Mechanisms of norcantharidin against renal tubulointerstitial fibrosis.

Renal tubulointerstitial fibrosis (RTIF) is a common feature and inevitable consequence of all progressive chronic kidney diseases, leading to end-stage renal failure regardless of the initial cause. Although research over the past few decades has greatly improved our understanding of the pathophysiology of RTIF, until now there has been no specific treatment available that can halt the progression of RTIF. Norcantharidin (NCTD) is a demethylated analogue of cantharidin, a natural compound isolated from 1500 species of medicinal insect, the blister beetle (Mylabris phalerata Pallas), traditionally used for medicinal purposes. Many studies have found that NCTD can attenuate RTIF and has the potential to be an anti-RTIF drug. This article reviews the recent progress of NCTD in the treatment of RTIF, with emphasis on the pharmacological mechanism of NCTD against RTIF.

Downregulation of hepcidin by norcantharidin in macrophage.

Norcantharidin (NCTD) is a demethylated analogue of cantharidin. It was recently demonstrated that NCTD reduces iron contents in the liver and spleen of mice in vivo, indicating that NCTD can affect iron metabolism via hepcidin. Here, we investigated the effects of NCTD on expression of iron storage protein ferritin-light chain (Ft-L), transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1), hepcidin, iron regulatory protein 1 (IRP1), IL-6, p-JAK2 and p-STAT3 in lipopolysaccharides (LPS)-treated RAW264.7 cells in vitro via Real-time PCR and Western blotting analysis. We demonstrate that NCTD down-regulates Ft-L, hepcidin, IL-6, pJAK2, pSTAT3 and up-regulates TfR1, DMT1, Fpn1 and IRP1 expression in LPS treated cells, showing that NCTD can inhibit hepcidin via the IL-6/JAK2/STAT3 signalling pathway and also increase TfR1, DMT1 and Fpn1 expression via down-regulating hepcidin and up-regulating IRP1. Our findings provide further evidence in vitro for the role of NCTD in iron metabolism.

Pharmacological mechanisms of norcantharidin against hepatocellular carcinoma.

Norcantharidin (NCTD) is a water-soluble synthetic small molecule drug that has been approved by the Chinese FDA for the treatment of cancer in China. Among these NCTD-treated cancers, hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide and one of the most extensively studied. Research over the past few decades has made great strides in understanding how NCTD induces mitotic arrest, anti-proliferation, anti-metastasis, apoptosis and cytotoxic autophagy or autophagic cell death in HCC. In this article, we review recent progress in the application of NCTD for the treatment of HCC, with emphasis on the pharmacological mechanism of NCTD against hepatocellular carcinoma. The accumulated results show that NCTD has the ability to induce mitotic arrest, anti-proliferation, anti-metastasis, apoptosis and cytotoxic autophagy or autophagic cell death in HCC by down-regulating the expression of ISG15, MMP-9, u-PA, Mcl-1 and the accumulation of regulatory T cells, up-regulating the expression of FAM46C, miR-214 and the expression and phosphorylation of p21Cip1/Waf1 and CDC25C, and by inhibiting the c-Met-mTOR and JAK/STAT3 signaling pathways, reversing the methylation of RASSF1A gene, and activating TRAIL-R2/DR5 signal transduction.

Norcantharidin down-regulates iron contents in the liver and spleen of lipopolysaccharide-treated mice.

OBJECTIVE: The inhibiting effect of Norcantharidin (NCTD) on IL-6 (interleukin-6) and STAT3 and the involvement of the IL-6/STAT3 pathway in hepcidin expression prompted us to speculate that NCTD could affect iron metabolism.We examined the effects of NCTD on serum iron (SI) and transferrin (Tf) saturation, iron and ferritin light chain (FTL), transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1), iron regulatory protein 1 (IRP1) and hepcidin, as well as IL-6 and STAT3 in the liver, spleen and duodenum of mice treated with lipopolysaccharide (LPS) in vivo, using RT-PCR, Western blotting and immunofluorescence analysis.NCTD could increase SI and Tf saturation and reduce tissue iron and FTL content by affecting expression of cell-iron transport proteins TfR1, DMT1 and Fpn1. The impact of NCTD on TfR1, DMT1 and Fpn1 expression is mediated by up-regulating IRP1 and down-regulating hepcidin expression, while NCTD-induced down-regulation of hepcidin is mediated by the IL-6/STAT3 signalling pathway in LPS-treated mice.NCTD affects iron metabolism by modifying the expression of IL-6/JAK2/STAT3/hepcidin and IRP1 and suggest that the ability of NCTD to reduce tissue iron contents may be a novel mechanism associated with the anti-cancer effects of NCTD.

The Role of Iron in Atherosclerosis in Apolipoprotein E Deficient Mice.

The role of iron in atherosclerosis is still a controversial and unsolved issue. Here, we investigated serum iron, expression of iron regulatory, transport and storage proteins, pro-inflammatory chemokines and cytokines in ApoE-/- mice. We demonstrated that ApoE-/- induced atherosclerosis and an increase in iron contents, expression of transferrin receptor 1 (TfR1), iron regulatory proteins (IRPs), heme oxygenase 1 (HO1), cellular adhesion molecules and pro-inflammatory cytokines, production of reactive oxygen species (ROS), and a reduction in expression of superoxide dismutase and glutathione peroxidase enzyme in aortic tissues. All of these changes induced by ApoE deficiency could be significantly abolished by deferoxamine. The data showed that the increased iron in aortic tissues was mainly due to the increased iron uptake via IRP/TfR1 upregulation. These findings plus a brief analysis of the controversial results reported previously showed that ApoE deficiency-induced atherosclerosis is partly mediated by the increased iron in aortic tissues.

Verapamil downregulates iron uptake and upregulates divalent metal transporter 1 expression in H9C2 cardiomyocytes.

Belgrade rats have a defect in divalent metal transport 1 (DMT1) with a reduced heart iron, indicating that DMT1 plays a physiological role in non-transferrin-bound iron (NTBI) uptake by cardiomyocytes. However, L-type voltage-dependent Ca2+ channel (LVDCC) blockers were recently demonstrated to significantly reduce NTBI uptake by cardiomyocytes, implying that LVDCC plays a dominant role in NTBI uptake by cardiomyocytes under iron-overloaded conditions. These findings led us to hypothesize that the LVDCC blocker-induced reduction in NTBI uptake might result not only from the inhibition of LVDCC-mediated NTBI uptake but also from the suppression of DMT1-mediated NTBI uptake. We therefore investigated the effects of the LVDCC blocker verapamil on NTBI uptake as well as DMT1 expression in H9C2 cells by the measurement of radio-labeled 55 Fe(II), reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. We demonstrated that verapamil induced a significant reduction in NTBI uptake by H9C2 cells but also unexpectedly a remarkable increase rather than decrease in the expression of DMT1 mRNA and protein in H9C2 cells. Our findings imply that the verapamil-induced reduction in NTBI uptake by H9C2 cells is not associated with DMT1 and also indicate that verapamil stimulates rather than inhibits DMT1 expression and DMT1-mediated iron uptake by heart cells.

The involvement of nuclear factor-κB in astroprotection against ischemia-reperfusion injury by ischemia-preconditioned neurons.

Ischemic preconditioned (IP) neurons protect astrocytes against ischemia/reperfusion (I/R)-induced injury by inhibiting oxidative stress. However, the relevant mechanisms are unknown. Based on the role of nuclear factor-κB (NF-κB) in cell survival and adaption to oxidative stress, we hypothesized that NF-κB might be associated with astroprotection induced by IP neurons via upregulation of antioxidant enzymes. Here, we investigated the effects of IP neurons on NF-κB activation, cell viability, reactive oxygen species (ROS), expression of antioxidant enzymes, erythropoietin (EPO), and tumor necrosis factor α (TNF-α), in the presence or absence of BAY11-7082 (an NF-κB inhibitor), anti-EPO, and anti-TNF-α antibodies, in astrocytes treated with or without I/R. We found that IP neurons could keep NF-κB activation at a relatively higher but beneficial level, and in turn, upregulated the activity of antioxidant enzymes and hence enhanced cell viability and reduced ROS in I/R treated astrocytes. The results collectively indicated that IP neurons are able to significantly inhibit the I/R-induced NF-κB overactivation, probably via EPO and TNF-α, being essential for IP neuron-induced astroprotection under the conditions of I/R. We concluded that NF-κB-mediated antioxidative stress is one of the mechanisms by which IP neurons protect astrocytes against I/R injury.

Hepcidin inhibits autophagy in intracerebral hemorrhage models in vitro and in vivo.

Iron has a key role in the activation of the autophagic pathway in rats with intracerebral hemorrhage (ICH), and hepcidin has the ability to reduce brain iron in ICH-rats. We therefore hypothesized that hepcidin might be able to inhibit autophagy by reducing iron in an ICH brain. Here, we investigated the effects of Ad-hepcidin and/or hepcidin peptide on autophagic activities in ICH models in vitro and in vivo. We demonstrated that ad-hepcidin and hepcidin peptide both inhibited hemin-induced increase in LC3-II/LC3-I conversion ratio and reversed the reduction in p62 content in cortical neurons in vitro. We also showed that ad-hepcidin inhibited ICH-induced increase in LC3-II/LC3-I conversion ratio and reversed ICH-induced reduction in p62 content in the brain cortex of rats in vivo. Based on these findings plus previous data on the effects of ad-hepcidin and/or hepcidin peptide on iron contents in ICH models, we suggested that hepcidin-induced inhibition of autophagy might be mediated via reducing iron in hemin-treated neurons in vitro and ICH-rat brain in vivo.

Apolipoprotein E deficiency induces a progressive increase in tissue iron contents with age in mice.

Association of both iron/hepcidin and apolipoprotein E (ApoE) with development of Alzheimer disease (AD) and atherosclerosis led us to hypothesize that ApoE might be required for body iron homeostasis. Here, we demonstrated that ApoE knock-out (KO) induced a progressive accumulation of iron with age in the liver and spleen of mice. Subsequent investigations showed that the increased iron in the liver and spleen was due to phosphorylated extracellular regulated protein kinases (pERK) mediated up-regulation of transferrin receptor 1 (TfR1), and nuclear factor erythroid 2-related factor-2 (Nrf2)-dependent down-regulation of ferroportin 1. Furthermore, replenishment of ApoE could partially reverse the iron-related phenotype in ApoE KO mice. The findings imply that ApoE may be essential for body iron homeostasis and also suggest that clinical late-onset diseases with unexplained iron abnormality may partly be related to deficiency or reduced expression of ApoE.

Kocuria soli sp. nov., an actinobacterium isolated from soil.

A Gram-stain-positive, aerobic, coccoid-shaped, non-spore-forming actinobacterial strain, designated M5W7-7T, was isolated from a hot spring soil sample collected from Anshan, Liaoning province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain M5W7-7T clustered closely with species of the genus Kocuria, and showed the highest sequence similarity of 97.1 % to Kocuria subflava YIM 13062T. Strain M5W7-7T grew at 10-37 °C (optimum, 37 °C), pH6.0-11.0 (pH 6.0-7.0) and in the presence of 0-7 % (w/v) NaCl (0 %). Substrate mycelia and aerial mycelia were not formed, and diffusible pigments were not observed on any media tested. Strain M5W7-7T contained MK-6(H2) and MK-7(H2) as the dominant menaquinones. The polar lipid profile of strain M5W7-7T contained diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids, an unidentified phospholipid and an unidentified lipid. The predominant whole-cell sugars were galactose and glucose. The predominant fatty acid was anteiso-C15 : 0. The DNA G+C content of strain M5W7-7T was 67.0 mol%. On the basis of phylogenetic relationships, phenotypic characterization and chemotaxonomic analyses, strain M5W7-7T represents a novel species of the genus Kocuria, for which the name Kocuriasoli sp. nov. is proposed. The type strain is M5W7-7T (=KCTC 49195T =CGMCC 1.13744T).

Brachybacterium endophyticum sp. nov., a novel endophytic actinobacterium isolated from bark of Scutellaria baicalensis Georgi.

A Gram-positive, aerobic, coccus-shaped, non-spore-forming actinobacterium, designated strain M1HQ-2T, was isolated from a surface-sterilized bark of Scutellaria baicalensis Georgi collected from Guizhou, China and tested using a polyphasic approach to determine its taxonomic position. Strain M1HQ-2T grew at 4-37 °C (optimum, 30 °C), pH 5.0-11.0 (pH 8.0) and in the presence of 0-15 % (w/v) NaCl (1-3 %). Substrate mycelia and aerial mycelia were not formed, and diffusible pigments were not observed on any media tested. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain M1HQ-2T belonged to the genus Brachybacterium and had the highest 16S rRNA gene sequence similarity of 97.6 % to Brachybacteriumsquillarum M-6-3T. Strain M1HQ-2T contained MK-7 as the dominant menaquinone. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The polar lipids profile of strain M1HQ-2T contained diphosphatidylglycerol, phosphatidylglycerol, an unidentified phospholipid and an unidentified lipid. The predominant fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content of strain M1HQ-2T was 71.0 mol%. The average nucleotide identity value between strain M1HQ-2T and type strain of Brachybacterium sacelli was 76.7 %. The estimated DNA-DNA hybridization value between strain M1HQ-2T and type strain of B. sacelli was 20.6 %. On the basis of phylogenetic analysis, chemotaxonomic characteristics and phenotypic data, strain M1HQ-2T represents a novel species of the genus Brachybacterium, for which the name Brachybacteriumendophyticum sp. nov. is proposed. The type strain is M1HQ-2T (=KCTC 49087T=CGMCC 1.16391T).

Different Characteristics of Hepcidin Expression in IL-6+/+ and IL-6-/- Neurons and Astrocytes Treated with Lipopolysaccharides.

A region-specific regulation of inflammation on the expression hepcidin in the brain has been demonstrated, however, it remains unknown whether there is also a cell-specific regulation of inflammation on hepcidin in the brain. Here, we investigated the effects of lipopolysaccharides (LPS) on the expression of hepcidin mRNA and also the expression of IL-6 mRNA, the phosphorylation of STAT3 and the expression of ferroportin 1 (Fpn1) and ferritin light chain (Ft-L) proteins in neurons and astrocytes obtained from wild type (IL-6+/+) and IL-6 knockout (IL-6-/-) mice. We demonstrated that the responses of the expression of hepcidin and IL-6 mRNAs, the phosphorylation of STAT3, and the expression of Fpn1 protein to LPS in IL-6+/+ astrocytes and also the responses of the expression of hepcidin mRNA, the phosphorylation of STAT3 and the expression of Fpn1 protein to IL-6 in IL-6-/- astrocytes were much stronger than those in IL-6+/+ and IL-6-/- neurons. A significant increase in Ft-L was found in LPS-treated IL-6+/+ and IL-6-treated IL-6-/- astrocytes, but not in LPS-treated IL-6+/+ and IL-6-treated IL-6-/- neurons. Our findings provide in vitro evidence for the existence of a cell-specific regulation of LPS on the expression of hepcidin and also Ft-L in the brain.

[Consumptions of Meat, Dietary Fat, and Fatty-acids and Prevalence of Overweight/Obesity in Children and Adolescents-a Cross-sectional Survey in Chengdu].

OBJECTIVES: To determine the prevalence of overweight and obesity in children and adolescents aged 7-15 years in Longquanyi District of Chengdu and its association with consumptions of meat, dietary fat and fatty-acids. METHODS: A total of 1 811 children and adolescents aged 7-15 years in Longquanyi District were selected using stratified cluster sampling strategy. Their body mass, height and waist circumference were measured. The prevalence of overweight/obesity was estimated based on body mass index (BMI), body mass index standard deviation score (BMI SDS), and waist-to-height ratio (WHtR). Daily consumptions of meat, dietary fat and fatty-acids were calculated using data collected through a food frequency questionnaire and 3-d 24 h dietary recall. The children with overweight/obesity were compared with those with normal body mass in food/nutrient consumptions using Wilcoxon tests. The BMI SDS, WHtR, and prevalence of overweight and obesity were also compared between those having low, moderate and high food/nutrient consumptions using Chi-square tests or Kruskal-Wallis tests. RESULTS: About 10.34% and 6.59% of participants were found to be overweight and obese, respectively. Boys had higher prevalence of overweight (12.05%) and overweight/obesity (18.97%) than girls (8.55%, 14.80%) ( P<0.05). Girls consumed more meat (including red meat and white meat), saturated fatty-acid (SFA) and monounsaturated fatty-acid (MUFA) than boys ( P<0.05). The consumptions of meat (both red meat and white meat), SFA and MUFA increased with age ( P<0.05). Overweight/obese girls consumed more SFA, MUFA and fat (%EN) than those of normal weight. The BMI SDS and WHtR of girls increased with fat (%EN) consumptions ( P<0.05). The BMI SDS of girls also increased with MUFA consumptions ( P<0.05). CONCLUSIONS: Consumptions of red meat, dietary fat, SFA, and MUFA are associated with overweight/obesity of girls aged 7-15 years in Chengdu. Further studies are needed to understand the gender differences.

[Dietary Fiber and Pubertal Development among Children and Adolescents--a Cross-sectional Study in Chengdu, Sichuan].

OBJECTIVE: To determine the association between intake of dietary fiber and pubertal development among children and adolescents in Chengdu. METHODS: A cross-sectional survey was undertaken in 1 340 children and adolescents aged 9-15 years. Data about dietary intake were collected through 24-h dietary self-recall. Pubertal development was measured by trained investigators using Tanner criteria. Consumptions of total fiber and fiber from different sources were compared among the participants with different stages of pubertal development. RESULTS: Data from 1 328 children and adolescents were analyzed. Boys (n = 667) at a later stage of pubertal development consumed less total fiber and fruit fiber than those at an earlier stage (P < 0.05). Similarly, girls (n = 651) at a later stage of pubertal development consumed less fruit fiber than those at an earlier stage (P < 0.05). CONCLUSION: Dietary fiber intake, especially fruit fiber, is lower in children and adolescents with early commencement of puberty development. Further studies are needed to establish the relationship between dietary fiber and pubertal development.

[Consumptions of Meat and Dairy Products, Zinc Intake and Pubertal Development in Adolescents in Chengdu].

OBJECTIVE: To determine the associations between meat, dairy and zinc intake and pubertal development in adolescents in Chengdu. METHODS: A total of 1320 children and adolescents aged 9-15 years in Chengdu were recruited using a stratified cluster sampling strategy. Dietary intake was assessed by the food frequency questionnaire (FFQ) and 3-day 24-hour dietary recall. Pubertal development was evaluated through physical examinations. Consumptions of meat and dairy, and zinc intake were compared between groups with different levels of pubertal development according to the Tanner criteria. RESULTS: The median age of spermarche was 13. 00 years. The boys who had had spermarche consumed more meat (including red meat) and dairy products than those who had not yet (P<0. 05). Daily consumption of total meat was positively correlated with the level of pubertal development (P<0. 05). The median age of menarche was 12. 11 years. The girls who had had menarche consumed more meat and less diiry products than those who had not yet (P<0. 05). Daily consumption of dairy products was negatively associated with breast development and the level of pubertal development (P < 0. 05). CONCLUSION: Consumptions of meat, red meat and dairy products are associated with pubertal development in adolescents in Chengdu. However, the differences between boys and girls warrant further studies.