Inhibition of SOCS3 signaling in the nucleus tractus solitarii and retrotrapezoid nucleus alleviates hypoventilation in diet-induced obese male mice.

The central leptin signaling system has been found to facilitate breathing and is linked to obesity-related hypoventilation. Activation of leptin signaling in the nucleus tractus solitarii (NTS) and retrotrapezoid nucleus (RTN) enhances respiratory drive. In this study, we investigated how medullary leptin signaling contributes to hypoventilation and whether respective deletion of SOCS3 in the NTS and RTN could mitigate hypoventilation in diet-induced obesity (DIO) male mice. Our findings revealed a decrease in the number of CO2-activated NTS neurons and downregulation of acid-sensing ion channels in DIO mice compared to lean control mice. Moreover, NTS leptin signaling was disrupted, as evidenced by the downregulation of phosphorylated STAT3 and the upregulation of SOCS3 in DIO mice. Importantly, deleting SOCS3 in the NTS and RTN significantly improved the diminished hypercapnic ventilatory response in DIO mice. In conclusion, our study suggests that disrupted medullary leptin signaling contributes to obesity-related hypoventilation, and inhibiting the upregulated SOCS3 in the NTS and RTN can alleviate this condition.

Whole-brain monosynaptic inputs and outputs of leptin receptor b neurons of the nucleus tractus solitarii in mice.

The nucleus tractus solitarii (NTS) is the primary central station that integrates visceral afferent information and regulates respiratory, gastrointestinal, cardiovascular, and other physiological functions. Leptin receptor b (LepRb)-expressing neurons of the NTS (NTSLepRb neurons) are implicated in central respiration regulation, respiratory facilitation, and respiratory drive enhancement. Furthermore, LepRb dysfunction is involved in obesity, insulin resistance, and sleep-disordered breathing. However, the monosynaptic inputs and outputs of NTSLepRb neurons in whole-brain mapping remain to be elucidated. Therefore, the exploration of its whole-brain connection system may provide strong support for comprehensively understanding the physiological and pathological functions of NTSLepRb neurons. In the present study, we used a cell type-specific, modified rabies virus and adeno-associated virus with the Cre-loxp system to map monosynaptic inputs and outputs of NTSLepRb neurons in LepRb-Cre mice. The results showed that NTSLepRb neurons received inputs from 48 nuclei in the whole brain from five brain regions, including especially the medulla. We found that NTSLepRb neurons received inputs from nuclei associated with respiration, such as the pre-Bötzinger complex, ambiguus nucleus, and parabrachial nucleus. Interestingly, some brain areas related to cardiovascular regulation-i.e., the ventrolateral periaqueductal gray and locus coeruleus-also sent a small number of inputs to NTSLepRb neurons. In addition, anterograde tracing results demonstrated that NTSLepRb neurons sent efferent projections to 15 nuclei, including the dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus, which are involved in regulation of energy metabolism and feeding behaviors. Quantitative statistical analysis revealed that the inputs of the whole brain to NTSLepRb neurons were significantly greater than the outputs. Our study comprehensively revealed neuronal connections of NTSLepRb neurons in the whole brain and provided a neuroanatomical basis for further research on physiological and pathological functions of NTSLepRb neurons.

Clinical and Imaging Characteristics of Primary Severe Community-Acquired Pneumonia Caused by Hypervirulent Klebsiella Pneumoniae.

BACKGROUND: To investigate the CT imaging features and microbial phenotypes of primary severe community-acquired pneumonia caused by hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Patients diagnosed with primary hvKp pneumonia were included, and their clinical data were analyzed, including the baseline characteristics and CT imaging results. After hypermucoviscosity phenotyping, the strains, serological types, and virulence genes of hvKp were identified using multiplex PCR. RESULTS: Twelve patients with primary hvKp pneumonia were included (11 males, 1 female). All patients were infected via respiratory tract inhalation. Ten patients were long-term drinkers. Four patients (33.3%), who were long-term alcohol abusers, died within 30 days after diagnosis. No extrapulmonary metastatic infection was found in any patient. The imaging of lung lesions at the early disease stage exhibited an extensive consolidation in the lungs. As the disease progressed, the most common imaging features were pleural effusion (9/12), cavitation and necrosis (8/12), and pneumothorax (3/12). The serological typing of the capsular polysaccharides on hvKp strains were K1 (6/12) and K2 (6/12). Furthermore, the virulence genotyping showed rmpA (11/12), magA (11/12), ureA (12/12), mrkD (12/12), fim-1 (12/12), wabG (12/12), ybtS (12/12), and iucB (11/12). CONCLUSIONS: Primary severe community-acquired hvKp-associated pneumonia is more common in men, especially those with a long-term history of alcohol consumption. CT scanning at the early disease stage mostly showed extensive pulmonary consolidation, which was prone to be combined with cavitation, necrosis, and pleural effusion. K1 and K2 serotypes were identified among the hvKp strains, which were not prone to form extrapulmonary metastasis via the bloodstream.

Immunological microenvironment alterations in follicles of women with proven severe endometriosis undergoing in vitro fertilization.

The purpose of this study was to test the hypothesis that different cytokine profiles may exist in the follicular fluid of endometriosis (EM) patients undergoing in vitro fertilization (IVF), as these differences may provide insights into the pathogenesis of the disease. This was a cross-sectional study conducted at the reproductive center of a medical university hospital. The study included 49 patients receiving IVF. 20 infertile women with proven EM and 29 women without diagnosed EM (control group) were evaluated. Follicular fluid (FF) and serum were collected at the time of follicle aspiration and the concentrations of 38 cytokines were determined by multiplexed immunoassay. The results indicated that the levels of IL-4, IL-13, IL-3 and IL-1α were significantly increased in the FF of women with EM, while levels of IFN-γ, IL-17A, MDC and MIP-1α were decreased compared with in the control subjects. In conclusions, the immune microenvironment of the FF in patients with EM is altered. This may contribute to the pathologic mechanism responsible for the poor outcome of IVF in patients with EM.

lncRNA FLVCR1-AS1 silencing inhibits lung cancer cell proliferation, migration, and invasion by inhibiting the activity of the Wnt/ß-catenin signaling pathway.

Long noncoding RNAs have been reported to be essential regulators in several human diseases, including tumorigenesis. A recent report revealed that FLVCR1-AS1 promotes the progression of hepatocellular carcinoma. However, whether FLVCR1-AS1 is involved in lung cancer remains unclear. In this study, we found that the expression of FLVCR1-AS1 was increased in lung cancer tissues according to The Cancer Genome Atlas database. Similarly, FLVCR1-AS1 was significantly upregulated in lung cancer cell lines. Knockdown of FLVCR1-AS1 dramatically reduced the cell proliferation, migration, and invasion of SPCA1 and A549. Mechanistically, we found that the expression levels of CTNNB1, SOX4, CCND1, CCND2, c-MYC, as well as nucleus ß-catenin were decreased in lung cancer cells after FLVCR1-AS1 silencing. Thus, FLVCR1-AS1 positively regulates the activation of the Wnt/ß-catenin pathway. Overexpression of CTNNB1 reversed the effect of FLVCR1-AS1 knockdown on A549 cells. In sum, FLVCR1-AS1 silencing inhibited the proliferation, migration, and invasion of lung cancer cells by inhibiting the activity of the Wnt/ß-catenin signaling pathway.

Expression of Bcl-2 and Bad in hippocampus of status epileptic rats and molecular mechanism of intervened recombinant human erythropoietin.

Injury of hippocampal neurons in status epilepticus (SE) SD rats kindled by pentylenetetrazol (PTZ) were studied, and the changes of apoptosis neurons, protein expression of Bad and Bcl-2 alone and combined application of phosphatidyl inositol 3-kinase (PI3K) inhibitor LY294002 and recombinant human erythropoietin (rHuEpo) were evaluated for the possible mechanisms of rHuEpo. The SE rats kindled by the PTZ were randomly divided into normal control group [normal saline (NS)], model group (PTZ + NS), rHuEpo treated group (PTZ + rHuEpo), LY294002 treated group (PTZ + LY294002 + rHuEpo) and LY294002 control group (rHuEpo + PTZ + DMSO). Apoptosis of hippocampal neurons was detected by TUNEL method; expression of phosphorylation protein kinase B (p-PKB/p-Akt), Bcl-2 and Bad were detected by immunohistochemistry; the expression of Bcl-2 mRNA, Bad mRNA in hippocampal neurons of rats were detected through reverse transcription polymerase chain reaction (RT-PCR); the expression of Akt, p-Akt and Bcl-2, Bad protein in hippocampal neurons of rats were detected by western blotting. The amount of apoptotic neurons was less in the rHuEpo treated group and the LY294002 control group than in the LY294002 treated group (P<0.05). The expression of p-Akt protein and Bcl-2 protein increased while the Bad protein decreased significantly in the rHuEpo treated group and the LY294002 control group compared with the LY294002 treated group (P<0.05). The expression of Bad protein and Bad mRNA in hippocampus increased while the p-Akt, Bcl-2, Bcl-2 mRNA decreased significantly in the LY294002 treated group compared with the rHuEpo treated group (P<0.05). The PI3K/Akt signaling pathway is one of the pathways of rHuEpo neuroprotective effects and was confirmed from both the of positive and negative aspects. rHuEpo regulates the expression of mitochondrial apoptotic pathway related factors Bad and Bcl-2 to inhibit apoptosis and promotes neuronal survival.

The role of mmu-miR-155-5p-NF-κB signaling in the education of bone marrow-derived mesenchymal stem cells by gastric cancer cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are important precursors of tumor stromal cells. Previously, we have demonstrated that miR-155-5p inhibition directly induced transition of BM-MSCs into gastric cancer-associated MSCs. Whether miR-155-5p is involved in the education of BM-MSCs by gastric cancer cells has not been established. Murine BM-MSCs (mMSCs) were isolated and grown in conditioned medium derived from gastric cancer cell line MFC (MFC-CM). The tumor-promoting phenotype and function of mMSCs were detected by immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cell colony formation assay, transwell migration, and invasion assays. Luciferase reporter assays and western blot analyses were conducted to reveal the relationship between nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 and mmu-miR-155-5p. miRNA mimics, inhibitor, and the NF-κB inhibitor pyrrolidine dithiocarbamic acid (PDTC) were used to evaluate the role of miR-155-5p-NF-κB signaling in the education of mMSCs by MFC-CM. We successfully established the education model of mMSCs by MFC-CM and found that mmu-miR-155-5p expression levels were reduced in mMSCs. Mimicking this deregulation by transfecting miRNA inhibitor into mMSCs produced a similar effect as that of MFC-CM on mMSCs. NF-κB p65 was validated as a target of mmu-miR-155-5p, which also negatively regulated NF-κB activation. Inhibition of NF-κB activation by PDTC abolished the effect of the miRNA inhibitor on mMSCs. mmu-miR-155-5p overexpression partially blocked the effect of MFC-CM in educating mMSCs, while PDTC treatment completely eliminated MFC-CM activity. These results indicate that miR-155-5p is not the sole miRNA mediating the education of BM-MSCs by gastric cancer cells, but downstream NF-κB signaling is indispensable for this process.

miR-155-5p inhibition promotes the transition of bone marrow mesenchymal stem cells to gastric cancer tissue derived MSC-like cells via NF-κB p65 activation.

Gastric cancer tissue-derived MSC-like cells (GC-MSC) share similar characteristics to bone marrow MSC (BM-MSC); however, the phenotypical and functional differences and the molecular mechanism of transition between the two cell types remain unclear. Compared to BM-MSC, GC-MSC exhibited the classic phenotype of reactive stroma cells, a stronger gastric cancer promoting capacity and lower expression of miR-155-5p. Inhibition of miR-155-5p by transfecting miRNA inhibitor induced a phenotypical and functional transition of BM-MSC into GC-MSC-like cells, and the reverse experiment deprived GC-MSC of tumor-promoting phenotype and function. NF-kappa B p65 (NF-κB p65) and inhibitor of NF-kappa B kinase subunit epsilon (IKBKE/IKKε) were identified as targets of miR-155-5p and important for miRNA inhibitor activating NF-κB p65 in the transition. Inactivation of NF-κB by pyrrolidine dithiocarbamic acid (PDTC) significantly blocked the effect of miR-155-5p inhibitor on BM-MSC. IKBKE, NF-κB p65 and phospho-NF-κB p65 proteins were highly enriched in MSC-like cells of gastric cancer tissues, and the latter two were correlated with the pathological progression of gastric cancer. In GC-MSC, the expression of miR-155-5p was downregulated and NF-κB p65 protein was increased and activated. NF-κB inactivation by PDTC or knockdown of its downstream cytokines reversed the phenotype and function of GC-MSC. Taken together, our findings revealed that miR-155-5p downregulation induces BM-MSC to acquire a GC-MSC-like phenotype and function depending on NF-κB p65 activation, which suggests a novel mechanism underlying the cancer associated MSC remodeling in the tumor microenvironment and offers an effective target and approach for gastric cancer therapy.

N-methyl-N-nitro-N'-nitrosoguanidine induces the expression of CCR2 in human gastric epithelial cells promoting CCL2-mediated migration.

Chronic inflammation has a decisive role in tumorigenesis, particularly in gastric carcinogenesis. The CC chemokine ligand 2 (CCL2), an important inflammatory cytokine, is involved in the initiation, development and progression of various types of cancer. However, the role of CCL2 in gastric cancer remains to be elucidated. The present study demonstrated that recombinant CCL2 stimulation caused no effect on the morphology, proliferation and migration of human GES-1 gastric mucosa epithelial cells, in which the protein expression of CC-chemokine receptor 2 (CCR2) was markedly low. However, the expression of CCR2 was significantly upregulated in the GES-1 cells following pretreatment with the chemical carcinogen, N-methyl-N-nitro-N'-nitrosoguanidine (MNNG), for 12 h or transformed with MNNG (MC cells). The present study used CCL2 to stimulate MNNG pretreated GES-1 cells and MC cells, and demonstrated that CCL2 clearly promoted their migration and the epithelial-mesenchymal transition (EMT). However, no effect was observed on the proliferative ability of the cells. Taken together, these findings suggested that the CCL2/CCR2 chemokine signaling may regulate the EMT in gastric epithelial cells and resulted in gastric carcinogenesis in response to the intake of the carcinogen, MNNG.

Pre-treatment of human umbilical cord-derived mesenchymal stem cells with interleukin-6 abolishes their growth-promoting effect on gastric cancer cells.

The inflammatory microenvironment contributes to cancer development and progression. Mesenchymal stem cells (MSCs), as important stromal cells, may be 'educated' by the inflammatory microenvironment to support the development of gastric cancer. Cytokines are a key component of cancer-related inflammation. Interleukin (IL)-6, as an inflammatory cytokine, has multiple roles in cancer. However, whether MSCs can be 'educated' by IL-6 to support gastric cancer remains unknown. In the present study, we focused on the phenotype and function of human umbilical cord-derived MSCs hUC­MSCs pre-treated with IL-6 in gastric cancer. We found that the protein levels of α-smooth muscle actin (α-SMA) were upregulated, and phosphorylated nuclear factor (NF)-κB protein levels were downregulated in the hUC­MSCs pre-treated with IL-6, as shown by western blot analysis. The levels of tumor­promoting cytokines, including chemokine (C-C motif) ligand 5 (CCL5), platelet-derived growth factor­BB (PDGF­BB), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α(TNFα), were markedly reduced in the hUC­MSCs following treatment with IL-6, as shown by RT-qPCR. In in vitro experiments, we co-cultured MSCs with N-methyl­N'­nitro­N­nitrosoguanidine (MNNG)­transformed GES-1 gastric epithelial cells or SGC-7901 gastric cancer cells. Transwell and colony-forming cell assays revealed that the hUC-MSCs significantly promoted gastric cellular migration and proliferation. However, following treatment with IL-6, the hUC-MSCs had no growth-promoting effect on the gastric epithelial cells and gastric cancer cells. In in vivo experiments, we co-transplanted MSCs and SGC-7901 cells into nude mice in order to establish a nude mouse model of gastric cancer. The hUC-MSCs significantly promoted the growth gastric tumors through the promotion of cell proliferation and the inhibition of cell apoptosis. On the contrary, pre-treatment with IL-6 provided the hUC­MSCs with the ability to inhibit cell proliferation and significantly induce cell apoptosis. Taken together, our findings indicate that pre-treatment with IL-6 significantly abolishes the ability of hUC-MSCs to promote gastric epithelial cell proliferation and migration and provide new insight into the effects of the inflammatory cytokine, IL-6, on the tumor-promoting ability of MSCs and its role in gastric cancer.

Antitumour activities of sesquiterpene lactones from Inula helenium and Inula japonica.

Eight sesquiterpene lactones were isolated from the roots of Inula helenium and flowers of I. japonica. Among them, isoalantolactone (3) and santamarine (6) exhibited significant growth inhibitory activities against gynecologic cancer cell lines, while others weakly inhibited the growth of the cell lines (IC50 < or = 100 microM). In addition, 3 significantly inhibited the tumour growth of S180 tumour-bearing mice. Compounds 3 and 6 were not toxic to human embryonic lung fibroblast cells in vitro. These results demonstrated that the antitumour activities are closely related to the structures of the compounds, that is, an alpha-exomethylene-gamma-lactone ring is necessary for these activities.