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. 2022 Dec 26;12(1):47.
doi: 10.3390/antiox12010047.

Anti-Osteoarthritic Effects of Prunella Vulgaris and Gentiana Lutea In Vitro and In Vivo

Jeonghyun Kim  1   2 , Chang-Gun Lee  1   2   3 , Seokjin Hwang  1   2 , Seung-Hee Yun  1   2 , Laxmi Prasad Uprety  1   2 , Kang-Il Oh  1   2 , Shivani Singh  1   2 , Jisu Yoo  1   2 , Hyesoo Jeong  4 , Yoonjoong Yong  4 , Subin Yeo  4 , Eunkuk Park  1   2 , Seon-Yong Jeong  1   2   4
Affiliations

Anti-Osteoarthritic Effects of Prunella Vulgaris and Gentiana Lutea In Vitro and In Vivo

Jeonghyun Kim et al. Antioxidants (Basel). .

Abstract

Osteoarthritis (OA) is the progressive destruction of articular cartilage with severe symptoms, including pain and stiffness. We investigated the anti-osteoarthritic effects of Prunella vulgaris (PV) and Gentiana lutea (GL) extract in primary cultured chondrocytes RAW 264.7 cells in vitro and destabilization of the medial meniscus (DMM)-induced OA mice in vivo. Primary chondrocytes were induced with IL-1β, and RAW 264.7 cells were treated with LPS and co-incubated with either individual extracts of PV and GL or different ratios of PV and GL mixture. For the OA animal model, the medial meniscus (DMM) was destabilized in 9-week-old male C57BL/6 mice. Treatment of individual PV and GL and combination of PV and GL extracts inhibited the mRNA expression level of COX2 in chondrocytes and RAW 264.7 cells. The optimized inhibitory effect was attained with a PV and GL combination at an 8:2 ratio (PG) without cytotoxic effects. PG extracts prevented the expression of catabolic factors (COX2, Mmp3, Mmp9, and Mmp13) and inflammatory mediator levels (PGE2 and collagenase). In addition, PG decreased subchondral sclerosis and increased BMD in the subchondral region of DMM-induced OA mice with protection of articular cartilage destruction by inhibiting inflammatory processes. This study suggests that PG may be an alternative medicinal herb for treatment of OA.

Keywords: Gentiana lutea; Prunella vulgaris; anti-inflammatory effect; destabilization of medial meniscus; osteoarthritis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of PV and GL extract on Raw 264.7 cells and primary chondrocytes. RAW 264.7 cells were incubated individually with PV and GL extract and their combined mixture (PG extract) with different ratios (7:3 and 8:2) for 24 h. Primary chondrocytes were incubated individually with PV and GL extract and their combined mixture (PG extract) with different ratios (7:3 and 8:2) for 48 h. (A,B) Relative COX2 expression was determined by qRT-PCR. (C,D) Cell viability was examined by WST assay. * p < 0.05 vs. Mock, # p < 0.05 vs. GL, $ p < 0.05 vs. PV. CONT; non-treated group, PV; Prunella vulgaris extract, GL; Gentiana lutea extract.
Figure 1
Figure 1
Effects of PV and GL extract on Raw 264.7 cells and primary chondrocytes. RAW 264.7 cells were incubated individually with PV and GL extract and their combined mixture (PG extract) with different ratios (7:3 and 8:2) for 24 h. Primary chondrocytes were incubated individually with PV and GL extract and their combined mixture (PG extract) with different ratios (7:3 and 8:2) for 48 h. (A,B) Relative COX2 expression was determined by qRT-PCR. (C,D) Cell viability was examined by WST assay. * p < 0.05 vs. Mock, # p < 0.05 vs. GL, $ p < 0.05 vs. PV. CONT; non-treated group, PV; Prunella vulgaris extract, GL; Gentiana lutea extract.
Figure 2
Figure 2
Effect of PG extract on inflammatory-associated factors in RAW 264.7 and primary chondrocytes. (A) RAW 264.7 cells were co-incubated with LPS and different concentrations (100 and 150 μg/mL) of PG extract (8:2 ratio) for 24 h. (B) Primary chondrocytes were co-incubated with IL-1β and different concentrations (100 and 150 μg/mL) of PG extract (8:2 ratio) for 48 h. Relative expression levels of COX2, Mmp3, Mmp9, and Mmp13 were determined by qRT-PCR. * p < 0.05 vs. Mock, # p < 0.05 vs. 100. CONT; non-treated group.
Figure 3
Figure 3
Effect of PG extract on inflammatory signaling cascade of OA in RAW 264.7 cells. RAW 264.7 cells were co-incubated with LPS and different concentrations (100 and 150 μg/mL) of PG extract (8:2 ratio) for 24 h. (A) Protein levels of COX2, NF-κB and p-NF- κB were subjected to Western blot analysis. β-actin was used as a loading control. (B) NO production was examined using nitrate/nitrite assay. * p < 0.05 vs. Mock, CONT; non-treated group.
Figure 4
Figure 4
Effect of PG extract on destructive mediators in IL-1β induced primary chondrocyte inflammation. Primary cultured mouse chondrocytes were incubated with different concentrations of PG extract (100 and 150 μg/mL) in the presence of IL-1β. (A) PGE2 and (B) collagenase levels in the cell supernatants were examined. * p < 0.05 vs. Mock. CONT; non-treated group.
Figure 5
Figure 5
Effects of PG extract on DMM-induced mouse OA model. Mice were induced OA by DMM, and different concentrations of PG extract (50, 100, and 200 mg/kg) were administered for 8 weeks. (A) Subchondral knee joint was scanned by micro-CT. (B) BMD of the subchondral knee joint was determined. * p < 0.05 vs. DMM, # p < 0.05 vs. DMM + 50, $ p < 0.05 vs. Sham. Sham; non-treated group.
Figure 6
Figure 6
Effects of PG extract on catabolic factors in a DMM-induced OA mouse model. (A) Representative images for safranin O staining and immunohistochemistry (IHC) for COX2, Mmp3 and Mmp13 of articular cartilage. (B) OARSI scoring and quantification of IHC for COX2, Mmp3 and Mmp13 was determined. * p < 0.05 vs. DMM, # p < 0.05 vs. DMM + 50. Sham; non-treated group.
Figure 7
Figure 7
Effect of PG extract on plasma levels of inflammatory cytokines in a DMM-induced OA mouse model. Plasma levels of inflammatory cytokines, such as IFN-γ, IL-1β, IL-6, and TNF-α, were determined using cytokine/chemokine magnetic bead panel immunology multiplex assay. * p < 0.05 vs. DMM, # p < 0.05 vs. DMM + 50. Sham; non-treated group.
Figure 8
Figure 8
Molecular mechanism for the inhibition of inflammatory cytokines using a PG extract.
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