大黄素对家兔实验性皮肤创伤的促愈合作用及其机制

目的探讨大黄素治疗皮肤创伤的可能性及其作用机制。方法采用家兔皮肤切除伤创伤模型,同时滴加绿脓杆菌或金黄色葡萄球菌。大黄素(100~200μg·g-1)敷于创面,每天1次,连续7d或14d。观察创面面积、创面细菌数量和病理组织学改变。生化测定创面组织蛋白和羟脯氨酸(OHP)含量;免疫组化S-P法检测转化生长因子(TGF-β1)含量;逆转录聚合酶链反应测定TGF-β1mRNA表达;Western印迹分析Smad2,3,4和7表达。结果大黄素(100~200μg·g-1)随药物剂量和时间的增加而提高创面愈合百分率和降低感染创面表面细菌数;创面组织蛋白和羟脯氨酸的含量也随着剂量的增加而增加。病理结果显示,大黄素200μg·g-1明显促进创面炎性渗出物吸收、肉芽组织形成和表皮增生。随大黄素浓度的增加,与基质对照组相比,TGF-β1基因和蛋白的表达逐渐增高,均有显著差异;对Smad4蛋白表达无影响,大黄素150和200μg·g-1使Smad7蛋白表达降低;大黄素200μg·g-1使Smad3和Smad2表达增加,其他剂量则无影响。与rhEGF组相比,大黄素100和(或)150μg·g-1组使Smad2和Smad3表达明显下降。结论大黄素促进实验性皮肤创面的修复,其机制可能与TGF-β1和Smads信号转导通路有关。     

Emodin induces cytotoxic effect in human breast carcinoma MCF-7 cell through modulating the expression of apoptosis-related genes

Abstract

Context: The poor prognostic outcome of breast cancer is largely due to its resistance to cancer therapies. Development of therapeutic agents that can inhibit growth and induce apoptosis in breast cancer cells can help solve the problem. Emodin is an active anthraquinone that has been reported to have diverse biological effects.

Objective: In this study, the anticancer effects of emodin on growth inhibition, apoptosis induction and the expression of apoptosis-related genes in MCF-7 cells were investigated.

Materials and methods: Growth inhibition induced by emodin was investigated by the MTS assay and the colony formation assay; while emodin-induced apoptosis was determined by the COMET assay and DNA fragmentation detection. Emodin (35?μM)-induced alterations in the expression of apoptotic-related genes were detected by using real-time PCR.

Results: Emodin had significant growth inhibitory effects on MCF-7 cells with IC₅₀?=?7.22?µg/ml (～30?μM). It also exerted a concentration-dependant inhibitory effect on the colony-forming ability of MCF-7 cells with IC₅₀?=?7.60?µg/ml (～30?µM). Hallmarks of apoptosis, such as single-strand DNA breakage and DNA fragmentation, were observed in emodin-treated MCF-7 cells. The gene expression of Fas ligand (FASL) was up-regulated (p?<?0.01) but those of MCL1, CCND1 and C-MYC were down-regulated (p?<?0.05) in emodin-treated MCF-7 cells.

Discussion and conclusion: This study indicated that emodin could induce growth inhibition and apoptosis in MCF-7 cells through the modulation of the expression of apoptosis-related genes. The growth inhibitory effects of emodin might involve both the intrinsic and the extrinsic apoptotic pathways and cell cycle arrest.     

Emodin induces apoptosis in human promyeloleukemic HL-60 cells accompanied by activation of caspase 3 cascade but independent of reactive oxygen species production

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent of Rheum palmatum, and showed inhibitory activity on lipopolysaccharide-induced NO production in our previous study. However, the apoptosis-inducing activity of emodin has remained undefined. Among three structurally related anthraquinones, including emodin, physcion, and chrysophanol, emodin showed the most potent cytotoxic effects on HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells. Emodin at apoptosis-inducing concentrations causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity, according to cleavage of caspase 3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase 3 fragments being detected in emodin- but not physcion- or chrysophanol-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in emodin-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bad remained unchanged. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated emodin-induced DNA ladders, associated with the blockage of PARP and D4-GDI cleavage. Free radical scavenging agents including NAC, catalase, SOD, ALL, DPI, L-NAME and PDTC showed no preventive effect on emodin-induced apoptotic responses, whereas NAC, CAT and PDTC prevented HL-60 cells from ROS (H(2)O(2))-induced apoptosis through inhibition of caspase 3 cascades. Induction of catalase, but not SOD, activity was detected in emodin-treated HL-60 cells by in gel activity assays, and H(2)O(2)-induced intracellular peroxide level was significantly reduced by prior treatment of emodin in HL-60 cells. Our experiments provide evidence that emodin is an effective apoptosis inducer in HL-60 cells through activation of the caspase 3 cascade, but that it is independent of ROS production.     

Concentration-dependent bifunctional effect of TGF-beta 1 on immunoglobulin production: a role for Smad3 in IgA production in vitro

Injury to the liver results in rapid induction of transforming growth factor-beta1 (TGF-beta(1)) consistent with a role for TGF-beta(1) in repairing damaged tissue. In addition to its ubiquitous role in injury repair, TGF-beta(1) is also well established as a critical regulator of immune homeostasis; however, its mechanisms of action remain enigmatic. We have previously demonstrated that the hepatotoxic chlorinated hydrocarbon, carbon tetrachloride, suppresses helper T-lymphocyte function in a TGF-beta(1)-dependent manner. Here, we report that, in opposition to its immunosuppressive effects at picomolar concentrations, femtomolar concentrations of TGF-beta(1) augment T cell-dependent anti-sRBC IgM antibody forming cell (AFC) and T cell-independent DNP-Ficoll-induced AFC responses. These data support a concentration-dependent bifunctional effect by TGF-beta(1) on humoral immune responses in vitro. We further investigated a putative mechanistic role for Smad3, an intracellular mediator of TGF-beta(1) signaling, in propagating the inhibitory effects of TGF-beta(1) on humoral immune responses. Relative to wild type littermates, splenocytes from mice homologous for a null mutation in the gene encoding the TGF-beta receptor-activated Smad3 (Smad3(Exon8-/-)) were less sensitive to inhibition by TGF-beta(1) following anti-sRBC- and LPS-sensitization in vitro. In agreement, inhibition of IgM protein production by TGF-beta(1) was also dampened in LPS-sensitized Smad3(Exon8-/-) splenic B cells. Moreover, stimulation of IgA by TGF-beta(1) was abrogated in LPS-sensitized Smad3(Exon8-/-) splenocytes suggesting an additional role for Smad3 in regulating IgA production in vitro. Our results suggest that the effects of TGF-beta(1) on humoral immune responses fundamentally differ in a concentration-dependent manner and are mediated, in part, through Smad3 signaling.     

Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery Vehicle Enhances Diabetic Wound Healing

PURPOSE: To accelerate diabetic wound healing with TGF-beta1 gene delivery system using a thermosensitive hydrogel made of a triblock copolymer, PEG-PLGA-PEG. METHODS: Two 7 x 7 mm full thickness excisional wounds were created in parallel at the back of each genetically diabetic mouse. The hydrogel containing plasmid TGF-beta1 was administered to the wound and formed an adhesive film in situ. Controls were either untreated or treated with the hydrogel without DNA. We used a commercial wound dressing, Humatrix, either with or without DNA, to compare the therapeutic effect with the thermosensitive hydrogel. RESULTS: We found that thermosensitive hydrogel alone is slightly beneficial for reepithealization at early stage of healing (day 1-5), but significantly accelerated repithelializaion, increased cell proliferation, and organized collagen were observed in the wound bed treated with thermosensitive hydrogel containing plasmid TGF-beta1. The accelerated reepithelialization was accompanied with enhanced collagen synthesis and more organized extracellular matrix deposition. Humatrix alone or with plasmid TGF-beta1, had little effect. CONCLUSIONS: Thermosensitive hydrogel made of PEG-PLGA-PEG triblock copolymer provides excellent wound dressing activity and delivers plasmid TGF-beta1 to promote wound healing in a diabetic mouse model.     

Fucoidan modulates the effect of transforming growth factor (TGF)-beta1 on fibroblast proliferation and wound repopulation in in vitro models of dermal wound repair

Aberrant wound healing, either causing scarring or chronic wounds, is a significant cause of morbidity. There is therefore, considerable interest in agents which can modulate certain aspects of the wound healing process. Fucoidans, sulphated polyfucose polysaccharides which may be extracted from Fucus spp., have been shown to modulate the effects of a variety of growth factors through mechanisms thought to be similar to the action of heparin. We investigated the interaction between two commercial preparations of fucoidan and transforming growth factor (TGF)-beta(1). These preparations of fucoidan, as well as heparin, inhibited fibroblast proliferation at concentrations from 0.01 to 100 mg/ml. The anti-proliferative effects of 1 ng/ml TGF-beta(1) on dermal fibroblasts were abrogated by fucoidan preparation F7 when used at concentrations over 1 mg/ml. In a three dimensional in vitro model of wound repair, the fibroblast populated collagen lattice or "dermal equivalent", TGF-beta(1) reduced the rate of fibroblast repopulation of a wound defect created by punch biopsy. Addition of fucoidan to the model in the presence of TGF-beta(1) increased the rate of fibroblast repopulation of the wound and at 10 mg/ml of fucoidan the number of cells which had migrated into the wounded defect was similar to that of control cultures. These data suggest that fucoidan has properties which may be beneficial in the treatment of wound healing.     

Fluvastatin decreases cardiac fibrosis possibly through regulation of TGF-beta(1)/Smad 7 expression in the spontaneously hypertensive rats

Statins ameliorate myocardial fibrosis after myocardial infarction. We designed this study to determine whether fluvastatin reduced hypertension-induced myocardial hypertrophy and fibrosis and whether these fluvastatin effects involved transforming growth factor beta1 (TGF-beta1) and Smad 7, factors known to play a role in the myocardial hypertrophy and fibrosis. We randomized 14 week old spontaneously hypertensive rats (SHRs) to receiving vehicle or 5-20 mg/kg/day fluvastatin for 8 weeks. Wistar Kyoto (WKY) rats receiving vehicle or 10 mg/kg/day fluvastatin were also studied. SHRs had an increased blood pressure, left ventricular hypertrophy and fibrosis compared with WKY rats. SHRs also had an elevated TGF-beta1 expression and a decreased Smad 7 expression. These changes in SHRs were dose-dependently attenuated by fluvastatin. For example, the hydroxyproline content was 3.2+/-0.1, 4.0+/-0.1 and 3.5+/-0.1 microg/mg heart and the Smad 7 protein expression was 5.1+/-0.6, 1.0+/-0.1 and 4.1+/-0.7 arbitrary units for WKY rats, SHRs and SHRs receiving 20 mg/kg/day fluvastatin, respectively. The hydroxyproline content in the SHRs treated with or without fluvastatin was positively correlated with the left ventricular mass index, systolic blood pressure and the amount of TGF-beta1 proteins and negatively correlated with the Smad 7 expression level. The left ventricular mass index was positively correlated with the systolic blood pressure. Fluvastatin did not alter the blood pressure, left ventricular mass index and collagen content of WKY rats. These results suggest that fluvastatin reduces hypertension-induced myocardial hypertrophy and fibrosis. These effects may involve an increased expression of Smad 7 and a decreased expression of TFG-beta1. Our results call for clinical studies to evaluate these fluvastatin effects in hypertensive patients.     

Effect of IFN-γ and dexamethasone on TGF-β1-induced human fetal lung fibroblast-myofibroblast differentiation

AIM: To study whether Smads signaling pathway was involved in human fetal lung fibroblast-myofibroblast differentiation induced by transforming growth factor (TGF)-beta1 and the role of interferon (IFN)-gamma, dexamethasone (DEX) in the fibroblast-myofibroblast differentiation. METHODS: Alpha-smooth muscle actin (alpha-SMA), Smad2/3, and Smad7 protein were assessed by Western blot. Collagen protein was analyzed by measuring hydroxyproline. Alpha-SMA and collagen III mRNA were assessed by RT-PCR. Myofibroblasts morphology and Smad2/3 nuclear translocation were assessed by immunohistochemistry. The overexpression of Smad7, a negative mediator of Smads signaling pathway, was acquired by transfection of Smad7 vector. RESULTS: During fibroblast-myofibroblast differentiation induced by TGF-beta1, IFN-gamma 200 microg/L markedly blocked TGF-beta1-induced alpha-SMA protein expression (P<0.01), collagen protein (P<0.01) and mRNA (P<0.05) expression, and myofibroblasts morphological transformation, but DEX 10 micromol/L augmented TGF-beta1-induced alpha-SMA expression (P<0.01). For myofibroblasts, both IFN-gamma 200 microg/L and DEX 10 micromol/L did not inhibit alpha-SMA expression (P>0.05) and collagen protein (P>0.05) and mRNA expression (P>0.05) and did not change myofibroblasts morphology. Transient transfection of Smad7 vector resulted in significant inhibition of TGF-beta1-induced alpha-SMA expression (P<0.01). IFN-gamma 200 microg/L did not block TGF-beta1-stimulated Smad2/3 phosphorylation and their nuclear translocation. CONCLUSION: TGF-beta1 induced fibroblast-myofibroblast differentiation in a Smad proteins-dependent manner. IFN-gamma could block this process but it was not mediated by interrupting smad2/3 phosphorylation and their nuclear translocation and DEX played a synergism with TGF-beta1. Differentiated myofibroblasts, however, were resistant to both IFN-gamma and DEX.     

Emodin upregulates urokinase plasminogen activator, plasminogen activator inhibitor-1 and promotes wound healing in human fibroblasts

Urokinase plasminogen activator (uPA) system is important for several biological processes that call for extracellular proteolysis, fibrinolysis, cell migration, proliferation and angiogenesis. The current study highlights the fibrinolytic and wound healing potential of emodin, an anthraquinone, with relevance to the uPA system. Emodin increased the fibrinolytic activity of fibroblast cells in a dose-dependent manner. Zymography linked the activity to increased uPA activity. Subsequent RT-PCR and western analyses demonstrated uPA gene upregulation. Interestingly, PAI-1, the inhibitor of uPA was also upregulated. EMSA showed the upregulation occurred independent of emodin's effect on nuclear factor kappa B (NFkappaB). The effect on uPA system is supposedly via generation of reactive oxygen species (ROS) since cotreatment with ascorbic acid, an anti-oxidant, attenuated the activity. In addition to profibrinolytic potential, emodin also demonstrated wound healing activity in in vitro wound models. Presence of emodin in the medium enhanced the rate of migration of fibroblasts into the wounded region. These in vitro experiments reveal that emodin is a potent profibrinolytic and wound healing agent.     

Effects and mechanisms of emodin on cell death in human lung squamous cell carcinoma

1. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active component from the root and rhizome of Rheum palmatum that has been reported to exhibit antitumour effects, but the mechanism is not known. The study investigated the effects and mechanisms of emodin-induced cell death in human lung squamous carcinoma cell line CH27. 2. Emodin (50 microM)-induced CH27 cell apoptosis was confirmed by cell morphological change, sub-G1 formation in flow cytometry analysis, viability assay and degradation of focal adhesion kinase in this study. 3. Emodin-induced apoptosis of CH27 cells does not involve modulation of endogenous Bcl-X(L) protein expression, but appears to be associated with the increased expression of cellular Bak and Bax proteins. This study also demonstrated the translocation of Bak and Bax from cytosolic to particulate fractions. 4. This study has shown that emodin-treated CH27 cells revealed the increases in the relative abundance of cytochrome c for the indicated time intervals in cytosolic fraction. 5. This study demonstrates that the activation of caspase-3, caspase-9 and caspase-8 is an important determinant of apoptotic death induced by emodin. 6. These results suggested that emodin induces CH27 cell death by Bax death pathway and Fas pathway.     

高效液相色谱法测定烧伤液中大黄素、大黄素甲醚、槲皮素的含量

目的建立高效液相色谱法测定烧伤液中大黄素、大黄素甲醚、槲皮素的含量。方法采用HypersilODSC18（4.0mm×250mm,5μm）,以甲醇一磷酸盐缓冲液（pH=4）（65：35）,流速1.0ml/ml,检测波长254nm。结果大黄素线性范围0.7～7.0μg/ml,平均回收率98.65%,RSD为1.24%;大黄素甲醚线性范围0.504～5.04μg/ml,平均回收率为98.70%,RSD为1.08%,槲皮素线性范围0.384～3.84μg/ml,平均回收率为99.36%,RSD为1.54%。结论该方法简便、准确,可用于烧伤液的质控。     

Wound healing acceleration of a novel transforming growth factor-beta inducer, SEK-1005

The studies were carried out to elucidate the effect of a novel cyclic peptide, SEK-1005 (C(45)H(70)N(8)O(13)), on wound healing. SEK-1005 (4-10 microg/wound) applied topically significantly accelerated the healing of a full-thickness wound on the dorsal skin of a rat. In a healing-impaired mouse, the peptide (2-10 microg/wound) had more potent activity, exerting an effect comparable to that of basic fibroblast growth factor (FGF). However, SEK-1005 (0.1-100 ng/ml) scarcely promoted the proliferation of cultured fibroblasts (NIH3T3 cells) while basic FGF (0.2-5 ng/ml) showed marked mitogenic activity. SEK-1005 (2-10 microg/wound) significantly increased the topical production of transforming growth factor (TGF)-beta1, a cytokine that is known to accelerate wound healing. This activity was closely correlated with the wound-repairing effect. From the above, SEK-1005 can be considered as a new type of wound healing agent with potent TGF-beta1-inducing activity.     

大黄素对血管生成的抑制作用

目的研究大黄素对血管生成的抑制作用及相关作用机制。方法用鸡胚观察药物对血管生成的影响;用培养的内皮细胞检测大黄素抑制细胞增殖和诱导细胞凋亡作用。结果大黄素150和300 μg/egg对鸡胚的血管生成的抑制率分别为37.6%和63.2%。大黄素抑制内皮细胞增殖，在有bFGF、无bFGF、有VEGF的条件下，其IC₅₀值分别为5.56，8.40和6.91 mg·L^-1。大黄素可诱导内皮细胞凋亡，并可干扰内皮细胞周期，出现G₂/M期阻滞；可导致Cyclin B1,P34^cdc2和Bcl-2等蛋白的表达下调，但对Bax的表达无影响。结论大黄素有抑制血管生成作用，有可能应用于肿瘤治疗。     

中药大黄的生化学研究.ⅪⅩ.蒽醌衍生物对线粒体呼吸链的抑制部位

本实验结果表明:大黄素、大黄酸和芦荟大黄素是线粒体呼吸链电子传递的抑制剂。(1)三药对NADH脱氢酶有不同程度的抑制作用,(2)大黄素对琥珀酸脱氢酶有较强的抑制作用,而大黄酸和芦荟大黄素对此酶仅有轻微的抑制作用;(3)三药对辅酶Q-细胞色素C还原酶及细胞色素C氧化酶也仅有轻微的抑制作用;(4)动力学观察表明:三药对NADH脱氢酶的抑制均为竞争性的。     

大黄素对大鼠体外颅骨成骨细胞的影响

目的观察大黄素对体外大鼠颅骨成骨细胞细胞周期、骨钙素含量、骨结节数目和面积、Ⅰ型胶原mRNA表达的影响。方法在成骨细胞体系中加入不同浓度的大黄素,应用流式细胞术、放射免疫测定法、茜素红染色法及RTPCR的方法观察大黄素对大鼠颅骨成骨细胞的增殖、骨钙素含量、骨结节数目和面积、Ⅰ型胶原mRNA表达的影响。结果大黄素可刺激成骨细胞分泌骨钙素。低浓度的大黄素可上调Ⅰ型胶原基因的表达,高浓度时则降低Ⅰ型胶原的基因表达。结论大黄素可以刺激成骨细胞分泌骨钙素和增加Ⅰ型胶原mRNA的表达。     

大黄素对豚鼠结肠带平滑肌细胞钾通道活性的影响

用检测平滑肌细胞电活动和张力技术,研究了大黄素对豚鼠结肠带平滑肌细胞电和收缩活动的影响并与 cromakalim, glybenclamide,四乙胺及BaCl₂的作用进行比较。结果表明,大黄素加强平滑肌细胞电和收缩活动,作用效果与剂量有关;大黄素与cromakalim的作用相互抑制。其促进平滑肌细胞电和收缩活动的作用与glybenclamide相似,而与四乙胺和BaCl₂有明显区别。提示大黄素的作用机制与抑制细胞膜K_ATP等钾通道的活性相关。     

SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7

Clinically, there is a great need for small molecule inhibitors that could control pathogenic effects of transforming growth factor (TGF-beta) and/or modulate effects of TGF-beta in normal responses. Inhibition of TGF-beta signaling would be predicted to enhance re-epithelialization of cutaneous wounds and reduce scarring fibrosis. Selective small molecule inhibitors of the TGF-beta signaling pathway developed for therapeutics will also be powerful tools in experimentally dissecting this complex pathway, especially its cross-talk with other signaling pathways. In this study, we characterized 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124), a member of a new class of small molecule inhibitors related to imidazole inhibitors of p38, which inhibit the TGF-beta type I receptor serine/threonine kinase known as activin receptor-like kinase (ALK) 5. We demonstrate that this compound selectively and concentration-dependently inhibits ALK4-, ALK5-, and ALK 7-dependent activation of downstream cytoplasmic signal transducers, Smad2 and Smad3, and of TGF-beta-induced mitogen-activated protein kinase pathway components but does not alter ALK1, ALK2, ALK3 or ALK6-induced Smad signaling. SB-505124 also blocks more complex endpoints of TGF-beta action, as evidenced by its ability to abrogate cell death caused by TGF-beta1 treatment. SB-505124 is three to five times more potent than a related ALK5 inhibitor described previously, SB-431542.     

Emodin, a naturally occurring anthraquinone derivative, suppresses IgE-mediated anaphylactic reaction and mast cell activation

The high-affinity receptor for IgE (Fc?RI)-mediated activation of mast cells plays an important role in allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. Emodin, a naturally occurring anthraquinone derivative in oriental herbal medicines, has several beneficial pharmacologic effects, such as anti-cancer and anti-diabetic activities. However, the anti-allergic effect of emodin has not yet been investigated. To assess the anti-allergic activity of emodin, in vivo passive anaphylaxis animal model and in vitro mouse bone marrow-derived mast cells were used to investigate the mechanism of its action on mast cells. Our results showed that emodin inhibited degranulation, generation of eicosanoids (prostaglandin D₂ and leukotriene C₄), and secretion of cytokines (TNF-α and IL-6) in a dose-dependent manner in IgE/Ag-stimulated mast cells. Biochemical analysis of the Fc?RI-mediated signaling pathways demonstrated that emodin inhibited the phosphorylation of Syk and multiple downstream signaling processes including mobilization of intracellular Ca²⁺ and activation of the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and NF-κB pathways. When administered orally, emodin attenuated the mast cell-dependent passive anaphylactic reaction in IgE-sensitized mice. Thus, emodin inhibits mast cell activation and thereby the anaphylactic reaction through suppression of the receptor-proximal Syk-dependent signaling pathways. Therefore, emodin might provide a basis for development of a novel anti-allergic drug.