Inhibitory Effects of Reynoutria japonica Houtt. on Pain and Cartilage Breakdown in Osteoarthritis Based on Its Multifaceted Anti-Inflammatory Activity: An In Vivo and In Vitro Approach

Abstract

In the past 30 years, the number of years lived with disability due to osteoarthritis (OA) has doubled, making it an increasing global health burden. To address this issue, interventions that inhibit the progressive pathology driven by age-related low-grade inflammation, the primary mechanism of OA, are being actively pursued. Recent investigations have focused on modulating the age-related low-grade inflammatory pathology of this disease as a therapeutic target. However, no agent has successfully halted the disease's progression or reversed its irreversible course. Reynoutria japonica Houtt. (RJ), a promising East Asian herbal medicine, has been utilized for several diseases due to its potent anti-inflammatory activity. This study aims to determine RJ's capacity to inhibit OA symptoms and associated inflammation, exploring its potential for further development. In vivo and in vitro experiments demonstrated RJ's anti-OA activity and modulation of multifaceted inflammatory targets. RJ significantly inhibited pain, gait deterioration, and cartilage destruction in a monosodium iodoacetate-induced OA rat model, with its analgesic effect further confirmed in an acetic acid-induced writhing model. RJ exhibited consistent anti-inflammatory activity against multiple targets in serum and cartilage of the OA rat model and lipopolysaccharide-induced RAW 264.7 cells. The inhibition of inflammatory cytokines, including interleukin-1β, interleukin-6, matrix metalloproteinase-13, tumor necrosis factor-α, and nitric oxide synthase 2, suggests that RJ's alleviation of OA manifestations relates to its multifaceted anti-inflammatory activity. These results indicate that RJ merits further investigation as a disease-modifying drug candidate targeting OA's inflammatory pathology. To further characterize the pharmacological properties of RJ, future studies with expanded designs are warranted.

Keywords: East Asian herbal medicine; Reynoutria japonica Houtt.; analgesic; anti-inflammatory; chondroprotective; osteoarthritis.

Figure 1

HPLC chromatogram of the RJ extract at 280 nm: polydatin, resveratrol, and emodin retention time = 11.997 min, 15.437 min, and 24.166 min, respectively. The x-axis shows the retention time; the y-axis shows the wavelength; the z-axis shows the absorbance unit. HPLC: High-performance liquid chromatography; RJ: Reynoutria japonica Houtt.

Figure 2

Assessment the discomfort about the weight-bearing capacity of the OA-induced model; (A) the weight bearing of OA rats in the RJ 100, RJ 300, and INDO 3 groups (n = 9) for 0 to 24 days; (B) the AUC was recorded using the incapacitance meter. ** p < 0.01 vs. MIA, *** p < 0.001 vs. MIA, ### p < 0.001 vs. sham Tukey’s multiple comparison test after Two-way ANOVA. AUC: area under the curve; INDO 3: indomethacin 3 mg/kg; MIA: monosodium iodoacetate, RJ: Reynoutria japonica Houtt., sham: non-treated group.

Figure 3

Photos of the right knee cartilage of OA-induced model. INDO 3, RJ 100, and RJ 300 (n = 9) were administered to OA-induced rats. (A) Representative images showing cartilage degradation. Arrows mean the cartilage-damaged site. (B) Macroscopic score. Grade 0: Typical appearance of the cartilage surface; grade 1: Slight yellow discoloration or mild fibrillation; grade 2: Erosion in the middle or superficial layer of cartilage is visible; grade 3: Severe deterioration reaching the subchondral bone; grade 4: Massive damage and extensive disclosure of subchondral bone. ** p < 0.01 vs. MIA, *** p < 0.001 vs. MIA using 1-way ANOVA and Dunnett’s test, ### p < 0.001 vs. sham by Unpaired T-test by Two-tailed p value. INDO 3: indomethacin 3 mg/kg; MIA: monosodium iodoacetate, RJ: Reynoutria japonica Houtt., sham: non-treated group.

Figure 4

Gait analysis of OA-induced model during 21 days. (A) Representative photo indicated to footprints. (B) Paw area (blue line) and (C) stride length (red line) was analyzed on day 0, 7, 14, and 21 in OA-induced rats (n = 9). INDO 3: indomethacin 3 mg/kg; MIA: monosodium iodoacetate, RJ: Reynoutria japonica Houtt., sham: non-treated group.

Figure 4

Gait analysis of OA-induced model during 21 days. (A) Representative photo indicated to footprints. (B) Paw area (blue line) and (C) stride length (red line) was analyzed on day 0, 7, 14, and 21 in OA-induced rats (n = 9). INDO 3: indomethacin 3 mg/kg; MIA: monosodium iodoacetate, RJ: Reynoutria japonica Houtt., sham: non-treated group.

Figure 5

Effect of cytokines level in the RJ groups. (A) IL-1β, (B) IL-6, and (C) TNF-α expression level in OA-induced rats. OA models were treated RJ 100 and RJ 300 (n = 9) during 0–24 days. *** p < 0.001 vs. MIA by One-way ANOVA and Dunnett’s test, ### p < 0.001 vs. sham by Unpaired T-test by Two-tailed p value. INDO 3: indomethacin 3 mg/kg; MIA: monosodium iodoacetate, RJ: Reynoutria japonica Houtt., sham: non-treated group.

Figure 5

Effect of cytokines level in the RJ groups. (A) IL-1β, (B) IL-6, and (C) TNF-α expression level in OA-induced rats. OA models were treated RJ 100 and RJ 300 (n = 9) during 0–24 days. *** p < 0.001 vs. MIA by One-way ANOVA and Dunnett’s test, ### p < 0.001 vs. sham by Unpaired T-test by Two-tailed p value. INDO 3: indomethacin 3 mg/kg; MIA: monosodium iodoacetate, RJ: Reynoutria japonica Houtt., sham: non-treated group.

Figure 6

Analgesic effect of RJ in AIW models. Mice were treated with RJ 200, RJ 600, and IBU 200 (n = 8). All mice were injected with 0.7% acetic acid intraperitoneally 10 min prior to the recorded. ** p < 0.01 vs. CT, *** p < 0.001 vs. CT by One-way ANOVA and Dunnett’s test. CT: control, IBU 200: ibuprofen 200 mg/kg; RJ: Reynoutria japonica Houtt.

Figure 7

Effects of RJ using RAW264.7 cells. (A) Cell viability (%) and (B) NO production. * p < 0.05 vs. CT by 1-way ANOVA and Dunnett’s test, ### p < 0.001 vs. NT by Unpaired T-test by Two-tailed p value. CT: control, DEX 1: dexamethasone 1 μg/mL, LPS: lipopolysaccharide, NT: non-treated, RJ: Reynoutria japonica Houtt.

Figure 8

Analysis of cytokine expression levels in RAW264.7. (A–G) mRNA analysis of COX-2, IL-1β, IL-6, NOS2, TNF-α, MMP8, and MMP13; (H) protein analysis of COX-2, IL-1β, IL-6, TNF-α, and MMP13. The cells were incubated with LPS and DEX 1, RJ 30, RJ 100, and RJ 300 during 24 h. * p < 0.05 vs. CT by 1-way ANOVA and Dunnett’s test, ** p < 0.01 vs. CT, *** p < 0.001 vs. CT, ### p < 0.001 vs. NT by Unpaired T-test by Two-tailed p value. CT: control, DEX 1: dexamethasone 1 μg/mL, LPS: lipopolysaccharide, NT: non-treated, RJ: Reynoutria japonica Houtt.

Figure 8

Analysis of cytokine expression levels in RAW264.7. (A–G) mRNA analysis of COX-2, IL-1β, IL-6, NOS2, TNF-α, MMP8, and MMP13; (H) protein analysis of COX-2, IL-1β, IL-6, TNF-α, and MMP13. The cells were incubated with LPS and DEX 1, RJ 30, RJ 100, and RJ 300 during 24 h. * p < 0.05 vs. CT by 1-way ANOVA and Dunnett’s test, ** p < 0.01 vs. CT, *** p < 0.001 vs. CT, ### p < 0.001 vs. NT by Unpaired T-test by Two-tailed p value. CT: control, DEX 1: dexamethasone 1 μg/mL, LPS: lipopolysaccharide, NT: non-treated, RJ: Reynoutria japonica Houtt.

Figure 9

RJ groups reduced inflammatory cytokine levels in OA-induced models. (A–F) mRNA levels of COX-2, IL-1β, IL-6, NOS2, TNF-α, and MMP13 analyzed using qRT-PCR; (G) protein expression levels of COX-2, IL-1β, IL-6, TNF-α, and MMP13 analyzed using Western blot analysis. ### p < 0.001 vs. sham by Unpaired T-test by Two-tailed p value, * p < 0.05 vs. MIA, ** p < 0.01 vs. MIA, *** p < 0.001 vs. MIA using one-way ANOVA and Dunnett’s test. INDO 3: indomethacin 3 mg/kg; MIA: monosodium iodoacetate, RJ: Reynoutria japonica Houtt., sham: non-treated group.

Figure 9

RJ groups reduced inflammatory cytokine levels in OA-induced models. (A–F) mRNA levels of COX-2, IL-1β, IL-6, NOS2, TNF-α, and MMP13 analyzed using qRT-PCR; (G) protein expression levels of COX-2, IL-1β, IL-6, TNF-α, and MMP13 analyzed using Western blot analysis. ### p < 0.001 vs. sham by Unpaired T-test by Two-tailed p value, * p < 0.05 vs. MIA, ** p < 0.01 vs. MIA, *** p < 0.001 vs. MIA using one-way ANOVA and Dunnett’s test. INDO 3: indomethacin 3 mg/kg; MIA: monosodium iodoacetate, RJ: Reynoutria japonica Houtt., sham: non-treated group.