Resveratrol analog, HS-1793 enhance anti-tumor immunity by reducing the CD4+CD25+ regulatory T cells in FM3A tumor bearing mice

Natural agents with the immunomodulating property have been gaining traction to be employed in the complementary therapy of cancer because the ineffectiveness of numerous therapeutic strategies may be related in part to the tumor-induced immunosuppressive phenotypes, especially regulatory T (Treg) cells found in the tumor microenvironment. The present study was undertaken to examine whether HS-1793, synthetic resvertrol analog free from the restriction of metabolic instability and high dose requirement of resveratrol, induces an in vivo anti-tumor effect in FM3A tumor bearing mice through the suppression of Treg cells, which contribute to an increase in tumor specific cytotoxic T cell responses. Intraperitoneal injections of HS-1793 showed not only therapeutic benefits on established tumors, but also preventive anti-tumor effects. Treg cells (CD4+CD25+Foxp3+ cells) were significantly reduced in the total splenocytes as well as tumor tissues from HS-1793-administered mice, and the production of TGF-β inducing Treg showed a similar pattern. On the contrary, the administration of HS-1793 increased IFN-γ-expressing CD8+ T cells, upregulated IFN-γ production, and enhanced the cytotoxicity of splenocytes against FM3A tumor cells both in therapeutic and preventive experimental animals. These results demonstrated the suppressive role of HS-1793 on the function of Treg cells contributing to tumor specific cytotoxic T lymphocyte responses in tumor-bearing mice, which explained the underlying mechanism of the anti-tumor immunity of HS-1793.     

肿瘤相关巨噬细胞在肿瘤发病过程及治疗中的研究进展

巨噬细胞是机体内参与免疫反应的重要免疫细胞之一, 随着人们对巨噬细胞研究的深入, 肿瘤相关巨噬细胞(TAMs)这一概念被提出。TAMs是肿瘤微环境(TME)中复杂而异质的细胞群, 与肿瘤相关炎症的发生有密切关系, 可影响肿瘤的生长、侵袭、转移和肿瘤内血管形成, 也可分泌多种细胞因子和趋化因子抑制抗肿瘤免疫反应的发生。由此可见, TAMs与肿瘤的发生发展有密切的关系, 因此成为肿瘤治疗的有趣靶点。本文就TAMs如何参与肿瘤进展、如何在临床中用于肿瘤治疗的靶点进行综述。     

Skin reactions induced by trimetrexate,an analog of methotrexate

We have observed three forms of skin toxicity induced by the new antifol trimetrexate in a Phase I trial. They are: radiation recall, cellulitis at the infusion site, and generalized skin eruptions with erythroderma. A total of 25 episodes of some form of skin reaction occurred in 31 patients. The generalized eruption began about four days after drug administration and cleared within a week. The mechanism of skin toxicity of trimetrexate and other antifols is unknown.The opinions expressed in this article are solely those of the authors and do not necessarily reflect those of any government agency.     

Changes in thymidine incorporation into tumor cells induced by supernatants of calcium-ionophore-treated macrophages incubated with tumor cells

Thioglycollate-elicited peritoneal M phi of mice were treated with calcium ionophore A23187 prior to incubation with tumor cells. Supernatants prepared from the incubation mixtures were assayed for their effects on incorporation of 3H-thymidine into tumor cells. Significant inhibition of incorporation was observed in most instances. Ingestion of indomethacin in the drinking water for 7 days by prospective M phi donors increased the inhibition by supernatants. The activity of the supernatants was neither specific for the strain and sex of M phi donors nor correlated with the genotype of the tumors. The activity even crossed the species barrier inasmuch as inhibitory supernatants were produced by murine M phi incubated with either murine or human tumor cells. Supernatants derived from murine as well as human tumor cells significantly depressed thymidine incorporation into murine and human tumor cells, although there were some differences in the response of the two kinds of tumor cells. In rare instances, supernatants enhanced, rather than inhibited, thymidine incorporation. Moreover, when supernatants, added to tumor cells, were removed and replaced by fresh medium, in most instances, significant enhancement of thymidine incorporated occurred. The formation of both growth-inhibiting and growth-promoting factors by M phi in the in vitro model used in this study, may explain the dual role of M phi in neoplasia in vivo.     

Potassium channels-mediated vasorelaxation of rat aorta induced by resveratrol

Resveratrol, a phenolic substance present in grapes and a variety of medical plants, has been reported to induce vasorelaxation, however the mechanisms are uncertain. In this paper we investigate the possible participation of K(+) channels in the endothelium-independent vasodilatation of rat aorta induced by resveratrol. Resveratrol induced concentration-dependent relaxation of rings with endothelium and without endothelium. We used different potassium channel inhibitors to determine whether the K(+) channels mediated endothelium-independent relaxation of rat aorta induced by resveratrol. Highly selective blocker of ATP-sensitive K(+) channels, glibenclamide, as well as non-selective blockers of K(+) channels, tetraethylammonium, did not block resveratrol-induced relaxation of rat aortic rings. Charybdotoxin, a blocker of calcium-sensitive K(+) channels did not affect the resveratrol-induced relaxation. 4-Aminopiridine, non-selective blocker of voltage-gated K(+) (Kv) channels, and margatoxin that inhibits Kv1 channels abolished relaxation of rat aortic rings induced by resveratrol. In conclusion, we have shown that resveratrol potently relaxed rat aortic rings with denuded endothelium. It seems that 4-aminopiridine and margatoxin-sensitive K(+) channels located in the smooth muscle of rat aorta mediated this relaxation.     

干扰素γ治疗肝和肺纤维化

实验室、动物试验和临床研究表明,干扰素γ(Interferon gamma,IFN-γ)对肝和肺纤维化有抑制作用.国内外学者报告用IFN-γ治疗各种原因引起的肝纤维化有效,临床症状明显改善,肝纤维化程度明显减轻.IFN-γ治疗特发性肺纤维化的初步结果显示,治疗后各种生物学指标有所改善,临床效果各家报道不一,多数学者认为IFN-γ治疗能降低病死率,对病情轻到中度的患者效果较好,用药时间应在1年以上.     

Clonidine reverses the amnesia induced by dopamine beta hydroxylase inhibition.

The role of noradrenergic (NE) mechanisms in amnesia induced by the dopamine-beta-hydroxylase (DBH) inhibitor, diethyldithiocarbamate (DEDTC) was examined by studying the antiamnestic characteristics of the alpha-NE receptor stimulator clonidine. DEDTC (250 mg/kg) administered 3 hr prior to training to C57BL/6J mice resulted in marked deficits when retention of a multiple trial food motivated spatial discrimination task was measured 24 hr after learning. Investigation of the temporal aspects of recovery indicated that the agonist was an effective antiamnestic agent when administered 0, 1, 3, 21 and 23 hr after training. No recovery was observed when the drug was administered 6 and 18 hr posttraining. A dose response study of the effectiveness of clonidine administered 1 hr prior to testing indicated recovery of memory at doses ranging from 10-500 microgram/kg. The clonidine induced recovery was not a result of general performance facilitation, but specific to the memory tested. In addition, the clonidine effect was pharmacologically specific to its actions on NE receptors, as recovery was blocked by pre-treatment with the alpha-NE antagonist, phentolamine. No recovery from DEDTC induced amnesia was seen with post-training or pre-test injection of d-amphetamine.     

Effects of resveratrol,curcumin and their derivatives on the activation of microglia induced by LPS

Objective To determine the inhibitory effects of 21 resveratrol derivatives and 3 natural curcuminoids on lipopolysaccharide(LPS)-induced Nitric oxide(NO)and tumor necrosis factor-alpha(TNF-α)production in microglia and their structure-activity relationships.Methods Cell viability was evaluated by the MTT reduction assay.Accumulation of nitrite(NO2-)in culture supernatant fluids was measured by the Griess reaction.Sodium nitroprusside(SNP)(2.5 mM)solution was used to determine the scavenging activities of these compounds.The levels of TNF-α in the culture medium were measured by using an ELISA kit.Semi-quantitative RT-PCR analysis was used to determine the mRNA levels of inducible NOS(iNOS)and TNF-α.Results It was found,for the first time,that certain resveratrol derivatives that have 3,5-dimethoxyl groups in the A-ring,such as(E)-4-(3,5-dimethoxystyryl)phenol(pterostilbene,compound 2),or have substituted the B-ring of resveratrol with quinolyl,such as(E)-5-[2-(quinolin-4-yl)vinyl]benzene-1,3-diol(compound 18)and(E)-4-(3,5-dimethoxystyryl)quinoline(compound 19),strongly inhibited NO production.Compounds 2,18,and 19 reduced LPS-induced protein and mRNA expression of inducible NO synthase(iNOS),but did not display direct NO-scavenging activity up to 30 μM in sodium nitroprusside(SNP)solution.Moreover,compounds 2,18,and 19 could also significantly inhibit the production of TNF-α by LPS-activated microglia.Furthermore,we found the demethoxy derivatives of curcumin have more potent inhibition activity on NO and TNF-α releasing in activated-microglia.Conclusions In the present study we compared the activated-microglia inhibition effect of resvertrol,curcumin and their derivatives and provided a glance of the structure-activity relationships of these compounds,the information is beneficial to design new potent compounds which can provide better therapeutic implications for various neurodegenerative diseases.     

Suppression of nitric oxide synthase and the down-regulation of the activation of NFkappaB in macrophages by resveratrol

Resveratrol, naringenin and naringin are naturally occurring flavonoids in grapes and grapefruits. The anti-inflammatory effects of these flavonoids have been well documented, but the mechanism is poorly characterized. High concentration of NO are produced by inducible NO synthase (iNOS) in inflammation, and the prevention of the expression of iNOS may be an important anti-inflammatory mechanism. In this study, the effects of these flavonoids on the induction of NO synthase (NOS) in RAW 264.7 cells activated with bacterial lipopolysaccharide (LPS, 50 ng ml(-1)) were investigated. Resveratrol was found strongly to inhibit NO generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic iNOS protein and steady state mRNA levels. However, the inhibitory abilities of naringenin were lower, and the inhibitory abilities of naringin were almost negligible. In electrophoretic mobility shift assays, the activation of NFkappaB induced by LPS for 1 h was inhibited by resveratrol (30 microM). Furthermore, in immunoblotting analysis, cells treated with LPS plus resveratrol showed an inhibition of phosphorylation as well as degradation of IkappaBalpha, and a reduced nuclear content of NFkappaB subunits. The flavonoids may be of value for inhibiting the enhanced expression of iNOS in inflammation through down-regulation of NFkappaB binding activity.     

Baclofen reverses the reduction in prepulse inhibition of the acoustic startle response induced by dizocilpine,but not by apomorphine

Rationale Since baclofen, the prototypical GABA_B receptor agonist, is known to reduce the activity of dopaminergic mesolimbic neurons, a putative antipsychotic property of this compound has been suggested, but the evidence for this is still controversial.Objectives The aim of the present study was to elucidate the effects of baclofen on the prepulse inhibition (PPI) of the acoustic startle response (ASR), a behavioral paradigm considered to be one of the most powerful tools for the evaluation of sensorimotor gating and for the screening of antipsychotics.Methods We tested the effects of baclofen (1.25, 2.5, 5 and 10 mg/kg IP) in rats, per se and in co-treatment with some of the substances known to induce a robust reduction of PPI, such as apomorphine (0.25 mg/kg SC) and dizocilpine (0.1 mg/kg SC). Finally, in order to ascertain whether the effects of baclofen could be ascribed to its activity on GABA_B receptors, we analyzed whether its action could be prevented by pretreatment with SCH 50911, a selective GABA_B receptor antagonist (20 mg/kg IP). All the experiments were carried out using standard procedures for the assessment of PPI of the ASR.Results Baclofen per se produced no significant change in PPI parameters. Moreover, while no effect on apomorphine-mediated alterations in PPI parameters was observed, baclofen proved able to reverse dizocilpine-induced PPI disruption, and this effect was significantly prevented by SCH 50911. On the other hand, this last compound exhibited no effects per se at the same dose.Conclusions These results indicate that GABA_B receptors are implicated in the neurobiological circuitry accounting for glutamatergic action in sensorimotor gating, and therefore can be proposed as putative new targets in the pharmacological therapy of psychotic disorders. Further studies should be addressed to evaluate more closely the clinical efficacy of baclofen in this respect.     

环孢素A逆转野百合碱诱导的肺动脉高压

目的:探讨环孢素A(cyclosporine A,CsA)对野百合碱(monocrotaline,MCT)诱发肺动脉高压的影响及其作用机制.方法:36只雄性Sprague-Dawley(SD)大鼠随机分为正常对照组(n=8)、肺动脉高压模型组(n=12)、CsA低、高剂量组(0.33和1 mg·kg-1,n=8).后3组大鼠颈背部皮下一次性注射MCT 50mg·kg-1诱导肺动脉高压模型,MCT注射后d 14～d 21,CsA组灌胃给药,模型组灌胃等体积的生理氯化钠溶液(5 mL·kg-1).MCT注射后d 22,右心导管术测肺动脉压,称肺湿重(wW)、右心室自由壁(RV)重和左心室加室间隔(LV S)重,计算右心肥大指数[RVHI=RV/(LV S)],肺湿重指数(LI=wW/BW).HE染色观察肺病理改变;免疫组化方法观察肺动脉平滑肌细胞增殖细胞核抗原(PCNA)阳性表达.用Image-ProPlus 5.1软件分析肺动脉中膜相对厚度及肺动脉平滑肌PCNA阳性细胞的比值.结果:CsA低、高剂量组均能逆转MCT诱导的肺动脉高压(P<0.05或P<0.01),降低RVHI及LI(P<0.05或P<0.01),改善肺动脉重构(P<0.05或P<0.01),减轻肺部炎症,抑制肺动脉平滑肌的增殖(P<0.05或P<0.01),且高剂量组更明显.结论:CsA能明显降低MCT诱导的肺动脉高压,逆转肺动脉重构,其机制与抑制肺动脉平滑肌增殖和抑制肺部炎症有关.     

青蒿琥酯诱导肿瘤细胞凋亡与抑制存活蛋白表达有关

目的 :探讨青蒿琥酯 (artesunate ,Art)诱导肿瘤细胞凋亡与存活蛋白 (survivinprotein)表达的关系。方法 :采用细胞荧光染色、流式细胞术、琼脂糖凝胶电泳法和测定细胞浆Caspase 3的活性等手段检测肿瘤细胞暴露于不同浓度的Art时 ,对肿瘤细胞凋亡的诱导作用 ;用RT PCR、WesternBlotting法 ,检测不同浓度的Art作用于肿瘤细胞时 ,对survivinmRNA和survivin蛋白表达的影响。结果 :HL6 0细胞暴露于Art时 ,呈现典型细胞凋亡特征 ,如 :胞核固缩、形成凋亡小体 ;凋亡细胞的比例呈浓度依赖性增高 ;琼脂糖电泳出现明显的“梯状”条带 ;细胞浆Caspase 3的活性呈浓度依赖性增高等。RT PCR检测表明 ,A5 49细胞暴露于Art 10和 5 0g·L-172h后 ,survivinmRNA的表达呈浓度依赖性降低 ,对照组、10和 5 0mg·L-1处理组的survivin条带和内标GAPDH条带灰度的比值分别为 1.74 5、0 .390和0 .0 2 3;WesternBlotting法也检测到Art抑制Survivin蛋白的表达。结论 :Art诱导肿瘤细胞发生凋亡 ,激活Caspase 3途径 ,可能与抑制survivin基因表达有关     

Folic acid reverses endothelial dysfunction induced by inhibition of tetrahydrobiopterin biosynthesis

While folic acid has been shown to reverse endothelial dysfunction, the exact underlying mechanism remains elusive. Here, folic acid reversed both the endothelial dysfunction and increased production of superoxide following depletion of rabbit aortic ring tetrahydrobiopterin (BH₄) levels with 2,4-diamino-6-hydroxy-pyrimidine (DAHP) and N-acetyl-5-hydroxy-tryptamine (NAS). Incubation with l-nitroarginine methyl ester also attenuated the production of superoxide. DAHP and NAS reduced BH₄ concentrations in both aorta and cultured porcine aortic endothelial cells. Folic acid had no effect on BH₄ concentrations in either preparation. The superoxide anion scavenger Tiron but not folic acid reversed the endothelial dysfunction produced in aortic rings by inhibition of copper–zinc superoxide dismutase with diethyldithiocarbamic acid. Neither folic acid nor its metabolite 5-methyltetrahydrofolate prevented the in vitro oxidation of BH₄. This study demonstrates that folic acid reverses the endothelial dysfunction induced by BH₄ depletion independently of either the regeneration or stabilization of BH₄ or an antioxidant effect.     

Modification of gamma radiation induced response of peritoneal macrophages and splenocytes by Hippophae rhamnoides (RH-3) in mice

Alcoholic extract of Hippophae rhamnoides, RH-3, reported to render >80% survival against lethal whole body Co-60-gamma irradiation (10 Gy) in mice, was investigated for its immunostimulatory effects. In comparison with un-irradiated control, whole body irradiation did not reduce peritoneal macrophage counts at 24 h post-irradiation. RH-3 treatment (30 mg kg(-1) body weight) alone or 30 min before whole-body irradiation enhanced viable counts of macrophages significantly (P< or =0.05) compared with both un-irradiated control and irradiated groups. Whole-body irradiation reduced the number of viable splenocytes significantly (P<0.05) compared with un-irradiated control at 24 h post-irradiation. RH-3 treatment alone or before whole-body irradiation appreciably countered radiation-induced decrease in splenocyte count. 3H-thymidine uptake method revealed that whole-body irradiation reduced splenocyte proliferation significantly (159 +/- 45 counts min(-1)/10(6) cells; P< or =0.05) in comparison with control (607 +/- 142 counts min(-1)) at 24 h after irradiation but RH3 treatment before irradiation reduced the steep decrease and maintained it as 444+/-153 counts min(-1). After whole-body irradiation, the ratio of spleen weight/mouse weight decreased to 1.5 +/- 04 compared with 2.9 +/- 0.32 in un-irradiated control at 24 h post-irradiation. Similarly, total protein content in splenocytes also decreased to 48 +/- 6 microg/10(6) cells in comparison with 368 +/- 16 microg/10(6) cells of un-irradiated control. RH-3 treatment before irradiation countered radiation-induced decrease in both spleen weight/mouse weight ratio (4.0 +/- 0.35) and total protein content (360 +/- 13 mug/10(6) splenocytes). In the supernatant of peritoneal macrophage cultures exposed to 2 Gy Co-60-gamma radiation ex-vivo, the total nitrite content was enhanced significantly (P<0.05) to 5.72 +/- 0.09 microM in comparison with un-irradiated control (1.64 +/- 0.09 microM). RH-3 treatment (30 microg mL(-1)) before irradiation reduced total nitrite significantly (0.93 +/- 0.3; P< or =0.05) in comparison with irradiated control group. At 24 h after whole body irradiation, the CD4+/CD8+ ratio reduced to 1.5 in comparison with un-irradiated control (1.9) but RH-3 treatment before irradiation restored the ratio to 2.1. These findings explicitly reveal the immunostimulatory activity of RH-3, which may play an important role in the manifestation of its radioprotective efficacy.     

白藜芦醇诱导人宫颈癌Hela细胞凋亡及其机制

目的探讨白藜芦醇诱导人宫颈癌Hela细胞的凋亡作用及其分子机制。方法应用不同浓度的白藜芦醇作用于人宫颈癌Hela细胞,采用四甲基偶氮唑蓝（MTT）法检测药物对细胞的增殖抑制率;流式细胞术（FCM）检测细胞凋亡、细胞周期分布：免疫组化法检测Survivin及Caspase-3的表达。结果白藜芦醇能明显抑制Hela细胞的增殖,并呈剂量和时间依赖性。经白藜芦醇处理Hela细胞后,FCM分析发现各实验组S期细胞比例增高,G2／M期细胞比例减少,凋亡率明显高于对照组,并呈剂量依赖性（P〈0．01）;各实验组Heal细胞Survivin的表达均低于对照组,而Caspase-3的表达均高于对照组（P〈0．01）。结论白藜芦醇能明显抑制人宫颈癌Hela细胞的增殖,诱导其凋亡,其机制可能与抑制Survivin的表达、上调Caspase-3的表达有关。