Immunomodulatory effect of Hibiscus cannabinus extract on macrophage functions

Hibiscus cannabinus L. (Malvaceae) (known as Kenaf) has long been used as a folk medicine in India and Africa for the treatment of blood and throat disorders, bilious conditions, fever and puerperium. In this study, therefore, we aimed either to demonstrate its ethnopharmacological activity by examining its macrophage function-regulating effects or to expand its therapeutic efficacy into other macrophage-mediated diseases. The total crude extract (EtOH extract) of Hibiscus cannabinus fresh leaves, prepared with 80% ethanol, significantly suppressed TNF-alpha production and the mRNA expression of interleukin (IL)-3 and IL-12 in the RAW264.7 cells, stimulated by lipopolysaccharide (LPS, 2.5 microg/ml). The secretion of inflammatory mediators (i.e., nitric oxide [NO], reactive oxygen species [ROS] and prostaglandin E(2) [PGE(2)]) was diminished by the EtOH extract. The extract induced the expression of heme oxygenase-1 (HO-1) mRNA, a potent cytoprotective molecule. The Kenaf extract suppressed both the phagocytic uptake and the expression of costimulatory molecules (CD80 and CD86) of LPS-activated RAW264.7 cells. It is interesting that Kenaf also down-regulated both the functional activation of beta1-integrin (CD29) and the LPS-induced up-regulation of the surface CD29 level. Taken together, these data suggest that Kenaf may be able to modulate macrophage-mediated responses and that some of the activities may contribute to expand its therapeutic usage.     

轮叶党参对老年小鼠益智及抗氧化的作用

本文用水迷宫实验及测定脂质过氧化物的方法研究了轮叶党参水提取液对实验动物益智作用及抗氧化作用。结果表明,4g/kg和8g/kg轮叶党参水提取液均明显缩短老年小鼠在水迷宫试验中潜伏期并明显减少水迷宫试验的错误次数。说明给予轮叶党参组小鼠学习记忆速度较老年对照组快。轮叶党参水提取物明显降低小鼠脑组织和红细胞及大鼠红细胞中脂质过氧化物(LPO)含量,且明显提高成年大鼠血清中的SOD活性。提示轮叶党参水提取液有改善老年小鼠学习记忆的作用,并能清除老年小鼠体内的脂质过氧化产物。     

Anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium on LPS-stimulated Raw264.7 cells

Aim of the study

Lilium lancifolium is commonly used to treat bronchitis, pneumonia, etc. In this study, we investigated the anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium (LL extracts) in LPS-stimulated Raw264.7 cells.

Material and methods

Levels of NO, PGE₂ and pro-inflammatory cytokines (IL-6 and TNF-α) in the supernatant fraction were determined using sandwich ELISA. Expression of COX-2 and iNOS, phosphorylation of MAPK subgroups (ERK and JNK), and NF-κB activation in extracts were detected via Western blot and immunocytochemistry assays.

Results

The LL extract significantly inhibited NO, PGE₂, IL-6 and TNF-α production in LPS-stimulated cells, and suppressed iNOS and COX-2 expression. A mechanism-based study showed that phosphorylation of ERK1/2 and JNK and translocation of the NF-κB p65 subunit into nuclei were inhibited by the LL extract. Furthermore, interleukin-4 and interleukin-13 production in Con A-induced splenocytes was suppressed.

Conclusion

These results indicate that anti-inflammatory effects of methanol extracts from Lilium lancifolium are due to downregulation of iNOS and COX-2 via suppression of NF-κB activation and nuclear translocation as well as blocking of ERK and JNK signaling in LPS-stimulated Raw264.7 cells.     

Treatment of THP-1 cells with Uncaria tomentosa extracts differentially regulates the expression if IL-1beta and TNF-alpha

Uncaria tomentosa, commonly known as cat's claw, is a medicinal plant native to Peru, which has been used for decades in the treatment of various inflammatory disorders. Uncaria tomentosa can be used as an antioxidant, has anti-apoptotic properties, and can enhance DNA repair, however it is best know for its anti-inflammatory properties. Treatment with Uncaria tomentosa extracts inhibits the production of the pro-inflammatory cytokine, TNF-alpha, which is a critical mediator of the immune response. In this paper, we showed that treatment of THP-1 monocyte-like cells with Uncaria tomentosa extracts inhibited the MAP kinase signaling pathway and altered cytokine expression. Using ELISA assays, we showed that treatment with Uncaria tomentosa extracts augmented LPS-dependent expression of IL-1beta by 2.4-fold, while inhibiting the LPS-dependent expression of TNF-alpha by 5.5-fold. We also showed that treatment of LPS-stimulated THP-1 cells with Uncaria tomentosa extracts blocked ERK1/2 and MEK1/2 phosphorylation in a dose-dependent manner. These data demonstrate that treatment of THP-1 cells with Uncaria tomentosa extracts has opposite effects on IL-1beta and TNF-alpha secretion, and that these changes may involve effects on the MAP kinase pathway.     

羊乳的本草考证

通过对羊乳的名称、植物形态、产地及功效的本草学考证,表明本草中关于羊乳的记载与现代研究结果基本一致;历代使用羊乳的药材来源于桔梗科党参属植物羊乳Codonopsis lanceolata。     

基于网络药理学的党参干预肠易激综合征作用机制研究

目的基于网络药理学探讨党参治疗肠易激综合征的作用靶点和作用机制。方法通过中药系统药理学数据库与分析平台(TCMSP)筛选出党参口服生物利用度≥30%和类药性≥0.18的化合物作为候选药效成分,利用HTDocking平台预测与候选药效成分匹配的靶蛋白,采用Cytoscape3.7.0软件构建化合物-靶点网络。通过GeneCards、NCBI和OMIM数据库查找肠易激综合征相关基因,利用Cytoscape3.7.0绘制疾病基因蛋白相互作用网络,并与党参化合物-靶点网络进行映射,发现党参治疗肠易激综合征的关键化合物和潜在作用靶点。使用Funrich3.1.3软件对潜在作用靶点进行GO功能注释和KEGG通路富集,对党参治疗肠易激综合征的作用机制做出合理推测。结果共得到党参候选药效成分17个,预测作用靶点111个,肠易激综合征相关基因1368个,党参治疗肠易激综合征的关键化合物15个,潜在作用靶点55个(包括IL6、PTGS2、AKT1、EGFR、IL2、IL10、IL4等),靶点参与的功能主要有免疫应答、炎症反应等,主要富集通路有TRAILA信号通路、TNF信号通路等448条。结论党参可能通过调节PTGS2等炎症因子,参与TRAIL信号通路、TNF信号通路等途径,调节免疫反应,从而达到治疗肠易激综合征的目的。     

藏药十八味党参丸提取物对脂多糖诱导的RAW264.7细胞Akt/p38MAPK信号通路及M1/M2极化影响的探究

目的探究藏药十八味党参丸(TEP)的提取物调控脂多糖(LPS)诱导小鼠巨噬细胞RAW 264.7的炎性反应及M1/M2极化分型的探究。方法 Alarmarblue法评价RAW 264.7活力;荧光酶标定量分析细胞活性氧(reactive oxygen species, ROS)水平;Griess法检测上清液中细胞一氧化氮(NO)的分泌情况;流式细胞术检测表面标志物CD206和CCR7蛋白的表达;RT-qPCR法检测细胞白介素(IL)-1β、IL-6、趋化因子2(CCL2)、一氧化氮合酶(iNOS)、精氨酸酶(ARG)-1和肿瘤坏死因子α(TNF-α)基因表达的情况;Western blot法检测TEP调控细胞合成iNOS、蛋白激酶B(Akt)和p38丝裂原活化激酶(p38MAPK)信号通路相关蛋白的表达情况。结果与模型组相比,TEP组一定程度上提高了RAW 264.7的细胞活性,同时抑制了LPS诱导的细胞内ROS水平的升高。经过LPS刺激后,模型组NO表达量显著升高,在24 h后表达量达到最高,而TEP组NO表达显著降低。模型组的CCR7表达升高,CD206表达下降,TEP干预后CCR7表达下降,CD206表达升高,且趋势呈现浓度依赖。与模型组相比,TEP组的IL-1β、IL-6、CCL2、iNOS、ARG和TNF-α基因表达以及iNOS、Akt和p38MAPK蛋白表达水平均显著降低。结论 TEP能够抑制细胞内ROS水平,降低NO分泌和炎性基因的表达水平,抑制细胞的M1表型,促进M2表型,其机制可能与细胞内iNOS、Akt和p38MAPK信号通路蛋白表达被抑制有关。     

藏药十八味党参丸提取物对脂多糖诱导的RAW264.7细胞Akt/p38MAPK信号通路及M1/M2极化影响的探究

目的探究藏药十八味党参丸(TEP)的提取物调控脂多糖(LPS)诱导小鼠巨噬细胞RAW 264.7的炎性反应及M1/M2极化分型的探究。方法 Alarmarblue法评价RAW 264.7活力;荧光酶标定量分析细胞活性氧(reactive oxygen species, ROS)水平;Griess法检测上清液中细胞一氧化氮(NO)的分泌情况;流式细胞术检测表面标志物CD206和CCR7蛋白的表达;RT-qPCR法检测细胞白介素(IL)-1β、IL-6、趋化因子2(CCL2)、一氧化氮合酶(iNOS)、精氨酸酶(ARG)-1和肿瘤坏死因子α(TNF-α)基因表达的情况;Western blot法检测TEP调控细胞合成iNOS、蛋白激酶B(Akt)和p38丝裂原活化激酶(p38MAPK)信号通路相关蛋白的表达情况。结果与模型组相比,TEP组一定程度上提高了RAW 264.7的细胞活性,同时抑制了LPS诱导的细胞内ROS水平的升高。经过LPS刺激后,模型组NO表达量显著升高,在24 h后表达量达到最高,而TEP组NO表达显著降低。模型组的CCR7表达升高,CD206表达下降,TEP干预后CCR7表达下降,CD206表达升高,且趋势呈现浓度依赖。与模型组相比,TEP组的IL-1β、IL-6、CCL2、iNOS、ARG和TNF-α基因表达以及iNOS、Akt和p38MAPK蛋白表达水平均显著降低。结论 TEP能够抑制细胞内ROS水平,降低NO分泌和炎性基因的表达水平,抑制细胞的M1表型,促进M2表型,其机制可能与细胞内iNOS、Akt和p38MAPK信号通路蛋白表达被抑制有关。     

Antispasmodic and relaxant effects of the hidroalcoholic extract of Pimpinella anisum (Apiaceae) on rat anococcygeus smooth muscle

The present work describes the mechanisms involved in the muscle relaxant effect of ethanol:water (40:60, 60:40 and 80:20) aerial parts extracts of Pimpinella anisum. Three hidroalcoholic extracts in which the proportion of ethanol was 40% (HA(40%)), 60% (HA(60%)) or 80% (HA(80%)) were tested for activity in the rat anococcygeus smooth muscle. The three extracts (50 microg/mL) inhibited acetylcholine-induced contraction. The extract HA(60%) (5-50 microg/mL) concentration dependently relaxed acetylcholine-pre-contracted tissues (31.55+/-3.56%). Conversely, HA(40%) and HA(80%) did not exert relaxant action. Pre-incubation of the preparations with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 3 microM) and oxyhemoglobin (10 microM) reduced the relaxation induced by HA(60%) (percentage of relaxation: 6.81+/-1.86%, 13.13+/-5.87% and 2.12+/-1.46%, respectively). Neither indomethacin (10 microM) nor tetraethylammonium (1 mM) affected the relaxation induced by HA(60%). Incubation of the tissues with L-NAME significantly enhanced the maximal contraction induced by acetylcholine, indicating an inhibitory role for NO in the modulation of the contractile response of anococcygeus smooth muscle to acetylcholine. However, simultaneous addition of L-NAME and HA(60%) resulted in an effect similar to that observed with L-NAME alone, further confirming the observation that Pimpinella anisum acts by realizing NO. Additionally, HA(60%) did not alter CaCl(2)-induced contraction. Collectively, our results provide functional evidence that the effects elicited by the hidroalcoholic extract of Pimpinella anisum involve the participation of NO and subsequent activation of the NO-cGMP pathway. The relaxant action displayed by Pimpinella anisum justifies its use in the folk medicine as an antispasmodic agent.     

The production of nitric oxide and prostaglandin E2 in peritoneal macrophages is inhibited by Andrographis paniculata, Angelica sinensis and Morus alba ethyl acetate fractions

Aim of the study

Traditional Chinese medicine herbs (TCMHs) are used in medicines as well as in daily dietary supplements in Asia. In this study, we employed pNF-κB-Luc or pIFN-γ-Luc and BALB/c mice peritoneal macrophages or splenocytes to investigate both the immune and inflammatory effects of six selected plant species.

Materials and Methods

Specifically, we used ethyl acetate fractions of Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao (Fabaceae) (AM), Andrographis paniculata (Burm. f.) Nees (Acanthaceae) (AP), Angelica sinensis (Oliv.) Diels (Apiaceae) (AS), Eucommia ulmodes Oliv. (Eucommiaceae) leaves (EU leaves), Isatis indigotica Fort. (Brassicaceae) (II) and Morus alba L. (Moraceae) (MA).

Results

We found that ethyl acetate fractions of AP, AS and MA significantly decreased NF-κB luciferase activity and also the secretion of NO and PGE₂ in LPS/IFN-γ stimulated mouse peritoneal macrophages (p < 0.05). In contrast, they did not affect IFN-γ luciferase activity or IFN-γ production in concanavalin A (Con A)-activated mouse splenocytes. Our results indicated that the anti-inflammatory properties of these plant extracts might be resulted from the inhibition of pro-inflammatory mediators (e.g., NO and PGE₂), at least in part via suppression of a signaling pathway such as NF-κB.

Conclusions

Collectively, we have found that three potent bioactive TCMH species exerted significant NF-κB inhibitory activity and acted in a cell type dependent fashion.     

基于网络药理学的党参增强免疫功能机制研究

目的运用网络药理学方法探究党参增强免疫功能的潜在作用机制。方法利用中药系统药理学数据库与分析平台(TCMSP)查找党参的活性成分及与活性成分相关的潜在靶点,并采用Cytoscape3.6.1软件构建党参活性成分-靶点网络。在NCBI网站下Gene数据库中以"immunosuppression"为关键词搜索免疫抑制靶点,使用装载ReactomeFIViz插件的Cytoscape3.6.1软件构建免疫抑制靶点相互作用网络,并将其与党参活性成分-靶点网络融合,获得党参活性成分的免疫作用靶点。使用DAVID数据库对党参免疫作用靶点进行KEGG通路富集分析,以探究党参增强免疫功能的作用机制。结果党参发挥免疫作用的主要靶点有肿瘤坏死因子(TNF),转录因子p65(RELA),白细胞介素10(IL10),白细胞介素6(IL6),白细胞介素2(IL2)等。其中木樨草素,芹菜素,棕榈酸甲酯,棕榈酸,白术内酯III主要作用于TNF;芹菜素,月桂酸,辣椒素,木樨草素,棕榈酸甲酯主要作用于RELA;木樨草素,棕榈酸甲酯,棕榈酸主要作用于IL-10;月桂酸,木樨草素,棕榈酸甲酯主要作用于IL-6;芹菜素,木樨草素主要作用于IL-2。KEGG分析得到与党参免疫作用有关的通路16条,主要涉及T细胞受体信号通路,NOD样受体信号通路等。结论党参中的免疫活性成分可能通过调控TNF、RELA、IL10、IL6等靶点,T细胞受体信号通路,NOD样受体信号通路等多个途径发挥增强机体免疫功能的作用。     

Immunomodulatory properties of Xylaria nigripes in peritoneal macrophage cells of Balb/c mice

Ethnopharmacological relevance

Wu Ling Shen, a folklore name for Xylaria nigripes (XN), is a high value medicinal fungus used in traditional Chinese medicine.

Aim of study

The present study aimed to examine the immunomodulatory properties of aqueous (XN-H) and ethanol (XN-E) XN extracts in lipopolysaccharide (LPS)-induced peritoneal macrophage cells of Balb/c mice.

Materials and methods

After treating the macrophage cells with LPS (1 μg/ml) and different XN extracts, the immunomodulatory properties were determined by the responses of inflammatory mediators, namely nitrite oxide (NO), prostaglandin E2 (PGE₂) and cytokine (IL-1β, IL-6, TNF-α and IFN-γ) production, iNOS, COX-2 and IκB-α expression, and NF-κB activation.

Results

Results showed that treatment of macrophages with 5-30 μg/ml of XN-H or XN-E plus 1 μg/ml LPS exhibited no cytotoxic effect on cell viability. At these concentrations, although both XN-H and XN-E showed a dose-dependent inhibitory effect on NO, PGE₂, IL-1β, IL-6, TNF-α and IFN-γ production in LPS-stimulated macrophages, a greater potency was noted in the XN-H treated group. RT-PCR assay also showed that XN-H possessed a greater inhibition than XN-E on iNOS and COX-2 RNA expression. Furthermore, XN-H also showed a significant stronger suppression than XN-E on the LPS-induced IκB-α phosphorylation and NF-κB activation. XN-E showed a higher total flavonoid and phenol contents but a lower β-glucan content than XN-H.

Conclusion

Taken together, these results conclude that XN-H possesses a stronger anti-inflammatory activity than XN-E, and its mechanism of action could be mediated by inhibiting iNOS and COX-2 expression via the NF-κB signaling pathway, and these activities could be contributed by the β-glucan content.     

淫羊藿苷拮抗脂多糖炎症模型的体内和体外研究

目的 从前炎症细胞因子、炎性介质、黏附分子等环节,评价淫羊藿苷的抗炎作用。     

The mechanism of vasorelaxation induced by Schisandra chinensis extract in rat thoracic aorta

Aim of the study

Schisandra chinensis (SC) is a known medical herb for the treatment of cardiovascular symptoms associated with menopausal symptoms in Korea. However, the pharmacological action mechanisms involved have not been well studied. This study was aimed to investigate the vascular effects of SC in rat thoracic aorta.

Materials and methods

We isolated the hexane, chloroform, and methanol extracts from SC and evaluated their vasodilatory effects in the rat thoracic aorta.

Results

Hexane extracts of SC (SCHE, 5 × 10⁻⁵ to 10⁻³ g/L) caused a concentration-dependent relaxation in both endothelium-intact and -denuded aortas. The relaxant effect of SCHE on the endothelium-intact aorta was more prominent than on the endothelium-denuded aorta. The former was significantly attenuated by L-NAME, a nitric oxide synthase inhibitor, and ODQ, a soluble guanyl cyclase inhibitor, but not by tetraethylammonium, a nonselective blocker of K⁺ channels, and indomethacin, a cyclooxygenase inhibitor. Furthermore, SCHE caused nitrite production as well as eNOS activation in aortic segments, suggesting implication of NO signal pathway in SCHE-induced relaxation. In endothelium-denuded aorta, SCHE-induced vasorelaxation was also attenuated by calyculin A, an inhibitor of myosin light chain (MLC) phosphatase, but not by ML-9, a MLC kinase inhibitor, suggestive of implication of MLC phosphatase activation. Phenylephrine-enhanced MLC phosphorylation ratio was significantly attenuated by SCHE, which was recovered to the control level by pretreatment with calyculin A.

Conclusions

Taken collectively, these findings suggest that the vascular relaxation evoked by SCHE was mediated by not only endothelium dependent NO pathway but also direct effect on vascular smooth muscle cell via dephosphorylation of MLC.     

Protective effect of Acanthopanax senticosus extract against endotoxic shock in mice

AIM OF THE STUDY: In this study, we evaluated protective effect of Acanthopanax senticosus extract (ASE) and a possible signaling pathway involved during endotoxic shock induced by intraperitoneal injection lipopolysaccharide (LPS) and D-galactosamine (D-GalN) in BALB/c mice. MATERIALS AND METHODS: Mice were intraperitoneal administrated with ASE (100, 200 or 400mg/kg) prior to injection of 50 microg/kg LPS and 1g/kg D-GalN. The levels of tumor necrosis-alpha (TNF-alpha) and interleukin-10 (IL-10) in serum and liver. Nitric oxide (NO) production in serum and inducible nitric oxide synthase (iNOS) protein level were investigated. Nuclear factor-kappa B (NF-kappaB) activation in liver was determined. Furthermore, we evaluated the effect of ASE pretreatment on infiltration of inflammatory cells into the heart, liver and lung of mice. RESULTS: Treatment of mice with ASE prior to LPS/D-GalN injection significantly improved the survival rate. ASE pretreatment inhibited the elevation of TNF-alpha in serum and liver. ASE also decreased iNOS level in liver and the overproduction of nitric oxide (NO) in serum. In addition, IL-10 levels in serum and liver were markedly enhanced. ASE pretreatment inhibited NF-kappaB activation in liver of mice. Moreover, infiltration of inflammatory cells into the heart, liver and lung of mice was also attenuated by ASE pretreatment. CONCLUSIONS: These results suggested that ASE protected mice against LPS/D-GalN-induced endotoxic shock involving inhibition of NF-kappaB activation, which caused down-regulation of TNF-alpha and involved up-regulation of IL-10. Acanthopanax senticosus may thus prove beneficial in the prevention of endotoxic shock.     

Differential Inhibition of T Lymphocyte Proliferation and Cytokine Synthesis by [6]‐Gingerol, [8]‐Gingerol,and [10]‐Gingerol

[6]‐Gingerol, [8]‐gingerol, and [10]‐gingerol are pungent components of fresh ginger, extracts of which inhibit various components of the inflammatory response. Because little is known regarding the effect of gingerols with different unbranched alkyl side chain lengths on the activation and effector function of T lymphocytes, we compared the effects of [6]‐gingerol, [8]‐gingerol, and [10]‐gingerol on murine T lymphocyte proliferation, expression of CD25 and CD69 activation markers, cytokine synthesis, and interleukin (IL)‐2 receptor signaling. All three gingerols inhibited DNA synthesis by T lymphocytes, as well as interferon‐γ synthesis. In contrast, only [8]‐gingerol and [10]‐gingerol inhibited CD25 and CD69 expression, and IL‐2 synthesis. None of the gingerols affected IL‐4 synthesis. Exogenous IL‐2 enhanced T lymphocyte proliferation in the presence of [6]‐gingerol but did not significantly increase T lymphocyte proliferation in the presence of [8]‐gingerol or [10]‐gingerol. In line with this finding, [8]‐gingerol and [10]‐gingerol impaired IL‐2‐induced proliferation of CTLL‐2 cells, but constitutive CD25 expression was unaffected, indicating inhibition of IL‐2 receptor signaling. In general, [10]‐gingerol and [8]‐gingerol were more potent inhibitors of T lymphocytes than [6]‐gingerol. Suppression of T lymphocyte responses by gingerols suggests that these phytochemicals may be beneficial in chronic inflammatory conditions associated with excessive or inappropriate T lymphocyte activation. Copyright © 2015 John Wiley & Sons, Ltd.