Chondroprotective effects of Protaetia brevitarsis seulensis larvae as an edible insect on osteoarthritis in mice

Abstract

Osteoarthritis (OA) is a common chronic joint inflammatory disease characterized by progressive destruction of the articular cartilage, bone remodeling, and excessive chronic pain. Most therapeutic approaches do not rescue the progression of OA effectively or provide relief of symptoms. Protaetia brevitarsis seulensis larva (PBSL), which is attracting attention, is an edible insect with very high nutritional value and herbal medicine for the treatment of blood stasis, hepatic disease, and various inflammatory diseases. However, the effect of PBSL on OA has not yet been investigated. This study aimed to demonstrate the effects of PBSL water extract on the progression of OA using monosodium iodoacetate (MIA)-induced mice and SW1353 chondrocytes or murine macrophages. We injected MIA into the intraarticular area of mice following pretreatment with either saline or PBSL (200 mg/kg) for 2 weeks, and then locomotor activity, microcomputed tomography and histopathological analysis, quantitative reverse transcriptase-polymerase chain reaction analysis, and western blot analysis were performed. To determine the molecular effects of PBSL, we used interleukin-1β (IL-1β)-induced SW1353 chondrosarcoma or lipopolysaccharide (LPS)-stimulated macrophages. Pretreatment with PBSL diminished the symptoms of OA. Physical activity, articular cartilage damage, and the generation of microfractures were rescued by pretreatment with PBSL in the mouse model. Pretreatment with PBSL suppressed the progress of OA through the regulation of articular cartilage degradation genes and inflammation in both in vivo and in vitro models. Our results demonstrated that PBSL has value as edible insect that can be used in the development of functional foods for OA.

Keywords: Protaetia brevitarsis seulensis larva; chondroprotective effects; edible insect; osteoarthritis.

FIGURE 1

Effects of Protaetia brevitarsis seulensis larva (PBSL) on osteoarthritis (OA) symptoms in monosodium iodoacetate (MIA)‐induced OA mouse models. (a) The experimental scheme used to induce OA and administer oral pretreatment of PBSL. (b) At 7 days post‐MIA injection, locomotor activity was monitored using an open‐field test with an automated monitoring system. Data were analyzed with one‐way analysis of variance (ANOVA) (n > 3 per group, *p < .05, **p < .01 ***p < .001 vs. MIA group). (c) Histological analysis of mouse knee joint sections was stained with hematoxylin and eosin and Safranin O‐fast green (insets show higher magnification, 100×). Scale bars are indicated. (d) Microcomputed tomography (CT) images of mouse knee joint tissue were scanned using an in vivo micro‐CT imaging system. (e) Quantification of micro‐CT analysis image was analyzed using AccuCT micro‐CT analysis software. Data were analyzed using one‐way ANOVA (n = 3 per group). Con, untreated with saline; MIA, MIA‐injected with pretreatment of saline; MIA + PBSL, MIA‐injected with pretreatment of PBSL mice.

FIGURE 2

Effects of Protaetia brevitarsis seulensis larva (PBSL) on levels of MMPs and their inhibitors in monosodium iodoacetate (MIA)‐induced osteoarthritis mice knee tissue. (a) qPCR analysis was performed for gene expression of MMPs and their inhibitors in mouse knee joint tissue. Data were analyzed using one‐way analysis of variance (ANOVA) (n > 3 per group, *p < .05, ***p < .001 vs. Control group, ***p < .001 vs. MIA alone group). (b) Western blot analysis of the knee joint tissue extracts was performed for the protein expression of matrix metalloproteinase (MMP)3 and MMP13. Con, untreated with saline; MIA, MIA‐injected with pretreatment of saline; MIA + PBSL, MIA‐injected with pretreatment of PBSL mice.

FIGURE 3

Effects of Protaetia brevitarsis seulensis larva (PBSL) on the expression of matrix metalloproteinases (MMPs) and their inhibitors in interleukin (IL)1β‐induced SW1353 cells. (a) Cell viability was determined via MTS assay. Cells were treated with various concentrations of PBSL (50–400 μg/mL) for 24 h. (b) Cells were pretreated with PBSL (50–400 μg/mL) for 2 h and stimulated with IL‐1β (20 ng/mL) or IL‐1β alone for 24 h. Cell viability was determined using MTS assay. (c) Quantitative polymerase chain reaction analysis was performed for gene expression of MMPs and their inhibitors in SW1353 cells after treatment. (d) Western blot analysis was performed for protein expression of metalloproteinases and their inhibitors in SW1353 cells. Data were analyzed using one‐way analysis of variance (ANOVA) (n > 3 per group, *p < .05, **p < .01, ***p < .001 vs. IL‐1β alone group).

FIGURE 4

Effects of Protaetia brevitarsis seulensis larva (PBSL) on inflammatory mediators in RAW264.7 cells. (a) Cell viability, (b) NO synthesis, and (c) expression of the iNOS protein after treatment of PBSL in lipopolysaccharide (LPS)‐induced RAW264.7 cells. The cells were treated with PBSL (100, 200, and 400 μg/mL) and LPS (100 ng/mL) for 18 h. The analysis of NO synthesis and iNOS expression by PBSL was measured using the Griess assay and western blot analysis. Data were analyzed using one‐way analysis of variance (ANOVA) [n > 3 per group, *p < .05, ***p < .001 vs. NC (a) or LPS alone group (b)].

FIGURE 5

Effects of Protaetia brevitarsis seulensis larva (PBSL) or PBSL‐derived components on inflammatory factors in SW1353 cells or monosodium iodoacetate (MIA)‐induced mice knee tissue. (a) mRNA expression of inflammatory cytokines and mediators in SW1353 cells. Data were analyzed using one‐way analysis of variance (ANOVA) (n > 3 per group, **p < .01, ***p < .001 vs. IL‐1β alone group). (b) mRNA expression of inflammatory cytokines and mediators in mice knee tissue 10 days after MIA induction. Data were analyzed using one‐way ANOVA (n > 3 per group, *p < .05, **p < .01 vs. Control group, *p < .05 vs. MIA alone group). (c) Protein levels of interleukin‐6, cyclooxygenase 2 (COX2), and tumor necrosis factor‐α in SW1353 cells. (d) Protein levels of COX2 and iNOS in mice knee tissue 10 days post‐MIA induction. (e) Nitrite oxide (NO) synthesis by PBSL compound treatment in lipopolysaccharide (LPS)‐induced RAW264.7 cells. Cells were treated with 100 μM of PBSL compounds (benzoic acid, adenosine, uridine, hypoxanthine, adenine, and inosine) with LPS (100 ng/mL) for 18 h. Inhibition effects of components on NO synthesis were analyzed using a Griess assay. Data were analyzed using one‐way ANOVA (n > 3 per group, *p < .05 vs. LPS alone group).