Phospholipase D2 acts as an important regulator in LPS-induced nitric oxide synthesis in Raw 264.7 cells

The purpose of this study was to identify the role of phospholipase D2 (PLD2) in lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis. LPS enhanced NO synthesis and inducible nitric oxide synthase (iNOS) expression in macrophage cell line, Raw 264.7 cells. When Raw 264.7 cells were stimulated with LPS, the expressions of PLDs were increased. Thus, to investigate the role of PLD in NO synthesis, we transfected PLD1, PLD2, and their dominant negative forms to Raw 264.7 cells, respectively. Interestingly, only PLD2 overexpression, but not that of PLD1, increased NO synthesis and iNOS expression. Moreover, LPS-induced NO synthesis and iNOS expression were blocked by PLD2 siRNA, suggesting that LPS upregulates NO synthesis through PLD2. Next, we investigated the S6K1-p42/44 MAPK-STAT3 signaling pathway in LPS-induced NO synthesis mechanism. Knockdown of PLD2 with siRNA also decreased phosphorylation of S6K1, p42/44 MAPK and STAT3 induced by LPS. Furthermore, we found that STAT3 bound with the iNOS promoter, and their binding was mediated by PLD2. Taken together, our results demonstrate the importance of PLD2 for LPS-induced NO synthesis in Raw 264.7 cells with involvement of the S6K1-p42/44 MAPK-STAT3 pathway.     

β-Phenylethyl and 8-methylsulphinyloctyl isothiocyanates, constituents of watercress, suppress LPS induced production of nitric oxide and prostaglandin E2 in RAW 264.7 macrophages

Beta-phenylethyl (PEITC) and 8-methylsulphinyloctyl isothiocyanates (MSO) represent two phytochemical constituents present in watercress Rorripa nasturtium aquaticum, with known chemopreventative properties. In the present investigation, we examined whether PEITC and MSO could modulate the inflammatory response of Raw 264.7 macrophages to bacterial lipopolysaccharide (LPS) by assessment of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Overproduction of both nitric oxide (NO) and prostaglandins (PGE) has been associated with numerous pathological conditions including chronic inflammation and cancer. Our results demonstrate that LPS (1 microg/ml approximately 24 h) induced nitrite and prostaglandin E2 (PGE-2) synthesis in Raw 264.7 cells was attenuated by both isothiocyanates (ITCs) in a concentration-dependent manner. Both PEITC and MSO decreased (iNOS) and (COX-2) protein expression levels leading to reduced secretion of both pro-inflammatory mediators. Interestingly, the reduction in both iNOS and COX-2 expression were associated with the inactivation of nuclear factor-kappaB and stabilization of IkappaBalpha. Taken together our data gives further insight into the possible chemopreventative properties of two dietary derived isothiocyanates from watercress.     

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.     

Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-kappaB pathways

We recently reported that lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus, stimulated inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release, and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. This study was carried out to further investigate the roles of COX-2 and prostaglandin E2 (PGE2) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Treatment of RAW 264.7 macrophages with LTA caused a time-dependent increase in PGE2 release. LTA-induced iNOS expression and NO release were inhibited by a non-selective COX inhibitor (indomethacin), a selective COX-2 inhibitor (NS-398), an adenylyl cyclase (AC) inhibitor (dideoxyadenosine, DDA), and a protein kinase A (PKA) inhibitor (KT-5720). Furthermore, both PGE2 and the direct PKA activator, dibutyryl-cAMP, also induced iNOS expression in a concentration-dependent manner. Stimulation of RAW 264.7 macrophages with LTA, PGE2, and dibutyryl-cAMP all caused p38 MAPK activation in a time-dependent manner. LTA-mediated p38 MAPK activation was inhibited by indomethacin, NS-398, and SB 203580, but not by PD 98059. The PGE2-mediated p38 MAPK activation was inhibited by DDA, KT-5720, and SB 203580, but not by PD 98059. LTA caused time-dependent activation of the nuclear factor-kappaB (NF-kappaB)-specific DNA-protein complex formation. The LTA-induced increase in kappaB-luciferase activity was inhibited by indomethacin, NS-398, KT-5720, and a dominant negative mutant of p38 alphaMAPK (p38 alphaMAPK DN). These results suggest that LTA-induced iNOS expression and NO release involve COX-2-generated PGE2 production, and AC, PKA, p38 MAPK, and NF-kappaB activation in RAW 264.7 macrophages.     

EP2 and EP4 receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE(2) in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP(2) and EP(4) agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP(2) or EP(4) knockout mice. In addition, LPS failed to decrease the motility of EP(2) and EP(4) knockout mice ileum. EP(2)- or EP(4)-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE(2) induces iNOS expression through cAMP/ERK pathways by activating EP(2) and EP(4) receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.     

人参皂苷Rg2抗MTD大鼠海马神经元凋亡的研究

目的:观察多发梗塞性痴呆模型大鼠CPU凋亡相关蛋白的改变,研究其在学习记忆障碍中的作用机制,并探讨人参皂苷Rg2的干预作用。方法:将复合血栓诱导剂经大鼠左颈总-颈内动脉注射入其左侧大脑半球,诱导半球内血栓形成,造成多发梗塞,从而建立大鼠的学习和记忆功能障碍模型。分别给予不同剂量的人参皂苷Rg2治疗7天,并以尼莫地平进行对照。用Y型电迷宫检测正常对照及痴呆模型大鼠学习和记忆能力的改变;对大鼠的脑组织切片作尼氏染色行病理学检查;以免疫组化方法检测Glu、CalpainⅡ、Caspase-3、Bax的表达。结果:颈内动脉注射复合血栓诱导剂可造成大鼠在Y型电迷宫中的学习和记忆成绩明显降低,与假手术对照组比较有显著差异(P<0.05)。尼氏染色可在大鼠脑片的CPU区发现胶质瘢痕化的微梗塞灶。在免疫组化结果中,Glu、CalpainⅡ、Caspase-3、Bax表达均增高。结论:单侧大脑注射复合血栓诱导剂可造成大鼠的学习记忆能力降低,可部分模拟MID的行为和病理学改变。人参皂苷Rg2能明显改善MID模型大鼠的学习记忆成绩,其作用机制可能与它的钙通道阻滞作用使凋亡蛋白CalpainⅡ、Caspase-3、Bax表达降低有关...     

Effect of progesterone on the expression of bax and bcl-2 and on caspase activity in bovine luteal cells

Bovine luteal cells from days 6-10 and 11-15 of the estrous cycle were exposed (6 h) to factors that support or disrupt steroidogenesis. The expression of bcl-2 and bax and level of active caspase-3 in cells was measured. Progesterone (P4) increased (P<0.01) while staurosporine decreased (P<0.01-P<0.001) bcl-2 expression at both stages of the estrous cycle studied. In cells from 11-15 days of the estrous cycle expression of bcl-2 was stimulated (P<0.05) by prostaglandin (PG)E2 and inhibited (P<0.01) by 3,3',4,4'-tertrachlorobiphenyl (PCB)-77. Treatment with aminoglutethimide (blocker of cytochrome P450scc; 1.5 x 10(-4)M), nitric oxide donor (spermine NONOate), and staurosporine increased bax expression in cells collected from both experimental periods. The influence of these factors was greater in cells from days 11-15 (P<0.001) than by cells on days 6-10 (P<0.05) of the estrous cycle. PCB-77 stimulated expression of bax in cells from 11-15 days of cycle (P<0.01) only. Treatment of luteal cells with P4 and PGE2 for 24 h decreased (P<0.05) level of active caspase-3 while aminoglutethimide (P<0.05), spermine NONOate (P<0.05), and staurosporine (P<0.001) increased caspase-3 activity in the cells. Moreover, P4 decreased (P<0.05) while staurosporine increased (P<0.01) the ratio of bax/bcl-2 at both stages of the cycle. Aminoglutethimide, spermine NONOate and PCB increased (0<0.05) this ratio in cells on days 11-15 of the cycle. These results suggest that P4 concentrations in luteal cells protects against apoptosis, while disruption of steroidogenesis and reduced ability of luteal cells to produce P4 can induce cell death.     

Hyperglycemia induces apoptosis of pancreatic islet endothelial cells via reactive nitrogen species-mediated Jun N-terminal kinase activation

Hyperglycemia significantly stimulates pancreatic islet endothelial cell apoptosis; however, the precise mechanisms are not fully understood. In the present study, treating pancreatic islet endothelial (MS-1) cells with high glucose (30 mmol/l) but not mannitol significantly increased the number of apoptotic cells as compared with a physiological glucose concentration (5.5 mmol/l). Hyperglycemia significantly stimulated the expression of inducible nitric oxide synthase (iNOS) and production of NO and peroxynitrite (ONOO⁻), relevant to MS-1 cell apoptosis. Moreover, induced reactive nitrogen species (RNS) significantly increased the expression of bax, cleaved caspase-3 and poly adenosine diphosphate (ADP)-ribose polymerase (PARP) via JNK activation, but the expression of bcl-2 was not altered. Furthermore, SP600125 (a specific inhibitor of JNK) and 1400W (a specific inhibitor of iNOS) significantly attenuated cell apoptosis induced by high glucose. Therefore, hyperglycemia triggers MS-1 cell apoptosis by activating an intrinsic-dependent apoptotic pathway via RNS-mediated JNK activation.     

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.     

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.     

Effect of bismuth subgallate on nitric oxide and prostaglandin E2 production by macrophages

Bismuth subgallate (BSG) is used widely in clinics, including Vincent's angina, syphilis, and adenotonsillectomy. This study examined the effects of BSG on nitric oxide (NO) and prostaglandin E2 (PGE2) production in activated RAW 264.7 cells. BSG suppressed production of NO and PGE2 in a dose-dependent manner. BSG could increase TGF-beta1 production, which in turn might promote degradation of iNOS mRNA, thus inhibiting NO production. Additionally, BSG inhibited mPGES protein expression and COX-2 activity in activated RAW 264.7 cells. Exogenous addition of SNP reversed the inhibition effect of PGE2 production by BSG. This behavior indicates that PGE2 inhibition by BSG exerts an indirect effect through NO inhibition.     

8-Hydroxyquinoline inhibits iNOS expression and nitric oxide production by down-regulating LPS-induced activity of NF-kappaB and C/EBPbeta in Raw 264.7 cells

In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, 8-hydroxyquinoline (8HQ) inhibited the LPS-induced expression of both iNOS protein and mRNA in a parallel dose-dependent manner. 8HQ did not enhance the degradation of iNOS mRNA. To investigate the mechanism by which 8HQ inhibits iNOS gene expression, we examined the activation of MAP kinases in Raw 264.7 cells. We did not observe any significant change in the phosphorylation of MAPKs between LPS alone and LPS plus 8HQ-treated cells. Moreover, 8HQ significantly inhibited the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), but not activator protein-1 and cAMP response element-binding protein. Taken together, these results suggest that 8HQ acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of C/EBPbeta DNA-binding activity and NF-kappaB activation.     

Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-kappa B activation

The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and p-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-kappa B. We found that the NF-kappa B inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-kappa B involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B.     

Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.     

Differential regulation by fucoidan of IFN‐γ‐induced NO production in glial cells and macrophages

Fucoidan has shown numerous biological actions; however, the molecular bases of these actions have being issued. We examined the effect of fucoidan on NO production induced by IFN‐γ and the molecular mechanisms underlying these effects in two types of cells including glia (C6, BV‐2) and macrophages (RAW264.7, peritoneal primary cells). Fucoidan affected IFN‐γ‐induced NO and/or iNOS expression both in macrophages and glial cells but in a contrast way. Our data showed that in C6 glioma cells both JAK/STAT and p38 signaling positively regulated IFN‐γ‐induced iNOS, which were inhibited by fucoidan. In contrast, in RAW264.7 cells JAK/STAT is a positive regulator whereas p38 is a negative regulator of NO/iNOS production. In RAW264.7 cells, fucoidan enhanced p38 activation and induced TNF‐α production. We also confirmed the dual regulation of p38 in BV‐2 microglia and primary peritoneal macrophages. From these results, we suggest that fucoidan affects not only IFN‐γ‐induced NO/iNOS production differently in brain and peritoneal macrophages due to the different roles of p38 but the effects on TNF‐α production in the two cell types. These novel observations including selective and cell‐type specific effects of fucoidan on IFN‐γ‐mediated signaling and iNOS expression raise the possibility that it alters the sensitivity of cells to the p38 activation. J. Cell. Biochem. 111: 1337–1345, 2010. © 2010 Wiley‐Liss, Inc.     

Dipyrithione inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in macrophages and protects against endotoxic shock in mice

Dipyrithione (PTS2) possesses anti-bacterial and anti-fungal activity. In the present study, we found that PTS2 dose-dependently inhibited the LPS-induced up-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein level in RAW264.7 cells. RT-PCR experiments showed that PTS2 suppressed LPS-induced iNOS but not COX-2 expression at the mRNA level. As expected, PTS2 prevented NO secretion in RAW264.7 cells. Furthermore, PTS2 administration significantly decreased LPS-induced mortality in mice. Mechanistically, PTS2 decreased expression and phosphorylation of STAT1, but did not interfere with the MAPK and NF-kappaB pathways. In conclusion, PTS2 protects mice against endotoxic shock and inhibits LPS-induced production of pro-inflammatory mediators, suggesting that PTS2 could play an anti-inflammatory role in response to LPS.     

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.     

长期酒精摄入对雄性大鼠生殖系统的损伤

摘要目的：研究长期酒精摄入对雄性大鼠生殖系统的损伤机制。方法：选用8 周龄的SD 大鼠,进行随机分组：对照组（5％蔗糖, 口服）;酒精组（4g/kg,口服）。连续12周后,分别取附睾考察精子数目、活力;取血清检测睾酮和促黄体生产素（LH）含量;计算睾 丸- 体重比,并检测睾丸中丙二醛（MDA）、谷胱甘肽（GSH）含量以及谷胱甘肽过氧化物酶（GPx）和超氧化物歧化酶（SOD）的活 性;同时检测凋亡相关蛋白bax,bcl-2 以及caspase-3 前体和剪切体的蛋白表达。结果：酒精组12 周后,大鼠的睾丸- 体重比明显 降低（P<0.05）,精子数目减少（P<0.01）,精子活力下降（P<0.01）;血清中睾酮含量下降（P<0.05）,LH 含量增加（P<0.05）;睾丸中 MDA 含量增加（P<0.01）,GSH 含量降低（P<0.05）,GPx 和SOD活性下降（P<0.01）;凋亡相关蛋白bax 表达增加（P<0.05）,caspase -3 剪切体与前体的比值增加（P<0.01）。结论：长期摄入酒精引起的大鼠睾丸内氧化应激水平的增加是其导致其生殖系统损伤的重 要因素之一。     

乳酸堆积和二氯乙酸钠对肝癌细胞凋亡及bax、bcl-2 表达 和caspase-3 活性的影响

目的：探讨乳酸堆积和二氯乙酸钠(DCA)对肝癌细胞(HepG2)凋亡和bax、bcl-2 表达及caspase-3 活性的影响。方法：通过体 外培养HepG2,建立稳定的体外培养模型,配制成终浓度分别为0 mmol/L、1.0 mmol/L、2.0 mmol/L、4.0 mmol/L、8.0 mmol/L的乳 酸培养液以及在不同浓度乳酸组中加入终浓度为10-3mmol/L DCA 培养液与HepG2共同培养,其中以0 mmol/L 乳酸组为对照 组。采用MTT法检测乳酸对HepG2 的抑制率,流式细胞仪检测乳酸和DCA 对HepG2的凋亡百分率,用Real-time PCR法测定 bax 及bcl-2 mRNA的表达,用免疫荧光法检测caspase-3 的活性。结果：乳酸对HepG2 的IC50值为13.6 mol/L,与对照组比较,随 着乳酸浓度的增加,HepG2 凋亡率增加,bax mRNA 表达升高,bcl-2 mRNA 的表达降低,caspase-3活性增加,其中1.0 mmol/L 乳 酸组与对照组比较(P>0.05),2.0 mmol/L,4.0 mmol/L 和8.0 mmol/L乳酸组与对照组比较差异有统计学意义(P<0.05)。加入DCA 后,HepG2 凋亡减少,2.0 mmol/L 乳酸+DCA 组、4.0 mmol/L乳酸+DCA 组、8.0 mmol/L乳酸+DCA 组与同浓度的乳酸组比较, bax mRNA 表达减少（P<0.05）,bcl-2 mRNA 表达增加（P<0.05）,caspase-3 活性减低（P<0.05）。结论：乳酸可诱导HepG2凋亡,且随 着乳酸浓度的增高,HepG2 的凋亡率增加,其机制可能是通过对bcl-2 及bax mRNA 表达的改变以及激活caspase-3 活性而实现, DCA可以降低HepG2 凋亡,对乳酸堆积造成的HepG2凋亡有抑制作用。