The expression of COX-2 in VEGF-treated endothelial cells is mediated through protein tyrosine kinase.

Cyclooxygenase (COX), existing as the COX-1 and COX-2 isoforms, converts arachidonic acid to prostaglandin H2, which is then further metabolized to various prostaglandins. Vascular endothelial growth factor (VEGF) has been shown to play important roles in inflammation and is upregulated by the prostaglandin E series through COX-2 in several cell types. Here, we have investigated the effects of VEGF on the COX isoform expressed in human umbilical vein endothelial cells (HUVEC). The signalling mechanism of the COX isoform expressed in endothelial cells activated with VEGF will be also investigated using the tyrosine kinase inhibitor, genistein, and protein kinase C inhibitor, staurosporine. The activity of COX-2 was assessed by measuring the production of 6-keto-prostaglandin F1alpha in the presence of exogenous arachidonic acids (10 microM, 10 min) by enzyme immunoassay. The expression of COX isoform protein was detected by immunoblot using specific antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC treated with VEGF (0.01-50 ng/ml), COX-2 protein, but not COX-1, and COX activity were increased in a dose-dependent manner. Interestingly, the increased COX-2 protein and activity in response to VEGF (10 ng/ml) was inhibited by the tyrosine kinase inhibitor, genistein (0.05-5 microg/ml), but not by the protein kinase C inhibitor, staurosporine (0.1-10 ng/ml). Thus, the induction of COX-2 by VEGF in endothelial cells was mediated through protein tyrosine kinase, and the uses of specific COX-2 inhibitors in these conditions, in which VEGF was involved, might have a role

Identification of Apigenin and Luteolin in Artemisia annua L. for the Quality Control

Objective: To identify active compounds and establish the chemical fingerprint of Artemisia annua L. for the quality control. Methods: Thin-layer chromatography (TLC) conditions were developed to screen for 2 common flavonoids (apigenin and luteolin). Three mobile phases were used to isolate these flavonoids in 80% ethanolic extract of A. annua. Hexane : ethyl acetate : acetic acid (31:14:5, v/v) and toluene : 1,4-dioxane : acetic acid (90:25:4, v/v) were used in normal phase TLC (NP-TLC), and 5.5% formic acid in water : methanol (50:50, v/v) were used in reverse phase TLC (RP-TLC). Chromatograms were visualized under visible light after spraying with Fast Blue B Salt. Apigenin and luteolin bands were checked by comparing their Rf values and UV-Vis absorption spectra with reference markers. Results: Apigenin and luteolin were simultaneously detected with good specificity in RP-TLC condition, while only apigenin was detected in NP-TLC condition. Apigenin band intensity was higher than luteolin band intensity in both conditions. Conclusion: This knowledge can be applied to the development of quality control assessments to ensure product efficacy and consistency

Identification of Apigenin and Luteolin in Artemisia annua L. for the Quality Control

Objective: To identify active compounds and establish the chemical fingerprint of Artemisia annua L. for the quality control. Methods: Thin-layer chromatography (TLC) conditions were developed to screen for 2 common flavonoids (apigenin and luteolin). Three mobile phases were used to isolate these flavonoids in 80% ethanolic extract of A. annua.Hexane : ethyl acetate : acetic acid (31:14:5, v/v) and toluene : 1,4-dioxane : acetic acid (90:25:4, v/v) were used in normal phase TLC (NP-TLC), and 5.5% formic acid in water : methanol (50:50, v/v) were used in reverse phase TLC (RP-TLC). Chromatograms were visualized under visible light after spraying with Fast Blue B Salt. Apigenin and luteolin bands were checked by comparing their Rf values and UV-Vis absorption spectra with reference markers. Results: Apigenin and luteolin were simultaneously detected with good specificity in RP-TLC condition, while only apigenin was detected in NP-TLC condition. Apigenin band intensity was higher than luteolin band intensity in both conditions. Conclusion: This knowledge can be applied to the development of quality control assessments to ensure product efficacy and consistenc

The Effects of Thai Herbal Ha-Rak Formula on COX Isoform Expression in Human Umbilical Vein Endothelial Cells Induced by IL-1 β

Objective. To investigate the modulated effects of HRF on cyclooxygenase isoform expression and its activity, using the human umbilical vein endothelial cell (HUVEC) model induced by interleukin-1 beta (IL-1β). Methods. Cells were treated with indomethacin (positive control), HRF, and its components at various concentrations prior to treatment with IL-1β at 24 h. Cell viability was determined by MTT assay. Moreover, the anti-inflammatory effects of HRF and its components through mRNA and protein expression were established using real-time quantitative PCR and Western blot, respectively. COX activity was identified via exogenous and endogenous PGE2 productions using the EIA. Result. There was no cytotoxicity in HUVECs treated with HRF. None of the experimental conditions used in the study affected the expression of COX-1, but COX-2 protein expression was inhibited at concentrations under 10 µg/mL. Despite the significantly increased levels of exogenous PGE2, HRF had no effect on COX-2 mRNA expression. However, the production of PGE2 was lower at a concentration of 100 µg/mL HRF than at a concentration below 10 µg/mL. Interestingly, each component of HRF revealed different effects of the Ha-Rak formula. Conclusion. Our preliminary findings suggest that HRF and its components provide diverse modulation of COX-2 and PGE2 at the in vitro level

Study the Effect of an Antipyretic Drug, Thai Herbal Ha-Rak Formula on Platelet Aggregation in Healthy Thai Volunteers: A Randomized, Placebo - Controlled Trial

Background: Fever may alter platelet homeostasis leading to bleeding tendency. Thai herbal Ha-rak formula (HRF), a traditional Thai remedy consisting of five medicinal plants, is indicated for relieving fever. However, its effect on platelet has not been evaluated. This study aims to investigate the effect of HRF on platelet aggregation. Methods: A randomized, placebo, controlled trial was carried out in 46-healthy Thai volunteers, both male and female. The subjects either received the maximum recommended dose per day of HRF (1,500 mg) or placebo. Platelet aggregation, using aggregometer (AggRam), was assessed in platelet rich plasma (PRP) in response to each of three different agonists including epinephrine, adenosine diphosphate (ADP) and collagen at pre-dose and 8, 32 hours and 7-10 days after the first dose. Results: All participants completed the study. Only few adverse events occurred which spontaneously improved without further treatment. Overall, analysis of platelet activity compared before and after HRF administration did not show significant difference of maximum percentage of platelet aggregation at any time point except the platelet response to collagen at 32 hours and 1 week after the first HRF dose. However, subgroup analysis characterized by sex, and platelet aggregation in response to all agonists did not reveal any significant change. The same results applied to subgroup analysis based on the different patterns of platelet aggregation. Conclusion: HRF at a dose of 1,500 mg/day is well tolerated and has a significant effect on platelet aggregation only when induced by collagen

The induction of cyclooxygenase-2 in IL-1b -treated endothelial cells is inhibited by prostaglandin E 2 through cAMP

PROSTAGLANDINS (PGS) h ave n um er ous car dio vas cular an d in flam m ator y effects. Cycloox yge nas e (COX), w h ic h e x is ts as COX-1 an d COX-2 is ofo r m s , is th e firs t en zym e in th e p ath w ay in w h ic h arach idon ic acid is con verted to PGs . Pr os taglan din E 2 (PGE Introduction Prostaglandins (PGs) have numerous cardiovasc ular and inflammatory e ffec ts. 1 Cycloox yge nase (COX) is the first enzyme in the pathw ay in w hich arachidonic acid is conve rted to PGs. 2 ,3 COX ex ists in at least tw o isoforms. One is the constitutiv e e nzyme , COX-1, producing re gulatory prostanoids under physiologic al conditions, 4 w hereas the other, COX-2, is induc ed by mitogens, The main PGs produc ed in the body are prostacyclin (PGI 2 ), PGE 2 , PGF 2 a , Thrombox ane A 2 (TXA 2 ) and PGD 2 . Each PGs has different charac te rs and functions. Among the PGs, PGE 2 is a potent lipid molecule w ith c omplex proinflammatory and immunoregulatory p rope rties. 9 PGE 2 is considered a major contributor to the production and maintenanc e of immunosuppression after overw he lming injury. 1 0 PGE 2 is believed to modulate biochemic al and immunologic al e ve nts le ading to p arturition. 11 PGE 2 also ex e rts a variety of biologic al ac tivities for the mainte nance of local homeostasis in the body. 1 2 Inte restingly, w e have show n in p re vious studie s that the induc tion of COX-2 e lic ite d by e ndotox in (lipopolysaccharide , LPS) in e ndothelial c ells is inhibite d by PGE 1 and 13,14-dihydro PGE 1 . 1 3 Eluc idation of the effects of PGE 2 on the signalling molec ule such as COX could lead to potential therape utic interve ntions and unde rstanding of the fee dback re gulation of COX in e ndothelial c ells. Here , w e have inve stigated the effects of PGE 2 on the induc tion of COX-2 in human umbilical ve in endothe lial ce lls (HUVEC) treate d w ith inte rleukin-1b (IL-1b ) (1 ng /ml). 287 Research Paper Mediators of Inflammation, 8, 287-294 (1999) Material and methods Cell culture Human umbilic al ve in e ndothelial ce lls (HUVEC) w ere obtaine d from babies born to normal pregnant w ome n as pre viously de sc ribe d 1 4 and c ulture d in 96-w ell plates w ith Human Endothelial-SFM Basal Grow th Medium (Gibco) containing 10% fetal calf se rum (Gibco), 100 units /ml pe nicillin G sodium and 100 m g /ml streptomycin. Ce lls w ere inc ubate d at 37°C in a humidified inc ubator and grow n to confluenc e before use. Measurement of COX activity Conflue nt HUVEC w e re gently w ashe d tw o times w ith phosp hate-buffe re d saline (PBS) and re plac ed w ith fresh medium (200 m l/w e ll) before use. Ce lls w ere tre ate d w ith no addition, IL-1b (1 ng /ml), IL-1b (1 ng / ml) plus PGE 2 (0.03, 0.3 or 3 m M) or PGE 2 (3 m M) alone for 24 h, afte r w hich time the medium w as remove d and w ashed tw ice w ith PBS. COX ac tivity w as me asured by the production of four COX me tabolites, e.g. 6-keto-PGF 1 a (a stable metabolite of PGI 2 ), PGE 2 , Prostaglandin F 2 a (PGF 2 a ) and thromobox ane B 2 (TXB 2 ; a stable metabolite of TXA 2 ) in the replace d fresh me dium c ontaining ex ogenous arachidonic ac id (10 m M for 10 min) using enzyme immunoassay (EIA). Briefly, 50 m l of standard PGs or samp le s w e re added to pre-c oate d mouse anti-rabbit IgG microtitre plates (96-w e ll). The n, PGs ac etylcholineste rase trac er (Clayman; 50 m l) and rabbit antiserum of PGs w e re adde d. The plate w as c overe d w ith plastic film and inc ubate d for 18 h at 4°C, after w hich time the w ells w ere emptied and rinse d five time s w ith w ash buffe r (PBS containing 0.05% Tw ee n). Ellman's reagent (Cayman; 200 m l) w as adde d to e ach w ell and the plates w ere shaken on a mic rotitre plate shaker. The duration of the reaction w as about 90 min. A yellow c olour develops w hich can be read using a mic roplate reader (BIORAD; OD 415 nm). Immunoblot (Western blot) analysis HUVEC w hich w e re untre ate d, treated w ith IL-1b (1 ng /ml), IL-1b (1 ng /ml) plus PGE 2 (0.03, 0.3 and 3 m M) or PGE 2 (3 m M) alone w ere cultured in six -w e ll culture plates (37°C; for 24 h). After 24 h inc ubation, ce lls w ere ex trac te d and analyse d by immunoblotting using spe cific antibodies for COX-1 and COX-2 prote in (a gene rous gift from Dr Gary O'Ne ill, Merck Frosst, Canada) as pre viously described. 15 The othe r ex periment w as performed to study the signalling mole cule in the e ffec ts of PGE 2 on COX ex pre ssion by using forskolin (cAMP activator). HUVEC w ere treated w ith no addition, IL-1b (1 ng / ml), IL-1b (1 ng /ml) plus PGE 2 (3 m M), IL-1b (1 ng /ml) plus PGE 2 (3 m M) w ith forskolin (10, 50 and 100 m M), IL-1b (1 ng /ml) plus forskolin (100 m M), PGE 2 (3 m M) plus forskolin (100 m M), forskolin (100 m M) alone or PGE 2 (3 m M) alone for 24 h, after w hich time, the me dium w as removed and re place d w ith fre sh me dium c ontaining ex oge nous arachidonic ac id (10 m M for 10 min). The me dium w as then removed to me asure COX activity by 6-ke to-PGF 1 a production. The re mained c ells w ere ex tracted and analyse d by immunoblotting using spe cific antibodies for COX-1 and COX-2 prote in. Measurement of cell viability Ce ll respiration, an indicator of c ell viability, w as asse sse d by the mitochondrial-depe ndent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. Statistical analysis The re sults are show n as mean standard e rror of the me an (SEM) of triplicate de te rminations (w e lls) from at least four separate ex perimental days (n =12). Student's paire d or unpaired t-te sts, as ap propriate, w ere use d for the de te rmination of significanc e of differences betw ee n me ans and a P value of less than 0.05 w as taken as statistically significant. Materials DMSO, phosphate buffe red saline (PBS; pH 7.4), Trizma base , EDTA, triton X-100, phenylme thylsulphonyl fluoride (PMSF), pe pstatin A, le upeptin, glycerol, bromphenol blue, 2-mercaptoethanol, sodium dodec yl sulphate (SDS), forskolin, anti-rabbit IgG antibody, goat IgG, premix e d BCIP/NBT solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe nyltetrazolium bromide (MTT), pe nicillin G sodium and stre ptomycin w ere supplied by Sigma Che mical Comp any (St Louis, MO, USA). PGs (6-keto-PGF 1 a , PGE 2 , PGF 2 a and TXB 2 ) and the ir respective ac etylcholineste rase trac er and rabbit antiserum, pre-coated mouse anti-rabbit IgG microtitre plates (96-w ell) and Ellman's reage nt w ere purchased from Cayman (Sapphire Biosc ie nc e, Australia). Human Endothelial-SFM Basal Grow th Me dium and fetal calf serum w as obtained from GibThai (Thailand). Rec ombinant human IL-1b , w ere purchased from Ge nzyme (USA). Pure nitroc ellulose me mbrane (0.45 mic ron) and filte r pape r w ere purchased from BIO-RAD (USA). P. Ak a ra s e re e n o n t et al. 288 Mediators of Inflammation · Vol 8 · 1999 Results The effect of PGE 2 on COX activity as measured by the production of 6-keto-PGF 1 a , PGE 2 , PGF 2 a and TXB 2 in HUVEC treated with IL-1b (1 ng/ml) Untreate d HUVEC in the presenc e of arachidonic acid (10 m M for 10 min) re le ase low er amounts of 6-ke to-PGF 1 a (3.36 ± 0.1 ng /ml), PGE 2 (0.4 ± 0.04 ng /ml), PGF 2 a (0.78 ± 0.01 ng /ml) and TXB 2 (0.04 ± 0.01 ng /ml). In IL-1b (0.01, 0.1 and 1 ng /ml) tre ate d HUVEC; the produc tion of 6-keto-PGF 1 a , PGE 2 and PGF 2 a w as increase d but not TXB 2 IL-1b alone , PGE 2 alone and IL-1b plus PGE 2 did not affec t on cells viability (97 ± 2, 98 ± 1 and 98 ± 1%, respective ly) w hen compare to the control untreate d ce lls over a 24-h incubation pe riod. The stability of PGE 2 (3 m M) in c ulture d me dium upto 24 h w as also te ste d and has not change d significantly be tw ee n 3 (2.97 ± 0.2), 6 ( 2.98 ± 0.1), 12 (2.95 ± 0.2) and 24 (2.97 ± 0.2) hours inc ubation of PGE 2 . The effect of PGE 2 on COX isoform expressed in HUVEC treated with IL-1b Untre ated HUVEC contained no COX-2 prote in The effect of forskolin on 6-keto-PGF 1 a production in HUVEC treated with IL-1b plus PGE 2 The COX ac tivity (as measured by 6-keto-PGF 1 a production) in HUVEC tre ate d w ith forskolin (100 m M) plus PGE 2 (3 m M) or forskolin (100 m M) alone w as not changed in comparison w ith untre ate d HUVEC ( PGE 2 in hibit COX-2 indu ctio n th ro u g h cAMP Mediators of P. Ak a ra s e re e n o n t et al. 290 Discussion He re, w e show ed that the induction of COX-2 e licite d by IL-1b in HUVEC c an be inhibited by PGE 2 in a dosedepende nt manne r. Moreover, PGE 2 had no affe ct on either COX-1 protein or ac tivity. Inte restingly, forskolin (cAMP activator) can re ve rse this inhibition of PGE 2 on COX-2 protein and activity in IL-1b tre ate d HUVEC. The results sugge ste d that (i) PGE 2 is a ne gative fe edback regulator through c AMP in the induc tion of COX-2 elic ited by IL-1b in e ndothelial ce lls and (ii) the use s of PGE 2 in the c ondition in w hich COX-2 has be en involved may be therapeutic. PGs induc e a w ide range of biological actions that are mediate d by spe cific membrane-bound rec eptors. Among the PGs, PGE 2 is considered to ex ert a varie ty of biologic al activities such as the maintenanc e of loc al homeostasis in the body, 12 it is a major contributor to the production and maintenanc e of immunosuppression afte r ove rw helming injury 10 and an imp ortant factor for implantation and dec idualization. 1 7 Therefore , PGE 2 is a lipid molec ule w ith complex inflammatory modulation and immunoregulatory prop erties. Our results have be en supported that PGE 2 c an ac t as anti-inflammation and immunosuppression in the induction of COX-2 in endothelial ce lls by IL-1b . The ex act mechanisms by w hich PGE 2 inhibite d COX-2 induction in e ndothelial c ells ac tivated w ith IL1b are not know n. These may involve binding to spe cific c ell surface rece ptors and influe nc ing sec ond me sse nger syste ms through G-prote ins. Inde ed, the se should be c omplex because the e ffec ts of PGE 2 are ex e rted by a varie ty of PGE re ceptors w hich are different in their signal transduc tion p rope rties. There are at least four subtypes of PGE rece ptors. The EP1 and EP3 re ce ptors are couple d to Ca 2 + mobiliz ation and the inhibition of ade nylate cyclase, re spe ctively, and the EP2 and EP4 re cep tors are coupled to the same signal transduc tion pathw ay, stimulation of ade nylate cyclase. 1 9 How e ver, our studies show e d that forskolin (cAMP activator) can re verse the inhibiton of PGE 2 on COX-2 induce d in IL-1b tre ate d HUVEC suggesting PGE 2 may inhibit COX-2 ex p re sse d in IL-1b treated HUVEC through c AMP inhibition via EP3 re ceptors. PGE 2 is one of the PGs or COX me tabolites, such as PGI 2 , PGE 2 , PGD 2 , PGF 2 a and TXA 2 , synthe size d by COX-1 and COX-2 w hich are involved in physiology and pathology, 4 -8 respe ctive ly. Each COX is oform c an produc e diffe rent COX me tabolites in different cell type s such as PGI 2 is a major COX-1 and COX-2 metabolite in endothelial c ells w hile PGE 2 is a major COX-2 metabolite in mac rophages. 2 0 These differences in COX metabolite produc tion in different c ell type s may be re sulted from the feedback regulation of each COX metabolite produce d. Our results show e d that PGE 2 (0.03 m M) inhibite d PGE 2 produc tion (30% inhibition; 13 PGE se rie s have bee n use d in clinical disorders such as peripheral vascular oc clusive dise ase s, 2 1 NSAIDsinduc ed gastric ulcer, 2 2 abortion 23 and impote nc e. Thus, w e propose d that uses of PGE 2 in the c ondition in w hich COX-2 has bee n involve d may be therapeutic and the e ffec ts of othe r COX me tabolites such as PGI 2 or PGF 2 a on COX-2 ex pressed in different cells should be e luc idate d. ACKNOWLEDGMENTS: This w ork w as supported by a grant from Siriraj China Medical Board to P. Akarase reenont

The Effects of Andrographis paniculata on Platelet Activity in Healthy Thai Volunteers

Background. Andrographis paniculata (AP) has been used in Thai traditional medicine to treat various infections, including the common cold and fever. Its bioactive compound, andrographolide, has shown antiplatelet activities in an in vitro study model. Since clinical studies of the effects of AP on the human platelet function have never been reported, we investigated its effect on platelet activity in ten healthy volunteers. Methods. Two grams of AP was taken 3 times within one day. The blood was withdrawn by venipuncture before and 2 and 24 hours after the AP administration to analyze the effects of AP on platelet aggregation, the expression of enzyme cyclooxygenase (COX) mRNA and protein, and TXB2, including P-selectin. Result. Even though there was no significant change in the studied parameters, this study exhibited patient-to-patient variability in platelet function. It was found that ADP-induced platelet aggregation tended to decrease after AP administration, while epinephrine-induced platelet aggregation in females tended to be higher than that in males for the entire study period. Moreover, COX-1 mRNA levels tended to decrease while P-selectin levels tended to rise after AP administration. Conclusion. These controversial results are possibly due to the multifactorial mechanisms of platelet aggregation as well as the multichemical composition of AP. Further study, probably at the molecular level, is needed to unveil the underlying mechanisms of action of AP