On the other hand, AACOCF3 did not influence the thermal hyperalgesia (Figure 5(a)). IN); and RNAlater (RNA stabilization solution) from Ambion (Austin, TX). 2.2. Animals All experiments were carried out according to protocols approved by the Institutional Animal Care Committee of Kyoto Prefectural University of Medicine. Rats were housed four per cage and maintained on a 12?h light/dark cycle (light on 8:00C20:00) with controlled temperature (25 3C) and humidity (55 15%). Animals were allowed free access to food and water at all times. 2.3. Pharmacological Treatment The plantar surface of the left paw received a subcutaneous injection of either 3?mg type carrageenan (Sigma-Aldrich, St. Louis, MO) dissolved in 100?(a metabolite of PGI2), the hind paws were coronally cut into 50?were measured using EIA kits according to the manufacturer’s instructions. Tissue pellet remaining in the plastic tube was heated in a heat block to completely evaporate the ethanol. The weight of dried pellet was considered to be the dry tissue weight of the paw from which the prostaglandins were extracted. 2.5. Real-Time RT-PCR Frozen paw sections were prepared as described above. Twenty of these sections were placed into a vial made up of RNA later (1?mL) and stored at ?30C until further processing. For RNA extraction, the samples were homogenized in 1?mL phenol-based RNA extraction solution (Isogen) with polytron for 30?s followed by sonication for 20?s. Total RNA was isolated according to the manufacturer’s instructions. cDNA was prepared from total RNA using M-MLV reverse transcriptase and random hexamer as the primer. The reverse-transcribed cDNA was Glabridin amplified using a light cycler (Roche Diagnostics). mRNAs of COX-2, mPGES-1, iPLA2 (IL1tt 0.05. Data are presented as mean SEM. 3. Results We examined the effects of PLA2 inhibitors on PGE2 and 6-keto-PGF1(a metabolite of PGI2) levels in inflamed foot pad. Carrageenan and PLA2 inhibitors/vehicle were injected into the right foot pad at the same time. Three hours after the injection, carrageenan significantly elevated PGE2 and 6-keto-PGF1levels compared to injection of saline alone in vehicle-, BEL- and AACOCF3-coinjected groups (= 4 in each group, = 0.0002C0.014) (Figure 1). BEL, an iPLA2 inhibitor, significantly suppressed carrageenan-induced increases in PGE2 by 57% (= 0.009) and 6-keto-PGF1by 49% (= 0.017) compared to vehicle. On the other hand, AACOCF3, a cPLA2 inhibitor and less potent iPLA2 inhibitor, did not suppress the prostaglandin levels compared to the vehicle-treated Ptprc rats. The two inhibitors did not exert significant effects around the prostaglandin levels Glabridin in the saline-injected foot pad. Open in a separate window Physique 1 Contents of Glabridin PGE2 (a) and 6-keto-PGF1(b) in rat hind paw. Carrageenan (Car) injection (filled bars) significantly elevated both prostaglandin levels compared to saline (Sal) injection (open bars) in vehicle-, BEL-, and AACOCF3- (AACO-) treated groups (= 0.0002C0.014,t= 4 in each group). BEL but not AACOCF3 significantly suppressed carrageenan-induced increases in PGE2 (a) and 6-keto-PGF1(b) compared to vehicle (= 0.009 for PGE2 and = 0.017 for 6-keto-PGF1tand iPLA2 and iPLA2 relative to that of an internal control gene (GAPDH) in rat hind paw. Their relative levels were not influenced by carrageenan-induced inflammation. Open bars and filled bars represent results from saline (Sal)-injected group and carrageenan (Car)-injected group, respectively. = 4 in each group. We then asked if BEL influences the induction of COX-2, mPGES-1, and PGIS, which are possibly involved in carrageenan-induced prostaglandin synthesis. COX-2 mRNA, mPGES-1 mRNA, and PGIS mRNA were increased by carrageenan injection though the increases did not reach the statistically Glabridin significant level except for COX-2 mRNA and PGIS mRNA in carrageenan + BEL group (Physique 3). There was no significant effect of BEL around the COX-2 mRNA, mPGES-1 mRNA, or PGIS mRNA levels (Physique 3). We also examined the effects of BEL on carrageenan-induced proinflammatory cytokine mRNAs (Physique 4). Carrageenan significantly elevated the mRNA levels of IL1and IL6. BEL slightly but significantly elevated carrageenan-induced IL1mRNA and did not change carrageenan-induced IL6 mRNA. Therefore, BEL seemed to act solely around the enzyme activity of iPLA2s resulting in suppression of prostaglandin levels in inflamed tissue. Open in a separate window Physique 3 Relative expression of COX-2 mRNA (a), mPGES-1.
On the other hand, AACOCF3 did not influence the thermal hyperalgesia (Figure 5(a))