Testosterone augments endotoxin-mediated cerebrovascular inflammation in male rats

Activation of inflammatory mechanisms contributes to cerebrovascular pathophysiology. Male gender is associated with increased stroke risk, yet little is known about the effects of testosterone in the cerebral circulation. Therefore, we explored the impact of testosterone treatment on cerebrovascular inflammation with both in vivo and in vitro models of inflammation. We hypothesized that testosterone would augment the expression of two vascular markers of cellular inflammation, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Using four groups of male rats [intact, orchiectomized (ORX), and ORX treated with either testosterone (ORXT) or the testosterone metabolite 17beta-estradiol (ORXE)], we determined effects of the sex hormones on cerebrovascular inflammation after intraperitoneal LPS injection. Western blot analysis showed that induction of inflammatory markers was increased in cerebral blood vessels from ORXT rats compared with intact or ORX rats. In contrast, in cerebral blood vessels from ORXE rats, there was a significant decrease in endotoxin-induced COX-2 and iNOS protein levels. Confocal microscopy of cerebral blood vessels from ORXT rats showed increased COX-2 and iNOS immunoreactivity in both endothelial and smooth muscle cells after LPS treatment. In vitro incubation with LPS also induced COX-2 in pial vessels isolated from the four animal treatment groups, with the greatest induction observed in ORXT vessels compared with the ORX and ORXE groups. Production of PGE2, a principal COX-2-derived prostaglandin end product, was also greatest in cerebral vessels isolated from ORXT rats. In conclusion, testosterone increases cerebrovascular inflammation; this effect may contribute to stroke differences between men and women.     

Estrogen and progestagens differentially modulate vascular proinflammatory factors

The potential benefit of ovarian hormone replacement therapy in cerebrovascular disease is well supported by experimental observations but not by recent large, randomized clinical trials. This discrepancy points out the need for better understanding of the vascular actions of ovarian hormones as well as medroxyprogesterone acetate (MPA), a synthetic analog of progesterone (P) widely prescribed in combination with estrogens. Therefore, we investigated whether in vivo exposure to 17beta-estradiol (E) and/or P or MPA modifies inflammation in the cerebral vasculature, a key process in the evolution of ischemic brain injury. Female rats were injected (ip) with LPS to induce inflammation, and 6 h later brains were taken for blood vessel isolation and Western blot analysis of the inflammatory enzymes inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). In ovariectomized (O) females, LPS induced cerebrovascular iNOS and COX-2; however, this effect was significantly decreased when O animals were treated for 3 wk with E. In contrast, treatment of O females with either MPA or P exacerbated the cerebrovascular inflammatory response to LPS. In intact females, LPS induction of iNOS and COX-2 in cerebral vessels was found to vary with the stage of the estrous cycle: LPS had the greatest effect during estrus, when circulating estrogen is low and progesterone is high. Thus exposure to endogenous or exogenous ovarian hormones appears to modulate cerebrovascular inflammation. Anti-inflammatory effects of estrogen would attenuate ischemic brain injury; however, this vasoprotective benefit may be diminished in the presence of progestagens.     

Selected contribution: cerebrovascular nos and cyclooxygenase are unaffected by estrogen in mice lacking estrogen receptor-alpha.

Estrogen alters reactivity of cerebral arteries by modifying production of endothelium-dependent vasodilators. Estrogen receptors (ER) are thought to be involved, but the responsible ER subtype is unknown. ER-alpha knockout (alphaERKO) mice were used to test whether estrogen acts via ER-alpha. Mice were ovariectomized, with or without estrogen replacement, and cerebral blood vessels were isolated 1 mo later. Estrogen increased levels of endothelial nitric oxide synthase and cyclooxygenase-1 in vessels from wild-type mice but was ineffective in alphaERKO mice. Endothelium-denuded middle cerebral artery segments from all animals constricted when pressurized. In denuded arteries from alphaERKO but not wild-type mice, estrogen treatment enhanced constriction. In endothelium-intact, pressurized arteries from wild-type estrogen-treated mice, diameters were larger compared with arteries from untreated wild-type mice. In addition, contractile responses to indomethacin were greater in arteries from wild-type estrogen-treated mice compared with arteries from untreated wild-type mice. In contrast, estrogen treatment of alphaERKO mice had no effect on diameter or indomethacin responses of endothelium-intact arteries. Thus ER-alpha regulation of endothelial nitric oxide synthase and cyclooxygenase-1 pathways appears to contribute to effects of estrogen on cerebral artery reactivity.     

Role of nuclear factor-kappaB in gastric ulcer healing in rats

We investigated the role of nuclear factor-kappaB (NF-kappaB) in gastric ulcer healing in rats. NF-kappaB was activated in ulcerated tissue but not in normal mucosa, and the level of the activation was decreased with ulcer healing. NF-kappaB activation was observed in fibroblasts, monocytes/macrophages, and neutrophils. Treatment of gastric fibroblasts, isolated from the ulcer base, with interleukin-1beta activated NF-kappaB and the subsequently induced cyclooxygenase-2 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA expression. Inhibition of activated NF-kappaB action resulted in suppression of both their mRNA expression and increases in PGE(2) and CINC-1 levels induced by interleukin-1beta. Persistent prevention of NF-kappaB activation caused an impairment of ulcer healing in rats. Gene expression of interleukin-1beta, CINC-1, cyclooxygenase-2, and inducible nitric oxide synthase in ulcerated tissue had been inhibited before the delay in ulcer healing became manifest. The increased levels of cyclooxygenase-2 protein and PGE(2) production were also reduced. These results demonstrate that NF-kappaB, activated in ulcerated tissue, might upregulate the expression of healing-promoting factors responsible for gastric ulcer healing in rats.     

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 by cell-permeable superoxide dismutase in lipopolysaccharide-stimulated BV-2 microglial cells

Oxidative stress plays a pivotal role in uncontrolled neuro-inflammation leading to many neurological diseases including Alzheimer’s. One of the major antioxidant enzymes known to prevent deleterious effects due to oxidative stress is Cu,Zn-superoxide dismutase (SOD). In this study, we examined the regulatory function of SOD on the LPS-induced signaling pathways leading to NF-kappaB activation, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in BV-2 cells using cell-permeable SOD. Treatment of BV-2 cells with cell-permeable SOD led to a decrease in LPS-induced reactive oxygen species (ROS) generation and significantly inhibited protein and mRNA levels of iNOS and COX-2 upregulated by LPS. Production of NO and PGE2 in LPS stimulated BV-2 cells was significantly abrogated by pretreatment with a cell-permeable SOD fusion protein. Furthermore, cell-permeable SOD inhibited LPS-induced NF-kappaB DNA-binding activity and activation of MAP kinases including ERK, JNK, and p38 in BV-2 cells. These data indicate that SOD has a regulatory function for LPS-induced NF-kappaB activation leading to expression of iNOS and COX-2 in BV-2 cells and suggest that cell-permeable SOD is a feasible therapeutic agent for regulation of ROS-related neurological diseases.     

Estrogen suppresses IL-1beta-mediated induction of COX-2 pathway in rat cerebral blood vessels

Interleukin (IL)-1beta is a potent inducer of inflammatory prostaglandins, which are important mediators of vascular response to cerebral injury, whereas estrogen reduces brain injury in models of ischemic stroke. Thus we examined the effects of in vivo IL-1beta exposure on cerebrovascular cyclooxygenase (COX)-2 expression and function in an animal model of chronic estrogen replacement. Estrogen-treated and nontreated ovariectomized female rats received IL-1beta injections (10 microg/kg i.p.), and then cerebral vessels were isolated for biochemical and contractile measurements. In estrogen-deficient rats, IL-1beta induced cerebrovascular COX-2 protein expression; a peak response occurred 3 h after injection. COX-2 was localized to arterial endothelium using confocal microscopy. IL-1beta increased PGE2 but not PGI2 production and decreased vascular tone as measured in isolated cerebral arteries; the latter effect was partially reversed by treatment with the selective COX-2 inhibitor NS-398 (10 micromol/l). In contrast, in animals treated with estrogen, IL-1beta had no significant effect on COX-2 protein levels, PGE2 production, or vascular tone. Combined treatment with 17beta-estradiol and medroxyprogesterone acetate also prevented increases in PGE2 production after IL-1beta treatment, but treatment with 17alpha-estradiol had no effect. IL-1beta induction of COX-2 protein was prevented by treatment with the nuclear factor-kappaB inhibitor caffeic acid phenethyl ester (20 mg/kg i.p.), and estrogen treatment reduced cerebrovascular nuclear factor-kappaB activity. Estrogen thus has potent anti-inflammatory effects with respect to cerebral vascular responses to IL-1beta. These effects may have important implications for the incidence and severity of cerebrovascular disease.     

17beta-estradiol decreases vascular tone in cerebral arteries by shifting COX-dependent vasoconstriction to vasodilation

We have previously shown that estrogen treatment increases cerebrovascular cyclooxygenase-1, prostacyclin synthase, and production of prostacyclin. Therefore, vascular tone and prostanoid production were measured to investigate functional consequences of estrogen exposure. Middle cerebral arteries were isolated from ovariectomized female Fischer-344 rats with or without chronic in vivo 17beta-estradiol treatment. In vivo 17beta-estradiol treatment increased cerebral artery diameter; functional endothelium was required for expression of these differences. The nonspecific cyclooxygenase inhibitor indomethacin constricted, whereas arachidonic acid dilated, cerebral arteries from estrogen-treated animals. Estrogen exposure increased production of prostacyclin by cerebral arteries. Conversely, in estrogen-deficient animals, indomethacin dilated and arachidonic acid constricted cerebral blood vessels. This correlated with vasorelaxation following inhibition of the thromboxane-endoperoxide receptor with SQ-29548 but not after selective blockade of thromboxane synthase with furegrelate, suggesting prostaglandin endoperoxide (i.e., PGH2) activity. Removal of the endothelium or selective blockade of cyclooxygenase-1 with SC-560 abolished estrogen-mediated differences in the effects of arachidonate on vessel diameter and on prostacyclin production by cerebral arteries. These data suggest 17beta-estradiol decreases cerebrovascular tone by shifting the primary end product of the endothelial cyclooxygenase-1 pathway from the constrictor prostaglandin PGH2 to the vasodilator prostacyclin. These effects of estrogen may contribute to the heightened thromboresistance and enhanced cerebral blood flow documented in pre-versus postmenopausal women.     

Fibronectin prevents endotoxin shock after partial hepatectomy in rats via inhibition of nuclear factor-kappaB and apoptosis

Fibronectins (Fns) are involved in a number of biologic processes, such as cellular adhesion, motility, differentiation, apoptosis, hemostasis, wound healing, and ischemic injury. We investigated the possible mechanism underlying the protective action of plasma Fn (pFn) on endotoxin shock following partial hepatectomy in rats. Lipopolysaccharide (LPS) was administered intravenously to male Sprague-Dawley rats within 48 hrs of 70% hepatectomy. Prior to LPS administration, pFn or human serum albumin was given intravenously. The survival rate of the pFn-treated group was improved markedly compared with that of the controls. The levels of inflammatory cytokines and nitric oxide (NO) in serum were significantly lower in the pFn-treated group than in the control group. Expression of inducible nitric oxide synthase (iNOS) in hepatocytes also was reduced following pFn treatment. The degree of apoptosis and necrosis in the remnant liver was significantly lower in the pFn-treated rats than the controls. Furthermore, pFn pretreatment greatly inhibited the activation of nuclear factor-kappaB (NF-kappaB), caspase 3 and 8 activities, and cytochrome c release, and caused a decrease in mitochondrial Bcl-x(L). Plasma Fn prevents endotoxin-induced liver injury at least in part through inhibition of NF-kappaB activation, which causes the reduction of iNOS expression and NO production by hepatocytes, and through the downregulation of inflammatory cytokines and promotion of Bcl-x(L) expression.     

Inhibitory effect of the coffee diterpene kahweol on carrageenan-induced inflammation in rats

Previous studies reported that kahweol, a coffee-specific diterpene, inhibits cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in cultured lipopolysaccharide-activated macrophages. The aim of this study was to confirm the anti-inflammatory effects of kahweol by examining its effect on the inflammatory response induced by carrageenan in a rat using an acute air pouch inflammation model. Kahweol significantly reduced the levels of the inflammatory process markers in the air pouch, such as the volume of exudates, the amount of protein and the number of leukocytes and neutrophils. The levels of nitrite, TNF-alpha and prostaglandin E2 (PGE2) were also markedly lower in the air pouch of the kahweol-treated animals than in the controls. Immunoblot analysis showed that kahweol reduced the COX-2 and iNOS expression level in the exudate cells. The histological examination showed that there was a lower inflammatory response in the pouch tissues from the kahweol-treated animals. In addition, kahweol significantly reduced the paw edema induced by carrageenan and also markedly reduced the level of PGE2 production in the inflamed paw. These results suggest that kahweol has significant anti-inflammatory effects in vivo, which might be due to the inhibition of iNOS and COX-2 expression in the inflammatory sites.     

Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages

Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and approximately 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 x g indicates that the COX-2 is distributed both in the 100,000 x g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor L-NMMA at concentrations that inhibit nitrite accumulation by /=85% with higher concentrations of L-NMMA shows 1) up-regulation of PGE2 production, 2) accumulation of COX-2 protein in the 100,000 x g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10-15% of the total) attenuate PGE2 production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.     

Integrin-linked kinase regulates inducible nitric oxide synthase and cyclooxygenase-2 expression in an NF-kappa B-dependent manner.

Nitric oxide (NO) and prostaglandins are produced as a result of the stimulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, respectively, in response to cytokines or lipopolysaccharide (LPS). We demonstrate that the activity of integrin-linked kinase (ILK) is stimulated by LPS activation in J774 macrophages. Inhibition of ILK activity by dominant-negative ILK or a highly selective small molecule ILK inhibitor, in epithelial cells or LPS-stimulated J774 cells and murine macrophages, resulted in inhibition of iNOS expression and NO synthesis. LPS stimulates the phosphorylation of IkappaB on Ser-32 and promotes its degradation. Inhibition of ILK suppressed this LPS-stimulated IkappaB phosphorylation and degradation. Similarly, ILK inhibition suppressed the LPS-stimulated iNOS promoter activity. Mutation of the NF-kappaB sites in the iNOS promoter abolished LPS- and ILK-mediated regulation of iNOS promoter activity. Overexpression of ILK-stimulated NF-kappaB activity and inhibition of ILK or protein kinase B (PKB/Akt) suppressed this activation. We conclude that ILK can regulate NO production in macrophages by regulating iNOS expression through a pathway involving PKB/Akt and NF-kappaB. Furthermore, we also demonstrate that ILK activity is required for LPS stimulated cyclooxygenase-2 expression in murine and human macrophages. These findings implicate ILK as a potential target for anti-inflammatory applications.     

Role of p38 in nitric oxide synthase and cyclooxygenase expression, and nitric oxide and PGE2 synthesis in human gingival fibroblasts stimulated with lipopolysaccharides

Periodontal disease, a gingival inflammatory disease caused by gram-negative bacteria, is the main cause of tooth loss. Lipopolysaccharides (LPS) present in bacterial cell walls induce human gingival fibroblasts' production of pro-inflammatory cytotoxins such as IL-1beta and TNFalpha. The goal of this study was to determine p38 role in the expression of inducible nitric oxide synthase enzyme (i-NOS) and cyclooxygenase (COX-2), as well as in PGE(2) and nitric oxide synthesis in human gingival fibroblasts challenged with LPS. We found that lipopolysaccharides induced a rapid and significant increase in p38 phosphorylation. After interruption of p38 transduction pathway by pre-treatment with inhibitor SB203580, no response to stimulation with LPS was observed; i-NOS expression and nitric oxide synthesis was completely blocked. However, p38 inhibition only partially blocked COX-2 expression and PGE2 synthesis. We conclude that p38 is critically involved in i-NOS induction, and that it participates in COX-2 expression and in PGE2 synthesis.     

Methanol extracts of Stewartia koreana inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression by blocking NF-kappaB transactivation in LPS-activated RAW 264.7 cells

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are involved in various pathophysiological processes such as inflammation and carcinogenesis. In a search for inhibitors of COX-2 and iNOS production we found that extracts of Stewartia koreana strongly inhibited NO and PGE2 production in LPS-treated macrophage RAW 264.7 cells. We have now shown that the mRNA and protein levels of iNOS and COX-2 are reduced by the Stewartia koreana extract (SKE). SKE inhibited expression of an NF-kappaB reporter gene in response to LPS, and gel mobility shift assays revealed that SKE reduced NF-kappaB DNA-binding activity. The extract also inhibited LPS-induced phosphorylation of IkappaB-alpha and nuclear translocation of p65. Administration of the extract reduced the symptoms of arthritis in a collagen-induced arthritic mouse model. These results indicate that Stewartia extracts contain potentially useful agents for preventing and treating inflammatory diseases.     

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.     

Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action

NF-kappaB that plays an important role in iNOS expression is one of the targets of various potential anti-inflammatory agents including resveratrol. Resveratrol contains a structural similarity with estrogen, and there has been speculation about resveratrol as estrogen agonist. In this study, the mechanism and structural requirements of resveratrol and related hydroxystilbenes for the inhibition of LPS-induced nitric oxide production were studied in macrophage cells (RAW 264.7 and J774) by comparing its effect on LPS-induced NF-kappaB translocation and nitric oxide production, and by considering the possibility of involvement of an estrogen receptor. LPS-induced nitric oxide production was inhibited only when cells were treated with resveratrol prior to stimulation with LPS, suggesting that resveratrol does not affect the enzyme itself. A higher concentration of resveratrol than needed for the inhibition of nitric oxide production was required for the inhibition of NF-kappaB mobilization or iNOS expression. Estrogen and diethylstilbesterol, an estrogen agonist, caused only weak inhibition of nitric oxide production, and the effects of resveratrol were not noticeably blocked by ICI-182780, an estrogen antagonist. Structure-activity analysis of resveratrol and nine hydroxystilbenes suggests that the structural balance between oxygen functional groups on the benzene rings is important for their activity. Our results suggest that resveratrol might act on other cellular targets as well as NF-kappaB at the initial stage of gene expression. Unique structural features of hydroxystilbenes are needed for suppression of nitric oxide production and it is unlikely that estrogen receptor is involved in it.