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

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.

Responses of Transgenic Melatonin-Enriched Goats on LPS Stimulation and the Proteogenomic Profiles of Their PBMCs

“…The LPS and MT treatment are described previously [80]. We collected PBMCs and serum in order to test MT levels and the blood physiological index.…”

“…We performed our experiments at CapitalBio Technology (Beijing, China); the method of proteome analysis was carried out according to the method of Tao et al described previously [80]. …”

AanatKnockdown and Melatonin Supplementation in Embryo Development: Involvement of Mitochondrial Function and DNA Methylation

“…These results were also consistent with the results of the Aanat knockdown experiment. In addition, an Aanat overexpression goat model was successfully established with somatic cell nuclear transfer (SCNT) technology, described previously (71). With the help of superovulation and artificial insemination, sufficiency embryos were collected for classification and statistics.…”

“…We also generated a melatonin-enriched animal model (goat) with AANAT transfer using SCNT technology. Basically, a pIRES2-EGFP-AANAT expression vector was constructed and was transferred into the female fetal fibroblast cells (FFCs) through electrotransfection and then the nuclear of the transgenic FFCs was transferred to the eggs of the donor goats (71).…”

<p>Role of Melatonin in the Regulation of Pain</p>

“…4,5 However, melatonin has antioxidant and anti-inflammatory properties, acting as a free radical scavenger during inflammation and injury. 6,7 For example, melatonin reduced the elevated expression of nuclear factor-kappa B (NF-κB) and inhibited the enhanced level of proinflammatory cytokines IL-6 or TNF-α to modulate neuroinflammation in a model of diabetic neuropathy. 8 Some evidence suggests that melatonin also has immunomodulatory properties.…”