PMID- 28903783 OWN - NLM STAT- MEDLINE DCOM- 20180516 LR - 20181113 IS - 1476-511X (Electronic) IS - 1476-511X (Linking) VI - 16 IP - 1 DP - 2017 Sep 13 TI - The purification of reduced beta2-glycoprotein I showed its native activity in vitro. PG - 173 LID - 10.1186/s12944-017-0555-x [doi] LID - 173 AB - BACKGROUND: New evidence has shown that reduced beta2-glycoprotein I (beta2GPI) has anti-oxidative stress and anti-inflammatory activity. However, the details are still poorly understood. This study aims to prepare stable reduced beta2GPI with its native bioactivity in vitro. METHODS: Human beta2GPI was purified from plasma first with perchloric acid precipitation and then purified with a series of chromatography methods including Sephadex G-25 desalting, SP HP, AF-heparin HC-650 M, and Sephacryl S-200. The purified human beta2GPI was reduced with thioredoxin-1 (TRX-1) activated by DL-dithiothreitol (DTT). Glutathione (GSH) was selected to block the free thiols in reduced beta2GPI. LC/MS was used to verify the location of free thiols. Western blot analysis was used to detect beta2GPI immunoreactivity. MTS and flow cytometry were conducted to investigate its biological effect on oxidative-stress-induced death of human umbilical vein endothelial cells (HUVECs). The levels of tumour necrosis factor-alpha (TNF-alpha),interleukin-6 (IL-6) interleukin-10 (IL-10),interleukin-12P70 (IL-12P70),interferon-gamma (IFN-gamma) and monocyte chemoattractant protein -1(MCP-1) in mouse serum were quantified to assess its anti-inflammatory activity in lipopolysaccharide (LPS)-mediated systemic inflammation. RESULTS: We obtained approximately 10 mg beta2GPI (purity 98.7%) from 200 ml plasma. The protein yield was 0.05 mg/ml plasma. beta2GPI was then reduced by TRX-1/DTT in vitro; the free thiols were detected on Cys288 and Cys326 in domain V of beta2GPI. The GSH blockage stabilized the reduced beta2GPI in vitro. This reduced beta2GPI can be recognized by the anti-beta2GPI antibody, can significantly reduce the death of HUVECs after H(2)O(2) treatment and can significantly decrease the levels of TNF-alpha, IL-6,IFN-gamma and MCP-1 in mice upon LPS stimulation. CONCLUSION: Stable reduced beta2GPI can be obtained in vitro by TRX-1 deoxidation followed by the blockage of thiols with GSH. This reduced beta2GPI maintains the same immunological activity as oxidized beta2GPI and has the ability to counter the oxidative stress induced by H(2)O(2) in HUVECs and inflammation in LPS-mediated inflammation in mice. FAU - Zhou, Saijun AU - Zhou S AD - Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China. FAU - Lu, Ming AU - Lu M AD - Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China. FAU - Zhao, Jiantong AU - Zhao J AD - Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China. FAU - Liu, Shuaihui AU - Liu S AD - Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China. FAU - Li, Xin AU - Li X AD - Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China. FAU - Zhang, Rui AU - Zhang R AD - Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China. FAU - Liu, Hongyan AU - Liu H AD - Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China. FAU - Yu, Pei AU - Yu P AUID- ORCID: 0000-0001-7992-0480 AD - Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China. yupei@tmu.edu.cn. LA - eng PT - Journal Article DEP - 20170913 PL - England TA - Lipids Health Dis JT - Lipids in health and disease JID - 101147696 RN - 0 (Anti-Inflammatory Agents, Non-Steroidal) RN - 0 (Lipopolysaccharides) RN - 0 (Sulfhydryl Compounds) RN - 0 (beta 2-Glycoprotein I) SB - IM MH - Animals MH - Anti-Inflammatory Agents, Non-Steroidal/*pharmacology MH - Cell Death/drug effects MH - Human Umbilical Vein Endothelial Cells MH - Humans MH - Inflammation/chemically induced/drug therapy MH - Lipopolysaccharides/toxicity MH - Male MH - Mice MH - Oxidation-Reduction MH - Oxidative Stress/drug effects MH - Sulfhydryl Compounds/chemistry MH - beta 2-Glycoprotein I/chemistry/immunology/*isolation & purification/*pharmacology PMC - PMC5597989 OTO - NOTNLM OT - Diabetic vascular disease OT - GSH OT - Oxidative stress OT - Reduced beta2-glycoprotein I OT - Thioredoxin-1 OT - beta2-glycoprotein I COIS- ETHICS APPROVAL AND CONSENT TO PARTICIPATE: All procedures were performed in accordance with the institutional guidelines for human and animal research and were approved by the Animal Care and Use Committee of Metabolic Diseases Hospital of Tianjin Medical University, Tianjin, China. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EDAT- 2017/09/15 06:00 MHDA- 2018/05/17 06:00 PMCR- 2017/09/13 CRDT- 2017/09/15 06:00 PHST- 2017/04/14 00:00 [received] PHST- 2017/08/28 00:00 [accepted] PHST- 2017/09/15 06:00 [entrez] PHST- 2017/09/15 06:00 [pubmed] PHST- 2018/05/17 06:00 [medline] PHST- 2017/09/13 00:00 [pmc-release] AID - 10.1186/s12944-017-0555-x [pii] AID - 555 [pii] AID - 10.1186/s12944-017-0555-x [doi] PST - epublish SO - Lipids Health Dis. 2017 Sep 13;16(1):173. doi: 10.1186/s12944-017-0555-x.