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

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-1ß). METHODS: Cells were treated with indomethacin (positive control), HRF, and its components at various concentrations prior to treatment with IL-1ß 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.

Initial response of endothelial cells to acute stimulation with a lipid component: increase cyclooxygenase activity by induction of COX-2 through activation of tyrosine kinase.

OBJECTIVE: To study the initial response of endothelial cells acutely stimulated with a lipid component in the aspect of cyclooxygenase (COX) function which needed for prostacyclin synthesis, an endogenous antiatherogenic agent secreted from endothelial cells. MATERIAL AND METHOD: 25 hydroxycholesterol (25OHC) was used as a representative lipid component for stimulating human umbilical vein endothelial cell (HUVEC) obtained from umbilical cords of healthy newborns with informed consent of their mothers. HUVEC were treated with 25OHC (0.1, 1 or 10 microgram/mL) at times 6, or 24 h. COX activity was measured from amount of 6-keto-PGF(1 alfa) production in the presence of exogenous arachidonic acids (10 micromolar; 10 min) by enzyme immunoassay. The amount of COX-1 and COX-2 protein were detected by Western blot. Cell viability was assessed by using MTT assay. RESULTS: 25OHC induced COX-2 protein production with increasing the activity of COX enzyme in HUVEC without change in amount of COX-1 protein. The induction of COX-2 or increasing in COX activity depended on concentration of 25OHC and time to exposure which seemed to be inhibited by genistein, a specific tyrosine kinase inhibitor. CONCLUSION: Acute stimulation of HUVEC with 25OHC, an atherosclerotic lipid component, increases the activity of COX by inducing COX-2 expression in a manner that depended on concentration and time. The induction of COX-2 expression might possibly mediated through activation protein tyrosine kinase. These responses may be an initial defensive mechanism of endothelial cells from lipid component attack.

Modulation of antioxidant defense by Alpinia galanga and Curcuma aromatica extracts correlates with their inhibition of UVA-induced melanogenesis.

Ultraviolet A (UVA) irradiation is suggested to contribute to melanogenesis through promoting cellular oxidative stress and impairing antioxidant defenses. An overproduction of melanin can be associated with melanoma skin cancer and hyperpigmentation. Therefore, developing effective antimelanogenic agents is of importance. Alpinia galanga (AG) and Curcuma aromatica (CA) are traditional medicinal plants widely used for skin problems. Hence, this study investigated the antimelanogenic effects of AG and CA extracts (3.8-30 microg/ml) by assessing tyrosinase activity, tyrosinase mRNA levels, and melanin content in human melanoma cells (G361) exposed to UVA. The roles in protecting against melanogenesis were examined by evaluating their inhibitory effects on UVA-induced cellular oxidative stress and modulation of antioxidant defenses including antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GPx), and intracellular glutathione (GSH). In addition, possible active compounds accountable for biological activities of the extracts were identified by thin layer chromatography (TLC)-densitometric analysis. Our study demonstrated that UVA (8 J/cm(2)) induced both tyrosinase activity and mRNA levels and UVA (16 J/cm(2))-mediated melanin production were suppressed by the AG or CA extracts at noncytotoxic concentrations. Both extracts were able to protect against UVA-induced cellular oxidant formation and depletion of CAT and GPx activities and GSH content in a dose-dependent manner. Moreover, TLC-densitometric analysis detected the presence of eugenol and curcuminoids in AG and CA, respectively. This is the first report representing promising findings on AG and CA extract-derived antityrosinase properties correlated with their antioxidant potential. Inhibiting cellular oxidative stress and improving antioxidant defenses might be the mechanisms by which the extracts yield the protective effects on UVA-dependent melanogenesis.

Effects of selective COX-inhibitors and classical NSAIDs on endothelial cell proliferation and migration induced by human cholangiocarcinoma cell culture.

OBJECTIVE: Experiments were designed to explore cellular mechanisms and effects of NSAIDs on human umbilical vein endothelial cells (HUVEC) induced by human cholangiocarcinoma (HuCCA). MATERIAL AND METHOD: HUVEC were incubated with HuCCA or HuCCA-conditioned medium (CM) for various times to determine cell proliferation and migration. Expression of cyclooxygenase (COX) proteins was measured using immunoblotting technique. VSA (selective COX-1 inhibitor), NS-398 (selective COX-2 inhibitor), and aspirin were used as pharmacological tools to explore signaling mechanisms of HuCCA-CM-induced endothelial cell functions. RESULTS: HuCCA could significantly induce proliferation and migration of HUVEC. COX-2, but not COX-1, was increased. NS-398, but not VSA, could significantly inhibit HuCCA-CM-induced endothelial cell proliferation. HuCCA-CM-induced endothelial cell proliferations could be also inhibited by aspirin. CONCLUSION: These findings suggest that HuCCA-CM-derived substances could induce HUVEC proliferation through COX-2 signaling mechanism. Classical NSAID and selective COX-2 inhibitors could also inhibit this step of HUVEC proliferation.

The effects of estrone, estradiol and estriol on platelet aggregation induced by adrenaline and adenosine diphosphate.

The impact of estrogens on the cardiovascular system and their ability to regulate platelet functions remains controversial. Changes in platelet functions could contribute to thrombotic risk associated with estrogen treatments. Here, we investigated the effects of various forms of estrogen, including estrone (E1), estradiol (E2) and estriol (E3), on platelet aggregation induced by standard agonists (adrenaline and adenosine diphosphate). Platelet-rich plasma (PRP) was prepared from citrated blood donated by 25 normal volunteers. The study on platelet aggregation was carried out in 96-well flat-bottom microtitre plates and assessed using a microplate reader. For studying the effects of each estrogen, PRP was preincubated with 1, 10 and 100 nM of E1, E2 and E3 at 37 degrees C for 20 min, and then coincubated with normal saline (control untreated PRP), adrenaline (ADR) or adenosine diphosphate (ADP) in the microplate. Platelet aggregation was then measured every minute for 8 min. None of the estrogens (E1, E2 and E3) affected platelet aggregation in untreated PRP. Interestingly, only E1 and E3 can synergize the increased platelet aggregation by either ADR or ADP, while the effects of E2 on the increased platelet aggregation by either ADR or ADP depended on internal factors such as endogenous estradiol and platelet aggregated state. Thus, for the rational use of these internal factors for estrogen use, especially E2, in clinical applications, such as hormone replacement therapy, may need evaluation of thrombotic risk.

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.