Phosphatidylcholine-specific phospholipase C and D in stimulation of RAW264.7 mouse macrophage-like cells by lipopolysaccharide.

The purpose of these studies was to identify the role of phospholipases in the activation of macrophages by lipopolysaccharide (LPS). Tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC); butanol, an inhibitor of phosphatidylcholine phospholipase D (PC-PLD); and propranolol, an inhibitor of phosphatidate phosphohydrolase, were used in the study. Treatment of RAW264.7 murine macrophage-like cells with LPS resulted in expression of inducible nitric oxide synthase and tumor necrosis factor-alpha. The expression was partially inhibited by D609, butanol, or propranolol and was completely blocked by the combination of D609 and butanol. RAW264.7 cells constitutively produced low basal levels of diacylglycerol and phosphatidic acid; production of both was significantly increased after stimulation with LPS, reaching a peak in 2-3 min and remaining elevated after 30 min. In LPS-induced RAW264.7 cells, diacylglycerol was suppressed by each of the three inhibitors alone and almost abolished by D609 plus butanol or D609 plus propranolol. Phosphatidic acid was reduced to basal level by butanol after LPS stimulation for 2.5 min and by butanol plus D609 after LPS stimulation for 2.5 or 10 min. Taken together, these data indicate that activation of RAW264.7 cells by LPS can be mediated by the activities of both PC-PLC and PC-PLD.     

The anti-inflammatory effect of Sonchus oleraceus aqueous extract on lipopolysaccharide stimulated RAW 264.7 cells and mice

Context: Sonchus oleraceus L. (Asteraceae) (SO) is a dietary and traditional medicinal plant in China. However, its underlying mechanism of action as an anti-inflammatory agent is not known.

Objective: This study evaluates the anti-inflammatory activity of aqueous extract of SO.

Materials and methods: The extract of SO was used to treat RAW 264.7 cells (in the working concentrations of 500, 250, 125, 62.5, 31.3 and 15.6?μg/mL) for 24?h. Pro-inflammatory cytokines and mediators produced in LPS-stimulated RAW 264.7 cells were assessed. Meanwhile, the expression level of TLR-4, COX-2, pSTATs and NF-κB was tested. Moreover, the anti-inflammatory activity of the extract in vivo was assessed using xylene-induced mouse ear oedema model and the anti-inflammatory compounds in the extracts were analyzed by HPLC-MS.

Results: SO extract significantly inhibited the production of pro-inflammatory cytokines and mediators at gene and protein levels with the concentration of 31.3?μg/mL, and suppressed the expression of TLR-4, COX-2, NF-κB and pSTAT in RAW 264.7 cells. The anti-inflammatory activity of SO in vivo has significant anti-inflammatory effects with the concentration of 250 and 125?mg/kg, and less side effect on the weights of the mice at the concentration of 250?mg/kg. Moreover, HPLC-MS analysis revealed that the anti-inflammatory compounds in the extract were identified as villosol, ferulaic acid, β-sitosterol, ursolic acid and rutin.

Discussion and conclusion: This study indicated that SO extract has anti-inflammatory effects in vitro and in vivo, which will be further developed as novel pharmacological strategies in order to defeat inflammatory diseases.     

Comparative analysis of the anti-inflammatory activity of Huang-lian extracts in lipopolysaccharide-stimulated RAW264.7 murine macrophage-like cells using oligonucleotide microarrays

In this study, we attempted to determine the anti-inflammatory activity of a medicinal plant huang-lian using gene expression profiles as an index. Huang-line extracts (CEXs) were prepared from seven different plant origins and compared for their chemical composition and biological activity. In order to achieve this, RAW264.7 cells were treated with CEXs in the absence or presence of LPS for 6 h, and the differential gene expression profiles were analyzed using oligonucleotide microarrays. The alkaloid content of CEXs was determined by high performance liquid chromatography analysis. Evaluation of anti-inflammatory activity of CEXs was by measuring a decrease in cytokines and nitric oxide production in LPS-stimulated RAW264.7 cells. Hierarchical clustering analysis revealed that three CEXs from Coptis chinensis formed a cluster separate from the other four CEXs in LPS-stimulated cells, and were the most effective anti-inflammatoryagents. The extract prepared from Picrorrhiza kurrooa neither induced any changes in gene expression profiles nor possessed any anti-inflammatory activity. The extract from Jeffersonia dubia, which exhibited the highest cytotoxicity among the CEXs tested, was most effective in suppressing LPS-induced nitric oxide production but was not able to inhibit proinflammatory cytokine production. The results obtained in this study demonstrate that overall gene expression profiles of the extracts correlated well with their biological activity and that CEXs prepared from plants of diverse origins vary in their biological activity. These data also suggest that gene expression profiles may serve as a good indicator for the pharmacological activities of medicinal plants arising from diverse origins.     

Safety of non-steroidal anti-inflammatory drugs during pregnancy and lactation

As inhibitors of cyclooxygenase NSAIDs given during pregnancy have the potential to cause adverse maternal and fetal effects. Maternal effects include prolongation of pregnancy and labour, whereas constriction of the ductus arteriosus, renal dysfunction and haemostatic abnormalities can occur in the fetus and neonate. As weak acids, NSAIDs are excreted in small amounts into human breast milk with little risk for adverse effects in the suckling infant.     

Mechanism of cell death induced by cationic dendrimers in RAW 264.7 murine macrophage-like cells

Cationic dendrimers possess attractive nano-sized architectures that make them suitable as targeted drug/gene delivery systems. However, very little is known about their mechanisms of cell death in cellular systems. In the current study, the apoptotic and necrotic effects of starburst polyamidoamine(PAMAM) and polypropylenimine (DAB) dendrimers in cultured RAW 264.7 murine macrophage-like cells were investigated. Cationic dendrimer treatment produced a typically dose-dependent cytotoxic effect on macrophage cells. RAW 264.7 cells exposed to cationic dendrimers exhibited morphological features of apoptosis. Apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with cationic dendrimers. Analysis from flow cytometry demonstrated an increase in hypodiploid DNA population (sub-G1) and a simultaneous decrease in diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells to cationic dendrimers. Also, cells treated with DAB dendrimer induced a higher percentage of sub-G1 population than those treated with PAMAM dendrimer at the same dose. In addition, it was shown that pre-treatment of RAW 264.7 cells with the general caspase inhibitor zVAD-fmk prevented some degree of apoptosis induced by cationic dendrimers, suggesting that apoptosis in macrophage cells involves a caspase dependent pathway. Macrophage cells were also found to be sensitive to induction of apoptosis by dendrimers, whereas NIH/3T3 cells (mouse fibroblast) and BNL CL.2 (mouse liver) cells did not undergo apoptosis. These results could be helpful for optimizing the biocompatibility of dendrimers used for targeted drug/gene delivery.     

Inhibitory activity of Chinese herbal medicines toward histamine release from mast cells and nitric oxide production by macrophage-like cell line, RAW 264.7

One hundred and six species of traditional Chinese herbs were collected from the northeast of China, extracted with 60% ethanol, and tested for activity inhibiting histamine release and nitric oxide (NO) production. We found that 18 of the 106 species showed strong histamine-release inhibitory activity (inhibition >80%, 100 g/ml), of which Bidens parviflora Willd. was recognized to present the strongest activity (inhibition 97.8%, 100 g/ml). Seven species exhibited strong NO-production inhibitory activity (inhibition >50%, 100 g/ml) with Pholidota chinensis Lindl. as the most active one (inhibition 86.2%, 100 g/ml).     

非甾体抗炎药诱发肠病的诊治

非甾体抗炎药是临床上常用的抗炎、解热镇痛药。长期以来,人们比较重视非甾体抗炎药引起的胃损害,而流行病学调查显示,由于服用非甾体抗炎药引起下消化道的损伤同样严重。据报道,在长期口服非甾体抗炎药病人中,小肠黏膜受损率高达70%以上,本文就非甾体抗炎药导致的非甾体抗炎药肠病进行综述     

Inhibitory effect of Chunghyuldan in prostaglandin E2 and nitric oxide biosynthesis of lipopolysaccharide-induced RAW 264.7 cells

Chunghyuldan (Daio-Orengedokuto in Japanese) (CHD) has been used as an antihyperlipidemic and antiischemic agent in Korea. To evaluate in vitro the efficacy of Chunghyuldans (CHDs) metabolized with and without human intestinal microflora against brain ischemia, we investigated its anti-inflammatory effect on LPS-induced RAW264.7 cells. Both metabolized CHD (MCHD) and CHD showed antioxidant activities in vitro, and inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) productions in lipopolysaccharide (LPS)-induced RAW264.7 cells. These also inhibited enzyme activities and protein expressions of inducible NO synthase and cyclooxygenase-2 in LPS-induced RAW264.7 cells. MCHD-inhibitory activity against NO and PGE2 productions in LPS-induced RAW264.7 cells was more potent than those of CHD. These results suggest that CHD may show potent anti-inflammatory activity in vivo and can improve brain ischemia.     

非甾体抗炎药与麻醉药的相互作用

非甾体抗炎镇痛药(Non-steroidal anti-inflammatory drugs,NSAIDs)主要用于围手术期超前镇痛。本文对NSAIDs与常用的麻醉药如阿片类药、吸入麻醉药、静脉麻醉药和苯二氮艹卓类药物的相互作用加以综述。     

原花青素对RAW264.7细胞膜相关前列腺素E2合成酶-1表达的影响

目的探讨原花青素对RAW264.7细胞膜相关前列腺素E2合成酶-1(mPGES-1)表达的影响。方法酶免疫测定法(EIA)检测原花青素对PGE2生成的影响,逆转录聚合酶链反应(RT-PCR)检测mPGES-1mRNA的表达,Western blotting检测mPGES-1蛋白的表达。结果脂多糖(LPS)可以促进RAW264.7细胞PGE2的生成同时上调mPGES-1mRNA和蛋白的表达,而原花青素(4、20 mg.L-1)下调LPS诱导的RAW264.7细胞mPGES-1mRNA和蛋白的表达,从而抑制LPS诱导的RAW264.7细胞PGE2的生成。结论原花青素在mRNA和蛋白水平抑制LPS诱导的RAW264.7细胞mPGES-1表达从而减少PGE2的合成,这可能是原花青素抗炎的机制之一。     

Anticancer effects of non-steroidal anti-inflammatory drugs against cancer cells and cancer stem cells

Certain non-steroidal anti-inflammatory drugs (NSAIDs) are known to have anticancer effects. However, it is unclear whether all NSAIDs have anticancer effects, and thus far, very few studies have compared the antitumor effects among multiple NSAIDs. Therefore, we aimed to identify NSAIDs that enhance the anticancer effect of cisplatin (CDDP); the effects of 17 NSAIDs in lung cancer cells and their spheroids as cancer stem cells (CSCs) were evaluated. Some of the NSAIDs showed cytotoxic effects against A549 and SBC-3 cells and their CDDP-resistant cell lines (A549/DDP and SBC-3/DDP cells, respectively). In addition, co-addition of CDDP and celecoxib, which showed cytotoxic effects, increased the resistance to CDDP by increasing SLC7A11, which is one of the CDDP resistance mechanisms, in A549/DDP and SBC-3/DDP cells. On the other hand, celecoxib also showed antitumor effects on the spheroids of A549/DDP and SBC-3/DDP cells, and enhanced the antitumor effect of CDDP while increasing the mRNA levels of SLC7A11. Moreover, diclofenac was also cytotoxic and enhanced the cytotoxic effect of CDDP in cancer cells and CSCs. In conclusion, some NSAIDs including celecoxib and diclofenac may enhance the therapeutic efficacy of CDDP.     

Renal effects of non-steroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors

Nonsteroidal antiinflammatory drugs (NSAID) are one of the most commonly used medications worldwide to inhibiting COX activity for the treatment of pain and inflammation. Their nephrotoxicity has been well documented. With the development and clinical implementation of new COX-2 inhibitors, the safety, including the effects on renal function and blood pressure, is attracting increasing attention. In the kidney, COX-2 is constitutively expressed and is highly regulated in response to alterations in intravascular volume. COX-2 metabolites have been implicated in mediation of renin release, regulation of sodium excretion and maintenance of renal blood flow. Similar to conventional NSAIDs, inhibition of COX-2 may cause edema and modest elevations in blood pressure in a minority of subjects. COX-2 inhibitors may also exacerbate preexisting hypertension or interfere with other antihypertensive drugs. Occasional acute renal failure has also been reported. Caution should be taken when COX-2 inhibitors are prescribed, especially in high-risk patients (including elderly and patients with volume depletion). Recently, agents with combined lipooxygenase/COX inhibition and agents that combine NSAIDs with a nitric oxide (NO) donor have been reported to reduce adverse renal effects.     

STAT5通路抑制剂对脂多糖诱导RAW264.7细胞的保护作用研究

目的观察信号传导及转录激活因子(STAT)5通路抑制剂对经脂多糖(LPS)诱导的小鼠巨噬细胞RAW264.7细胞株一氧化氮(NO)和诱导型一氧化氮合成酶(iNOS)表达的影响。方法将处于对数期的RAW264.7细胞分为空白对照组、STAT5通路抑制剂对照组、LPS诱导组和STAT5通路抑制剂低、中、高浓度组,实时定量聚合酶链式反应(RT-PCR)法测定iNOS mRNA的表达量,Western blot测定iNOS以及磷酸化STAT5(p-STAT5)的蛋白表达量。结果RAW264.7细胞培养24 h后,空白对照组、STAT5通路抑制剂对照组、LPS诱导组以及STAT5通路抑制剂低、中、高浓度组细胞中NO的含量差异有统计学意义(F=25.69,P<0.05);低、中、高浓度STAT5通路抑制剂对RAW264.7细胞释放NO的抑制率差异有统计学意义(F=132.49,P<0.05)。空白对照组、STAT5通路抑制剂对照组、LPS诱导组和STAT5通路抑制剂低、中、高浓度组细胞iNOS mRNA以及蛋白质的表达量比较差异有统计学意义(F=123.59、23.37,P<0.05)。空白对照组、STAT5通路抑制剂对照组、LPS诱导组和STAT5通路抑制剂低、中、高浓度组细胞p-STAT5/STAT5表达比较差异有统计学意义(F=12.07,P<0.05)。结论STAT5通路抑制剂能够抑制RAW264.7细胞iNOS的表达以及NO的产生,其机制可能与抑制STAT5磷酸化的表达有关。     

Effects of melanocortin peptides on lipopolysaccharide/interferon-gamma-induced NF-kappaB DNA binding and nitric oxide production in macrophage-like RAW 264.7 cells: evidence for dual mechanisms of action

The pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) mediates broad anti-inflammatory and immunomodulatory effects, which include inhibition of the production and release of proinflammatory cytokines and nitric oxide (NO) from macrophages. We investigated the effects of alpha-MSH, alpha-MSH(1-10), and alpha-MSH(11-13) on NO production and nuclear factor-kappaB (NF-kappaB) translocation in RAW 264.7 macrophages. After stimulation of the cells with bacterial lipopolysaccharide/interferon-gamma (LPS/IFN-gamma), all three peptides inhibited NO production with an order of potency alpha-MSH > or = alpha-MSH(11-13) > alpha-MSH(1-10). All three MSH peptides inhibited NF-kappaB nuclear translocation with the maximal effect of alpha-MSH and alpha-MSH(11-13) being seen in the range 1 nM-1 microM, and that of alpha-MSH(1-10) at 1 microM. By use of (125)I-(Nle(4),D-Phe(7))alpha-MSH(NDP-MSH) radioligand binding, MC(1) receptor-binding sites were demonstrated on RAW 264.7 cells. alpha-MSH and alpha-MSH(1-10) competed with the (125)I-NDP-MSH binding at these MC(1) receptor-binding sites, but alpha-MSH(11-13) even in concentrations up to 1 mM did not. Moreover, alpha-MSH and alpha-MSH(1-10) caused powerful stimulation of cyclic 3',5'-adenosine monophosphate (cAMP) in the RAW 264.7 cell, whereas alpha-MSH(11-13) was ineffective. Forskolin stimulated cAMP and inhibited NO production to the same extent as alpha-MSH and alpha-MSH(1-10), but did not modify the translocation of NF-kappaB. Whereas the protein kinase A inhibitor H89 did not modify the effect of alpha-MSH on NF-kappaB translocation, H89 caused a partial inhibition of the inhibitory effect of alpha-MSH, alpha-MSH(1-10), alpha-MSH(11-13), and forskolin on NO production. In addition alpha-MSH, alpha-MSH(1-10), alpha-MSH(11-13), and forskolin also inhibited the activity of an NF-kappaB-dependent luciferase reporter and these effects were partially counteracted by H89. We suggest that melanocortin peptides act via dual mechanisms of action: one cAMP-independent and causing inhibition of NF-kappaB translocation and the other dependent on MC(1) receptor/cAMP activation.     

Organ tolerance of non-steroidal anti-inflammatory drugs: effect on liver cells

Experimental studies in mice and in rats showed the good tolerance of a new non-steroidal anti-inflammatory drug, flunoxaprofen, by normal and CCl4 damaged liver. The activity of an enzyme-inducing drug, such as phenobarbital, showed a greater increase after pretreatment with indomethacin than with flunoxaprofen or benoxaprofen. High doses of benoxaprofen and indomethacin significantly decreased bromosulphonphthalein excretion in rats with normal or CCl4 damaged liver; the effect was not observed with flunoxaprofen administered at the same dose as benoxaprofen. Moreover, benoxaprofen and indomethacin but not flunoxaprofen induced a significant increase of some serum liver enzymes in CCl4 poisoned rats.