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  • Effects of AANAT overexpression on the inflammatory responses and autophagy activity in the cellular and transgenic animal levels.

Effects of AANAT overexpression on the inflammatory responses and autophagy activity in the cellular and transgenic animal levels.

Autophagy (2018-07-10)
Jingli Tao, Minghui Yang, Hao Wu, Teng Ma, Changjiu He, Menglong Chai, Xiaosheng Zhang, Jinlong Zhang, Fangrong Ding, Sutian Wang, Shoulong Deng, Kuanfeng Zhu, Yukun Song, Pengyun Ji, Haijun Liu, Zhengxing Lian, Guoshi Liu
ABSTRACT

To explore the anti-inflammatory activity of endogenous produced melatonin, a melatonin-enriched animal model (goat) with AANAT transfer was successfully generated with somatic cell nuclear transfer (SCNT) technology. Basically, a pIRES2-EGFP-AANAT expression vector was constructed and was transferred into the female fetal fibroblast cells (FFCs) via electrotransfection and then the nuclear of the transgenic FFC was transferred to the eggs of the donor goats. The peripheral blood mononuclear cells (PBMCs) of the transgenic offspring expressed significantly higher levels of AANAT and melatonin synthetic function than those PBMCs from the wild-type (WT) animals. After challenge with lipopolysaccharide (LPS), the transgenic PBMCs had increased autophagosomes and LC3B expression while they exhibited suppressed production of the proinflammatory cytokines, IL1B and IL12 (IL12A-IL12B/p70), compared to their WT. The mechanistic analysis indicated that the anti-inflammatory activity of endogenous melatonin was mediated by MTNR1B (melatonin receptor 1B). MTNR1B stimulation activated the MAPK14 signaling pathway to promote cellular macroautophagy/autophagy, thus, suppressing the excessive inflammatory response of cellular. However, when the intact animals challenged with LPS, the serum proinflammatory cytokines were significantly higher in the transgenic goats than that in the WT. The results indicated that endogenous melatonin inhibited the MAPK1/3 signaling pathway and ROS production, subsequently downregulated gene expression of BECN1, ATG5 in PMBCs and then suppressed the autophagy activity of PBMCs and finally elevated levels of serum proinflammatory cytokines in transgenic animals, Herein we provided a novel melatonin-enriched animal model to study the potential effects of endogenously produced melatonin on inflammatory responses and autophagy activity.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3-Methyladenine, autophagy inhibitor
Sigma-Aldrich
Lipopolysaccharides from Escherichia coli O55:B5, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
Dounce tissue grinder pestle, Small clearance, working volume 2 mL
Sigma-Aldrich
Bovine Serum Albumin, powder, BioXtra
Sigma-Aldrich
D.E.R. 332, used as embedding medium
Sigma-Aldrich
Triethylammonium bicarbonate buffer, 1.0 M, pH 8.5±0.1
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Hyaluronidase from bovine testes, Type I-S, lyophilized powder, 400-1000 units/mg solid
Sigma-Aldrich
Tetraethylammonium borohydride, technical, ≥95% (T)
Sigma-Aldrich
4-P-PDOT, ≥98% (HPLC)