Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation.

Chuwen Li,Simon M Y Lee,Fan Tang,Chao Zhang,Dan Ma,Hefeng Zhou,Yu Feng,Maggie P M Hoi,Chengwei He,Chao Zhao

doi:10.3389/fnmol.2018.00098

Abstract

In response to the microenvironment, microglia may polarize into either an M1 pro-inflammatory phenotype, exacerbating neurotoxicity, or an M2 anti-inflammatory phenotype, conferring neuroprotection. Betulinic acid (BA) is a naturally pentacyclic triterpenoid with considerable anti-inflammatory properties. Here, we aim to investigate the potential effects of BA on microglial phenotype polarization and to reveal the underlying mechanisms of action. First, we confirmed that BA promoted M2 polarization and inhibited M1 polarization in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Then, we demonstrated that the effect of BA on microglial polarization was dependent on AMP-activated protein kinase (AMPK) activation, as evidenced by the fact that both AMPK inhibitor compound C and AMPK siRNA abolished the M2 polarization promoted by BA. Moreover, we found that calmodulin-dependent protein kinase kinase β (CaMKKβ), but not liver kinase B1, was the upstream kinase required for BA-mediated AMPK activation and microglial M2 polarization, via the use of both the CaMKKβ inhibitor STO-609 and CaMKKβ siRNA. Finally, BA enhanced AMPK phosphorylation and promoted M2 microglial polarization in the cerebral cortex of LPS-injected mice brains, which was attenuated by pre-administration of the AMPK inhibitor. This study demonstrated that BA promoted M2 polarization of microglia, thus conferring anti-neuroinflammatory effects via CaMKKβ-dependent AMPK activation.

Highlights

Neuroinflammation is an inevitable and important pathological process involved in all types of damages to, and disorders of, the central nervous system (CNS) (Ransohoff and Perry, 2009; Gemma, 2010; Dhama et al, 2015)
We found that betulinic acid (BA) induced AMP-activated protein kinase (AMPK) activation and promoted microglia polarization toward the M2 phenotype, inhibiting neuroinflammation
AMPKα, p-AMPK (Thr172), liver kinase B1 (LKB1), p-LKB1 (Ser428), p-calmodulin-dependent protein kinase kinase β (CaMKKβ) (Ser511), acetyl-CoA carboxylase (ACC), p-ACC (Ser79), β-actin, GAPDH, and HRP-linked secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, United States); inducible nitric oxide synthase (iNOS), CaMKKβ, and Arg-1 were purchased from Invitrogen (Carlsbad, CA, United States); cluster of differentiation 68 (CD68), mannose receptor (CD206), and YM1/2 were purchased from Abcam (Cambridge, MA, United States).The donkey anti-goat, Alex Fluor 488, and donkey anti-rabbit, Alex Fluor 647, secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, United States)

Summary

Introduction

Neuroinflammation is an inevitable and important pathological process involved in all types of damages to, and disorders of, the CNS (Ransohoff and Perry, 2009; Gemma, 2010; Dhama et al, 2015). Accumulating evidence has established activated microglia as major cellular elements of neuroinflammatory responses, executing specific immune functions to maintain physiological homoeostasis (Ransohoff and Perry, 2009; Gemma, 2010; Dhama et al, 2015). In response to various microenvironmental disturbances, microglia can be phenotypically polarized into a classical (pro-inflammatory; M1) or an alternative (antiinflammatory; M2) phenotype (David and Kroner, 2011; Saijo and Glass, 2011; Joseph and Venero, 2013; Mantovani et al, 2013). (David and Kroner, 2011; Saijo and Glass, 2011; Joseph and Venero, 2013; Mantovani et al, 2013). There are few compounds reported to regulate microglia polarization toward the M2 phenotype (Lu et al, 2010; Zhou et al, 2014)

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