The Effect of the Mixed Extract of Kalopanax pictus Nakai and Achyranthes japonica Nakai on the Improvement of Degenerative Osteoarthritis through Inflammation Inhibition in the Monosodium Iodoacetate-Induced Mouse Model

Abstract

Osteoarthritis is a chronic inflammatory disease, and, due to the lack of fundamental treatment, the main objective is to alleviate pain and prevent cartilage damage. Kalopanax pictus Nakai and Achyranthes japonica Nakai are herbal plants known for their excellent anti-inflammatory properties. The objective of this study is to confirm the potential of a mixture extract of Kalopanax pictus Nakai and Achyranthes japonica Nakai as a functional raw material for improving osteoarthritis through anti-inflammatory effects in macrophages and MIA-induced arthritis experimental animals. In macrophages inflamed by lipopolysaccharide (LPS), treatment of Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture inhibits NF-κB and mitogen-activated protein kinase (MAPK) activities, thereby inhibiting inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), inflammatory factors PGE2, MMP-2, and MMP-9, and nitric oxide (NO) was reduced. In addition, in an animal model of arthritis induced by MIA (monosodium iodoacetate), administration of Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture reduced blood levels of inflammatory cytokines TNF-α and IL-6, inflammatory factors prostaglandin E2(PGE2), matrix metalloproteinase-2(MMP-2), and NO. Through these anti-inflammatory effects, MIA-induced pain reduction (recovery of clinical index, increase in weight bearing, and increase in area and width of the foot), recovery of meniscus damage, loss of cartilage tissue or inflammatory cells in tissue infiltration reduction, and recovery of the proteglycan layer were confirmed. Therefore, it is considered that Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture has the potential as a functional raw material that promotes joint health.

Keywords: Achyranthes japonica Nakai; Kalopanax pictus Nakai; MMPs; anti-inflammation; osteoarthritis.

Figure 1

Chromatograms of liriodendrin-standard solution (A), ecdysterone-standard solution (B), liriodendrin-test solution (C), and ecdysterone-test solution (D). (1): Liriodendrin and (2): Ecdysterone.

Figure 1

Chromatograms of liriodendrin-standard solution (A), ecdysterone-standard solution (B), liriodendrin-test solution (C), and ecdysterone-test solution (D). (1): Liriodendrin and (2): Ecdysterone.

Figure 2

In RAW 264.7 cells, the (A) cell viability of SHP-47B and the physiological activity of (B) NO and (C) iNOS were measured. Cell viability was measured by WST-1 assay, and after measuring NO production, iNOS was measured by Western blot analysis. The data are expressed as the mean ± SD (n = 3), and different letters (g > f > e > d > c > b > a) indicate a significant difference at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 3

Analysis of the physiological activity of SHP-47B on the activation of NF-κB and MPAK pathways in LPS-induced inflammation in RAW 264.7 macrophages. Proteins obtained from RAW 264.7 cells were analyzed by Western blot for the expression of proteins related to each of the NF-κB and MAPK pathways.

Figure 4

Effects of SHP-47B treatment on (A) weight bearing and (B) clinical arthritis in MIA-induced arthritis mice model. The data are expressed as the mean ± SD (n = 7), and different letters (f > e > d > c > b > a) indicate a significant difference at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 5

The effects of SHP-47B treatment on plantar (A) area and (B) width in gait analysis of an mice model of arthritis induced by MIA. Gait analysis results were comprehensively expressed in the (C) mouse plantar measurement picture. The data are expressed as the mean ± SD (n = 7), and different letters (d > c > b > a) indicate a significant difference at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 6

Effect of SHP-47B treatment on hematological analysis, (A) tumor necrosis factor-α (TNF-α), (B) interleukin-6 (IL-6), (C) prostaglandin E2 (PGE2), (D) matrix metalloproteinase-2 (MMP-2), and (E) nitric oxide (NO) in a mice model of arthritis induced by MIA. The data are expressed as the mean ± SD (n = 7), and different letters (d > c > b > a) indicate a significant difference at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 6

Effect of SHP-47B treatment on hematological analysis, (A) tumor necrosis factor-α (TNF-α), (B) interleukin-6 (IL-6), (C) prostaglandin E2 (PGE2), (D) matrix metalloproteinase-2 (MMP-2), and (E) nitric oxide (NO) in a mice model of arthritis induced by MIA. The data are expressed as the mean ± SD (n = 7), and different letters (d > c > b > a) indicate a significant difference at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 7

Effects of SHP-47B treatment on (A) meniscus volume and (B) micro-CT images (blue-purple: meniscus) in an MIA-induced arthritis mice model. N: normal, C: control, L: SHP-47B 50 mg/kg, M: SHP-47B 100 mg/kg, H: SHP-47B 300 mg/kg, P: celecoxib. The data are expressed as the mean ± SD (n = 3), and different letters (b > a) indicate a significant difference at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 8

Analysis of synovial cell changes and inflammatory cell infiltration in the knee joint by H&E staining in an MIA-induced arthritis mouse model. (A) normal group, (B) control group, (C) SHP-47B 50 mg/kg group, (D) SHP-47B 100 mg/kg group, (E) SHP-47B 300 mg/kg group, and (F) positive control (celecoxib) group. Histological analysis result image magnification = 4×, and scale bar = 300 μm.

Figure 9

Analysis of cartilage tissue damage through Safranin-O staining in MIA-induced arthritis mouse model. (A) normal group, (B) control group, (C) SHP-47B 50 mg/kg group, (D) SHP-47B 100 mg/kg group, (E) SHP-47B 300 mg/kg group, and (F) positive control (celecoxib) group. Histological analysis result image magnification = 4×, and scale bar = 300 μm.