PMID- 30309411 OWN - NLM STAT- MEDLINE DCOM- 20190416 LR - 20190416 IS - 2095-4352 (Print) VI - 30 IP - 9 DP - 2018 Sep TI - [Effect of caveolin-1 scaffolding domain peptides on heme oxygenase-1 activity increasing and M1/M2 phenotype polarization in rat alveolar macrophages induced by lipopolysaccharide]. PG - 855-860 LID - 10.3760/cma.j.issn.2095-4352.2018.09.007 [doi] AB - OBJECTIVE: To investigate the effect of caveolin-1 scaffolding domain (CSD) peptides on heme oxygenase-1 (HO-1) activity increasing and M1/M2 phenotype polarization in rat alveolar macrophages (AMs) induced by lipopolysaccharide (LPS). METHODS: Bioinformatics was used to analyze the binding of full-length wild-type CSD polypeptide and 101 amino acid deleted truncated mutant CSD polypeptide (Delta101CSD) to HO-1. Primary AMs were isolated from rats, when cell fusion reached 80%, they were synchronized with serum-free medium and divided into five groups: no treatment was given to the blank control group; LPS group was treated with 100 mug/L LPS for 16 hours; LPS + hemin group was treated with 100 mug/L LPS and 20 mumol/L hemin for 16 hours; wild-type CSD polypeptide + LPS + hemin group was pretreated with 10 mumol/L wild-type CSD polypeptide 6 hours before LPS treatment; Delta101CSD + LPS + hemin group was pretreated with 10 mumol/L Delta101CSD polypeptide 6 hours before LPS treatment. After treatment for 16 hours, the co-localization between caveolin-1 (Cav-1) and HO-1 was displayed by confocal microscope; the mRNA expressions of inflammatory cytokines interleukin-1beta (IL-1beta) and monocyte chemoattractant protein-1 (MCP-1) and M1/M2 polarization cytokines tumor necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), leukocyte differentiation antigen 206 (CD206) and IL-10 were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR); the HO-1 activity and nitric oxide (NO) production were determined by spectrophotometry. RESULTS: Bioinformatics analysis showed: both wild-type CSD and Delta101CSD peptides could bind to HO-1, and there was no significant difference in the binding ability between the two peptides, but the deletion of 101 Arg resulted in the disappearance of part of the binding region between Delta101CSD and HO-1. The results of laser confocal microscopy showed: the expressions of Cav-1 and HO-1 were lowed in the blank control group, and Cav-1 was bound to HO-1 in LPS group and LPS + hemin group. Both wild-type CSD and Delta101CSD peptides pretreatment could significantly reduce the binding of HO-1 to Cav-1 induced by LPS. HO-1 activity analysis showed: after LPS stimulation, the activity of HO-1 was significantly higher than that of the blank control group; the activity of HO-1 induced by LPS was increased by hemin; after pretreatment with two kinds of CSD peptides, the activity of HO-1 was further increased, and the effect of wild-type CSD peptide was more significant, which showed a statistically significant difference as compared with that of LPS + hemin group (pmolxmg(-1)xh(-1): 3 683+/-266 vs. 2 408+/-132, P < 0.05). RT-qPCR results showed: LPS could induce elevation of cytokines and M1 markers and decrease of M2 markers, while hemin could inhibit LPS-induced inflammatory response and M1/M2 phenotypic polarization. Compared with LPS + hemin group, after pretreatment with wild-type CSD peptide, the levels of inflammatory factors in AMs were decreased, and the mRNA expression levels of TNF-alpha and iNOS, M1 markers, were decreased [TNF-alpha mRNA (2(-DeltaDeltaCt)): 6.82+/-0.05 vs. 8.70+/-0.24, iNOS mRNA (2(-DeltaDeltaCt)): 331.50+/-32.05 vs. 506.70+/-0.10, both P < 0.05], and IL-10 mRNA expression level was increased (2(-DeltaDeltaCt): 269.09+/-6.54 vs. 119.05+/-3.30, P < 0.05). The deletion of 101 site partially weakened the inhibitory effect of CSD peptides on inflammatory factors and only reduced the expression of iNOS mRNA (2(-DeltaDeltaCt): 429.11+/-8.92 vs. 506.70+/-0.10, P < 0.05), indicating that its ability to transform AMs from M1 phenotype to M2 phenotype was poor. The two peptides had no effect on the expression of CD206. CONCLUSIONS: Wild-type CSD had beneficial effects of anti-inflammation by reducing Cav-1 binding to HO-1 induced by LPS, restoring the HO-1 activity and driving M2 phenotype in alveolar macrophages. FAU - Hong, Kan AU - Hong K AD - Department of Geriatric Medicine, Wuxi People's Hospital, Wuxi 214043, Jiangsu, China (Hong K); Department of Cardiology, Wuxi People's Hospital, Wuxi 214043, Jiangsu, China (Yu ZM); Department of Pathophysiology, Wuxi School of Medicine, Jiangnan University, Wuxi 214122, Jiangsu, China (Sun XQ, Wu C, Weng P, Wei MX, Zuo J, Chen JL, Pang QF). Corresponding author: Pang Qingfeng, Email: qfpang@jiangnan.edu.cn. FAU - Yu, Zhiming AU - Yu Z FAU - Sun, Xueqian AU - Sun X FAU - Wu, Chen AU - Wu C FAU - Weng, Ping AU - Weng P FAU - Wei, Mingxia AU - Wei M FAU - Zuo, Jing AU - Zuo J FAU - Chen, Junliang AU - Chen J FAU - Pang, Qingfeng AU - Pang Q LA - chi PT - Journal Article PL - China TA - Zhonghua Wei Zhong Bing Ji Jiu Yi Xue JT - Zhonghua wei zhong bing ji jiu yi xue JID - 101604552 RN - 0 (Caveolin 1) RN - 0 (Lipopolysaccharides) RN - 0 (Peptide Fragments) RN - 0 (caveolin-1 (82-101)) RN - EC 1.14.14.18 (Heme Oxygenase-1) SB - IM MH - Animals MH - Caveolin 1/*metabolism MH - *Cell Polarity MH - Heme Oxygenase-1/*metabolism MH - Lipopolysaccharides/metabolism MH - *Macrophages, Alveolar MH - Peptide Fragments/*metabolism MH - Phenotype MH - Rats EDAT- 2018/10/13 06:00 MHDA- 2019/04/17 06:00 CRDT- 2018/10/13 06:00 PHST- 2018/10/13 06:00 [entrez] PHST- 2018/10/13 06:00 [pubmed] PHST- 2019/04/17 06:00 [medline] AID - 10.3760/cma.j.issn.2095-4352.2018.09.007 [doi] PST - ppublish SO - Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2018 Sep;30(9):855-860. doi: 10.3760/cma.j.issn.2095-4352.2018.09.007.