CircKDM5B sponges miR-128 to regulate porcine blastocyst development by modulating trophectoderm barrier function.

Circular RNAs (circRNAs), which exert critical functions in the regulation of transcriptional and post-transcriptional gene expression, are found in mammalian cells but their functions in mammalian preimplantation embryo development remain poorly understood. Here, we showed that circKDM5B mediated miRNA-128 (miR-128) to regulate porcine early embryo development. We screened circRNAs potentially expressed in porcine embryos through an integrated analysis of sequencing data from mouse and human embryos, as well as porcine oocytes. An authentic circRNA originating from histone demethylase KDM5B (referred to as circKDM5B) was abundantly expressed in porcine embryos. Functional studies revealed that circKDM5B knockdown not only significantly reduced blastocyst formation but also decreased the number of total cells and trophectoderm (TE) cells. Moreover, the knockdown of circKDM5B resulted in the disturbance of tight junction assembly and impaired paracellular sealing within the TE epithelium. Mechanistically, miR-128 inhibitor injection could rescue the early development of circKDM5B knockdown embryos. Taken together, the findings revealed that circKDM5B functions as a miR-128 sponge, thereby facilitating early embryonic development in pigs through the modulation of gene expression linked to tight junction assembly.

Nerve growth factor promotes expression of costimulatory molecules and release of cytokines in dendritic cells involved in Th2 response through LPS-induced p75NTR.

INTRODUCTION: Nerve growth factor (NGF) plays an important role in asthmatic inflammatory responses. However, the effects of NGF on dendritic cells (DCs) in asthmatic inflammation remain unknown. Therefore, we examined the effects of NGF on co-stimulatory molecules and the release of cytokines after ovalbumin (OVA) and a low dose of LPS (low LPS) stimulation of dendritic cells. METHODS: Bone-marrow-derived dendritic cells (BMDCs) were collected from 6- to 8-week-old wide or TLR4(-/-) mice. BMDCs were treated with OVA and/or low LPS for 12h, and then stimulated with NGF for 24h. ELISA and flow cytometry were performed to measure TSLP, IL-6, IL-10, and IL-12 production and MHCII and CD86 expression on BMDCs. BMDCs were exposed to p75 neurotrophin receptor (p75NTR) inhibitor (TAT-Pep5) or NF-kB inhibitor (QNZ) 30 min prior to NGF 1 h after NGF intervention, the levels of RelA and RelB in cytoplasmic and nuclear were detected by west blot. Co-cultured BMDCs with naïve CD4(+) T cells, and ELISA was used to detect IL-4 and INF-γ levels. RESULTS: NGF was found to markedly promote OVA and low LPS-induced expression of MHCII, CD86, secretion of TSLP and IL-6, and Th2-response-stimulating capacity of BMDCs. NGF affected BMDCs through LPS-induced p75NTR expression. TAT-Pep5 or QNZ could attenuate the promotive effect of NGF. CONCLUSIONS: NGF facilitates OVA with lowLPS-induced maturation of mouse BMDCs through LPS-up-regulated p75 NTR via activation of NF-κB pathways, providing another mechanism for the involvement of NGF in the Th2 response.

[Anti-nerve growth factor antibody reduces airway hyperresponsiveness in a mouse model of asthma by down-regulating the level of autophagy in lungs].

OBJECTIVE: To study the effects of anti-nerve growth factor (NGF) antibody on the level of autophagy in lung tissue antigen presenting cells in a mouse model of asthma. METHODS: The 24 BALB/c mice aged 6 weeks were divided into control group (PBS sensitization, PBS challenge) , asthma group [ovalbumin (OVA) sensitization, OVA challenge] and anti-NGF group (OVA sensitization, OVA challenge) using the random number table, 8 mice in each group. Sensitization was carried out by intraperitoneal injection method while challenge by aerosol inhalation. The anti-NGF group received intraperitoneal injection of anti-NGF antibody for intervention 30 min before the atomization, while the control group and asthma group received intraperitoneal injection of PBS 30 min before the atomization. The lung tissue and serum were harvested 24 h after the last sensitization. Immunoelectron microscopy was used to observe the level of autophagy in mouse airway dendritic cells. Western blot assay was used to detect the autophagy marker molecule MHP1 LC3-II level in lung tissue. ELISA was used to detect the serum levels of IL-4, INF-γ and NGF. Immunohistochemical technique was used to detect the expression levels of costimulatory molecules CD80, CD86 and CD40, and the major histocompatibility complex class II molecule (MHC-II) on dendritic cells of lung tissue. RESULTS: The level of autophagy in lung tissue was lower in anti-NGF group (0.28 ± 0.07) than in asthma group (0.46 ± 0.10) but higher than in control group (0.17 ± 0.08) (F = 15.571, P < 0.05) ; and Pairwise comparison showed that all the intergroup differences were statistically significant (all P values<0.05) . The serum NGF concentration was lower in anti-NGF group [(0.65 ± 0.04) µg/L] than in asthma group [(0.75 ± 0.10) µg/L] but still higher than the control group [(0.52 ± 0.05) µg/L] (F = 61.972, P < 0.05); and Pairwise comparison showed that all the intergroup differences were statistically significant (all P values<0.05) . Both the costimulatory molecules CD80, CD86 and CD40 and the MHC-II on antigen presenting cells of lung tissue were significantly lower in anti-NGF group than in asthma group. Anti-NGF group had lower serum IL-4 but higher IFN-γ level than asthma group. CONCLUSIONS: Anti-NGF antibody attenuates Th2-dominant airway inflammation in this mouse model of asthma by down-regulating the level of autophagy in lung tissue to inhibit the antigen-presenting cell functions.

[Influence of electroacupuncture of "Dazhui"（GV14） and "Mingmen" （GV4） on expressions of NL1 and NF-200 in spinal cord injury rats].

OBJECTIVE: To observe the effect of electroacupuncture(EA) on activities of A2 type astrocytes(A2s)and A1 type astrocytes (A1s) , expressions of neurofilament protein 200 (NF-200, a marker of axon regeneration), nexin 1(NL1, a marker of synaptic regeneration), and regeneration of Nissl bodies in rats with spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of SCI. METHODS: A total of 75 male SD rats were rando-mized into sham operation, model, antibody neutralizing (AN), EA and EA+AN groups, with 15 rats in each group. The SCI model was established by using an infinite field impactor to deliver an about 200 k dyne weight onto the exposed spinal cord after making a dorsal laminectomy at vertebral level T10. EA (2 Hz, 1 mA) was applied to"Dazhui"(GV14) and "Mingmen"(GV4) for 20 min, once daily for 28 days. After modeling, intraspinal injection of neutralizing antibodies IL-1α, TNF-α and complement 1q (C1q, 2 µL) to the injured spinal locus for inhibition of A1 type astrocytes (A1s) was conducted on the 1st, 7th , 14th and 21st day for rats of AN and EA+AN groups. BBB rating scale was used to evaluate hindlimb locomotor function on day 1, 7, 14, 21 and 28 after modeling. The activation of A2s (its specific marker S100a10), astrocyte (its specific marker glial fibrillary acidic protein, GFAP), and A1s (its specific marker C3) in the spinal cord was detected by immunofluorescence, and the protein expressions of NF-200 and NL1 in the spinal cord detected by Western blot and immunohistochemistry, separately, and the neuronal regeneration was observed after Nissl staining. RESULTS: After SCI, the BBB scores at 1 , 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200 were significantly decreased (P<0.01), and the fluorescence intensity of double labelled S100a10 (A2s)/GFAP and C3, and the expression of NF-200 were considerably increased in the model group (P<0.05, P<0.01). In contrast to the model group, the BBB scores at 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200, and the fluorescence intensity of A2s/GFAP in the AN, EA and AN+EA groups, and the expressions of NL1 in the EA and AN+EA groups, and expression of NF-200 protein in the AN+EA group were evidently increased (P<0.05, P<0.01), and the fluorescence intensity of C3 was strikingly decreased in the EA group (P<0.01). The effect of AN+EA was significantly superior to that of single AN and EA in increasing BBB scores at 14, 21 and 28 day, and in up-regulating the immunoactivity of NF-200(P<0.01, P<0.05). Nissl staining showed damaged structure of the gray matter of the spinal cord, atrophy of the Nissl body, and pyknosis of neurons, which was milder in the AN and EA groups, particularly in the AN+EA group. CONCLUSION: EA at GV14 and GV4 may promote activation of A2s and promote regeneration of axons and synapses in SCI model rats.

Vitamin B6 regulates IL-33 homeostasis to alleviate type 2 inflammation.

Interleukin-33 (IL-33) is a crucial nuclear cytokine that induces the type 2 immune response and maintains immune homeostasis. The fine-tuned regulation of IL-33 in tissue cells is critical to control of the type 2 immune response in airway inflammation, but the mechanism is still unclear. Here, we found that healthy individuals had higher phosphate-pyridoxal (PLP, an active form of vitamin B6) concentrations in the serum than asthma patients. Lower serum PLP concentrations in asthma patients were strongly associated with worse lung function and inflammation. In a mouse model of lung inflammation, we revealed that PLP alleviated the type 2 immune response and that this inhibitory effect relied on the activity of IL-33. A mechanistic study showed that in vivo, pyridoxal (PL) needed to be converted into PLP, which inhibited the type 2 response by regulating IL-33 stability. In mice heterozygous for pyridoxal kinase (PDXK), the conversion of PL to PLP was limited, and IL-33 levels were increased in the lungs, aggravating type 2 inflammation. Furthermore, we found that the mouse double minute 2 homolog (MDM2) protein, an E3 ubiquitin-protein ligase, could ubiquitinate the N-terminus of IL-33 and sustain IL-33 stability in epithelial cells. PLP reduced MDM2-mediated IL-33 polyubiquitination and decreased the level of IL-33 through the proteasome pathway. In addition, inhalation of PLP alleviated asthma-related effects in mouse models. In summary, our data indicate that vitamin B6 regulates MDM2-mediated IL-33 stability to constrain the type 2 response, which might help develop a potential preventive and therapeutic agent for allergy-related diseases.

Comparison of traditional methods and high-throughput genetic sequencing in the detection of pathogens in pulmonary infectious diseases.

BACKGROUND: The major causes of pulmonary infections are various microorganisms. This study aimed to compare the positive rates of pathogenic microorganism DNA/RNA high-throughput genetic sequencing (PMseq), which is an emerging technique, with traditional methods for pulmonary disease detection, and to investigate the differences in different sample types. METHODS: Bronchoalveolar lavage fluid (BALF) and venous blood samples from 104 patients were collected for detection. RESULTS: The positive rates of PMseq in BALF and venous blood were both significantly higher than those of traditional methods in the same sample (P<0.001). For BALF, the detection sensitivities were 96.9% for non-febrile patients and 100% for febrile patients. For venous blood, the detection sensitivities were 50.0% for non-febrile patients and 81.3% for febrile patients. There was no statistical difference in the sensitivity of venous blood samples with or without fever (P=0.075). For patients without fever, the sensitivity of BALF was much higher than venous blood samples (P<0.001). In patients with fever, there were no significant differences between different samples. CONCLUSIONS: This study showed that PMseq has a higher positive rate for the detection of pulmonary diseases. For patients without fever, it is recommended to use BALF instead of venous blood samples because of the higher sensitivity. However, for patients with fever, venous blood samples can be used when bronchoalveolar lavage is inconvenient.

Chromatin remodeler INO80 mediates trophectoderm permeability barrier to modulate morula-to-blastocyst transition.

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.

Inhibition of Alveolar Macrophage Pyroptosis Reduces Lipopolysaccharide-induced Acute Lung Injury in Mice.

BACKGROUND: Pyroptosis is the term for caspase-1-dependent cell death associated with pro-inflammatory cytokines. The role of alveolar macrophage (AM) pyroptosis in the pathogenesis of the acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains unclear. METHODS: C57BL/6 wild-type mice were assigned to sham, lipopolysaccharide (LPS) + vehicle, LPS + acetyl-tyrosyl-valyl- alanyl-aspartyl-chloromethylketone (Ac-YVAD-CMK) and LPS + Z-Asp-Glu-Val-Asp-fluoromethylketone groups. Mice were given intraperitoneal (IP) injections of LPS. Drugs were IP injected 1 h before LPS administration. Mice were sacrificed 16 h after LPS administration, and AMs were isolated. Western blot analysis for active caspase-1 and cleaved caspase-3, evaluation of lung injury and a cytokine release analysis were performed. AMs were treated with LPS and adenosine triphosphate (ATP); caspase-1-dependent cell death was evaluated using flow cytometry; the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosomes were examined by immunofluorescence. RESULTS: The expression of activated caspase-1 in AMs was enhanced following LPS challenge compared with the sham group. In the ex vivo study, the caspase-1/propidium iodide-positive cells, caspase-1 specks and ASC pyroptosomes were up-regulated in AMs following LPS/ATP stimulation. The specific caspase-1 inhibitor Ac-YVAD-CMK inhibited the activation of caspase-1 and pyroptotic cell death. Ac-YVAD-CMK also reduced the lung injury, pulmonary edema and total protein in bronchoalveolar lavage fluid (BALF). In addition, Ac-YVAD-CMK significantly inhibited interleukin-α2 (IL-1α2) release both in serum and BALF and reduced the levels of IL-18, tumor necrosis factor-α± (TNF-α±), High Mobility Group Box 1 (HMGB1) in BALF during LPS-induced ALI/ARDS. CONCLUSIONS: This study reported AM pyroptosis during LPS-induced ALI/ARDS in mice and has demonstrated that Ac-YVAD-CMK can prevent AM-induced pyroptosis and lung injury. These preliminary findings may form the basis for further studies to evaluate this pathway as a target for prevention or reduction of ALI/ARDS.

Lung dendritic cells undergo maturation and polarization towards a T helper type 2-stimulating phenotype in a mouse model of asthma: Role of nerve growth factor.

Nerve growth factor (NGF) and dendritic cells (DCs) have been hypothesized to modulate T cell responses in a mouse model of asthma. However, whether NGF plays a role in regulating the maturation and polarization of lung DCs remains unclear. In the present study, the effect of NGF inhibition on the maturation and phenotype of lung DCs was investigated in a mouse model of asthma. BALB/c mice were sensitized and challenged with ovalbumin (OVA), and subsequently received anti-NGF treatment. At 24 h following the last challenge, airway responsiveness and inflammation were examined. The concentrations of NGF, interferon (IFN)-γ and interleukin (IL)-4 were analyzed. In addition, maturation and CD103 expression in the lung DCs were investigated. Anti-NGF treatment was found to significantly reduce airway hyperreactivity and inflammation in asthmatic mice. In addition, a subdued T helper 2 (Th2) response was observed, characterized by the downregulation of IL-4 and the upregulation of IFN-γ. Furthermore, the expression of the DC surface molecules, CD80, CD86 and major histocompatibility complex class II, as well as the proportion of lung CD103+ DCs, decreased in the OVA-sensitized and challenged mice. The proportion of lung CD103+ DCs also exhibited a positive correlation with the levels of plasma NGF in the mice. These results may provide an explanation for the role of NGF in amplifying the Th2 response in allergic diseases. Therefore, NGF may promote the maturation and polarization towards a Th2-stimulating phenotype of activated DCs, contributing to an amplification of the Th2 response in asthma.

[Study on reliability and validity of the Tinnitus Evaluation Questionnaire].

OBJECTIVE: To investigate the value of the Tinnitus Evaluation Questionnaire (TEQ) in clinical application. METHODS: Cronbach's α coefficient was used to examine the reliability of the TEQ internal consistency. Examined the re-measured reliability by the correlation coefficient by two doctors' 1 - 3 hours interval questionnaires' scores. And inspected criteria validity according to the correlation coefficient of the TEQ and Tinnitus Handicap Inventory (THI). RESULTS: In the 202 tinnitus patients, the TEQ Cronbach's α coefficient was 0.76 and re-measured reliability was 0.938. The THI correlation coefficient was 0.769. Among which, 99 patients feel tinnitus alleviated obviously after the treatment, the TEQ scores were significantly lower than that before the treatment (t = 21.42, P < 0.001). CONCLUSIONS: The TEQ reflects the severity of tinnitus completely, and has preferable reliability and validity. The characteristics are concise, practical and exact. It is worthy of clinical application.