# Anti-Gingivitis and Anti-Osteoclastogenesis Effects of Ethanol Extract from Hippophae rhamnoides L. Fruits in IL-1β-Induced Human Gingival Fibroblasts and RANKL-Induced RAW 264.7 Macrophages

## TL;DR

This study shows that an extract from sea buckthorn fruits can reduce inflammation and prevent bone loss in periodontal disease.

## Contribution

The novel finding is that DKB140, an ethanol extract from Hippophae rhamnoides, has dual anti-gingivitis and anti-osteoclastogenesis effects.

## Key findings

- DKB140 reduced inflammatory markers like COX-2 and cytokines in macrophages and fibroblasts.
- The extract inhibited osteoclast differentiation by suppressing key proteins like NFATc1 and CTSK.
- Mechanistically, DKB140's effects were linked to the NF-κB/MAPK/NLRP3 signaling pathway.

## Abstract

Periodontal disease is a chronic inflammatory disorder that irreversibly damages soft tissue. Current treatment options often face challenges such as drug resistance, highlighting the need for alternative therapies with fewer side effects. This study was conducted to explore the therapeutic potential of Hippophae rhamnoides extracted using 70% ethanol (DKB140, with kaempferol as the marker compound) for periodontal disease treatment. The anti-gingivitis and anti-periodontitis effects of DKB140 were demonstrated in RAW 264.7 macrophages and human gingival fibroblasts (hGF-1) using enzyme-linked immunosorbent assay (ELISA), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blot analysis by investigating the NLRP3 signaling pathway and its inhibitory effects on matrix metalloproteinase (MMP) expression and osteoclast differentiation. In RAW 264.7 macrophages, DKB140 attenuated the production of prostaglandin E2 and COX-2 mRNA expression. Furthermore, DKB140 inhibited inflammatory cytokine (IL-6, IL-8, and IL-1β) production and mRNA expression. In hGF-1s, DKB140 downregulated prostaglandin E2, COX-2, and inflammatory cytokine levels. Mechanistic investigations demonstrated that the anti-gingivitis effects of DKB140 were mediated through the NF-κB/MAPK/NLRP3 signaling pathway. These findings were confirmed using an NLRP3 inhibitor. Moreover, DKB140 reduced the expression of collagenases (MMP-1 and MMP-13), gelatinases (MMP-2 and MMP-9), and stromelysins (MMP-3 and MMP-10) in hGF-1s. Furthermore, DKB140 decreased the expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), dendritic cell-specific transmembrane protein (DCstamp), and cathepsin K (CTSK), which are responsible for osteoclast differentiation in receptor of nuclear factor kappa-B ligand (RANKL)-stimulated osteoclasts. These findings suggest that DKB140 can function as a dual-functional agent with anti-gingival and anti-periodontal properties.