Cannabinoid receptors promote chronic intermittent hypoxia-induced breast cancer metastasis via IGF-1R/AKT/GSK-3ß.

The progression of breast cancer is closely related to obstructive sleep apnea-hypopnea syndrome (OSAHS). Low concentrations of cannabinoids promote tumor proliferation. However, the role of cannabinoid receptors (CBs) in chronic intermittent hypoxia (CIH)-induced breast cancer has not been reported. The migration and invasion of breast cancer cell lines (MCF-7 and T47D) were measured by scratch assay and transwell assay. Gene and protein expressions were analyzed by qPCR and western blotting. Tumor xenograft mice model were established to evaluate the function of CBs. We observed that chronic hypoxia (CH) and CIH increased CBs expression and promoted migration and invasion in breast cancer. Mice grafted with MCF-7 exhibited obvious tumor growth, angiogenesis, and lung metastasis in CIH compared with CH and control. In addition, CIH induced CBs expression, which subsequently activated insulin-like growth factor-1 receptor (IGF-1R)/AKT/glycogen synthase kinase-3ß (GSK-3ß) axis. Knockdown of CBs alleviated CIH-induced migration and invasion of breast cancer in vitro. Furthermore, CIH exaggerated the malignancy of breast cancer and silencing of CBs suppressed tumor growth and metastasis in vivo. Our study contributed to understanding the role of CIH in breast cancer development modulation.

Four new species of Panophrys (Anura, Megophryidae) from eastern China, with discussion on the recognition of Panophrys as a distinct genus.

The diversity of Panophrys horned toads is considered highly underestimated with a large number of undescribed cryptic species. In this work, we describe four Panophrys species from eastern China which were proposed as cryptic species by molecular data in previous study, additionally provide new information on the biogeography of these four species. Panophrys daiyunensis sp. nov. from southern Fujian, Panophrys daoji sp. nov. from eastern Zhejiang, Panophrys sanmingensis sp. nov. from the hilly area among Fujian, Jiangxi and Guangdong, and Panophrys tongboensis sp. nov. from northeastern Jiangxi, can be distinguished from all recognized congeners by a combination of morphological characteristics. The descriptions of these four new species take the recognized species of Panophrys to 51, which is the largest genus within the Asian horned toads subfamily Megophryinae. Considered as an appropriate arrangement for the Asian horned toads currently and applied in this study to describe the new species, the generic recognition of Panophrys is also discussed.

Obstructive sleep apnea syndrome and causal relationship with female breast cancer: a mendelian randomization study.

Although observational studies have reported a positive association between obstructive sleep apnea syndrome (OSAS) and breast cancer (BC) risk, causality remains inconclusive. We aim to explore whether OSAS is associated with etiology of BC by conducting a two-sample Mendelian randomization (MR) study in a Chinese population and Asian population from the Breast Cancer Association Consortium (BCAC). We found a detrimental causal effect of OSAS on BC risk in the primary analysis of our samples (IVW OR, 2.47 for BC risk per log-odds increment in OSAS risk, 95% CI = 1.86-3.27; P = 3.6×10-10). This was very similar to results of the direct observational case-control study between OSAS and BC risk (OR = 2.80; 95% CI = 2.24-3.50; P =1.4×10-19). Replication in the Asian population of the BCAC study also supported our results (IVW OR, 1.33 for BC risk per log-odds increment in OSAS risk, 95% CI = 1.13-1.56; P = 0.0006). Sensitivity analyses confirmed the robustness of our findings. We provide novel evidence that genetically determined higher risk of OSAS has a causal effect on higher risk of BC. Further studies focused on the mechanisms of the relationship between OSAS and breast carcinogenesis are needed.

Toll-Like Receptor 4 (TLR-4) Pathway Promotes Pulmonary Inflammation in Chronic Intermittent Hypoxia-Induced Obstructive Sleep Apnea.

BACKGROUND Studies have shown that intermittent hypoxia mimics obstructive sleep apnea in causing pulmonary inflammation, but the mechanism is not yet clear.TLR-4 is a recognized proinflammatory factor, so the purpose of this study was to assess the function of TLR-4 in pulmonary inflammation induced by chronic intermittent hypoxia simulating obstructive sleep apnea. MATERIAL AND METHODS Healthy male Wistar rats were divided into 3 groups (8 in each group): the normoxia control group (CG), the intermittent hypoxia group (IH), and the TLR4 antagonist TAK242 treatment group (3 mg/kg, daily), with exposure durations of 12 weeks and 16 weeks (HI). The morphological changes of lung tissue were determined with hematoxylin-eosin (HE) staining. The expressions of the TLR-4 pathway in lung tissue were tested by Western blotting and RT-PCR. The levels of IL-6 and TNF-a in serum and lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). The levels of SOD and MDA in lung tissue were detected by use of SOD and MDA kits, respectively. RESULTS After TAK242 treatment, damage to lung tissue was increased, and the expressions of TLR-4, MYD88, P65, IL-6, TNF-α, MDA, and SOD were decreased. Intermittent hypoxic exposure caused alveolar expansion, thickening of alveolar septum, and fusion of adjacent alveoli into larger cysts under intermittent hypoxia in a time-dependent manner. Compared with the CG and HI groups, the mean lining interval (MLI) become more thickened and the alveolar destruction index (DI) increased significantly in the IH group. CONCLUSIONS Chronic intermittent hypoxia causes pulmonary inflammatory response and the inflammatory pathway involved in TLR4 receptor may be one of the mechanisms that trigger lung inflammation.

Bone Metabolic Markers in Patients with Obstructive Sleep Apnea Syndrome.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is prevalent in obesity and is associated with many metabolic abnormalities. The relationship between OSAS and bone metabolism is still unclear. The aim of this study was to investigate the relationship between the severity of OSAS and bone metabolic markers. METHODS: A total of 119 obese males were enrolled in this study in spring months from 2015 to 2017. All candidates underwent polysomnography, and their bone mineral density (BMD) and the serum levels of total procollagen type 1 N-terminal propeptide (t-P1NP), N-terminal midfragment of osteocalcin (N-MID), ß-C-terminal telopeptide of type 1 collagen (ß-CTX), vitamin D (VD), and parathyroid hormone (PTH) were measured. The analysis of variance and Pearson correlation analysis were performed for data analyses. RESULTS: No significant differences in the mean values of BMD were observed among the obesity, mild-to-moderate OSAS, and severe OSAS groups; and the serum levels of t-P1NP and ß-CTX in the severe OSAS group were significantly higher than those in the obesity group (48.42 ± 23.78 ng/ml vs. 31.98 ± 9.85 ng/ml, P < 0.001; 0.53 ± 0.24 ng/ml vs. 0.41 ± 0.13 ng/ml, P = 0.011, respectively). The serum level of VD in the obesity group was significantly higher than those in the mild-to-moderate and severe OSAS groups (both P < 0.001), and decreased as the severity of OSAS increased (P < 0.001). The serum level of PTH in the severe OSAS group was significantly higher than those in the obesity and mild-to-moderate OSAS groups (both P < 0.001). The results of correlation analysis indicated that the level of apnea-hypopnea index (AHI) was correlated with the levels of t-P1NP (r = 0.396, P < 0.001), VD (r = -0.404, P < 0.001), and PTH (r = 0.400, P < 0.001), whereas the level of minimum O2saturation (SaO2min) was correlated with the levels of VD (r = 0.258, P = 0.016) and PTH (r = -0.376, P < 0.001). CONCLUSIONS: The levels of bone resorption and formation markers in patients with severe OSAS were significantly increased compared to obese men, and the severity of OSAS was correlated with the serum levels of t-P1NP, VD, and PTH.

Effects of RNA interference-mediated silencing of toll-like receptor 4 gene on proliferation and apoptosis of human breast cancer MCF-7 and MDA-MB-231 cells: An in vitro study.

Breast cancer is known as the most prevalent cancer in women worldwide, and has an undeniable negative impact on public health, both physically, and mentally. This study aims to investigate the effects of toll-like receptor 4 (TLR4) gene silencing on proliferation and apoptosis of human breast cancer cells to explore for a new theoretical basis for its treatment. TLR4 small interference RNA (siRNA) fragment recombinant plasmids were constructed, including TLR4 siRNA-1, TLR4 siRNA-2, and TLR4 siRNA-3. Human breast cancer MCF-7 and MDA-MB-231 cells were assigned into blank, negative control (NC), TLR4 siRNA-1, TLR4 siRNA-2, and TLR4 siRNA-3 groups. MCF-7 and MDA-MB-231 cell growth was detected by MTT assay. Apoptosis and cell cycle were determined by flow cytometry. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were conducted to determine the expression of TLR4, CDK4, cyclin D1, Livin, Bcl-2, p53, c-FLIP, and caspase-3. In comparison with the NC and blank groups, the TLR4 siRNA-1, TLR4 siRNA-2, and TLR4 siRNA-3 groups showed decreased the expression of TLR4, inhibited proliferation of MCF-7 and MDA-MB-231 cells and promoted MCF-7 and MDA-MB-231 cell apoptosis, and the cells were blocked in G1 phase. In comparison with the NC and blank groups, in the TLR4 siRNA-1, TLR4 siRNA-2, and TLR4 siRNA-3 groups, siRNA-TLR4 significantly increased expression of p53 and caspase-3 in MCF-7 and MDA-MB-231 cells, while it decreased the expressions of CDK4, cyclinD1, Livin, Bal-2, and c-FLIP. The study demonstrates that TLR4 gene silencing inhibits proliferation and induces apoptosis of MCF-7 and MDA-MB-231 cells.

Upregulation of microRNA-335-5p reduces inflammatory responses by inhibiting FASN through the activation of AMPK/ULK1 signaling pathway in a septic mouse model.

Sepsis, as a systemic inflammatory response syndrome (SIRS) subtype, is generally characterized by infection. Emerging evidence has highlighted dysregulated microRNAs (miRNAs) are involved in the progression of sepsis. The aim of the study was to investigate the effects of miR-335-5p on inflammatory responses in a septic mouse model. The hypothesis was subsequently asserted that the FASN gene and AMPK/ULK1 signaling pathway may participate in the regulation of miR-335-5p. A septic mouse model was established in order to validate the effect of miR-335-5p on the inflammatory response by means of suppressing the endogenous expression of FASN by siRNA against FASN in endothelial cells. A target prediction program and luciferase activity was employed to ascertain as to whether miR--335-5p targets FASN. The levels of inflammatory factors including IL-6 and IL-1ß were determined by means of ELISA assay. RT-qPCR and western blot analysis were used to determine the AMPK/ULK1 signaling pathway-, apoptosis- and autophagy-related genes. Flow cytometry was employed in order to evaluate sepsis-induced cell apoptosis in response to miR-335-5p and FASN alternations. FASN was identified as a target gene of miR--335-5p. Gain- and loss-of-function studies revealed that miR-335-5p acted to enhance autophagy, reduce cell apoptosis, promote cell cycle entry in endothelial cells, and reduce inflammatory response through the modulation of pro- and anti-apoptotic factors in endothelial cells. The effect of miR-335-5p on endothelial cells was increased when FASN was suppressed by siRNA as well as when the AMPK/ULK1 signaling pathway was activated, suggesting that miR-335-5p influences sepsis by targeting and inhibiting FASN, and activating the AMPK/ULK1 signaling pathway. Our study provides evidence indicating that overexpressed miR-335-5p enhances autophagy by targeting FASN through activation of the AMPK/ULK1 signaling pathway working to alleviate the inflammatory response in septic mouse models, emphasizing the value of the functional upregulation of miR-335-5p as therapeutic strategy for sepsis.

Differential immunolocalization between lingual antimicrobial peptide and lactoferrin in mammary gland of dairy cows.

Lingual antimicrobial peptide (LAP), one of the ß-defensins in bovines, and lactoferrin (LF) are synthesized in mammary epithelium and have bactericidal and bacteriostatic functions. However, it is not known whether they have similar expression patterns. Therefore, the present study was undertaken to compare (1) immunolocalization of LAP and LF in the mammary gland and (2) changes in concentration of these two components in milk after lipopolysaccharide (LPS) challenge. Bovine mammary tissues without LPS challenge were collected and their sections were immunostained with antibodies to LAP or LF. Milk from our previous study was collected every hour up to 12h and twice daily from d 1 to 7 after LPS challenge (the day of infusion was considered as d 0). These milk samples were measured for LAP but not LF in our previous report. Therefore, concentration of LF was measured by enzyme immunoassay in the present study. Epithelial cells of some alveoli showed immunopositive reaction for LF, but negative for LAP. Conversely, some alveoli were LAP positive in their epithelial cells but LF negative. Many alveoli had immunoreactions for neither LAP nor LF. The concentration of LAP in milk was elevated significantly at 3h after LPS infusion compared with pre-infusion values and remained at a high level until 12h. However, LF concentration in milk remained low at d 0 and increased at d 2. These results suggest that LAP and LF were mostly differentially localized in the alveolar epithelium in mammary glands. The different spatial expressions between them may be associated with their different temporal expression mechanisms.