43, and 2.84 in NDR, NPDR and PDR group respectively compared with normal control group. CONCLUSION: Serum proteomic analysis of 2D-DIGE combined with MALDI-TOF-TOF MS is feasible to be applied in the study of DR. 13 2-GPI probably takes part in the process of DR occurrence and development and it could be a candidate biomarker on DR diagnosis in early phase.

T cells from induced and spontaneous models of SLE recognize a common T cell epitope on β2-glycoprotein Ⅰ

Systemic lupus erythematosus is a prototypic model for B-cell epitope spread in autoimmunity.Autoantibodies to numerous molecularly distinct self-antigens emerge in a sequential manner over several years,leading to disease manifestations.Among the earliest autoantibodies to appear are those targeting phospholipid-binding proteins,particularlyβ2-glycoprotein Ⅰ.Notably,mice immunized with β2-glycoprotein Ⅰ and lipopolysaccharide develop a strong T cell response to β2-glycoprotein Ⅰ that is associated with autoantibody production and renal disease,similar to that seen in human SLE.Here we hypothesized that mice with murine systemic lupus erythematosus,whether induced or spontaneous,should have T cells that recognizeβ2-glycoprotein I.We evaluated the response of splenic T cells from mice with induced(C57BL/6 and C3H/HeN)and spontaneous(MRL/lpr)systemic lupus erythematosus to peptides spanning the entire sequence of humanβ2GPI.We found that mice with induced and spontaneous systemic lupus erythematosus recognize a common T cell epitope(peptide 31;LYRDTAVFECLPQHAMFG)in domain Ⅲ ofβ2-glycoprotein I.β2GPI-reactive CD4^(+)T cells from the two models differed primarily in cytokine production:T cells from mice with induced SLE expressed IFN-γ,while T cells from MRL/lpr mice expressed both IL-17 and IFN-γ,indicating that IL-17-expressing T cells are not necessary for generating aβ2GPI-reactive T cell response.These data suggest that the generation of aβ2-glycoprotein Ireactive T cell response is shared by both induced and spontaneous models of systemic lupus erythematosus and that this T cell response may mediate epitope spread to autoantibodies in both models.

LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4

AIM:To investigate the effect of leucine-rich-alpha-2-glycoprotein 1(LRG1)on epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE)cells,and to explore the role of NADPH oxidase 4(NOX4).METHODS:RPE cells(ARPE-19 cell line)were treated with transforming growth factor-β1(TGF-β1)to induce EMT.Changes of the m RNA and protein expression levels of LRG1 were tested in the TGF-β1 treated cells.The recombinant human LRG1 protein(r LRG1)and si RNA of LRG1 were used to establish accumulation of exogenous LRG1 model and the down-regulation of LRG1 model in ARPE-19 cells respectively,and to detect EMT-related markers including fibronectin,α-smooth muscle actin(α-SMA)and zonula occludens-1(ZO-1).The m RNA and protein expression level of NOX4 were measured according to the above treatments.VAS2870 was used as a NOX4 inhibitor in r LRG1-treated cells.EMT-related markers were detected to verify the effect of NOX4 in the process of EMT.RESULTS:TGF-β1 promoted the expression of LRG1 at both the m RNA and protein levels during the process of EMT which showed the up-regulation of fibronectin andα-SMA,as well as the down-regulation of ZO-1.Furthermore,the r LRG1 promoted EMT of ARPE-19 cells,which manifested high levels of fibronectin andα-SMA and low level of ZO-1,whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin andα-SMA and increasing the expression of ZO-1 in ARPE-19 cells.Besides,the r LRG1 activated and LRG1 si RNA suppressed NOX4 expression.EMT was inhibited when VAS2870 was used in the r LRG1-treated cells.CONCLUSION:These results for the first time demonstrate that LRG1 promotes EMT of RPE cells by activating NOX4,which may provide a novel direction to explore the mechanisms of subretinal fibrosis.

Role of β2-glycoprotein I in the pathogenesis of the antiphospholipid syndrome

Antiphospholipid syndrome(APS)is typically characterized by increased levels of three classes of antiphospholipid antibodies,namely lupus anticoagulant,anti-β2-glycoprotein I(anti-β2GPI),and anticardiolipin antibodies.β2-Glycoprotein(β2GPI)is a phospholipid-binding protein composed of five domains(DI-V)and a major antigen in APS.β2GPI is expressed on the surfaces of several cell types,including endothelial cells,monocytes,trophoblast cells,and platelets.Its binding to the anti-β2GPI antibody triggers downstream signaling events and ultimately exerts a variety of cellular effects.β2GPI modulates hemostasis and the complement system,as well as playing an important role in APS-associated vascular injury.Therefore,studying β2GPI will help elucidate the pathogenesis of APS and improve the treatment of patients with this condition.This review will mainly focus on the structure and function of β2GPI,as well as its implication in the pathogenesis of APS.

锌-α_2-糖蛋白(ZAG)在雄性大鼠生殖系统中表达差异的研究

目的探讨(研究)锌-α 2-糖蛋白(ZAG)在成年雄性SD大鼠生殖系统中的表达情况。方法应用商品化试剂盒分别抽提RNA和蛋白质,利用实时荧光定量PCR(Realtime PCR)和免疫印迹(Westernblot)的方法研究ZAG在SD大鼠生殖系统各器官中的表达情况。结果雄性SD大鼠生殖系统各器官中均有ZAG蛋白的存在,不同组织表达有一定的差异,尤其是在尾部附睾及附睾脂肪组织中表达较高,其它组织相对较低。结论大鼠生殖系统中ZAG主要分布在尾部附睾及附睾脂肪组织。通过对ZAG蛋白在雄性SD大鼠生殖系统表达差异的研究,有助于进一步了解该蛋白在雄性生殖系统中的生物学功能。

2型糖尿病患者血清锌α2糖蛋白水平的变化及其相关因素分析

目的探讨T2DM患者血清锌α2糖蛋白(ZAG)的水平与胰岛素抵抗(IR)的相关性,探索其潜在的作为肥胖或代谢综合征血清指标的可能性。方法 166名受试者分为T2DM组和正常糖耐量(NGT)组,根据BMI又分为正常体重组(NW)和超重肥胖组(OW-OB)两个亚组,均测定空腹血清ZAG水平和相关临床指标。结果 T2DM组比NGT组空腹血清ZAG水平明显升高(t=2.228,P=0.028)。T2DM组患者空腹血清ZAG水平分别与HOMA-IR,WC,HbA1c,TG正相关(r分别为0.543,0.442,0.385,0.437,P<0.05),与HDL-C,TNF-a负相关(r分别为-0.447,-0.501,P<0.05)。多元逐步线性回归显示在T2DM组,WC和HDL-C是其的独立影响因素。结论 T2DM患者血清ZAG显著高于NGT患者,超重肥胖和正常体重亚组间差异无统计学意义。T2DM组中ZAG与HOMA-IR,WC,HbA1c,TG正相关,与HDL-C,TNF-α负相关;其中WC和HDL-C是血清ZAG的独立影响因素。

锌-α2糖蛋白在人精子顶体反应过程中的作用

目的：初步探讨锌-α2糖蛋白（ZAG）在人精子顶体反应中的作用。方法采用ZAG抗体对人精子进行免疫荧光染色，激光共聚焦显微镜观察ZAG在顶体反应前后定位的变化，并通过精子透明带结合实验进一步证明ZAG在顶体反应中的作用。结果顶体反应后精子顶体头部的ZAG荧光明显下降甚至消失。精子经ZAG抗体处理后，其透明带结合能力显著下降。结论 ZAG在人精子顶体反应中发挥一定的作用，并影响人精子的透明带结合能力。本研究为探索ZAG在受精过程中的功能及分子作用机制奠定基础。

Identification of LRG1 targeting peptide and its application in targeted imaging for breast cancer

Breast cancer remains a leading cause of morbidity and mortality among women worldwide,emphasizing the urgent need for enhanced diagnostic and therapeutic approaches.Leucine-rich-alpha-2-glycoprotein 1(LRG1)has emerged as a notable target due to its markedly elevated expression in breast tumors,suggesting the viability of LRG1 as a theranostic target.In our study,we employed phage display technology to identify a peptide,termed ET,that binds to LRG1 with a dissociation constant of 48.4μM.After modified with fluorescent cyanine dye,the ET peptide showcased effective tumor-targeting imaging across three different primary breast tumor models and a metastatic breast tumor model.We also undertook a comprehensive safety evaluation,which verified the good biosafety credentials of ET peptide.In summary,the ET peptide identified in this study shows effective LRG1-targeting ability both in vitro and in vivo,thus exhibiting immense potential for clinical translation.