Dual Regulating Effect of Shaoyao-Gangcao-Tang on COX- 2 Expression in Acute and Resolution Phases of Carrageenin-Induced Pleurisy in Rats

Purpose: To investigate the effects and potential mechanisms of Shaoyao-Gangcao-Tang (SGT) on acute and resolution phases of carrageenin-induced pleurisy in rats. Methods: To determine the effects of SGT at 2 h, Sprague-Dawley rats received injection of 0.2 ml of 1 % λ-carrageenin into the pleural cavity after treatment with 4.0, 13.3 and 40.0 g/kg SGT for three days. At 2 h after pleurisy induction, exudate volume, total cell number, prostaglandin E2 (PGE2) production and cyclooxygenase-2 (COX-2) protein expression were measured. To determine the effects at 48 h, the rats were treated with SGT at 24, 36 and 46 h after injection of λ-carrageenin into the pleural cavity, and the exudate volume, total cell number, 15-deoxy-Δ 12, 14 -PGJ2 (15d-PGJ2) production and COX-2 protein expression were evaluated. Results: At 2 h after pleurisy induction, 13.3 and 40.0 g/kg SGT significantly decreased exudate volume by 34 (p < 0.05) and b 4 0% (p < 0.01), total cell number by 27 (p < 0.05) and 41 % (p < 0.01), PGE2 production by 17 (p < 0.05) and 35 % (p < 0.01), as well as COX-2 protein expression by 21 (p < 0.01) and 43 % (p < 0.01) compared with control group treated with saline. At 48 h after pleurisy induction, 13.3 and 40 g/kg SGT also significantly decreased exudate volume by 36 (p < 0.05) and 55 % (p < 0.01), as well as total cell number by 31 (p < 0.05) and 43 % (p < 0.01), but markedly increased 15dPGJ2 production by 26 (p < 0.05) and 51 % (p < 0.01), as well as COX-2 protein expression by 50 (p < 0.01) and 100 % (p < 0.01) compared with control group. Conclusion: The findings suggest that SGT has dual regulating effect in acute and resolution phases of inflammation, involving inhibiting acute inflammation through down-regulation of pro-inflammatory mediators, and promoting inflammatory resolution through up-regulation of pro-resolution mediators.


INTRODUCTION
Inflammation, which occurs in response to the harmful stimuli, is beneficial for our body to clean the harmful stimuli and restore the tissue structure and function.Therefore, the hallmark of a successful inflammatory response is not only the clearance of injurious stimuli, but also the restoration of normal physiology.If acute inflammation cannot resolve normally, it will switch to chronic inflammation resulting in ongoing tissue damage [1].Thus, promoting inflammatory resolution may be a promising method for the treatment of inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel diseases and asthma [2].
New evidence shows that the resolution of acute inflammation is a highly coordinated and active process which is regulated by endogenous proresolving mediators, such as lipoxin, protectin and 15d-PGJ 2 [3].15d-PGJ 2 was initially identified as a high-affinity natural ligand for peroxisome proliferator activated receptor gamma (PPARγ) [4].It is now considered to exert its biological functions through PPARγdependent as well as -independent mechanisms.Studies have revealed that 15d-PGJ 2 can inhibit NF-κB [5] and AP-1 [6] activation, resulting in the reduction of pro-inflammatory mediators generation including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and COX-2, as well as the induction of apoptosis in activated macrophages [7] and myofibroblasts [8].15d-PGJ 2 also preferentially inhibits monocytes rather than polymorphonuclear leukocytes trafficking through the differential regulation of cell adhesion molecules and chemokines expression [9].These data indicate that 15d-PGJ 2 can tightly regulate the resolution of acute inflammation.
As the key enzyme of regulating PGE 2 generation, COX-2 has been thought to be a proinflammatory mediator.However, Gilroy et al [10] suggested that COX-2 might promote resolution of inflammation for its contribution to 15d-PGJ 2 production.PGD 2 is a major product from the COX-catalyzed reaction.PGD 2 undergoes dehydration to yield biologically active PGs of the J 2 series, including PGJ 2 , Δ 12, 14 -PGJ 2 and 15d-PGJ 2 [11].Therefore, COX-2 has a dual role in inflammation progress, which is pro-inflammatory in acute phase and pro-resolving in resolution phase [12].SGT, a widely used traditional Chinese formula, has been applied to various clinical symptoms associated with analgesia, anti-spasm in China for hundreds of years.Feng et al demonstrated that SGT could inhibit PGE 2 production [13], but the underlying mechanisms were not fully understood.Our previous work found that SGT markedly inhibited the pro-inflammatory mediators production and bone erosion in rats with adjuvant-induced arthritis (AIA) [14].Considering that ongoing production of proinflammatory mediators and bone erosion in AIA resulted from abnormal resolution of inflammation, we thought that SGT might regulate the resolution of inflammation.To prove our presumption, in this study, we investigated the impact of SGT on exudate volume, total cell number, PGE 2 and 15d-PGJ 2 production, as well as COX-2 expression in acute and resolution phases of carrageenin-induced pleurisy in rats.

EXPERIMENTAL SGT preparation
Shaoyao ( Paeonia lactiflora Pall.) and Gancao (Glycyrrhiza uralensis Fisch.) were purchased from Chongqing Tongjunge Pharmacy (Chongqing, China) and were identified by Dr Ji-Fen Zhang, College of Pharmaceutical Sciences, Southwest University (Chongqing, China).SGT was prepared as we previously described [14].The content of paeoniflorin in SGT extract used in this study was quantitatively analyzed by high performance liquid chromatography (HPLC) method as described previously [15].In this study, SGT was found to contain 9.17 mg peoniflorin per g SGT extract.

Animals
Male Sprague-Dawley (SD) rats weighing 200 ± 20 g were purchased from Chongqing Medical University (Chongqing, China).All rats were housed in a temperature-controlled room (23 ± 2 °C) under a light/dark cycle with lights on from 7:00 am to 7:00 pm.They were allowed food and water ad libitum.The animals adapted to experimental environment for 1 week before experiments were carried out.All animal procedures were approved by the Ethical Committee in Animal Research of Chongqing Technology and Business University (approval no.2011-6-6/CTBU).The procedures involving animals and their care conformed to international guidelines for the use of laboratory animals [16].
Carrageenin-induced pleurisy 0.2 ml of 1 %w/v λ-carrageenin (Sigma, MO, USA) suspended in saline was injected into the right pleural cavity of 30 rats under light ether anesthesia.Pleural exudate were collected at 2, 6, 18, 24 and 48 hours after injection.At each time point, pleural cavities of 6 rats washed out with 1.0 ml saline containing heparin (5 U/ml).Exudate and washing solutions were removed.The volume of the exudate was calculated by subtracting the volume of the washing solution (1.0 ml) from the total volume recovered.The cells were counted using a hemocytometer.The fluid was centrifuged at 1500 rpm for 5 min at 4 °C, the supernatant collected and stored at -70 °C for analysis of PGE 2 or 15d-PGJ 2 .The pellets were subjected to total protein extraction for Western blot assay.

Animal treatment
To determine the effects at 2 h, rats were randomly divided into 4 groups with 10 rats in each group: (1) Control group, (2) 4 g/kg SGT group, (3) 13.3 g/kg SGT group, (4) 40 g/kg SGT group.Rats of groups ( 2), ( 3) and ( 4) groups were intragastrically treated with SGT twice a day for 3 consecutive days.Rats of group (1) group were treated with an equal volume of saline.One hour after the last treatment, pleurisy was induced by carrageenin injection into the right pleural cavity of rats of all groups.Two hours after this injection, inflammatory exudate was collected for analysis.
To determine the effects of SGT at 48 h, the rats were randomly divided into 4 groups as described above, and treated with SGT or saline at 24, 36 and 46 h after the injection of carrageenin.Inflammatory exudate was collected for analysis 48 h after this injection.

PGE 2 and 15d-PGJ 2 levels analysis
Inflammatory exudate was centrifuged at 1500 rpm for 5 min and total PGE 2 and 15d-PGJ 2 levels determined in the cell-free inflammatory exudate.PGE 2 was measured by a commercial radioimmunoassay kit (The General Hospital of the People's Liberation Army, Beijing, China).15d-PGJ 2 was measured by a commercial enzyme-linked immunosorbent (ELISA) kit (Cayman Chemical, Michigan, USA).Assays were performed according to the manufacturer's instructions.

Statistical analysis
All data were presented as mean ± standard error of mean (SEM).Significant differences between data were evaluated by ANOVA test using SPSS 16 software.P < 0.05 was considered as statistical significant.

RESULTS
In the carrageenin-induced pleurisy, exudate volume and total cell number increased quickly from 2 to 24 h after carrageenin injection, and thereafter declined from 24 to 48 h (Fig 1A and  B).Maximal PGE 2 production occurred at 2 h, and declined from 2 to 48 h (Fig 1C).High COX-2 protein expression occurred at 2, 6 and 48 h during the carrageenin-induced pleurisy (Fig 1D).However, this protein could not be detected at 18 or 24 h.Interestingly, the expression of COX-2 at 48 h was much higher than that at 2 and 6 h.COX-2 protein expression at 48 h was approximately 41 and 63 % higher than at 2 and 6 h, respectively (Fig 1E).

DISCUSSION
In the present study we found that SGT significantly decreased the exudate volume, total cell number, prostaglandin E 2 (PGE 2 ) production and COX-2 protein expression at 2 h of λcarrageenin-induced pleurisy.We also found that SGT markedly decreased the exudate volume and total cell number 48 h after pleurisy innduction.Interestingly, we found that 15d-PGJ 2 production and COX-2 protein expression increased 48 h after SGT administration induced pleurisy.These data suggest that SGT has dual regulating action on acute and resolution phases of inflammation, involving inhibiting the acute inflammation and promoting the inflammatory resolution.
Carrageenin-induced pleurisy is commonly used in studies of acute inflammation and inflammatory resolution [10].In this study, we found that exudate volume and total inflammatory cell number peaked at 24 h and resolved by 48 h in carrageenin-induced pleurisy in rats.Another study demonstrated that cellular infiltrate was initially dominated by neutrophil granulocytes up to 12 h [10].With the increasing amount of mononuclear cells as inflammation progresses, neutrophil granulocytes were replaced by migrating monocytes, which differentiated into macrophages.These monocytes became dominant up to resolution at 48 h.Since there was a fast alternation of acute inflammation and inflammatory resolution during 48 h in carrageenin-induced pleurisy, we used this model for our investigations.First, we investigated the impact of SGT on acute phase of carrageenin-induced pleurisy.We selected the two indices of exudate volume and total cell number because they were positively associated with the severity of inflammation [18].We selected the 2 h of pleurisy because, at this time, we discovered that there existed considerable COX-2 protein expression, and maximal PGE 2 production.We found that, at 2 hours of carrageenin-induced pleurisy, doses of 13.3 and 40 g/kg SGT significantly reduced t exudate volume and total cell number in exudate in a dose-dependent manner.However, 4 g/kg did not show statistically inhibitory effects.These results suggest that SGT could inhibit the acute inflammation.Considerable amounts of PGE 2 , mainly synthesized by COX-2, is generated at sites of inflammation where it acts as a potent vasodilator and synergistically with other mediators such as histamine and bradykinin, resulting in enhancement in vascular permeability and edema [19].The results indicate that SGT might inhibit the acute inflammation through suppressing the COX-2 protein expression and PGE 2 production.
Since SGT administration did not interfere with the progress of acute inflammation, we could directly and exactly discover the effects and mechanisms of SGT on inflammatory resolution.SGT significantly decreased the exudate volume and total cell number at 48 hours of carrageenininduced pleurisy, indicating that SGT promotes inflammatory resolution.Interestingly, accompanying the reduction of PGE 2 , COX-2 protein expression was clearly enhanced at 48 h.Although this seems implausible, the following mechanism may make it more likely: In the resolution phase of inflammation, COX-2 catalyzes the production of cyclopentenone prostaglandins of the J-series (15d-PGJ 2 ) in macrophages, which act as a pro-resolving mediator in inflammation [20].# p < 0.05, ## p < 0.01 compared with control group

CONCLUSION
SGT has dual regulating action on acute and resolution phases of carrageenin-induced pleurisy, involving inhibiting acute inflammation and promoting inflammatory resolution.The mechanisms of this action may decrease COX-2 protein expression and PGE 2 production in acute phase of inflammation, as well as increase COX-2 protein expression and 15d-PGJ 2 production in the resolution phase of inflammation.To our knowledge, this study is the first one to explore the dual regulating action of traditional Chinese formula on acute inflammation and inflammatory resolution.These findings suggest that SGT is a promising anti-inflammatory drug candidate that requires further investigation.

Fig 3 :
Fig 3: Effect of SGT on PGE2 production and COX-2 protein expression at 2 h after injection of carrageenin.A: Effect of SGT on PGE2 production at 2 hours after injection of carrageenin (n = 10).B: Effect of SGT on COX-2 protein expression by Western blot analysis at 2 hours after injection of carrageenin.β-actin was used as loading control.The result presented was representative of four independent experiments.C: Bar charts show quantitative evaluation of COX-2 bands by densitometry from four independent experiments; # p < 0.05, ## p < 0.01 compared with control group.

Fig 4 :
Fig 4: Effects of SGT on exudate volume (A) and total cell number (B) at 48 h after injection of carrageenin (n = 10).#p < 0.05, ## p < 0.01 compared with control group

Fig 5 :
Fig 5: Effects of SGT on 15d-PGJ2 production and COX-2 protein expression at 48 h after injection of carrageenin.A: Effect of SGT on 15d-PGJ2 production at 48 hours after injection of carrageenin (n = 10).B: Effect of SGT on COX-2 protein expression by Western blot analysis at 48 hours after injection of carrageenin.β-actin is used as loading control.The result presented was representative of four independent experiments.C: Bar charts show quantitative evaluation of COX-2 bands by densitometry from four independent experiments.# p < 0.05, ## p < 0.01 compared with control group