Cinnamic aldehyde, an anti-inflammatory component in Du-Huo-Ji-Sheng-Tang, ameliorates arthritis in II collagenase and monosodium iodoacetate induced osteoarthritis rat models

Abstract

Background and aim: Du-Huo-Ji-Sheng-Tang (DHJST) is a Chinese herbal formula used for arthralgia and arthritis treatment clinically. This study aims to evaluate the joint-protecting efficacy of DHJST and to identify the active constituents as the evaluation marker.

Experimental procedure: DHJST can be categorized into three recipes: Blood-tonifying-herbs Si-Wu-Tang (SWT), Wind-dampness-dispelling-herbs (WDH) and Qi-tonifying-herbs (TH). All formulas were used to explore the joint-protecting efficacies.

Results and conclusion: s: Firstly, DHJST could decrease the arthritis progression in the monosodium-iodoacetate-induced rat and cure arthritis in the type II collagenase-induced rat. Further, in lipopolysaccharide-stimulated RAW 264.7 cells, DHJST, TH and Cinnamomum cassia (CC), an ingredient in TH, were the most potent nitric oxide (NO) and prostaglandin E₂ (PGE₂) inhibitors. The major components, cinnamic aldehyde, showed the strongest NO and PGE₂ inhibition. Up-regulated inducible NO synthase (iNOS) and cyclooxygenase-2 were inhibited by DHJST, TH, CC, and cinnamic aldehyde. In interleukin-1β-stimulated primary chondrocytes, upregulated iNOS was inhibited by DHJST, TH, Cinnamomum cassia, and cinnamic aldehyde. Upregulated matrix metalloprotease-13 was only inhibited by DHJST and TH and Eucommia ulmoides (EU) extract. Results suggest that DHJST presented joint-protective and cure arthritis effects. TH presented equal joint-protective effects as DHJST. The major anti-inflammatory ingredient in TH was Cinnamomum cassia in TH. And cinnamic aldehyde was the potent anti-inflammatory active compound in Cinnamomum cassia. Therefore, this study may facilitate the modern use of DHJST with TH as a simplified version but equally effective anti-osteoarthritic agents with cinnamic aldehyde as a quality control marker of DHJST and TH in osteoarthritis prevention or treatment.

Keywords: Arthritis; Cinnamic aldehyde; Cinnamomum cassia; Monosodium iodoacetate; Type II collagenase.

Graphical abstract

Fig. 1

Effects of DHJST on MIA-induced arthritis. Experimental flow chart (A). Effect of DHJST on MIA-induced paw edema swelling on days 1 and 3 (B). hind-limb weight-bearing on day 5 (C); hot-plate latent pain responses on day 10 (D). ∗p < 0.05, compared to the control. n = 3.

Fig. 2

Effect of DHJST on CIA-induced arthritis. Experimental flow chart (A). Changes in hind limb weight bearing in rats were measured (B). The weight distribution ratio was calculated as the left hind-limb (collagenase injection side) weight divided by the right hind-limb (control side) weight. Values are the mean ± SD. ∗p < 0.05, compared to the control; #p < 0.05, compared to day 0. n = 3.

Fig. 3

Effect of DHJST on pathologic changes in CIA-induced arthritis model. Representative images were presented. (A), normal with normal cartilage thickness (double head arrow); (B), control with cartilage thinning (double head arrow); (C), DHJST treated (25 mg/kg/day) with cartilage thickness similar to the negative control (double head arrow); (D), DHJST treated (50 mg/kg/day) with cartilage thickness restored (double head arrow).

Fig. 3

Effect of DHJST on pathologic changes in CIA-induced arthritis model. Representative images were presented. (A), normal with normal cartilage thickness (double head arrow); (B), control with cartilage thinning (double head arrow); (C), DHJST treated (25 mg/kg/day) with cartilage thickness similar to the negative control (double head arrow); (D), DHJST treated (50 mg/kg/day) with cartilage thickness restored (double head arrow).

Fig. 4

HPLC profiles of DHJST (A), SWT (B); TH (C); WDH (D); Cinnamomum cassia (E). Abbreviations: Peak (1) 2-methylcinnamic acid; Peak (2) cinnamic acid; Peak (3) cinnamic aldehyde.

Fig. 5

Inhibitory Effect DHJST, SWT, WDH and TH on LPS-induced nitric oxide (NO) production in RAW 264.7 cells. (A), DHJST (300 μg/mL) and combined extracts of mixture of SWT, WDH and TH (100 μg/mL for each); (B), DHJST, SWT, WDH and TH. ∗p < 0.05, compared to DHJST; (C), TH (100 μg/mL), mixture of TH and SWT (50 μg/mL for each), TH and WDH (50 μg/mL for each), SWT and WDH (50 μg/mL for each) ∗p < 0.05, compared to TH; #p < 0.05, compared to SWT and WDH; (D), effect of TH ingredients on NO inhibition; (E), effect of three major compounds in Cinnamomum cassia on NO inhibition. Abbreviations: PG, Panax ginsen; EU, Eucommia ulmoides; GU, Glycyrrhiza. Uralensis; CC, Cinnamomum cassia; (1), 2-methylcinnamic acid; (2), cinnamic acid; (3), cinnamic aldehyde. Values are the mean ± SD. n = 3.

Fig. 5

Inhibitory Effect DHJST, SWT, WDH and TH on LPS-induced nitric oxide (NO) production in RAW 264.7 cells. (A), DHJST (300 μg/mL) and combined extracts of mixture of SWT, WDH and TH (100 μg/mL for each); (B), DHJST, SWT, WDH and TH. ∗p < 0.05, compared to DHJST; (C), TH (100 μg/mL), mixture of TH and SWT (50 μg/mL for each), TH and WDH (50 μg/mL for each), SWT and WDH (50 μg/mL for each) ∗p < 0.05, compared to TH; #p < 0.05, compared to SWT and WDH; (D), effect of TH ingredients on NO inhibition; (E), effect of three major compounds in Cinnamomum cassia on NO inhibition. Abbreviations: PG, Panax ginsen; EU, Eucommia ulmoides; GU, Glycyrrhiza. Uralensis; CC, Cinnamomum cassia; (1), 2-methylcinnamic acid; (2), cinnamic acid; (3), cinnamic aldehyde. Values are the mean ± SD. n = 3.

Fig. 6

Regulatory effect on lipopolysaccharide (LPS)-induced iNOS and COX-2 production in RAW 264.7 cells by DHJST (A), TH (B), Cinnamomum cassia; (C), Eucommia ulmoides (D), and cinnamic aldehyde (E). Abbreviations: CC, Cinnamomum cassia; EU, Eucommia ulmoides.

Fig. 7

Regulatory effect on IL-1β-induced iNOS and MMP-13 in PRCs by DHJST (A), TH (B), Cinnamomum cassia (C), Eucommia ulmoides (D), and cinnamic aldehyde (E). Effect of 50 μg/mL of DHJST, TH, Cinnamomum cassia and Eucommia ulmoides on PGE₂ level (F), data represented as the mean (±S.D.) of at least three independent experiments, each performed in triplicate. Abbreviations: CC, Cinnamomum cassia; EU, Eucommia ulmoides.

Fig. 7

Regulatory effect on IL-1β-induced iNOS and MMP-13 in PRCs by DHJST (A), TH (B), Cinnamomum cassia (C), Eucommia ulmoides (D), and cinnamic aldehyde (E). Effect of 50 μg/mL of DHJST, TH, Cinnamomum cassia and Eucommia ulmoides on PGE₂ level (F), data represented as the mean (±S.D.) of at least three independent experiments, each performed in triplicate. Abbreviations: CC, Cinnamomum cassia; EU, Eucommia ulmoides.

Fig. 8

Effects of DHJST and TH on MIA-induced paw edema swelling on days 1 and 3 (A). hind-limb weight-bearing on day 5 (B); hot-plate latent pain responses on day 10 (C). ∗p < 0.05, compared to the control. n = 3.