Scutellaria baicalensis Georgi (Huang Qui), a Chinese traditional medicinal herb, is widely used as an antiinflammatory, antibacterial, and hepatoprotective drug (1,2). Baicalin (7-glucuronic acid,5,6-dihydroxy-flavone), a flavonoid compound isolated from Huang Qui, possesses antioxidant properties (3) and has an inhibitory effect on carrageenan-induced rat paw edema (1), suggesting that it may be a potential antiinflammatory drug.
The carrageenan-evoked thermal hyperalgesia resulting from the combined effect of the release of proinflammatory cytokines, cyclooxygenase (COX) products, and sympathomimetic amines (4) is a common model used to study inflammatory pain. Tumor necrosis factor-α (TNF-α) has an early and crucial role in the cascade of proinflammatory cytokine production and subsequent inflammatory processes (5). COX catalyzes the conversion of arachidonic acid to many biologically active mediators, such as prostaglandin E2 (PGE2). It has been reported that overproduction of inflammatory prostaglandins by an inducible form of COX (COX-2) plays an important pathophysiological role in the development of inflammatory pain (6,7).
Nitric oxide (NO), synthesized by the enzyme NO synthase (NOS), is an important mediator in the regulation of cell functions (8). However, overproduction of NO derived from inducible NOS (iNOS) activated by proinflammatory cytokines, free radicals, and lipopolysaccharide (LPS) may cause the pathogenesis of inflammatory diseases, including carrageenan-evoked inflammatory pain (6,9). Inhibition of COX-2 activity in a castor-oil-induced diarrhea model, as well as NO formation and iNOS expression in LPS-treated RAW 264.7 macrophages by baicalin (10,11), suggests that baicalin may suppress the COX-2 and iNOS pathways, a critical mediator accounting for the carrageenan-induced inflammatory pain.
In this study, we first demonstrate that baicalin exhibits an analgesic effect in a rat model of carrageenan-evoked thermal hyperalgesia. To further investigate the analgesic mechanisms involved, the effect of baicalin on the formation of important inflammatory mediators, including cytokine, NO, and PGE2 formation, as well as neutrophil infiltration at inflammatory sites, was also studied.
Male Sprague-Dawley rats (8–9 wk) weighing 200–250 g, purchased from the National Animal Center (Taipei, Taiwan), were used in this study. This study was approved by the local institutional animal care and use committee. Animals were housed in a standard environment and maintained on tap water and rodent food ad libitum throughout the investigation.
To induce local inflammation, 2 mg of λ-carrageenan (Sigma, St. Louis, MO; 100 μL of 2% [wt/vol] in saline) was injected subcutaneously into the plantar surface intraplantar injection (i.pl.) of rat right hind paws at Time 0 (T = 0). Rats were allowed 30 min to acclimate to the device before testing. Baicalin, purchased from Aldrich Chemical Co. (Milwaukee, WI), was dissolved in saline and delivered in a volume of 0.2 mL. Baicalin (10, 30, or 100 mg/kg intraperitoneally [IP]), vehicle (saline, IP), or ibuprofen (30 mg/kg IP) (Sigma) was injected at 30 min before (T = −30) or 165 min after (T = 165) carrageenan injection. Hyperalgesia was assessed by measuring the paw withdrawal latency with a nonnoxious heat stimulus by using a device (7370 Plantar Test; Ugo Basile, Comerio, Italy). The paw withdrawal latencies were evaluated every 30 min for 240 min starting 60 min after carrageenan injection, and the baseline latencies were assessed at 40 min before (T = −40) carrageenan injection. Each test was calculated as a mean of three repeated measurements. Each rat was used once. The vehicle (IP)-treated and the saline (i.pl.)-injected rats acted as a control group. Each group contained six rats.
After the analgesic experiments were finished, rats were killed by exsanguination. Then the hind paws were cut at the level of the calcaneus bone and centrifuged at 400 g for 15 min at 4°C to collect the exudates (edema fluid) for measuring the levels of TNF-α, interleukin (IL)-10, IL-1β, and IL-6 in paw exudates by using rat enzyme immunoassay kits (Genzyme Corp., Cambridge, MA). Concentrations of nitrate and PGE2 were determined in paw exudates collected as described previously, 1.5 or 4 h after carrageenan injection. The levels of nitrate (total nitrite and nitrate) in paw exudates were measured by using a Sievers nitric oxide analyzer (280 NOA; Sievers, Boulder, CO). The levels of PGE2 were measured by a rat enzyme immunoassay kit.
For myeloperoxidase (MPO) activity assay, 4 h after the injection of carrageenan, rats were killed. The tissues of carrageenan-injected paws were removed, washed with sterile normal saline, and homogenized in ice-cold 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer (pH 6.0; 5 mL of HTAB per gram of tissue) on ice by using a homogenizer (Pro Scientific Inc., Pro Model 200; Monroe, CT). They were then sonicated and centrifuged at 15,000 g for 15 min at 4°C. Then the supernatant was mixed 1:30 (supernatant/assay buffer) and read at 460 nm. The assay buffer consisted of 100 mM potassium phosphate (pH 6.0), 0.083 mL of H2O2 (Sigma; 30% stock diluted 1:1000), and 0.834 mL of o-dianisidine hydrochloride (Sigma; 10 mg/mL). MPO activity was calculated and expressed as ΔA460 per minute per milligram of protein.
Data were expressed as mean ± sem. Statistical analysis was performed by one-way analysis of variance. If significant results were revealed by this analysis, then differences between group means were evaluated by the Student’s t-test for unpaired observations. A P value <0.05 was considered statistically significant.
Basal withdrawal latencies for all experimental groups were similar, at approximately 22.0 ± 0.3 s. Contralateral left (carrageenan-uninjected) hind paw withdrawal latencies remained constant at basal levels for the entire experiment. Injection of carrageenan into the right hind paw of rats evoked thermal hyperalgesia with a significant decrease of withdrawal latencies that began at 60 min and reached the smallest value of 3.9 ± 0.5 s at 3 h after carrageenan injection. The administration of baicalin (10–100 mg/kg IP) either 30 min before or 165 min after carrageenan injection significantly inhibited carrageenan-evoked thermal hyperalgesia (P < 0.01 versus vehicle-treated and carrageenan-injected rats) in a dose-dependent manner (Fig. 1). Pre- or posttreatment with the same dose (30 mg/kg IP) of baicalin or ibuprofen, a nonsteroidal antiinflammatory drug (NSAID), had a similar analgesic effect.
The administration of baicalin 30 min before carrageenan injection dose-dependently inhibited the increased formation of TNF-α, IL-6, and IL-1β but enhanced the IL-10 formation in paw exudates at 4 h after carrageenan injection compared with that of vehicle-treated and carrageenan-injected rats (Fig. 2). Injection of carrageenan (i.pl.) led to a time-dependently increased production of nitrate and PGE2 in the paw exudates. The administration of baicalin 30 min before carrageenan injection dose-dependently inhibited the nitrate and PGE2 production at 4 h rather than at the early phase (1.5 h) after carrageenan injection (Fig. 3). Four hours after the injection of carrageenan, the increased MPO activity in carrageenan-injected paws was dose-dependently depressed by baicalin (Fig. 4).
In carrageenan-evoked inflammatory pain, the proinflammatory cytokines—including TNF-α, IL-1β, and IL-6—play an early and crucial role in the subsequent inflammatory responses (5). In contrast, an “antagonist cytokine,” IL-10, exerts its antiinflammatory effect by inhibiting the production of proinflammatory cytokines (12) and by inducing the formation of the IL-1 receptor antagonist (13). Injection of a monoclonal antibody to mouse IL-10 enhances carrageenan- or TNF-α-evoked inflammatory pain (14), suggesting that IL-10 may be a potential therapeutic drug for inhibiting inflammatory responses. In this study, we first demonstrated that baicalin has a preventive and therapeutic analgesic effect in inflammatory pain. In addition, baicalin inhibits the formation of TNF-α, IL-1β, and IL-6 but enhances the production of IL-10, thus resulting in an overall attenuation of the proinflammatory/antiinflammatory cytokine ratio in carrageenan-injected paws, which may contribute to its antiinflammatory and analgesic effects.
It was proposed that peripheral COX-1 plays a primary role in early inflammatory responses; after inflammation is fully established (when COX-2 is activated), peripheral prostaglandin production synthesized by COX-2, and perhaps also COX-1, may account for the maintenance of carrageenan-evoked thermal hyperalgesia (15). Systemic administration of COX-2 inhibitors or PGE2 monoclonal antibodies resulted in an attenuation of carrageenan-induced inflammatory hyperalgesia (7,16), further indicating that overproduction of PGE2 derived from COX-2 plays a critical role in the generation and maintenance of inflammatory pain. Similarly, NO produced by constitutive NOS is involved in the early phase and NO produced by iNOS is involved in the late phase during the development of the carrageenan-evoked inflammatory pain (9). Thus, overproduction of NO and PGE2 mainly derived from iNOS and COX-2, respectively, may play an important pathophysiological role in the development of carrageenan-induced inflammatory pain. Our results demonstrated that baicalin inhibited PGE2 and nitrate overproduction in carrageenan-injected paws only at the late phase (4 hours) (when COX-2 and iNOS activity were activated) rather than at the early phase (1.5 hours), suggesting that baicalin may be a relatively selective inhibitor for COX-2 and iNOS, which is possibly due to baicalin’s initial attenuation of the formation of proinflammatory cytokines. The increased MPO activity (an indicator of neutrophil infiltration) seen at sites of inflammation exerting an important source of oxygen-derived free radicals and many proinflammatory mediators (17) was significantly inhibited by baicalin, which may be another mechanism of its antiinflammatory effect.
Interestingly, the analgesic effect of baicalin was similar to that of ibuprofen, a traditional NSAID. Although NSAIDs are the most commonly used remedy for treating inflammation, they cause several serious adverse effects because of their nonselective inhibition of COX (18). Many studies have shown that the effect of ibuprofen on the production of proinflammatory cytokines is controversial. The administration of ibuprofen led to a pronounced increase of TNF-α and IL-6 levels in serum and to increased mortality in murine endotoxic shock (19,20). Ibuprofen also increased secretion of IL-6 and TNF-α but reduced production of IL-10 in LPS-treated mononuclear cells (21). Systemic administration of iloprost, a prostacyclin analog, inhibited serum TNF-α levels after IP LPS (21), indicating that TNF-α production is modulated by endogenous prostaglandins in vivo. The enhanced production of TNF-α by COX inhibitors may lead to exacerbation of some inflammatory processes. However, two studies (22,23) reported that treatment with ibuprofen attenuated cardiopulmonary dysfunction and LPS-induced lung injury through inhibiting TNF-α and macrophage inflammatory protein-2 production and proinflammatory cytokine gene expression but increased lung iNOS messenger RNA expression without affecting the MPO activity in the lung. The reasons causing the inconsistent effects of ibuprofen are still unknown. On the basis of these differences between baicalin and ibuprofen, we proposed that the analgesic effect of the two drugs may be through different mechanisms.
Baicalin has been used as an antiinflammatory and antiallergic drug in a variety of inflammatory diseases (2). Baicalin also possesses therapeutic activity against prostate and bladder cancers (24,25), as well as the infection of human peripheral blood mononuclear cells by acquired human immunodeficiency virus Type 1 (26). No serious side effects of baicalin have been reported. Recently, a study of metabolic pharmacokinetics of baicalin in rats showed that when baicalin was administered orally, glucuronides and sulfates of baicalein were exclusively circulating in the plasma, implying that baicalin is absorbed as conjugated metabolites of baicalein by the gastrointestinal tract (27).
This study is the first to demonstrate that baicalin exhibits a potent analgesic effect on carrageenan-evoked thermal hyperalgesia. Furthermore, we propose that the mechanisms of baicalin may be associated with the inhibition of inflammatory mediator overproduction, including proinflammatory cytokines, NO, and PGE2. The other possibilities are an increase of IL-10 and an inhibition of neutrophil infiltration in carrageenan-injected paws. These findings suggest that baicalin may be therapeutically useful for mitigating inflammatory pain.
We thank Ting-Hui Chu for technical assistance.
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