一种无孢灵芝菌丝体的活性成分

利用制备型高效液相、凝胶层析、制备型薄层色谱等方法对无孢灵芝龙芝2号的固体发酵菌丝体进行分离纯化,通过波谱分析,化合物分别鉴定为灵芝酸P（1）,灵芝酸T1（2）,灵芝酸Mk（3）,灵芝酸S（4）,灵芝酸T（5）,ganodermanondiol（6）,灵芝酸Me（7）,5α, 8α-epidioxyergosta-6, 22-dien-3β-ol（8）,灵芝酸R（9）,lanosta-7, 9(11), 24-trien-3α-hydroxy-26-oic acid（10）,ganodermenonol（11）。其中灵芝酸T1为首次发现的天然产物。体外细胞实验证实,11种化合物对肿瘤细胞L1210的增殖均有很强的抑制作用,其增殖抑制的IC₅₀值均在39.69μmol/L以下。     

Protective effect of ganoderic acid against the streptozotocin induced diabetes,inflammation, hyperlipidemia and microbiota imbalance in diabetic rats

Diabetes mellitus (DM) is a metabolic disorder with numerous symptoms categorized via serves hyperglycemia effect along with altered fat, protein and carbohydrate metabolism mainly resultant from defects in insulin action/secretion or both. The aim of the current experimental study was to comfort the neuroprotective effect of ganoderic acid against the streptozotocin (STZ)-induced type I diabetes mellitus in mice and explore the underlying mechanism. Differentiation of 3T3-L1 preadipocytes effect; hepatic and glucose consumption effect of ganoderic acid was estimated on HepG2 cell lines and peroxisome proliferator-activated receptor (PPAR). FFA content was estimated in adipose and hepatic tissues. Ganoderic acid induced the 3T3-L1 preadipocytes differentiation. The mRNA expression of PPAR was increased in the high glucose-treated group in HepG2 and ganoderic acid treatment down-regulated the mRNA expression of PPAR. Ganoderic acid exhibited the inhibitory effect of α-glucosidase and α-amylase. Ganoderic acid demonstrated the reduced blood glucose and increase insulin level and also reduced the free fatty in hepatic and adipose tissue. Histopathological study showed the enhancement of β-cells in ganoderic acid-treated mice. Finally, their prebiotic effects on gut microbiota were illustrated via enhancing the population of diabetes resistant bacteria and also reducing the quantity of diabetes sensitive bacteria. Ganoderic acid attenuated STZ induced T1DM in mice via inflammatory pathways.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.     

Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells

The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tert-butyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of SOD isoforms caused a 2- and 3.5-fold elevation in CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria, respectively, and 3-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-SOD expression similarly prevented PC-12 apoptosis, this was associated with increases in SOD activity but not GSH, indicating that cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-SOD overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-3 activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-SOD overexpression and is independent of cellular GSH levels per se.     

Anti-hepatitis B activities of ganoderic acid from Ganoderma lucidum

Ganoderic acid, from Ganoderma lucidum, at 8 μg/ml inhibited replication of hepatitis B virus (HBV) in HepG2215 cells over 8 days. Production of HBV surface antigen and HBV e antigen were 20 and 44% of controls without ganoderic acid. Male KM mice were significantly protected from liver injury, induced with carbon tetrachloride, by treatment with ganoderic acid at 10 mg and 30 mg/kg·d (by intravenous injection) 7 days. Ganoderic acid at the same dosage also significantly protected the mice from liver injury induced by M. bovis BCG plus lipopolysaccharide (from Escherichia coli 0127:B8).     

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.     

Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells

The proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have been shown to participate in signaling cascades regulating proliferation induced by platelet-derived growth factor (PDGF), a powerful smooth muscle mitogen. We sought to determine the role of Nox5 in the regulation of PDGF-stimulated human aortic smooth muscle cell (HASMC) proliferation. Cultured HASMC were found to express four isoforms of Nox5. When HASMC stimulated with PDGF were pretreated with N-acetyl cysteine (NAC), proliferation was significantly reduced. Proliferation induced by PDGF was also heavily dependent on JAK/STAT activation, as the JAK inhibitor, AG490, was able to completely abolish PDGF-stimulated HASMC growth. Specific knockdown of Nox5 with a siRNA strategy reduced PDGF-induced HASMC ROS production and proliferation. Additionally, siRNA to Nox5 inhibited PDGF-stimulated JAK2 and STAT3 phosphorylation. ROS produced by Nox5 play an important role in PDGF-induced JAK/STAT activation and HASMC proliferation.     

乙烯调控灵芝酸生物合成关键基因的筛选

灵芝是我国著名的药用真菌,灵芝酸是其主要活性成分,具有多种药理活性。乙烯可以促进灵芝酸的生物合成,但其调控机理尚不明确。本实验利用非靶向代谢组研究发现Top 20差异代谢物中含有6种灵芝的活性成分(灵芝酸η、赤芝酸F、赤芝酸N、丹芝酸A、灵芝酸V1和灵芝酸δ),其中有4种灵芝酸(灵芝酸η、赤芝酸F、赤芝酸N和灵芝酸V1)为上调积累,2种灵芝酸(灵芝酸δ和丹芝酸A)为下调积累。通过非靶向代谢组与转录组的关联分析发现基因GL23307、GL25546和GL29595同时与3种灵芝酸积累显著相关,并通过启动子顺式元件预测,发现分别编码泛素蛋白和抑肽酶基因GL25546、GL23307的启动子区域含有响应乙烯信号的顺式作用元件GCC-box,因此,推测这两个基因在乙烯调控灵芝酸生物合成中发挥重要作用。     

SOD3 Ameliorates Aβ<Subscript>25–35</Subscript>-Induced Oxidative Damage in SH-SY5Y Cells by Inhibiting the Mitochondrial Pathway

This study was designed to investigate the protective effects of extracellular superoxide dismutase (SOD3) against amyloid beta (Aβ_25–35)-induced damage in human neuroblastoma SH-SY5Y cells and to elucidate the mechanisms responsible for this beneficial effect. SH-SY5Y cells overexpressing SOD3 were generated by adenoviral vector-mediated infection and Aβ_25–35 was then added to the cell culture system to establish an in vitro model of oxidative stress. Cell viability, the generation of intracellular reactive oxygen species (ROS), the expression and activity of antioxidant enzymes, the levels of lipid peroxidation malondialdehyde (MDA), the expression of mitochondrial apoptosis-related genes and calcium images were examined. Following Aβ_25–35 exposure, SOD3 overexpression promoted the survival of SH-SY5Y cells, decreased the production of ROS, decreased MDA and calcium levels, and decreased cytochrome c, caspase-3, caspase-9 and Bax gene expression. Furthermore, SOD3 overexpression increased the expression and activity of antioxidant enzyme genes and Bcl-2 expression. Together, our data demonstrate that SOD3 ameliorates Aβ_25–35-induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial pathway. These data provide new insights into the functional actions of SOD3 on oxidative stress-induced cell damage.     

Endoplasmic reticulum stress,autophagic and apoptotic cell death,and immune activation by a natural triterpenoid in human prostate cancer cells

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC₅₀s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 ⁺ T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.     

Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer

BackgroundAberrant activation of STAT3 is frequently encountered and promotes survival, cellular proliferation, migration, invasion and angiogenesis in tumor cell. Convallatoxin, triterpenoid ingredient, exhibits anticancer pharmacological properties.PurposeIn this work, we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells.MethodsIn vitro, the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking, luciferase reporter assay, MTT assay, RT-PCR, Western blotting and immunofluorescence assays. Changes in cellular proliferation, apoptosis, migration, invasion and angiogenesis were analyzed by EdU labeling assay, colony formation assay, flow cytometry assay, wound-healing assay, matrigel transwell invasion assay and tube formation assays. And in vivo, antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells.ResultsConvallatoxin decreased the viability of colorectal cancer lines. Moreover, convallatoxin reduced the P-STAT3 (T705) via the JAK1, JAK2, and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells. Interestingly, we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways, which collaboratively regulated STAT3 activation and convallatoxin play a role in it. Convallatoxin also downregulated the expression of target genes involved cell survival (e.g., Survivin, Bcl-xl, Bcl-2), proliferation (e.g., Cyclin D1), metastasis (e.g., MMP-9), and angiogenesis (e.g., VEGF). Indeed, we found that convallatoxin inhibited tube formation, migration, and invasion of endothelial cells, and inhibited the proliferation. Finally, in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model.ConclusionThe result of the current study show that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.     

Synthesis,biological evaluation and molecular docking study of 1-amino-2-aroylnaphthalenes against prostate cancer

A series of functionalized naphthalene was synthesized and screened against human prostate cancer cell line (PC-3). The in vitro antiproliferative activity of the synthesized compounds was evaluated by monitoring their cytotoxic effects against PC-3 cells by using MTT assay. We observed that compound 5f resulted in more than 50% cell death at 14?µM. Treatment of PC-3 cells with 5f provides apoptosis by flow cytometry. Western blotting showed decreased expression of pro-caspase 8 and 9. Our study shows that cancer cell treated with 5f has higher concentration of reactive oxygen species as compare to untreated sample, which facilitate cancerous cell to enter apoptosis. Exact mechanism by which ROS is generated after 5f treatment is still under study. Molecular docking study further strengthens the results obtained from in vitro experiments. Compound 5f can be considered as a promising leads for anticancer agent against prostate cancer cells due to its potent cytotoxic activity and apoptotic effect.     

异槲皮苷对Aβ25-35导致的PC12细胞损伤的保护作用及机制研究

探讨异槲皮苷对β-淀粉样蛋白(Aβ25-35)导致的PC12细胞氧化损伤的保护作用。首先通过分子对接技术分析异槲皮苷与AMPK的结合情况。采用Aβ25-35(20μmol/L)损伤PC12细胞建立细胞氧化损伤模型,采用甲基噻唑蓝(MTT)法检测细胞活力,通过试剂盒检测乳酸脱氢酶(LDH)漏出量、活性氧(ROS)含量、丙二醛(MDA)含量以及抗氧化物酶超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力,采用Western blot法检测磷酸化腺苷酸活化蛋白激酶(p-AMPK)、过氧化物增殖体受体辅激活子-1α(PGC-1α)、沉默信息调节因子3(Sirt3)和异柠檬酸脱氢酶(IDH2)的蛋白表达。结果显示异槲皮苷与AMPK的结合力为-9.48 kJ/mol,提示AMPK可能为异槲皮苷的潜在作用靶点。异槲皮苷(1、10和100μmol/L)能够浓度依赖性的显著抑制Aβ25-35导致的PC12细胞死亡,减少ROS和MDA含量,升高SOD和GSH-Px活力。异槲皮苷抑制Aβ25-35导致的细胞氧化损伤并上调p-AMPK、PGC-1α、Sirt3和IDH2的蛋白表达。以上结果表明异槲皮苷可能通过调控AMPK/Sirt3信号通路发挥抗Aβ25-35导致的PC12细胞氧化损伤作用。     

Bioconversion of a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid by a crude enzyme from Ganoderma lucidum

Ganoderic acids (GAs) are oxygenated lanostane-type triterpenoids from the traditional medicinal mushroom Ganoderma lucidum and of significant biological activities. Although a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA) was found to be biosynthesized from lanosterol, further post-modification of HLDOA is yet unclear. In this work, by using HLDOA as the substrate and a crude enzyme from G. lucidum as the biocatalyst, we observed a new peak in liquid chromatography from the reaction system. The product was purified and identified to be 3-oxo-lanosta-8,24-dien-26-oic acid (OLDOA), which may be converted from HLDOA by a putative dehydrogenase of G. lucidum. The work is useful to future manufacture of GAs as well as their biosynthetic pathway elucidation.     

抑瘤素M受体对慢性自身免疫性荨麻疹发病机制的影响

本研究旨在探讨抑瘤素M受体(OSMR)在慢性自身免疫性荨麻疹(CAU)发病机制中的作用。本研究分别检测30例CAU患者及30名健康受试者的皮肤组织中OSMR、JAK和STAT3的表达,研究显示OSMR、JAK和STAT3在CAU患者皮肤组织中高表达(p<0.05)。转染OSMR-siRNA可显著降低CAU模型小鼠血清炎症因子IL-1、IL-6和IFN-γ水平,而转染JAK/STAT3信号通路激动剂Tyr705则可显著升高炎症因子水平(p<0.05)。转染OSMR-siRNA可显著降低CAU小鼠瘙痒次数、瘙痒时间和嗜酸性粒细胞计数,而转染Tyr705则可显著升高CAU小鼠瘙痒次数、瘙痒时间和嗜酸性粒细胞计数(p<0.05)。转染OSMR-siRNA促进了CAU小鼠上皮细胞的增殖能力,并抑制了细胞凋亡(p<0.05)。而转染Tyr705则抑制了CAU小鼠上皮细胞的增殖能力,并促进了细胞凋亡(p<0.05)。转染OSMR-siRNA下调了上皮细胞中OSMR、JAK和STAT3的表达,而转染Tyr705则上调了OSMR、JAK和STAT3的表达(p<0.05)。总之,本研究表明OSMR基因在CAU患者皮肤组织中高表达。OSMR基因沉默可通过抑制JAK/STAT3信号通路来抑制炎症因子表达及嗜酸性粒细胞数量,促进上皮细胞增殖并抑制细胞凋亡。     

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway.     

A ganoderic acid derivative,a highly oxygenated lanostane-type triterpenoid from Ganoderma lucidum

Ganoderic acid C, a new lanostane-type triterpenoid was isolated from the fruit body of Ganoderma lucidum. The structure of ganoderic acid C was elucidated by spectroscopic data and X-ray analysis of methyl ganoderate C acetate.