Betulinic Acid Inhibits RANKL-Induced Osteoclastogenesis via Attenuating Akt, NF-κB, and PLCγ2-Ca²⁺ Signaling and Prevents Inflammatory Bone Loss

Abstract

The increase of bone-resorbing osteoclast activity in bone remodeling is the major characteristic of various bone diseases. Thus, inhibiting osteoclastogenesis and bone-resorbing function may be an effective therapeutic target for bone diseases. Betulinic acid (BA), a natural plant-derived pentacyclic triterpenoid compound, is known to possess numerous pharmacological and biochemical properties including anti-inflammatory, anticancer, and antiadipogenic activity. However, the effect of BA on osteoclast differentiation and function in bone metabolism has not been demonstrated so far. In this study, we investigated whether BA could suppress RANKL-induced osteoclastogenesis and bone resorption. Interestingly, BA significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt and IκB, as well as PLCγ2-Ca2+ signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos and NFATc1. The inhibition of these pathways by BA was once more confirmed by retrovirus infection of constitutively active (CA)-Akt and CA-Ikkβ retrovirus and measurement of Ca2+ influx. BA also significantly inhibited the expression of osteoclastogenesis-specific marker genes. Moreover, we found that BA administration restored the bone loss induced through acute lipopolysaccharide injection in mice by a micro-CT and histological analysis. Our findings suggest that BA is a potential therapeutic candidate for bone diseases involving osteoclasts.

Lipopolysaccharide-Induced Bone Loss in Rodent Models: A Systematic Review and Meta-Analysis

“…Experimental durations less than 2 weeks generally consisted of two to three injections of LPS ranging from 5 to 10 mg/kg body weight per injection with bones collected for ex vivo analysis within 2 weeks of the first LPS exposure. This type of experimental design consistently demonstrated negative effects of LPS on trabecular bone structure, including BV/TV, Tb.N, and Tb.Sp, in the femur ( 42‐118 ) and tibia. ( 68,119‐124 ) In studies that reported negative effects of LPS exposure on trabecular bone structure, approximately half (22/47) also reported a decreased Tb.Th in the femur ( 48,55,59,70,77,81,88,89,94,97‐101,107,108,114,117,123 ) and tibia.…”

“…Experimental durations less than 2 weeks generally consisted of two to three injections of LPS ranging from 5 to 10 mg/kg body weight per injection with bones collected for ex vivo analysis within 2 weeks of the first LPS exposure. This type of experimental design consistently demonstrated negative effects of LPS on trabecular bone structure, including BV/TV, Tb.N, and Tb.Sp, in the femur ( 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 ,…”

Restraint upon Embryonic Metatarsal Ex Vivo Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway

“…Despite this, no effects on endochondral growth were seen with addition of betulinic acid to metatarsal cultures. Other studies using bone cells in vitro have found that 10-15 µM and 10 mM betulinic acid induce bone formation by enhancing BMP2 [49], and inhibit osteoclastogenesis via RANKL [50], respectively. Herein, betulinic acid was used at 2.5 µM, which might be too low to exert any effects on endochondral growth in the context of an ex vivo organ culture system, which likely shows different requirements to cultured cells in vitro.…”

Receptor activator of nuclear factor-κB ligand-mediated osteoclastogenesis signaling pathway and related therapeutic natural compounds

“…Betulinic acid (BA) is a pentacyclic triterpene compound that is derived from natural plants and is known to possess many pharmacological and biochemical properties, including anti-inflammatory and anti-cancer activities. Jeong et al (2020) found that BA inhibited RANKL-mediated intracellular levels of Ca 2+ through inhibition of PLCγ2 phosphorylation and reduced OCs production and bone resorption through the combined effect of Akt and NF-κB phosphorylation, and meanwhile BA reversed inflammatory bone loss induced by LPS injection in mice.…”