黄芪提取物免疫调节活性的体外实验研究

目的：研究黄芪提取物（Astragalus membranaceus extract,AME)对人的外周血免疫细胞（PBIC）免疫功能的调节作用。方法：采用^3H-TdR掺入法分析AME对外周血单个核细胞（PBMC）的增殖活性及对外周血粘附单核细胞（PBAM）吞噬肿瘤细胞的影响；采用^51Cr释放法测定AME对杀伤性T细胞（CTL）杀伤肿瘤细胞的影响；用ELISA法和生物学法研究AME对外周血B细胞（PBBC）产生IgG及对PBAM产生细胞因子的影响；采用SDS－PAGE分析AME的蛋白组成。结果：AME能促进PBMC的增殖；提高CTL对肿瘤细胞的杀伤活性；增强PBAM对肿瘤细胞的吞噬和产生细胞因子的功能；促进了PBBC产生IgG;AME含有多种蛋白成分。结论：AME对人免疫功能有增强作用，提高了抗肿瘤免疫效应，可应用于临床调节免疫功能和治疗肿瘤等疾病。     

姜黄素对Raji细胞体外抗癌作用的实验研究

目的研究姜黄素对Raji细胞体外抗癌作用及其机制,对比研究其对RaIi细胞和人单个核细胞的细胞毒性。方法用MTT法检测姜黄素对Raji细胞及人单个核细胞增殖的影响,Annexin—V／PI双标流式术、缺口末端标记法检测姜黄素对Raji细胞及人单个核细胞凋亡的影响,PI单标流式细胞术测定姜黄素对Raji细胞DNA含量分布的影响。结果(1)姜黄素对Raji细胞具有明显的增殖抑制作用。(2)姜黄素可以时间和剂量依赖性方式诱导Raji细胞凋亡。(3)姜黄素组Raji细胞周期发生变化,细胞周期被阻滞于G0／G1和G2／M期,S期比例减少。(4)姜黄素对人单个核细胞无明显抑制增殖和诱导凋亡作用。结论姜黄素能够调控Raji细胞的细胞周期并诱导其凋亡,从而抑制Raji细胞增殖;姜黄素对人单个核细胞无明显细胞毒作用,而选择性作用于肿瘤细胞。     

Seselin from Plumbago zeylanica inhibits phytohemagglutinin (PHA)-stimulated cell proliferation in human peripheral blood mononuclear cells

Effects of seselin (C(14)H(12)O(3); MW 228) identified from Plumbago zeylanica on phytohemagglutinin (PHA)-stimulated cell proliferation were studied in human peripheral blood mononuclear cells (PBMC). The data demonstrated that seselin inhibited PBMC proliferation-activated with PHA with an IC(50) of 53.87+/-0.74 microM. Cell viability test indicated that inhibitory effects of seselin on PBMC proliferation were not through direct cytotoxicity. The action mechanisms of seselin may involve the regulation of cell cycle progression, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production in PBMC. Since cell cycle analysis indicated that seselin arrested the cell cycle progression of activated PBMC from the G(1) transition to the S phase. Seselin suppressed IL-2 and IFN-gamma production in a concentration-dependent manner. Furthermore, seselin significantly decreased the IL-2 and IFN-gamma gene expression in PHA-activated PBMC. Therefore, results elucidated for the first time that seselin is likely an immunomodulatory agent for PBMC.     

Mountain grown ginseng induces apoptosis in HL-60 cells and its mechanism have little relation with TNF-alpha production

The root of ginseng is one of the most popular natural tonics in Oriental countries. Ginseng grown in the wild, deep in the mountains, is known as Sansam (mountain grown ginseng, MGG). MGG belongs to Araliaceae and Panax. In this study, we investigated the effects of MGG on the cytotoxicity, induction of apoptosis and the putative pathways of its actions in human promyelocytic leukemia cells, HL-60. Using apoptosis analysis, we found that MGG is a potent inducer of apoptosis, but it has less effect on human peripheral blood mononuclear cells. Caspase-3 activation and subsequent apoptotic cell death in MGG-treated cells were partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK. MGG also inhibited the caspase-8 activity. To determine whether MGG-induced apoptosis is involved in tumor necrosis factor-alpha (TNF-alpha) secretion, TNF-alpha secretion was quantified by enzyme-linked immunosorbent assay (ELISA) method. Unexpectedly, MGG significantly decreased the TNF-alpha secretion compared to the control. These results suggest that MGG-induced cytotoxicity have little relation with the secretion of TNF-alpha in HL-60 cells. Furthermore, MGG with rIFN-gamma synergistically increased nitric oxide (NO) production in mouse peritoneal macrophages. Taken together, our data indicate that MGG is a potent inducer of apoptosis on HL-60 cells and these abilities could be used clinically for the treatment of cancer.     

白附子对人T细胞和单核细胞的调节活性

目的：研究中药白附子的免疫学调节活性。方法：用^3H介入法测定了中药白附子提取物（Rhizoma typhonii extract,RTE)对小鼠脾细胞和人淋巴细胞的增殖活性；用绵羊红细胞花结形成试验分离纯化淋巴细胞后，检测了RTE的效应细胞；用^51Cr游离实验测定了人杀伤细胞和自然杀伤细胞的杀伤活性，用ELISA法和生物法研究了单核细胞的细胞因子产生，并用^3H介入法分析了单核细胞对肿瘤细胞的吞噬功能；用层析法分析了白附子的化学成分，并用SDS－PAGE法测定了其有效成分的分子量。结果：RTE对小鼠脾细胞和人淋巴细胞的增殖活性有很强的促进作用，并有较好的量效关系；RTE的效应细胞是T细胞；RTE增强人杀伤细胞对肿瘤细胞的特异性杀伤活性和自然杀伤细胞的非特异性杀伤活性、增强单核细胞的细胞因子（肿瘤坏死因子和白细胞介素1）产生，并增强单核细胞对肿瘤细胞的吞噬功能。白附子的化学成分为66道尔顿的糖蛋白。结论：RTE对人的T细胞和单核细胞有免疫增强作用，并通过刺激机体的免疫系统杀伤或吞噬肿瘤细胞和外来抗原，这些活性可应用于临床调节免疫功能和治疗肿瘤等疾病。     

黄酮类化合物调控肿瘤细胞自噬的研究进展

自噬是一种保守的细胞自我降解方式,是将受损细胞器或大分子物质由自噬泡包绕再通过自噬溶酶体降解再利用的过程。自噬在细胞的生物合成、营养和代谢平衡及应激与存活中具有重要作用。调控自噬能够引发多重级联反应,可以应用于肿瘤治疗。黄酮类化合物作用于不同的肿瘤细胞系会诱导作用各异的自噬。黄酮类化合物可以诱导自噬通过非凋亡或非典型凋亡途径较为直接地抑制肿瘤细胞增殖,也可以在诱导自噬后进一步引发凋亡等其他致死方式间接抑制肿瘤细胞增殖,或通过抑制保护性自噬从而强化其他形式的杀伤作用,但有时也会诱导肿瘤细胞发生保护性自噬,弱化细胞杀伤作用。对近10年来黄酮类化合物调控肿瘤细胞自噬的研究进展进行了综述,为中药调控细胞自噬的相关研究提供参考。     

Radix bupleuri containing compound (KY88 liver-livo) induces apoptosis and production of interleukin-4 and tumor necrosis factor-alpha in liver cancer cells in vitro

Hepatocellular carcinoma is an important health problem in Asia. A blend of herbal extracts containing radix bupleuri (KY88) was tested for its effects on liver cancer cells. A hepatocellular carcinoma cell line (HB8064) was cultured with methanol extract of KY88. We were able to produce a dose-dependent inhibition of cancer cell proliferation. At IC50 and IC100, KY88 induces a DNA ladder pattern, indicating the presence of apoptosis. We also checked the changes of the levels of interleukin (IL)-2, -4 and -6, interferon (INF)-gamma and tumor necrosis factor (TNF)-alpha by ELISA kits. After 24 hours of culture, there was activation of IL-2 and -4 and TNF-alpha. However, significant changes were observed only for IL-4 and TNF-alpha. Therefore, we concluded that KY88 is able to induce apoptosis, which may be regulated through changes in IL-4 and TNF-alpha.     

Effects of Paeonia lactiflora root extracts on the secretions of monocyte chemotactic protein-1 and -3 in human nasal fibroblasts

In the field of Oriental medicine, the root of Paeonia lactiflora (Paeoniae Radix) has been prescribed usually to treat the common cold, and it was thought to alleviate upper respiratory infection or nasal inflammation. Monocyte chemotactic proteins (MCP)-1 and MCP-3 are known as the most potent chemokines to mediate allergic inflammation. The object of the study was to investigate the effect of Paeoniae Radix on the release of the chemokines such as MCP-1 and MCP-3. To detect the secretion of MCPs, an enzyme-linked immunosorbent assay was performed for human nasal mucosal fibroblasts after stimulation with several cytokines such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta). The secretion of MCP-1 was increased after stimulation with TNF-alpha or IL-1beta treatment. Co-treatment of TNF-alpha and IFN-gamma, or IL-1beta and IFN-gamma increased the secretion of MCP-1 and MCP-3. Paeoniae Radix was tested with inflammatory cytokines, and the results showed that the Paeoniae Radix significantly decreased the secretion of MCP-1 and MCP-3. In our study, Paeoniae Radix may play an important role in nasal inflammation with a modulation of MCP-1 and MCP-3.     

健脾化瘀方对人肝癌细胞SMMC-7721增殖和凋亡的影响

[目的]探讨健脾化瘀方对人肝癌细胞系SMMC-7721的抑制率及药物浓度和作用时间对细胞抑制率的影响,以及IC_(50)药物浓度下对SMMC-7721凋亡的影响。[方法]采用MTT法,观察不同浓度的健脾化瘀方对人肝癌细胞株SMMC-7721增殖的影响及与药物作用时间之间的关系。用AnnexinV/PI双染色法,用流式细胞仪检测健脾化瘀方对肝癌细胞株SMMC-7721凋亡的影响。[结果]健脾化瘀方浓度在5mg/mL时,对SMMC-7721细胞的增殖有明显的抑制作用,呈剂量依赖和时间依赖效应关系。健脾化瘀方对人肝癌细胞SMMC-7721作用24h后,肝癌细胞凋亡率升高,并有一定的浓度依赖性。[结论]健脾化瘀方有抑制SMMC-7721细胞的增殖,能诱导人肝癌细胞株SMMC-7721的凋亡。     

Pretreatment with neem (Azadirachta indica) leaf preparation in Swiss mice diminishes leukopenia and enhances the antitumor activity of cyclophosphamide

Cancer chemotherapy is associated with several life threatening complications, including bone marrow suppression and leucopenia. To overcome this problem, colony stimulating factor (CSF), granulocyte colony stimulating factor (GCSF) and granulocyte macrophage colony stimulating factor (GMCSF), can be used, however, these therapeutics are expensive and have several disadvantages, including tumor growth promoting activities. This study attempted to use an immunostimulatory neem (Azadirachta indica) leaf preparation (NLP) to prevent the cyclophosphamide (CYP) induced reduction in the WBC count. Pretreatment of mice with NLP reduced the extent of leucopenia and neutropenia in normal and tumor bearing CYP treated mice. NLP pretreatment enhanced in vitro tumor cell cytotoxicity by peripheral blood mononuclear cells (PBMC) from CYP treated mice in either normal or tumor bearing conditions. Similarly, NLP pretreatment of mice enhanced the CYP mediated in vivo tumor growth inhibition and survivability of the host. Based on these observations, it is concluded that NLP would be an effective tool to reduce CYP-induced hematological complications.     

Effects of matrine on proliferation and apoptosis in gallbladder carcinoma cells (GBC-SD)

Although matrine, a primary active component of dried Sophora flavescens root (ku shen), is known to induce apoptosis in a variety of tumor cells in vitro, the molecular mechanism of such apoptosis remains elusive. This analysis of the cell cycle and apoptosis in matrine-treated human gallbladder carcinoma cells (GBC-SD) showed that matrine can indeed inhibit cell proliferation and induce G1 cell cycle arrest and apoptosis in a dose- and time-dependent manner. An additional western blot analysis of matrine-treated cells also showed caspase-3 and Bcl-2 activation, as well as cyclinE down-regulation. Overall, the results indicate that matrine perturbs gallbladder cancer cell progression during the G1 phase by down-regulating cyclinE and induces apoptosis by decreasing the expression of the antiapoptotic protein Bcl-2 and increasing expression of the proapoptotic protein Bax.     

Brazilian green propolis and its constituent, Artepillin C inhibits allogeneic activated human CD4 T cells expansion and activation

Ethnopharmacological relevance

Propolis has long been used as a popular folk medicine by various ethnic groups due to its wide spectrum of alleged biological and pharmaceutical properties including anti-microbial, anti-cancer and anti-inflammatory functions. All these can be linked to the modulation of immune function. Therefore, it will be relevant for us to find out whether there is any novel compound that can account for such action and the mechanism involved.

Aim of the study

We investigated the immune modulating effect of Brazilian green propolis (PBrazil) and its constituent Artepillin C (Art-C) by using mixed leukocytes reaction.

Materials and methods

The cytotoxic effect of Art-C on non-tumorigenic human liver cell line miHA and non-tumorigenic human kidney cell line HK-2 as well as human peripheral blood mononuclear cells (PBMCs) were measured by XTT cell proliferation assay. The effect of PBrazil and Art-C on T cell proliferation and activation were determined by using carboxyfluorescein succinimidyl ester (CFSE) and by CD25 expression, respectively. Cytokines including tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), interleukins such as IL-2, IL-17 were measured by intracellular cytokine staining and IL-10 was measured by ELISA. The effect of PBrazil and Art-C on regulatory T cells (Treg) induction was determined by the Foxp3 expression. The apoptotic effect of these compounds on CFSE labeled alloreactive T cells was measured by using Annexin V.

Results

Using mixed leukocytes reaction we demonstrated for the first time that both Art-C and PBrazil significantly inhibited the alloreactive CD4 T cell proliferation, activation, and suppressed the expressions of IL-2, IFN-γ and IL-17 in these alloreactive CD4 T cells. The inhibitions of Art-C and PBrazil on CD4 T cells were not due to direct cytotoxic effect on PBMC or inducing regulatory T cells differentiation. Both Art-C and PBrazil were found to selectively induce apoptosis in proliferating T cells. The anti-proliferative effect of Art-C and PBrazil were reversible and were also applied to the activated T cells.

Conclusions

In conclusion, our results indicated that Art-C and PBrazil can suppress alloreactive CD4 T cell responses in vitro, suggesting that Art-C could be used as a potential immunosuppressant, either solely or as adjunct agent in treating graft versus host disease.     

Inhibition of pro-inflammatory mediators and tumor cell proliferation by Anisomeles indica extracts

Anisomeles indica (L.) Kuntze (Labiatae), is a traditional anti-inflammatory herb used in Taiwan. The aqueous and methanolic extracts of whole plants, leaves, flowers and stems; and chloroform and n-butanol fractions of methanol extract, from A. indica were investigated for their anti-inflammatory activity on murine peritoneal macrophages. In addition, the tumor cells proliferation inhibition activities of these extracts were also evaluated against a panel of tumor cell lines such as Colon 205, PC 3, HepG2 and MCF 7. Treatment with A. indica extracts did not reduce cell viability at any dose used. However, all the extracts significantly inhibited the enhanced production of NO radicals, and pro-inflammatory cytokines (TNF-alpha, and IL-12) induced by LPS/IFN-gamma in a dose-dependent manner. Furthermore, methanolic extracts of leaves and flowers significantly and dose-dependently arrest mitogen-stimulated spleen cells in G0/G1 stage, in addition to their cell proliferation inhibition against Colon 205, MCF 7 and PC 3 by 94, 82; 98, 71; 82, 98%, respectively, at 200 microg/mL concentration. This is the first report on A. indica extracts for their growth inhibitory activities, against inflammatory mediator production, and human tumor cell lines, colon, prostate, hepatoma and breast cells proliferation.     

Induction of apoptosis in human acute leukemia Jurkat T cells by Albizzia julibrissin extract is mediated via mitochondria-dependent caspase-3 activation

To understand antitumor activity of Albizzia julibrissin Durazz (Leguminosae), which has been used as a traditional oriental medicine, the mechanism underlying cytotoxic effect of its extract on human acute leukemia Jurkat T cells were investigated. The methanol extract of the stripped barks (3kg) of Albizzia julibrissin was evaporated, dissolved in water, and then sequentially extracted by chloroform, ethyl acetate, and n-butanol. The substance in the butanol extract containing the most cytotoxic activity was further purified by a series of preparative column chromatography. The active substance obtained (723mg) was designated as HaBC18. When Jurkat T cells were treated with HaBC18 (0.5-2microg/ml), apoptosis along with several biochemical events such as mitochondrial cytochrome c release, activation of caspase-9 and -3, degradation of PARP, and DNA fragmentation was induced in a dose-dependent manner. However, the HaBC18-induced apoptosis was abrogated by an ectopic overexpression of Bcl-xL, which is known to block mitochondrial cytochrome c release. Primary cultures of human PBMC were less sensitive to the cytotoxicity relative to Jurkat T cells. These results demonstrate that the cytotoxicity of HaBC18 toward Jurkat T cells is attributable to apoptosis mediated by mitochondria-dependent death-signaling pathway regulated by Bcl-xL.     

Extracts from the roots of Lindera strychifolia induces apoptosis in lung cancer cells and prolongs survival of tumor-bearing mice

Lindera strychifolia, a scandent shrub Lauraceous medicinal plant, has been used in Chinese traditional medicine as a palliative and an anti-spasmodic. It also shows cytotoxic effects against several tumor cell lines and inhibits marcromolecule biosynthesis. This study investigated the anti-tumor effects of L. strychifolia extract against lung cancer cells using in vitro and in vivo models. Two human lung cancer cell lines A549 (adenocarcinoma) and SBC-3 (small cell carcinoma), and a non-tumor cell line 3T3-L1 (mice fibroblasts) were subjected to L. strychifolia extract treatment. On lung cancer cells, L. strychifolia induced cell growth inhibition in a dose-dependent manner. Conversely, the extract did not show any significant cytotoxic effect on 3T3-L1 cells. Therefore, the extract is specific for tumor cells. Tumor cells treated with L. strychifolia extract showed typical morphological appearance of apoptosis including nuclei fragmentation and cell condensation. The in vivo effects of L. strychifolia extract were investigated in C57BL/6 mice transplanted with Lewis lung cancer (LL-2) cells, and in BALB/c nude mice transplanted with A549 or SBC-3 human lung cancer cells. Oral administration of L. strychifolia extract prolonged survival time and inhibited tumor growth in a dose-dependent manner by inducing apoptosis in the LL-2 cell mice model. Furthermore, in A549 or SBC-3 cell nude mice models, oral administration of L. strychifolia extract also significantly inhibited tumor growth at the 5.0 mg/ml concentration. These findings suggested that the components of L. strychifolia have anticancer activity and may contribute to clinical applications in the prevention and treatment of lung cancer.     

Aqueous extract of Phyllanthus urinaria induces apoptosis in human cancer cells

Cell apoptosis is now known to play an important role in the maintenance of cellular homeostasis and anti-carcinogenesis. The anticancer effect of aqueous extract prepared from Phyllanthus urinaria (P. urinaria) was investigated by analyzing its potential to induce apoptosis in human cancer cells. We showed that the aqueous extract of P. urinaria could reduce the viability by inducing the apoptosis in human cancer cells derived from several different origins as demonstrated by morphological changes and DNA fragmentation. Yet, P. urinaria extract exhibited no cytotoxic effect on normal human cells, including vascular endothelial cells and liver cells under the same conditions. It suggests that the aqueous extract of P. urinaria is substantially useful in treating various kinds of human cancer cells without toxic side effect on normal cells.     

Effect of Rhaphidophora korthalsii methanol extract on human peripheral blood mononuclear cell (PBMC) proliferation and cytolytic activity toward HepG2

The study of bioactivity of natural product is one of the major researches for drug discovery. The aim of this finding was to study the proliferation effect of Rhaphidophora korthalsii methanol extract on human PBMC and subsequently the cytotoxic effect of activated PBMC toward HepG2 human hepatocellular carcinoma. In this present study, MTT assay, cell cycle study and Annexin 5 binding assay were used to study the immunomodulatory and cytotoxic effects. In vitro cytotoxic screening of Rhaphidophora korthalsii methanol extract showed that the extract was non-toxic against hepatocellular carcinoma (HepG2). In contrast, the extract was able to stimulate the proliferation of human PBMC at 48 h and 72 h in MTT assay and cell cycle progress study. The application of immunomodulator in tumor research was studied by using MTT microcytotoxicity assay and flow cytometric Annexin V. Results indicated that pre-treated PBMC with Rhaphidophora korthalsii methanol extract induced the highest cytotoxicity (44.87 ± 6.06% for MTT microcytotoxicity assay and 51.51 ± 3.85% for Annexin V) toward HepG2. This finding demonstrates that Rhaphidophora korthalsii methanol extract are potent to stimulate the cytotoxic effect of immune cells toward HepG2.     

Inhibition of tumor necrosis factor-alpha-induced apoptosis by Asparagus cochinchinensis in Hep G2 cells

A human hepatoma cell line, Hep G2 cells, is a reliable system for the study of alcohol-induced hepatotoxicity. In this study, we investigated the effect of an aqueous extract of Asparagus cochinchinensis(MERRIL) (Liliaceae) roots (ACAE) on ethanol (EtOH)-induced cytotoxicity in Hep G2 cells. ACAE (1-100 microg/ml) dose-dependently inhibited the EtOH-induced tumor necrosis factor-alpha (TNF-alpha) secretion. ACAE (1-100 microg/ml) also inhibited the EtOH and TNF-alpha-induced cytotoxicity. Furthermore, we found that ACAE inhibited the TNF-alpha-induced apoptosis of Hep G2 cells. These results suggest that ACAE may prevent the EtOH-induced cytotoxicity through inhibition of the apoptosis of Hep G2 cells.