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With the aging of the population and the extension of life expectancy, osteoporosis is becoming a global epidemic. Although there are several drugs used to treat osteoporosis in clinical practice, such as parathyroid hormone or bisphosphonates, they all have some serious side effects. Therefore, a safer drug is called for osteoporosis, especially for the prevention in the early stage of the disease, not only the treatment in the later stage. Panax notoginseng saponin (PNS), a traditional Chinese herb, has been used as anti-ischemic drug due to its function on improving vascular circulation. In order to verify whether Panax notoginseng saponins (PNS) could be used to prevent osteoporosis, ovariectomy (OVX) was induced in female C57BL/C6J mice, followed by orally administration with 40 mg/kg/d, 80 mg/kg/d, and 160 mg/kg/d of three different dosages of PNS for 9 weeks. Serum biochemical analysis, micro-CT, histological evaluation, and immunostaining of markers of osteogenesis and angiogenesis were performed in the sham, osteoporotic (OVX), and treatment (OVX+PNS) groups. Micro-CT and histological evaluation showed that compared to sham group, the bone mass of OVX group reduced significantly, while it was significantly restored in the moderate-dose PNS (40 mg/kg and 80 mg/kg) treatment groups. The expression of CD31 and osteocalcin (OCN) in the bone tissue of treatment group also increased, suggesting that PNS activated osteogenesis and angiogenesis, which subsequently increased the bone mass. These results confirmed the potential function of PNS on the prevention of osteoporosis. However, in the high dose of PNS (160 mg/kg) group, the antiosteoportic effect had been eliminated, which also suggested the importance of proper dose of PNS for the prevention and treatment of osteoporosis in postmenopausal women. [ABSTRACT FROM AUTHOR]

Although hepatitis B virus (HBV)-related cirrhosis and hepatocellular carcinoma (HCC) cause a sever health problem worldwide, the underlying mechanisms are still elusive. This study aimed to investigate the responses of different cell types isolated from HBV transgenic mice. A cross-sectional set of hepatocytes and oval cells were obtained from HBV transgenic and control mice. Flow cytometry, immunohistochemistry and microarray were applied to investigate the cell biology of the hepatocytes and oval cells. Our results showed that HBV induced the proliferation of both cell oval cells and hepatocytes, and induced cell death of HBV hepatocytes while had minimal effects on oval cells. Further molecular and pathways analysis identified some genes and signaling pathways may be responsible for the different responses between oval cells and hepatocytes. In addition, analyses of selectively ten genes by IHC staining in human samples were consistent with microarray data. In summary, HBV transgenic mice is a useful model for studying the biological behaviors of oval cells affected by HBV and HBV-cirrhosis. Also, our results help better understand the mechanisms of HBV induced cirrhosis, and provide novel progenitor markers or prognostic/therapeutic markers. [ABSTRACT FROM AUTHOR]

microRNA (miRNA) play important roles in drug addiction and act as a post-transcriptional regulator of gene expression. We previously reported extensive downregulation of miRNAs in the nucleus accumbens (NAc) of methamphetamine (METH)-sensitized mice. However, the regulatory mechanism of this METH-induced downregulation of miRNAs has yet to be elucidated. Thus, we examined METH-induced changes in the expression of miRNAs and their precursors, as well as the expression levels of mRNA and the proteins involved in miRNA biogenesis such as Dicer1 and Ago2, in the nucleus accumbens of METH-induced locomotor sensitized mice. miRNAs and Ago2 were significantly downregulated, while the expression of miRNA precursors remained unchanged or upregulated, which suggests that the downregulation of miRNAs was likely due to a reduction in Ago2-mediated splicing but unlikely to be regulated at the transcription level. Interestingly, the expression level of Dicer1, which is a potential target of METH-induced decreased miRNAs, such as miR-124, miR-212 and miR-29b, was significantly increased. In conclusion, this study indicates that miRNA biogenesis (such as Ago2 and Dicer1) and their miRNA products may have a role in the development of METH addiction. [ABSTRACT FROM AUTHOR]

Numerous evidence indicates that inflammation in adipose tissue is the primary cause of systemic insulin resistance induced by obesity. Obesity-associated changes in circulating LPS level and hypoxia/HIF-1α activation have been proposed to be involved in boosting obesity-induced inflammation. However, there is poor understanding of what triggers obesity-induced inflammation. In this study, we pinpoint lactate as a key trigger to mediate obesity-induced inflammation and systemic insulin resistance. Specific deletion of Slc16a1 that encodes MCT1, the primary lactate transporter in adipose tissues, robustly elevates blood levels of proinflammatory cytokines and aggravates systemic insulin resistance without alteration of adiposity in mice fed high-fat diet. Slc16a1 deletion in adipocytes elevates intracellular lactate level while reducing circulating lactate concentration. Mechanistically, lactate retention due to Slc16a1 deletion initiates adipocyte apoptosis and cytokine release. The locally recruited macrophages amplify the inflammation by release of proinflammatory cytokines to the circulation, leading to insulin resistance in peripheral tissues. This study, therefore, indicates that lactate within adipocytes has a key biological function linking obesity to insulin resistance, and harnessing lactate in adipocytes can be a promising strategy to break this link. [ABSTRACT FROM AUTHOR]

Choline metabolite trimethylamine N-oxide (TMAO) has been recognized as a risk factor of gestational diabetes mellitus (GDM), but its exact role in GDM has not been reported. In this study, we focused on the placenta development to reveal the role of TMAO in GDM. We found that the TMAO levels in peripheral and cord plasma were increased in women with GDM and that TMAO levels were positively correlated with newborn weight and placental thickness. Neutrophil extracellular traps (NETs) in the peripheral and cord plasma and the myeloperoxidase expression in the placenta of women with GDM also increased. NETs could inhibit the proliferation, migration, invasion, and angiogenesis of HTR-8/Svneo cells. However, TMAO not only could inhibit the formation of NETs but also could enhance the biological function of HTR-8/Svneo cells. With induction of GDM in NETs-deficient PAD4-/- and wild-type mice, the placental weight of PAD4-/- mice increased significantly. TMAO feeding also inhibited the formation of NETs and further increased the weight of the placenta and fetuses, and this increase did not affect the placental structure. Our data indicate that higher TMAO levels and the formation of abnormal NETs were associated with GDM. TMAO not only could promote the development of the placenta and fetuses but also could inhibit the formation of NETs. [ABSTRACT FROM AUTHOR]

The unc-13 homolog B (UNC13B) gene encodes a presynaptic protein, mammalian uncoordinated 13-2 (Munc13-2), which is highly expressed in the brain-predominantly in the cerebral cortex-and plays an essential role in synaptic vesicle priming and fusion, potentially affecting neuronal excitability. However, the functional significance of the UNC13B mutation in human disease is not known. In this study, we screened for novel genetic variants in a cohort of 446 unrelated cases (families) with partial epilepsy without acquired causes by trio-based whole-exome sequencing. UNC13B variants were identified in 12 individuals affected by partial epilepsy and/or febrile seizures from eight unrelated families. The eight probands all had focal seizures and focal discharges in EEG recordings, including two patients who experienced frequent daily seizures and one who showed abnormalities in the hippocampus by brain MRI; however, all of the patients showed a favourable outcome without intellectual or developmental abnormalities. The identified UNC13B variants included one nonsense variant, two variants at or around a splice site, one compound heterozygous missense variant and four missense variants that cosegregated in the families. The frequency of UNC13B variants identified in the present study was significantly higher than that in a control cohort of Han Chinese and controls of the East Asian and all populations in the Genome Aggregation Database (gnomAD). Computational modelling, including hydrogen bond and docking analyses, suggested that the variants lead to functional impairment. In Drosophila, seizure rate and duration were increased by Unc13b knockdown compared to wild-type flies, but these effects were less pronounced than in sodium voltage-gated channel alpha subunit 1 (Scn1a) knockdown Drosophila. Electrophysiological recordings showed that excitatory neurons in Unc13b-deficient flies exhibited increased excitability. These results indicate that UNC13B is potentially associated with epilepsy. The frequent daily seizures and hippocampal abnormalities but ultimately favourable outcome under anti-epileptic therapy in our patients indicate that partial epilepsy caused by UNC13B variant is a clinically manageable condition. [ABSTRACT FROM AUTHOR]

Background: Immunoglobulin E (IgE) belongs to a class of immunoglobulins involved in immune response to specific allergens. However, the roles of IgE and IgE receptor (FcεR1) in pathological cardiac remodeling and heart failure are unknown.Methods: Serum IgE levels and cardiac FcεR1 expression were assessed in diseased hearts from human and mouse. The role of FcεR1 signaling in pathological cardiac remodeling was explored in vivo by FcεR1 genetic depletion, anti-IgE antibodies, and bone marrow transplantation. The roles of the IgE-FcεR1 pathway were further evaluated in vitro in primary cultured rat cardiomyocytes and cardiac fibroblasts (CFs). RNA sequencing and bioinformatic analyses were used to identify biochemical changes and signaling pathways that are regulated by IgE/FcεR1.Results: Serum IgE levels were significantly elevated in patients with heart failure as well as in 2 mouse cardiac disease models induced by chronic pressure overload via transverse aortic constriction and chronic angiotensin II infusion. Interestingly, FcεR1 expression levels were also significantly upregulated in failing hearts from human and mouse. Blockade of the IgE-FcεR1 pathway by FcεR1 knockout alleviated transverse aortic constriction- or angiotensin II-induced pathological cardiac remodeling or dysfunction. Anti-IgE antibodies (including the clinical drug omalizumab) also significantly alleviated angiotensin II-induced cardiac remodeling. Bone marrow transplantation experiments indicated that IgE-induced cardiac remodeling was mediated through non-bone marrow-derived cells. FcεR1 was found to be expressed in both cardiomyocytes and CFs. In cultured rat cardiomyocytes, IgE-induced cardiomyocyte hypertrophy and hypertrophic marker expression were abolished by depleting FcεR1. In cultured rat CFs, IgE-induced CF activation and matrix protein production were also blocked by FcεR1 deficiency. RNA sequencing and signaling pathway analyses revealed that transforming growth factor-β may be a critical mediator, and blocking transforming growth factor-β indeed alleviated IgE-induced cardiomyocyte hypertrophy and cardiac fibroblast activation in vitro.Conclusions: Our findings suggest that IgE induction plays a causative role in pathological cardiac remodeling, at least partially via the activation of IgE-FcεR1 signaling in cardiomyocytes and CFs. Therapeutic strategies targeting the IgE-FcεR1 axis may be effective for managing IgE-mediated cardiac remodeling. [ABSTRACT FROM AUTHOR]

Objectives: To characterize the genetic environment of the carbapenem resistance determinant in Proteus vulgaris of swine origin.Methods: The carbapenem-resistant P. vulgaris strain BC22 was isolated from a faecal swab from a diseased pig with diarrhoea in Sichuan Province of China in 2018. The presence of carbapenemase genes was screened by PCR. WGS and bioinformatics analysis were performed to analyse the genetic environment of the carbapenem resistance determinant.Results: P. vulgaris strain BC22 was found to harbour the carbapenemase gene blaNDM-1. WGS data revealed that blaNDM-1 was located in a truncated ISAba125 composite transposon. The carbapenem resistance gene blaNDM-1 and 20 other resistance genes, including the multiresistance gene cfr and the bifunctional aminoglycoside/quinolone resistance gene aac(6')-lb-cr, were located in a novel SXT/R391 integrative and conjugative element (ICE). This new SXT/R391 ICE of 148.7 kb was chromosomally located, and could be transferred to Escherichia coli.Conclusions: Here, we report a carbapenemase gene, blaNDM-1, integrated into an SXT/R391 ICE. Our study highlights that this SXT/R391 ICE may facilitate the dissemination of clinically important resistance genes such as blaNDM-1, cfr and aac(6')-lb-cr. [ABSTRACT FROM AUTHOR]

Gastrointestinal cancer causes countless deaths every year due to therapeutic resistance. However, whether metabolic alterations contribute to chemoresistance is not well understood. In this study, we report that fatty acid (FA) catabolism was activated in gastrointestinal cancer cells treated with oxaliplatin, which exhibited higher expression of the rate-limiting enzymes carnitine palmitoyltransferase 1B (CPT1B) and CPT2. The clinical analysis also showed that high expression of these enzymes was associated with poor oxaliplatin-based chemotherapy outcomes in patients. Furthermore, genetic or pharmacological inhibition of CPT2 with perhexiline disturbed NADPH and redox homeostasis and increased reactive oxygen species (ROS) generation and cell apoptosis in gastrointestinal cancer cells following oxaliplatin treatment. Specifically, the combination of oxaliplatin and perhexiline significantly suppressed the progression of gastrointestinal cancer in cell-based xenograft and patient-derived xenograft (PDX) models. Mechanistically, CPT2 was transcriptionally upregulated by nuclear factor of activated T cells 3 (NFATc3), which translocated to the nucleus in response to oxaliplatin treatment. In summary, our study suggests that the inhibition of CPT-mediated FA catabolism combined with conventional chemotherapy is a promising therapeutic strategy for patients with gastrointestinal cancers. [ABSTRACT FROM AUTHOR]

Background: As a potential tumor suppressor gene, Claudin-7 (Cldn7), which is a component of tight junctions, may play an important role in colorectal cancer occurrence and development.Aims: To generate a knockout mouse model of inducible conditional Cldn7 in the intestine and analyze the phenotype of the mice after induction with tamoxifen.Methods: We constructed Cldn7-flox transgenic mice and crossed them with Villin-CreERT2 mice. The Cldn7 inducible conditional knockout mice appeared normal and were well developed at birth. We induced Cldn7 gene deletion by injecting different dosages of tamoxifen into the mice and then conducted a further phenotypic analysis.Results: After induction for 5 days in succession at a dose of 200 µl tamoxifen in sunflower oil at 10 mg/ml per mouse every time, the mice appeared dehydrated, had a lower temperature, and displayed inactivity or death. The results of hematoxylin-eosin staining showed that the intestines of the Cldn7 inducible conditional knockout mice had severe intestinal defects that included epithelial cell sloughing, necrosis, inflammation and hyperplasia. Owing to the death of ICKO mice, we adjusted the dose of tamoxifen to a dose of 100 µl in sunflower oil at 10 mg/ml per mouse (aged more than 8 weeks old) every 4 days. And we could induce atypical hyperplasia and adenoma in the intestine. Immunofluorescent staining indicated that the intestinal epithelial structure was destroyed. Electron microscopy experimental analysis indicated that the intercellular gap along the basolateral membrane of Cldn7 inducible conditional knockout mice in the intestine was increased and that contact between the cells and matrix was loosened.Conclusions: We generated a model of intestinal Cldn7 inducible conditional knockout mice. Intestinal Cldn7 deletion induced by tamoxifen initiated inflammation and hyperplasia in mice. [ABSTRACT FROM AUTHOR]

Abstract: Four new C18-diterpenoid alkaloids, weisaconitines A–D (1–4), were isolated from Aconitum weixiense. Based on extensive UV, IR, MS, 1D and 2D NMR analyses, their structures were elucidated as 8-O-ethyldolaconine (1), 4-demethylgenicunine B (2), 14-oxoaconosine (3), and 8-O-ethylaconosine (4). The analgesic activity of compound 4 was studied with CH3COOH-induced writhing model in mice. Compound 4 showed writhing inhibitions of 24% (50mg/kg), 26% (100mg/kg) and 34% (200mg/kg), respectively, as compared to the reference drug aspirin (63%) at a dose of 200mg/kg. [Copyright &y& Elsevier]

This study delineates a mechanism for antiapoptotic signaling initiated by atrial natriuretic peptide (ANP) stimulation leading to elevation of cGMP levels and subsequent nuclear accumulation of Akt kinase associated with zyxin, a cytoskeletal LIM-domain protein. Nuclear targeting of zyxin induces resistance to cell death coincident with nuclear accumulation of activated Akt. Nuclear translocation of zyxin triggered by cGMP also promotes nuclear Akt accumulation. Additional supportive evidence for nuclear accumulation of zyxin-enhancing cardiomyocyte survival includes the following: (a) promotion of zyxin nuclear localization by cardioprotective stimuli; (b) zyxin association with phospho-Akt473 induced by cardioprotective stimuli; and (c) recruitment of zyxin to the nucleus by activated nuclear-targeted Akt as well as recruitment of Akt by nuclear-targeted zyxin. Nuclear accumulation of zyxin requires both Akt activation and nuclear localization. Potentiation of cell survival is sensitive to stimulation intensity with high-level induction by ANP or cGMP signaling leading to apoptotic cell death rather than enhancing resistance to apoptotic stimuli. Myocardial nuclear accumulation of zyxin and Akt responds similarly in vivo following treatment of mice with ANP or cGMP. Thus, zyxin and activated Akt participate in a cGMP-dependent signaling cascade leading from ANP receptors to nuclear accumulation of both molecules. Nuclear accumulation of zyxin and activated Akt may represent a fundamental mechanism that facilitates nuclear-signal transduction and potentiates cell survival. [ABSTRACT FROM AUTHOR]

Background: Angiogenesis has a key role in numerous disease processes. One of the most important angiogenic factors is vascular endothelial growth factor (VEGF-A), whereas thrombospondin-1 (TSP-1) is a major antiangiogenic factor. Recent studies have shown that VEGF-A as well as TSP-1 is regulated by transforming growth factor-beta1 (TGF-beta1), but the mechanism remains unclear.Methods: We examined the role of TGF-beta1 and its signaling pathways in mediating expression of these two molecules. Rat proximal tubular cells (NRK52E) were stimulated with TGF-beta1 to induce VEGF-A and TSP-1 synthesis. To clarify roles of receptor-activated Smads (R-Smads), we blocked Smad signaling using overexpression of the inhibitory Smad, Smad7, and by using fibroblasts from wild-type or knockout mice. To confirm the antiantigenic role of Smads, soluble Flt-1 regulation in response to TGF-beta1 was also examined. In addition, the effect of conditioned media from NRK52E and Smad knockout cells was examined on endothelial cell proliferation.Results: Induction of VEGF-A and TSP-1 by TGF-beta1 in NRK52E cells was associated with activation of pathway-restricted R-Smads (Smad2 and 3) and blocking these Smads by overexpression of Smad7 blocked their induction. By using of Smad knockout cells, Smad3 was shown to have a key role in the stimulation of VEGF-A expression whereas Smad2 was critical for TSP-1 expression. Consistent with the hypothesis that Smad2 has an antiangiogenic function, we also demonstrated that Smad2, but not Smad3, mediated the expression of VEGF-A antagonist, soluble VEGF-A receptor sFlt-1, in response to TGF-beta1. Conditioned media from NRK52E, which was stimulated by TGF-beta1 for 24 hours, did not induce endothelial cell proliferation. However, conditioned media from Smad2 knockout induced endothelial cell proliferation, whereas endothelial cell proliferation was inhibited by Smad3 knockout-derived conditioned media.Conclusion: R-Smads have distinct roles in mediating the expression of pro- and antiangiogenic growth factors in response to TGF-beta1. [ABSTRACT FROM AUTHOR]

Dietary interventions such as fasting have been proved to be effective in the prevention of metabolic and autoimmune diseases as well as aging-related conditions. The complicated interaction between nutrition and immunity has drawn wide attention in recent years. In this study, we investigated the therapeutic effect of intermittent caloric restriction on autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, in mice. EAE was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein 35-55 peptide. After the EAE symptoms became obvious at the 4th week post-immunization, the mice were administered with a modified fasting-mimicking diet (FMD) at 1/3 cal of control for 3 days, followed by ad libitum with normal chow for 4 days. A total of two cycles of FMD was applied. Compared with the mice without receiving caloric restriction, the mice using FMD had significant decreases in EAE severity, immune cell infiltration in spinal cord and CNS demyelination. FMD administration also reversed EAE-mediated CNS accumulation of total CD4+ T cells and in particular, IFN-γ-producing CD4+ T cells. Moreover, FMD application elevated the cell proliferation rate in CNS and enhanced expression of brain-derived neurotrophic factor (BDNF) and remyelination markers. In conclusion, our results indicate that intermittent caloric restriction using the modified FMD was effective in the treatment of EAE through ameliorating inflammatory response and promoting recovery of the damaged tissue. [ABSTRACT FROM AUTHOR]

Background: Calorie restriction (CR) has been well proved to be a powerful tool to improve metabolic health associated with aging; and many types of CR have been proposed. Intermittent CR has become a trend in recent years due to its better compliance than continuous CR every day. However, there are few studies that directly compare the interventional activity of intermittent CR vs continuous CR in metabolic disorders such as diabetes. Methods: In this study, we analyzed two protocols of intermittent CR with the calorie-matched continuous CR in two diabetic mouse models including db/db and streptozotocin-treated mice. Intermittent CR was carried out by a fasting-mimicking diet (FMD, with 30% calorie intake of the control per day) for 2 days or 5 days (i.e., 2–5 or 5–9 regimes followed by free eating for 5 or 9 days respectively). Results: In the two diabetic mouse models, both intermittent CR and continuous CR significantly reduced fasting blood glucose level and improved insulin sensitivity. However, intermittent CR performed significantly better than continuous CR in improving glycemic control and insulin sensitivity in db/db mice. In addition, intermittent CR improved the glucose homeostasis of the db/db mice without causing loss of body weight. Analyses with the pancreatic islets reveal that intermittent CR profoundly elevated the number of insulin-positive cells in both diabetic mouse models. Conclusions: Our study indicated that both intermittent CR and continuous CR can lower fasting blood glucose level in the diabetic mice, while intermittent CR is better than the latter in improving glucose homeostasis in db/db mice. [ABSTRACT FROM AUTHOR]