Anti-inflammatory effects of the root, stem and leaf extracts of Chloranthus serratus on adjuvant-induced arthritis in rats

Abstract

Context: Chloranthus serratus [(Thunb.) Roem. et Schult, (Chloranthaceae)] is a folk medicine used for the treatment of rheumatoid arthritis.Objective: The aim of this study was to investigate anti-arthritic effects of the ethanol extracts of the roots (ER), stems (ES) and leaves (EL) of C. serratus on adjuvant arthritis rats and related mechanisms.Materials and methods: The rats were immunized by intradermal injection of complete Freund's adjuvant (CFA, 0.18 mL) into the right hind feet, and received intragastric administrations of the ER, ES and EL (2.07, 1.61 and 0.58 g/kg/d, respectively) for 14 days. The anti-arthritic activity was assessed by swelling rates, serum indicators, antioxidant capacity, histopathological and immunohistochemical analyses.Results: The LD₅₀ of the ER, ES and EL was higher than 10.35, 8.05 and 2.90 g/kg/p.o., respectively. Extract treatments decreased swelling rates, tumour necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF), interleukin 1 beta (IL-1β), migration inhibitory factor 1 (MIF-1), immunoglobulin G (IgG) and immunoglobulin M (IgM) levels and positive expression of VEGF in the arthritic rats (p < 0.01 or p < 0.05). The ER significantly decreased NO (3.91 ± 0.61 µmol/L), IL-6 (75.67 ± 16.83 pg/mL) and malondialdehyde (MDA) (2.28 ± 0.32 nmol/mL) contents and clearly increased IFN-γ (2082 ± 220.93 pg/mL) and superoxide dismutase (SOD) (601.98 ± 38.40 U/mL) levels. The ES and EL did not reverse the changes in some indicators. All the extracts alleviated inflammatory cell infiltration and synovial cell proliferation. Among them, the ER was the most pronounced.Discussion and conclusions: ER exerts the most promising effects, as shown by inhibiting the releases of inflammatory cytokines and enhancing antioxidant capacity, which provides a scientific basis for further research on C. serratus and its clinical applications.

Keywords: Rheumatoid arthritis; arthritic score; articular cartilage; complete Freund’s adjuvant; oxidative stress; pro-inflammatory mediators.

Figure 1.

UV fingerprints of the ER (A), ES (B) and EL (C) of C. serratus.

Figure 2.

Effects on the serum levels of TNF-α (A) and VEGF (B). The rats were treated with the ER (2.07 g/kg/d), ES (1.61 g/kg/d), EL (0.58 g/kg/d) and Tripterygium-polyglycosides (35 mg/kg/d) for 14 consecutive days after modelling. After administration, the levels of TNF-α and VEGF in each extract group were decreased to different extents, among them, the ER group was the most significant, followed by the EL group. All data were represented as mean ± SD (n = 6). *p < 0.05, **p < 0.01 vs. the Con group; ^#p < 0.05, ^##p < 0.01 vs. the MD group; ^$p < 0.05, ^$$p < 0.01 vs. the EL group.

Figure 3.

Effects on the levels of NO (A), IL-1β (B), IL-6 (C) and MIF-1 (D). The rats were treated with the ER (2.07 g/kg/d), ES (1.61 g/kg/d), EL (0.58 g/kg/d) and Tripterygium-polyglycosides (35 mg/kg/d) for 14 consecutive days after modelling. After administration, the expression of NO, IL-1β, IL-6 and MIF-1 in each extract group was decreased to different extents, and the ER group was the most obvious, followed by the EL group. All data were represented as mean ± SD (n = 6). *p < 0.05, **p < 0.01 vs. the Con group; ^#p < 0.05, ^##p < 0.01 vs. the MD group; ^$p < 0.05, ^$$p < 0.01 vs. the EL group.

Figure 4.

Effects on the levels of IFN-γ (A), IgG (B) and IgM (C). The rats were treated with the ER (2.07 g/kg/d), ES (1.61 g/kg/d), EL (0.58 g/kg/d) and Tripterygium-polyglycosides (35 mg/kg/d) for 14 consecutive days after modelling. After treating with the different extracts, the level of IFN-γ was increased, whereas the levels of IgG and IgM were decreased compared with those of the MD group. Moreover, the ER group was the most obvious, followed by the EL group. All data were represented as mean ± SD (n = 6). *p < 0.05, **p < 0.01 vs. the Con group; ^#p < 0.05, ^##p < 0.01 vs. the MD group; ^$p < 0.05, ^$$p < 0.01 vs. the EL group.

Figure 5.

Effects on the levels of SOD (A) and MDA (B). The rats were treated with the ER (2.07 g/kg/d), ES (1.61 g/kg/d), EL (0.58 g/kg/d) and Tripterygium-polyglycosides (35 mg/kg/d) for 14 consecutive days after modelling. Compared with the MD group, the level of SOD increased under the treatment of the different extracts in all the groups. Except for the ES group, the level of MDA decreased in all the groups. All data were represented as mean ± SD (n = 6). *p < 0.05, **p < 0.01 vs. the Con group; ^#p < 0.05, ^##p < 0.01 vs. the MD group; ^$p < 0.05, ^$$p < 0.01 vs. the EL group.

Figure 6.

Histopathological effects of the different part extracts of C. serratus on CFA-induced arthritic rats (HE, × 200). The rats were treated with the ER (2.07 g/kg/d), ES (1.61 g/kg/d), EL (0.58 g/kg/d) and Tripterygium-polyglycosides (35 mg/kg/d) for 14 consecutive days after modelling. (A) Representative photos of HE staining. (B) Histopathology score (points). The damage was the most serious in the ES group, followed by the EL group. All data were represented as mean ± SD (n = 6). *p < 0.05, **p < 0.01 vs. the Con group; ^#p < 0.05, ^##p < 0.01 vs. the MD group; ^$p < 0.05, ^$$p < 0.01 vs. the EL group.

Figure 7.

Effects on the positive expression of VEGF (× 200). The rats were treated with the ER (2.07 g/kg/d), ES (1.61 g/kg/d), EL (0.58 g/kg/d) and Tripterygium- polyglycosides (35 mg/kg/d) for 14 consecutive days after modelling. (A) Immunohistochemistry results of VEGF. (B) Column chart represented the positive expression rate of VEGF. All data were represented as mean ± SD (n = 6). *p < 0.05, **p < 0.01 vs. the Con group; ^#p < 0.05, ^##p < 0.01 vs. the MD group; ^$p < 0.05, ^$$p < 0.01 vs. the EL group.