Elsevier

Neuropeptides

Volume 85, February 2021, 102109
Neuropeptides

Adrenomedullin 2 attenuates LPS-induced inflammation in microglia cells by receptor-mediated cAMP-PKA pathway

https://doi.org/10.1016/j.npep.2020.102109Get rights and content

Abstract

Inflammation plays a critical role in the development of neurodegenerative diseases. Adrenomedullin 2 (AM2), a member of the calcitonin gene-related peptide family, has been known to have anti-inflammatory effects. Here, we evaluated the anti-inflammatory effects of AM2 in LPS-activated microglia and BV2 cells. The endogenous mRNA and protein expressions of AM2, calcitonin receptor-like receptor (CLR), receptor activity-modifying proteins (RAMPs) including RAMP1, RAMP2 and RAMP3 and the production of inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected by RT-PCR and Western blot. Our results revealed that LPS (1 μg/mL) significantly stimulated CLR, RAMP1, RAMP2 and RAMP3 protein expressions in BV2 microglia cells, but AM2 had a significant decrease. However, the mRNA levels of AM2, CLR, and RAMP1/2/3 were all markedly increased. LPS also induced obvious increases in mRNA and protein levels of the inflammatory mediators (TNF-α, IL-1β, COX2 and iNOS). More importantly, AM2 (10 nM) administration effectively inhibited the mRNA and protein expressions of these mediators induced by LPS and increased the cAMP content in LPS-stimulated BV2 cells. Furthermore, the antagonism with AM2 receptor antagonist IMD17-47, adrenomedullin (AM) receptor antagonist by AM22-52 or the inhibition of protein kinase A (PKA) activation by P1195 effectively prevented the inhibitory role of AM2 in LPS-induced production of the above inflammatory mediators. In conclusion, AM2 inhibits LPS-induced inflammation in BV2 microglia cells that may be mainly through AM receptor-mediated cAMP-PKA pathway. Our results indicate AM2 plays an important protective role in microglia inflammation, suggesting therapeutic potential for AM2 in neuroinflammation diseases caused by activated microglia.

Introduction

Activated microglia can participate in and aggravate the development of inflammatory reactions of the neurodegenerative diseases in the brain, particularly Alzheimer's and Parkinson diseases (Arranz and De Strooper, 2019). Overactivation of microglia results in the production of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) which play a critical role in the development of neuroinflammation (Kirkley et al., 2017). The inflammatory state resulting from activated microglia has been identified as an important mechanism responsible for the pathological processes of neurodegenerative diseases (Kirkley et al., 2017; Ransohoff et al., 2015). However, currently available agents that target neuroinflammation have failed to achieve significant clinical results in the amelioration of CNS injury (Lee and Suk, 2017). Therefore, there is still need of a lot research work to do for preventing neuroinflammation progression of neurodegenerative diseases caused by activated microglia (Tan et al., 2019; Mendonca et al., 2019).

Adrenomedullin 2 (AM2), also named as intermedin, is a member belonging to the calcitonin, amylin, calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) peptide family (Roh et al., 2004; Takei et al., 2004). It is widely distributed in peripheral organs and the central nervous system (Hay et al., 2018). At present, no unique receptor has yet been identified for AM2 which shares the receptor system consisting of calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMPs). The CLR/RAMP1 acts as the CGRP receptor, whereas CLR/RAMP2 or CLR/RAMP3 complex forms the AM receptor (Roh et al., 2004; Takei et al., 2004; Hay et al., 2018). AM2 has extensive protective role in tissue injury, such as injuries to the pulmonary, cardiovascular and renal systems (Holmes et al., 2013; Telli et al., 2018; Ni et al., 2018; Yu et al., 2018; Qiao et al., 2017). Moreover, AM2 can protect rat cerebral endothelial cells from oxidative damage in vitro (Chen et al., 2006). Furthermore, we previously reported that AM2 alleviates LPS-induced sympathetic activation in PVN in rats with obesity-related hypertension (Sun et al., 2019). Previous study also demonstrated that AM2 attenuates LPS-induced inflammation in the rat testis (Li et al., 2013a).

AM2 can also inhibit inflammation in rats with diabetes, hyperlipidemia or salt-sensitive hypertension (Hagiwara et al., 2008; Li et al., 2013b; Yang et al., 2014). However, whether AM2 inhibits inflammation caused by LPS in microglia remains unknown. It would be interesting to know whether or not the mRNA and protein expressions of AM2 and receptor system CLR/PAMP1/2/3 change in the microglia and whether AM2 plays a similar protective role in microglia inflammation stimulated by LPS.

Therefore, this study was undertaken to investigate the possible expression of AM2 and CLR/RAMP1/2/3 in the microglia treated with LPS, and its potential roles and mechanisms in microglia inflammation.

Section snippets

Experiment 1

The CCK8 cell cytotoxicity test was used to determine the effect of AM2 (10 nM) and LPS (1 μg/mL) on cell growth for 24 h.

Experiment 2

The endogenous mRNA CGRP, AM, AM2, CLR and RAMP1/2/3 levels, and AM2 content, AM2, CLR and RAMP1/2/3 protein expressions were determined under LPS (1 μg/mL) stimulation.

Experiment 3

The effect of AM2 on LPS-induced inflammation was explored in microglial and BV2 cells. AM2 (10 nM) was pretreated for 30 min and then LPS (1 μg/mL) was added to the cell culture medium to observe the protein

The effects of AM2 (10 nM) and LPS (1 μg/mL) on cell growth, and LPS stimulation on endogenous mRNA and protein expressions of AM2, CLR and RAMP1/2/3.

The result of CCK8 cytotoxicity assay showed that the AM2 (10 nM), LPS (1 μg/mL), and AM2 plus LPS had no significant influence on cell growth compared with the control group (Fig. 1A). After stimulation of LPS (1 μg/mL) for 24 h, RT-PCR and Western Blot were used to detect the changes of CGRP, AM, AM2, CLR and RAMP1/2/3 mRNA levels and protein expressions of AM2, CLR and RAMP1/2/3. The results showed that the mRNA levels of endogenous AM, AM2, CLR and RAMPs are significantly increased after

Discussion

Abnormal microglia activation accelerates inflammation that plays an essential role in the pathogenesis of neurodegenerative disorders such as Parkinson's and Alzheimer's disease (AD) (Graeber et al., 2011; Neumann et al., 2009; Dansokho and Heneka, 2018). LPS induces inflammation by activating a number of signaling pathways including NF-κB via the phosphorylation of MAPK. AM2 has anti-inflammatory actions that may be related to the activation of MAPK and NF-kB. In our study, LPS treatment

Declaration of competing interest

The authors claim that there are no conflicts of interest.

Acknowledgements

We gratefully acknowledge the generous support of the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine. This work was supported by the National Natural Science Foundation of China (81970356 and 81470539).

Author contribution

Ye-Bo Zhou designed the study. Jing Sun, Pei Qian, Ying Kang, Hang-Bing Dai, Fang-Zheng Wang and Hong-Yu Wang performed experiments. Hong Zhou and Qing Gao performed the data analysis. Ye-Bo Zhou wrote the manuscript and decided to submit the manuscript.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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