Introduction
Powderized dried roots of the plants
Acanthopanax senticosus (Siberian ginseng),
Angelica sinensis (Dong Quai) and
Scutellaria baicalensis (Baikal Skullcap) are commonly used in oriental medicine for a variety of indications based on traditional concepts.
A. senticosus is used as a general tonic to stimulate Qi forces [
1].
A. sinensis is used, for instance, to treat blood deficiency with wind–damp painful obstruction [
2,
3], and
S. baicalensis is used to clear heat, remove toxins and restrain bleeding [
4,
5]. All three plants are contained in herbal mixtures used for the treatment of chronic inflammatory disorders, including arthritis [
6]. Pharmacologic studies in animals have documented the anti-inflammatory effects of all three plants.
A. senticosus has been shown to reduce the expression of cyclo-oxygenase (COX)-2 and complement type 3 receptor (a marker for microglia in the central nervous system) in cerebral ischemia [
7] and to inhibit mast cell-dependent anaphylaxis [
8].
A. sinensis root polysaccharides inhibited neutrophil migration in ethanol-induced gastrointestinal inflammation in rats [
9] and reduced expression of pro-inflammatory factors in experimental colitis in rats [
10]. The flavonoids baicalein, which binds to chemokine ligands and inhibits leukotriene C4 synthesis, and wogonin have been implicated as the principal anti-inflammatory active ingredients of
S. baicalensis [
11,
12].
Considering their anti-inflammatory properties, extracts or mixtures of extracts from these plants might be suitable for the treatment or prevention of inflammatory arthropathies. Mixtures of medicinal herbs containing root preparations from these three herbs are indeed used in traditional oriental medicine for this purpose [
6], and there is anecdotal evidence from clinical experience in traditional oriental medicine that these herbs might be effective in treating musculoskeletal pain and arthritis (H.C. Kim, S.M. Jung, unpublished data). However, these herbs have not been validated for the treatment of acute or chronic synovitis in clinical studies or animal models of arthritis. As a first step, we therefore wanted to investigate the efficacy and mode of action of a mixture of standardized root extracts from the three plants in a simple animal model that resembles acute synovitis in humans.
The murine air pouch model represents an easily manipulable animal model of acute inflammation that has been used extensively in studies of a variety of anti-inflammatory agents. In contrast to animal models of chronic arthritis, the murine air pouch model lends itself well to the study of orally administered agents because it does not require prolonged gavage feedings of test substances to the animals. The air pouch is a newly formed, bursa-like tissue that grows around subcutaneously injected air and resembles the human synovial lining [
13]. For the purposes of definition, we shall refer to this newly formed tissue as the 'pouch membrane'. Depending on the pro-inflammatory agent instilled into the pouch, distinct forms of inflammation can be elicited [
14]. Injection of monosodium urate (MSU) crystals results in transient neutrophilic inflammation that resembles acute gouty arthritis in humans [
15,
16] and induces major pro-inflammatory cytokines that are active in chronic inflammatory arthropathies, such as TNF-α and IL-1 and -6 [
17‐
19]. Here, we show that the root extracts strongly inhibit inflammation in this model by decreasing neutrophil immigration into the pouch membrane, reducing expression of pro-inflammatory factors, including prostaglandin E
2 (PGE
2), and raising the level of the potentially anti-inflammatory prostaglandin D
2 (PGD
2), thereby normalizing the PGD
2:PGE
2 ratio. These findings suggest elevation of PGD
2 levels as a novel mechanism of action for an anti-inflammatory agent.
Discussion
A mixture of root extracts from A. senticosus, A. sinensis and S. baicalensis demonstrated strong anti-inflammatory properties in this model of MSU crystal-induced neutrophilic inflammation. These results agree well with previous reports that each herb exhibited some form of anti-inflammatory property in other experimental models.
The mode of action of this mixture seems to be owing to both a reduction of pro-inflammatory factors and a stimulation of at least one potentially anti-inflammatory factor, PGD
2. TNF-α, IL-6 and PGE
2 all have important roles in inflammatory arthropathies, including gout [
26‐
28]. Moreover, the levels PGE
2 and TNF-α are elevated in the inflamed rat air pouch [
14], and, in preliminary studies of the microarray analysis of isolated murine air pouch membranes stimulated with MSU crystals, we have recently identified IL-6 as an MSU crystal-induced cytokine in the air pouch membrane and localized its expression to membrane fibroblasts and inflammatory cells [
18]. Reductions in the levels of all these pro-inflammatory factors paralleled the reduction of the leukocyte count in the pouch exudate of mice treated with the root extracts. A reduction in neutrophil numbers within the pouch membrane was also observed, proving that the root extracts inhibited neutrophil recruitment and/or migration into the pouch membrane and not just their exit into the pouch exudate. We cannot explain fully why treatment with the extracts completely prevented the rise in the level of IL-6 mRNA in the pouch membrane, whereas a reduced level of IL-6 protein was still detected in the pouch exudate. The level of IL-6 mRNA peaks in MSU-stimulated air pouch membranes 1–4 hours after MSU injection and is up to tenfold higher than the level at 9 hours (F. Pessler, S.M. Jung, H.R. Schumacher, unpublished data). It is, therefore, possible that the low level at 9 hours reflects an overall reduction of IL-6 transcription throughout the time course and that some elevation of IL-6 mRNA would still be detectable at the earlier time points in MSU-stimulated mice treated with the extracts. Considering the short half-life of IL-6 mRNA and strong role of mRNA stabilization in regulation of IL-6 expression [
29,
30], another possible explanation is that the root extracts increased turnover of IL-6 mRNA, whereas the stability of the IL-6 protein was unaffected. Alternatively, active ingredients from the root extracts perhaps achieved higher concentrations in the pouch membrane than the exudate, in which leukocytes continued to synthesize IL-6. We did not test for potential effects of the extracts on IL-1β expression in the air pouch membrane. However, in ongoing investigations into the effects of the extracts on inflammatory mediator synthesis by cultured murine macrophages, we have detected a >95% reduction of MSU crystal-induced IL-1β and IL-6 mRNA synthesis (F. Pessler, H.C. Kim, H.R. Schumacher, unpublished results). It, therefore, seems probable that the extracts reduce the major pro-inflammatory cytokines nonselectively and thus do not affect any one cytokine specifically.
The effects of the extracts were assessed after four doses (feedings). This regimen was chosen because it was probably the earliest point at which steady-state serum levels of gastrointestinally absorbed substances could be expected. It will now be important to determine in greater detail the effective dose(s), time to onset of the anti-inflammatory effects and effects on established inflammation and to test other routes of administration.
The rise in the level of PGD
2, the precursor of the anti-inflammatory PGJ
2, following treatment with the root extracts, represents an intriguing observation. To our knowledge, elevation of PGD
2 levels has not been described as the effect of an anti-inflammatory agent. Although it is also involved in acute inflammatory states, such as asthma [
31,
32], PGD
2 is now increasingly recognized as an important mediator of the resolution of inflammation. For instance, h-PGDS mRNA [
33] and PGD
2 levels [
34] rise during the resolution phase of an acute inflammatory response and h-PGDS knock-out mice fail to resolve a delayed-type hypersensitivity reaction [
35]. Moreover, administration of PGD
2 or its metabolite PGJ
2 reduces the severity of carrageenan-induced pleurisy [
34,
36]. The prophylactic anti-inflammatory properties of PGD
2 have also been demonstrated in the murine air pouch [
17]. Injection of MSU crystals led to a decrease of endogenous PGD
2 synthase, whereas intrapouch injection of fibroblasts overexpressing the enzyme resulted in decreased inflammation and expression of pro-inflammatory mediators. It is thus tempting to speculate that the root extracts reduced inflammation, in part, by raising the level of PGD
2. The modest increase in h-PGDS mRNA argues that this might be partly owing to an elevated h-PGDS level, but other mechanisms, such as enhanced h-PGDS activity or PGD
2 stability, are also plausible. It is unclear whether PGD
2 itself or its degradation product PGJ
2 mediates the apparent anti-inflammatory effect of the root extracts. We have been unable to detect PGJ
2 in lavaged air pouch exudates by ELISA. This might be because of the instability of PGJ
2 in this model [
17] or because the PGJ
2 level rises later during the resolution phase of inflammation. Interestingly, TNF-α raises PGE
2, but decreases PGD
2, synthesis by zymosan-stimulated murine macrophages [
37]. The normalization of the PGD
2:PGE
2 ratio by the root extracts paralleled the inhibition of TNF-α mRNA synthesis in the pouch membrane, thus raising the possibility that inhibition of TNF-α might be part of the mechanism for PGD
2 stimulation in this model. Consistent with this hypothesis, in addition to neutrophils, monocytes and macrophages (cell types capable of high levels of TNF-α synthesis) represent the predominant inflammatory cells in the air pouch membrane. Transforming growth factor (TGF)-β is strongly associated with the resolution of crystal-induced inflammation [
38,
39]. Although we did not assay TGF-β levels, it is possible that treatment with the extracts might affect levels of anti-inflammatory substances in general and thus raise the level of TGF-β in parallel with that of PGD
2. It would, therefore, be interesting to measure TGF-β levels in future studies that aim to define the mechanism of action of the extracts further.
How do commonly used anti-inflammatory agents, such as NSAIDs and corticosteroids, affect PGD
2 levels? In an endotoxin-based mouse model of inflammation, administration of aspirin or indomethacin nearly abolished both PGE
2 and PGD
2 synthesis, whereas PGD
2 levels rose during the natural resolution of inflammation in untreated animals [
33]. Dexamethasone inhibited PGD
2 synthesis in zymosan-stimulated murine macrophages [
37]. Prednisone did not alter PGD
2 synthesis during the cutaneous late-phase allergic response in humans [
40]. It is, therefore, unlikely that NSAIDs or corticosteroids commonly function by raising the level of PGD
2.
Our results do not enable us to determine whether the root extracts predominantly blunted the inflammatory response at its onset or whether they also expedited its resolution. Considering their dual effects on pro- and anti-inflammatory factors, we favor a combination of the two possibilities. As commonly practiced in traditional oriental medicine, a mixture of herbs was used. Future studies should be directed towards determining the relative contribution(s) of each herb, to assess potential synergistic effects and isolate the active ingredient(s).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SMJ performed most of the experiments. HRS oversaw the project, edited the manuscript and gave initial instruction on the air pouch model. HCK provided the root extracts and Figure
2. MK performed the HPLC analysis. SHL assisted with the ELISA assays and statistical analysis. FP oversaw the project, performed part of the experiments, composed the illustrations and wrote the manuscript.