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

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.

Hypothalamic Microglial Activation in Obesity: A Mini-Review

“…In this context, promoting an M1-M2 switch could be an interesting approach to attenuate inflammation of the neural tissue ( Hu et al, 2015 ; Xu et al, 2015 ). In line, a recent study showed that betulinic acid (BA), a naturally pentacyclic triterpenoid with anti-inflammatory properties, promotes M2 polarization and inhibits M1 polarization in LPS-stimulated BV2 microglial cells and in the cerebral cortex of LPS-injected mice through calmodulin-dependent protein kinase kinase β (CaMKKβ)-dependent AMP-activated protein kinase (AMPK) activation ( Li et al, 2018 ). In another study, Xu et al (2015) showed that telmisartan, a potent angiotensin II receptor blocker, also prevents LPS-induced microglia activation throughout M2 microglia polarization through CaMKKβ-dependent AMPK activation.…”

“…Microglial cells express triggering receptor of myeloid cell-2 (TREM2), which is an important negative regulator of NF-κB. In vitro studies showed that TREM2 overexpression inhibits neuroinflammation by down-regulating PI3K/AKT and NF-κB signaling in BV2 microglia ( Zhong et al, 2017 ; Li et al, 2018 ). Curiously, LPS downregulates the expression of TREM2 through the activation of JNK and NF-κB signaling pathways, resulting in a pro-inflammatory vicious cycle ( Zhong et al, 2017 ).…”

Molecular Targets Involved in the Neuroprotection Mediated by Terpenoids

“…Interestingly, it has also been shown to promote M2 anti-inflammatory phenotype polarization of lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and to inhibit M1 proinflammatory phenotype. This study was carried out via calmodulin-dependent protein kinase β (CaMKKβ)-dependent AMPK activation, which was further confirmed in mice brains treated with betulinic acid [127].…”

Involvement of AMP‐activated protein kinase in neuroinflammation and neurodegeneration in the adult and developing brain

“…Accumulating data from experiments using LPS as a trigger for pro‐inflammatory reactions indicate that AMPK activation induces a microglial phenotypic shift from pro‐inflammatory to anti‐inflammatory in AMPK activation‐dependent manner. In primary cultured microglia or BV2, LPS‐induced pro‐inflammatory reaction is associated with reduction in phospho (p)‐AMPK (activated AMP) (Li et al, 2018b; Park et al, 2018; Xu et al, 2015; Zhou et al, 2014), and AMPK activators including metformin and 5‐aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (AICAR) suppress pro‐inflammatory and enhance anti‐inflammatory reactions (Giri et al, 2004; Lee et al, 2015; Lu et al, 2010; Li et al, 2018a; Xu et al, 2015; Park et al, 2018; Wang et al, 2018d; Zhou et al, 2014; Velagapudi et al, 2017; Chen et al, 2014). For example, AICAR (an analogue of AMP) attenuates LPS‐induced activation of NF‐κB and inhibits expression of proinflammatory cytokines (TNF‐α, IL1β, IL‐6) and iNOS, and these effects of AICAR are inhibited by the dominant negative form of AMPK and anti‐sense AMPK treatment in primary rat astrocytes, microglia, and peritoneal macrophages (Giri et al, 2004).…”

“…For example, AICAR (an analogue of AMP) attenuates LPS‐induced activation of NF‐κB and inhibits expression of proinflammatory cytokines (TNF‐α, IL1β, IL‐6) and iNOS, and these effects of AICAR are inhibited by the dominant negative form of AMPK and anti‐sense AMPK treatment in primary rat astrocytes, microglia, and peritoneal macrophages (Giri et al, 2004). Betulinic acid (BA), a naturally occurring pentacyclic triterpenoid, attenuates LPS‐induced pro‐inflammation reaction in BV‐2 microglial cells via CaMKKβ‐dependent AMPK activation, and compound C (an AMPK inhibitor) and AMPK siRNA abolish the effects, although BA does not affect phagocytic activation induced by LPS (Li et al, 2018a). ENERGI‐F704, an AMPK agonist, decreases the protein level and nuclear translocation of NF‐κB, leading to reduction of pro‐inflammatory mediators, such as IL‐6, TNF‐α, iNOs, and COX‐3 in LPS‐stimulated microglia (BV2) (Chen et al, 2014).…”