Zinc Deficiency Promotes Testicular Cell Apoptosis in Mice

Biological Trace Element Research - Zinc (Zn) plays an important role in spermatogenesis, and carbon tetrachloride (CCl4) induces testicular oxidative damage and cell death. The objective of the...     

Urokinase-Type Plasminogen Activator Deficiency Promotes Neoplasmatogenesis in the Colon of Mice

Urokinase-type plasminogen activator (uPA) participates in cancer-related biologic processes, such as wound healing and inflammation. The present study aimed to investigate the effect of uPA deficiency on the long-term outcome of early life episodes of dextran sodium sulfate (DSS)–induced colitis in mice. Wild-type (WT) and uPA-deficient (uPA^−/−) BALB/c mice were treated with DSS or remained untreated. Mice were necropsied either 1 week or 7 months after DSS treatment. Colon samples were analyzed by histopathology, immunohistochemistry, ELISA, and real-time polymerase chain reaction. At 7 months, with no colitis evident, half of the uPA^−/− mice had large colonic polypoid adenomas, whereas WT mice did not. One week after DSS treatment, there were typical DSS-induced colitis lesions in both WT and uPA^−/− mice. The affected colon of uPA^−/− mice, however, had features of delayed ulcer re-epithelialization and dysplastic lesions of higher grade developing on the basis of a significantly altered mucosal inflammatory milieu. The later was characterized by more neutrophils and macrophages, less regulatory T cells (Treg), significantly upregulated cytokines, including interleukin-6 (IL-6), IL-17, tumor necrosis factor-α, and IL-10, and lower levels of active transforming growth factor–β1 (TGF-β1) compared to WT mice. Dysfunctional Treg, more robust protumorigenic inflammatory events, and an inherited inability to produce adequate amounts of extracellular active TGF-β1 due to uPA deficiency are interlinked as probable explanations for the inflammatory-induced neoplasmatogenesis in the colon of uPA^−/− mice.     

Mushroom Ganoderma lucidum Prevents Colitis-Associated Carcinogenesis in Mice

Background

Epidemiological studies suggest that mushroom intake is inversely correlated with gastric, gastrointestinal and breast cancers. We have recently demonstrated anticancer and anti-inflammatory activity of triterpene extract isolated from mushroom Ganoderma lucidum (GLT). The aim of the present study was to evaluate whether GLT prevents colitis-associated carcinogenesis in mice.

Methods/Principal Findings

Colon carcinogenesis was induced by the food-borne carcinogen (2-Amino-1-methyl-6-phenylimidazol[4,5-b]pyridine [PhIP]) and inflammation (dextran sodium sulfate [DSS]) in mice. Mice were treated with 0, 100, 300 and 500 mg GLT/kg of body weight 3 times per week for 4 months. Cell proliferation, expression of cyclin D1 and COX-2 and macrophage infiltration was assessed by immunohistochemistry. The effect of GLT on XRE/AhR, PXR and rPXR was evaluated by the reporter gene assays. Expression of metabolizing enzymes CYP1A2, CYP3A1 and CYP3A4 in colon tissue was determined by immunohistochemistry. GLT treatment significantly suppressed focal hyperplasia, aberrant crypt foci (ACF) formation and tumor formation in mice exposed to PhIP/DSS. The anti-proliferative effects of GLT were further confirmed by the decreased staining with Ki-67 in colon tissues. PhIP/DSS-induced colon inflammation was demonstrated by the significant shortening of the large intestine and macrophage infiltrations, whereas GLT treatment prevented the shortening of colon lengths, and reduced infiltration of macrophages in colon tissue. GLT treatment also significantly down-regulated PhIP/DSS-dependent expression of cyclin D1, COX-2, CYP1A2 and CYP3A4 in colon tissue.

Conclusions

Our data suggest that GLT could be considered as an alternative dietary approach for the prevention of colitis-associated cancer.     

Estrogen Deficiency Promotes Hepatic Steatosis via a Glucocorticoid Receptor-Dependent Mechanism in Mice

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Modulation of the Intestinal Microbiota Alters Colitis-Associated Colorectal Cancer Susceptibility

It is well established that the intestinal microbiota plays a key role in the pathogenesis of Crohn''s disease (CD) and ulcerative colitis (UC) collectively referred to as inflammatory bowel disease (IBD). Epidemiological studies have provided strong evidence that IBD patients bear increased risk for the development of colorectal cancer (CRC). However, the impact of the microbiota on the development of colitis-associated cancer (CAC) remains largely unknown. In this study, we established a new model of CAC using azoxymethane (AOM)-exposed, conventionalized-Il10^−/− mice and have explored the contribution of the host intestinal microbiota and MyD88 signaling to the development of CAC. We show that 8/13 (62%) of AOM-Il10^−/− mice developed colon tumors compared to only 3/15 (20%) of AOM- wild-type (WT) mice. Conventionalized AOM-Il10^−/− mice developed spontaneous colitis and colorectal carcinomas while AOM-WT mice were colitis-free and developed only rare adenomas. Importantly, tumor multiplicity directly correlated with the presence of colitis. Il10^−/− mice mono-associated with the mildly colitogenic bacterium Bacteroides vulgatus displayed significantly reduced colitis and colorectal tumor multiplicity compared to Il10^−/− mice. Germ-free AOM-treated Il10^−/− mice showed normal colon histology and were devoid of tumors. Il10^−/−; Myd88^−/− mice treated with AOM displayed reduced expression of Il12p40 and Tnfα mRNA and showed no signs of tumor development. We present the first direct demonstration that manipulation of the intestinal microbiota alters the development of CAC. The TLR/MyD88 pathway is essential for microbiota-induced development of CAC. Unlike findings obtained using the AOM/DSS model, we demonstrate that the severity of chronic colitis directly correlates to colorectal tumor development and that bacterial-induced inflammation drives progression from adenoma to invasive carcinoma.     

Endogenous Siderophore 2,5-Dihydroxybenzoic Acid Deficiency Promotes Anemia and Splenic Iron Overload in Mice

Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.     

溃疡性结肠炎相关结直肠癌风险因素与预防进展

结直肠癌(CRC)是溃疡性结肠炎(UC)严重并发症之一,是UC患者死亡的重要原因。UC相关结直肠癌(UC-CRC)风险因素包括诊断时年龄、病程、炎症的程度及范围、CRC家族史、原发性硬化性胆管炎的合并,及吸烟、假性息肉、结肠持续炎症、结肠狭窄等。更多学者认为UC的癌变与肠黏膜反复炎症-修复密切相关,尤其是炎症的氧化应激、基因突变可能起到重要作用。预防上推荐结肠镜监测以减少CRC风险和UC的死亡率,新技术如放大内镜、色素内镜、共聚焦显微内镜均可能提高靶向活检效率。抗炎治疗是化学预防UC-CRC的主要方向,生物治疗、中医中药等对UC-CRC预防有广泛的应用前景。     

Hepatic Deficiency of Augmenter of Liver Regeneration Exacerbates Alcohol-Induced Liver Injury and Promotes Fibrosis in Mice

Why only a subpopulation (about 15%) of humans develops liver cirrhosis due to alcohol is a critical as yet unanswered question. Liver-specific depletion of augmenter of liver regeneration (ALR) protein in mice causes robust steatosis and hepatocyte apoptosis by 2 weeks; these pathologies regress subsequently with return of ALR expression even at lower than control levels, but the mice develop modest steatohepatitis by 8 weeks. We aimed to investigate whether chronic alcohol ingestion promotes excessive hepatic fibrosis in these ALR-deficient mice. Liver-specific ALR-deficient and wild type (WT) female mice (8–10 weeks old) were placed on 4% alcohol-supplemented or isocaloric diet for 4 weeks. Liver sections were examined for histopathology, and parameters of steatosis and fibrosis were quantified. The mRNA expression of alcohol dehydrogenase-1, acetaldehyde dehydrogenase-1 and cytochrome P450-2E1 increased in WT mice but decreased in ALR-deficient mice upon alcohol ingestion. While alcohol induced steatosis and mild inflammation in WT mice, ALR-deficient mice showed minimal steatosis, strong hepatocellular injury and inflammation, prominent ductular proliferation, and robust fibrosis. Compared to the WT mice, alcohol feeding of ALR-deficient mice resulted in significantly greater increase in hepatic TNFα and TGFβ, and oxidative stress; there was also hepatic iron accumulation, robust lipid peroxidation and mitochondrial DNA damage. Importantly, similar to ALR-deficient mice, lower hepatic ALR levels in human alcoholic liver cirrhosis were associated with increased iron content, reduced expression of alcohol dehydrogenase and acetaldehyde dehydrogenase, and elevated fibrogenic markers. We conclude that ALR deficiency or anomaly can play a critical role in alcohol-induced hepatic fibrosis/cirrhosis, mechanisms of which may involve dysregulation of alcohol metabolism and iron homeostasis, mitochondrial damage and oxidative injury.     

Crucial Involvement of Tumor-Associated Neutrophils in the Regulation of Chronic Colitis-Associated Carcinogenesis in Mice

Ulcerative colitis (UC) is a major form of chronic inflammation that can frequently progress to colon cancer. Several studies have demonstrated massive infiltration of neutrophils and macrophages into the lamina propria and submucosa in the progression of UC-associated colon carcinogenesis. Macrophages contribute to the development of colitis-associated colon cancer (CAC). However, the role of neutrophils is not well understood. To better understand the involvement of tumor-associated neutrophils (TANs) in the regulation of CAC, we used a mouse CAC model produced by administering azoxymethane (AOM), followed by repeated dextran sulfate sodium (DSS) ingestion. This causes severe colonic inflammation and subsequent development of multiple tumors in mice colon. We observed that colorectal mucosal inflammation became increasingly severe with AOM and DSS treatment. Macrophages infiltrated the lamina propria and submucosa, together with a marked increase in neutrophil infiltration. The chemokine CXCL2 increased in the lamina propria and submucosal regions of the colons of the treated mice, together with the infiltration of neutrophils expressing CXCR2, a specific receptor for CXCL2. This process was followed by neoplastic transformation. After AOM and DSS treatment, the mice showed enhanced production of metalloproteinase (MMP)-9 and neutrophil elastase (NE), accompanied by excessive vessel generation and cell proliferation. Moreover, CXCL2 promoted neutrophil recruitment and induced neutrophils to express MMP-9 and NE in vitro. Furthermore, administration of neutrophil-neutralizing antibodies after the last DSS cycle markedly reduced the number and size of tumors and decreased the expression of CXCR2, CXCL2, MMP-9, and NE. These observations indicate a crucial role for TANs in the initiation and progression of CAC and suggest that the CXCL2–CXCR2 axis might be useful in reducing the risk of UC-associated colon cancer.     

IL-6 Signaling Promotes Tumor Growth in Colorectal Cancer

Recent investigations support an important role for TGF-? in the development of colorectal cancer. However, the molecular consequences ofTGF-? signaling in the colon remains incompletely understood. In a recent study in Immunity, we analyzed the role of TGF-? in a murine model of colon cancer. Using transgenic mice overexpressing TGF-? or a dominant negative TGF-? receptor II under control of the CD2 minigene, we show that TGF-? signaling in tumor infiltrating T lymphocytes regulates the growth of dysplastic colon epithelial cells, as determined by histology and a novel system for high resolution chromoendoscopy in vivo. At the molecular level, TGF-? signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-?-dependent IL-6 trans-signaling prevented tumor progression in vivo. Similar to these observations in mice, here we show that human colon cancer tissue expressed only low amounts of membrane bound IL-6R. In contrast, expression and activity of the matrix metalloproteinase TACE were increased. In summary, our data provide novel insights into the role of TGF-? signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on an inhibition of TGF-?-dependent IL-6 trans-signaling.     

Recombinant Interleukin-1 Promotes Leishmaniasis in Susceptible Mice

Interleukin-1 exacerbates leishmanial lesions in Leishmania major-infected BALB/c mice. Indomethacin can modulate the effect of IL-1, so at least part of the IL-1 effect on disease progression is due to the induction of prostaglandin synthesis.     

Dysbiosis-Induced Secondary Bile Acid Deficiency Promotes Intestinal Inflammation

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Bovine Lactoferrin Decreases Cholera-Toxin-Induced Intestinal Fluid Accumulation in Mice by Ganglioside Interaction

Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea.     

不同来源乳铁蛋白对幼鼠肠道发育的影响

目的 探究哺乳期乳铁蛋白（lactoferrin，LF）的缺失及不同来源LF补充后对幼鼠肠道发育的影响。方法 以LF基因敲除型雌鼠作为哺乳母鼠造成幼鼠哺乳期无LF的摄入，且从幼鼠出生第3~21天每日人工饲喂100 mg/kg 牛血清白蛋白（BSA）、牛源乳铁蛋白（bovine Lactoferrin，bLF）及重组人源乳铁蛋白（recombinant human Lactoferrin，rhLF），于幼鼠21日龄取样，测定各组小鼠小肠发育指标。结果 在本实验周期下，哺乳期rhLF的补充显著性增加小鼠回肠绒毛长度/隐窝深度值（P<0.05），且上调回肠Occludin和ZO-1基因的表达（P<0.05），增加小鼠十二指肠、空肠和回肠麦芽糖酶酶活/乳糖酶酶活比值（P<0.05），表明哺乳期rhLF的补充能够增强小鼠肠道消化吸收能力和肠屏障功能；哺乳期bLF的补充显著增加小鼠十二指肠及回肠麦芽糖酶活性/乳糖酶活性比值（P<0.05）。结论 对于哺乳期无LF摄入的乳鼠来说，哺乳期间LF的补充能够增强乳鼠肠道对营养物质的消化吸收能力、促进肠道的发育成熟、增强肠道屏障功能，并且，本实验中rhLF表现出比bLF更加有效的作用。     

Calpain-6 Deficiency Promotes Skeletal Muscle Development and Regeneration

Calpains are Ca²⁺-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6''s in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.     

Diet-Induced Obesity Promotes Colon Tumor Development in Azoxymethane-Treated Mice

Obesity is an important risk factor for colon cancer in humans, and numerous studies have shown that a high fat diet enhances colon cancer development. As both increased adiposity and high fat diet can promote tumorigenesis, we examined the effect of diet-induced obesity, without ongoing high fat diet, on colon tumor development. C57BL/6J male mice were fed regular chow or high fat diet for 8 weeks. Diets were either maintained or switched resulting in four experimental groups: regular chow (R), high fat diet (H), regular chow switched to high fat diet (RH), and high fat diet switched to regular chow (HR). Mice were then administered azoxymethane to induce colon tumors. Tumor incidence and multiplicity were dramatically smaller in the R group relative to all groups that received high fat diet at any point. The effect of obesity on colon tumors could not be explained by differences in aberrant crypt foci number. Moreover, diet did not alter colonic expression of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, interleukin-1β, and interferon-γ, which were measured immediately after azoxymethane treatment. Crypt apoptosis and proliferation, which were measured at the same time, were increased in the HR relative to all other groups. Our results suggest that factors associated with obesity – independently of ongoing high fat diet and obesity – promote tumor development because HR group animals had significantly more tumors than R group, and these mice were fed the same regular chow throughout the entire carcinogenic period. Moreover, there was no difference in the number of aberrant crypt foci between these groups, and thus the effect of obesity appears to be on subsequent stages of tumor development when early preneoplastic lesions transition into adenomas.     

小鼠结直肠自发肿瘤模型的建立

目的:建立小鼠结直肠自发肿瘤模型。方法:用雄性Ap~(Mn/ )基因突变小鼠与雌性BubR1~( /-)基因敲除小鼠交配。得到的子代小鼠,按基因型分为Wild Type,BubR1~( /-),Apc(Mn/ ),和BubR~(1 /-)Apc(Mn/ 4)组,每组7只,观察各组肠组织肿瘤数目、大小和位置,行HE染色后观察病理学改变,并用免疫组化染色法研究各组结直肠β-catenin的表达。结果:①BubR1~( /-)Apc(Mn/ )组小鼠结直肠肿瘤的发病率显著增加,较Apc(Mn/ )组有显著差异(P=0．008)。②BubR1或Apc单独缺失组小鼠结直肠中β-catenin蛋白除在细胞膜表达外,在细胞质也有弱阳性表达;而BubR1~( /-)Apc(Mn/ )组小鼠结直肠β-catenin的表达水平明显增高,且多为细胞质表达和(或)膜浆共表达,部分合并细胞核表达。结论建立小鼠结直肠自发肿瘤模型对于筛选抗肿瘤药物和研究肿瘤发生机制具一定的应用价值。     

HMGA2通过激活Wnt信号通路促进结直肠癌细胞增殖

目的:HMGA2(high-mobility group AT-hook 2)一个染色质蛋白,被报道在多种癌症中都发挥重要作用。本文研究染色体蛋白HMGA2对Wnt信号传递的影响,及对结直肠癌细胞增殖的影响。方法:本文通过q RT-PCR和免疫印迹法检测HMGA2在结直肠癌样本中m RNA和蛋白水平。用荧光素酶报告基因系统研究HMGA2对Wnt信号通路的作用。用细胞增殖实验检测HMGA2对结直肠癌细胞增殖的作用。结果:在我们检测的大部分结直肠癌样本里,HMGA2表达水平升高;HMGA2蛋白可上调Wnt信号通路荧光素酶报告基因TOPflash-luciferase的表达,并呈现剂量依赖的形式。降低HMGA2表达可抑制由Wnt3a、Dvl(Dishevelled)、Li Cl以及βcatenin(S37A)引起的TOPflash-luciferase报告基因表达上调作用。此外,SW480中过量表达HMGA2可以促进细胞增殖。结论:HMGA2在结直肠癌中表达升高,HMGA2在结直肠癌中通过增强Wnt信号来促进结直肠癌细胞的增殖。