Metabolic Regulation of Gene Expression by Histone Lysine ß-Hydroxybutyrylation.

Here we report the identification and verification of a ß-hydroxybutyrate-derived protein modification, lysine ß-hydroxybutyrylation (Kbhb), as a new type of histone mark. Histone Kbhb marks are dramatically induced in response to elevated ß-hydroxybutyrate levels in cultured cells and in livers from mice subjected to prolonged fasting or streptozotocin-induced diabetic ketoacidosis. In total, we identified 44 histone Kbhb sites, a figure comparable to the known number of histone acetylation sites. By ChIP-seq and RNA-seq analysis, we demonstrate that histone Kbhb is a mark enriched in active gene promoters and that the increased H3K9bhb levels that occur during starvation are associated with genes upregulated in starvation-responsive metabolic pathways. Histone ß-hydroxybutyrylation thus represents a new epigenetic regulatory mark that couples metabolism to gene expression, offering a new avenue to study chromatin regulation and diverse functions of ß-hydroxybutyrate in the context of important human pathophysiological states, including diabetes, epilepsy, and neoplasia.

Propranolol inhibits the angiogenic capacity of hemangioma endothelia via blocking ß-adrenoceptor in mast cell.

BACKGROUND: Propranolol, a non-selective blocker of the ß-adrenoceptor (AR), is a first-line treatment for infantile hemangioma (IH). Mast cells have been implicated in the pathophysiology of propranolol-treated hemangioma. However, the function of mast cells remains unclear. METHODS: HMC-1s (Human mast cell line) having been treated with propranolol for 24 h were centrifuged, washed with PBS twice, and maintained in cell culture medium for another 24 h. The supernatants with propranolol which were named as propranolol-treated HMC-1s supernatants were obtained. The expression of cytokines and mediators was examined among HMC-1s dealt with propranolol. HemECs (hemangioma endothelial cells) were co-cultured with propranolol-treated HMC-1s supernatants, and their proliferation and apoptosis were investigated. The autophagic-related protein was examined in HemECs using immunoblot. RESULTS: In propranolol-treated HMC-1s, the expressions of ADRB1 (ß1-AR) and ADRB2 (ß2-AR) were reduced by 70% and 60%, respectively, and that of cytokines and mediators were reduced. The proliferation was decreased, but apoptosis and autophagy were induced in HemECs treated with propranolol-treated HMC-1s supernatants. However, propranolol can work well in shRNA-ADRB1 or shRNA-ADRB2 transfected HMC-1s. CONCLUSIONS: Propranolol inhibit the proliferation of HemECs and promote their apoptosis and autophagy through acting on both ß1 and ß2 adrenoceptor in mast cell. IMPACT: Treated with propranolol, ß1, and ß2 adrenoceptor on human mast cell expression was reduced significantly. After hemangioma endothelial cell treated with the supernatants from propranolol-treated human mast cell, its proliferation was decreased, but apoptosis and autophagy were significantly induced. Propranolol can work well in shRNA-ADRB1 or shRNA-ADRB2 transfected HMC-1s. Mast cells may have a role in the action of propranolol in infantile hemangioma through both ß1 and ß2 adrenoceptors to inhibit the angiogenic capacity of hemangioma endothelial cells.

Activation of unfolded protein response overcomes Ibrutinib resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most widespread type of non-Hodgkin lymphoma (NHL). As the most aggressive form of the DLBCL, the activated B-cell-like (ABC) subtype is often resistant to standard chemotherapies. Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a potential therapeutic approach for the DLBCL but fails to improve the outcome in the phase III trial. In the current study, we investigated the molecular mechanisms underlying ibrutinib resistance and explored new combination therapy with ibrutinib. We generated an ibrutinib-resistant ABC-DLBCL cell line (OCI-ly10-IR) through continuous exposure to ibrutinib. Transcriptome analysis of the parental and ibrutinib-resistant cell lines revealed that the ibrutinib-resistant cells had significantly lower expression of the unfolded protein response (UPR) marker genes. Overexpression of one UPR branch-XBP1s greatly potentiated ibrutinib-induced apoptosis in both sensitive and resistant cells. The UPR inhibitor tauroursodeoxycholic acid (TUDCA) partially reduced the apoptotic rate induced by the ibrutinib in sensitive cells. The UPR activator 2-deoxy-D-glucose (2-DG) in combination with the ibrutinib triggered even greater cell growth inhibition, apoptosis, and stronger calcium (Ca2+) flux inhibition than either of the agents alone. A combination treatment of ibrutinib (15 mg·kg-1·d-1, po.) and 2-DG (500 mg/kg, po, b.i.d.) synergistically retarded tumor growth in NOD/SCID mice bearing OCI-ly10-IR xenograft. In addition, ibrutinib induced the UPR in the sensitive cell lines but not in the resistant cell lines of the DLBCL. There was also a combined synergistic effect in the primary resistant DLBCL cell lines. Overall, our results suggest that targeting the UPR could be a potential combination strategy to overcome ibrutinib resistance in the DLBCL.

HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia.

BACKGROUND: Microglial mediated neuroinflammation in the rostral ventrolateral medulla (RVLM) plays roles in the etiology of stress-induced hypertension (SIH). It was reported that autophagy influenced inflammation via immunophenotypic switching of microglia. High-mobility group box 1 (HMGB1) acts as a regulator of autophagy and initiates the production of proinflammatory cytokines (PICs), but the underlying mechanisms remain unclear. METHODS: The stressed mice were subjected to intermittent electric foot shocks plus noises administered for 2 h twice daily for 15 consecutive days. In mice, blood pressure (BP) and renal sympathetic nerve activity (RSNA) were monitored by noninvasive tail-cuff method and platinum-iridium electrodes placed respectively. Microinjection of siRNA-HMGB1 (siHMGB1) into the RVLM of mice to study the effect of HMGB1 on microglia M1 activation was done. mRFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors were transfected into the RVLM to evaluate the process of autolysosome formation/autophagy flux. The expression of RAB7, lysosomal-associated membrane protein 1 (LAMP1), and lysosomal pH change were used to evaluate lysosomal function in microglia. Mitophagy was identified by transmission electron microscopic observation or by checking LC3 and MitoTracker colocalization under a confocal microscope. RESULTS: We showed chronic stress increased cytoplasmic translocations of HMGB1 and upregulation of its receptor RAGE expression in microglia. The mitochondria injury, oxidative stress, and M1 polarization were attenuated in the RVLM of stressed Cre-CX3CR1/RAGEfl/fl mice. The HMGB1/RAGE axis increased at the early stage of stress-induced mitophagy flux while impairing the late stages of mitophagy flux in microglia, as revealed by decreased GFP fluorescence quenching of GFP-RFP-LC3-II puncta and decreased colocalization of lysosomes with mitochondria. The expressions of RAB7 and LAMP1 were decreased in the stressed microglia, while knockout of RAGE reversed these effects and caused an increase in acidity of lysosomes. siHMGB1 in the RVLM resulted in BP lowering and RSNA decreasing in SIH mice. When the autophagy inducer, rapamycin, is used to facilitate the mitophagy flux, this treatment results in attenuated NF-κB activation and reduced PIC release in exogenous disulfide HMGB1 (ds-HMGB1)-stimulated microglia. CONCLUSIONS: Collectively, we demonstrated that inhibition of the HMGB1/RAGE axis activation led to increased stress-induced mitophagy flux, hence reducing the activity of microglia-mediated neuroinflammation and consequently reduced the sympathetic vasoconstriction drive in the RVLM.

Infiltrating T helper 17 cells in the paraventricular nucleus are pathogenic for stress-induced hypertension.

Central neuroinflammation produced by both innate and adaptive immunities plays a major role in the development of stress-induced hypertension (SIH), but successful T cell immunoregulation for SIH requires that the T cells can access brain tissues. So far, both the effects of T helper 17 (Th17) cells on SIH and the pathway for T cells entry into the brain were unknown. Here we show that the blood pressure (BP), heart rate (HR) and the norepinephrine(NE) of the SIH rats were considerably higher, the numbers of Th17 cells and IL-17 were higher, relative to control. Anti-IL-17 attenuated the elevation of BP and HR of the SIH rats when microinjected into the paraventricular nucleus (PVN).Alb-FITC, after infusion into the carotid artery, were found in the brain parenchyma of the PVN in the SIH rats. We concluded that Th17 cells infiltrated the parenchyma of the paraventricular nucleus (PVN) via a compromised blood brain barrier (BBB) in response to stress and Th17 cells and IL-17 play an important role in the pathophysiology of SIH.

Neuroinflammation contributes to autophagy flux blockage in the neurons of rostral ventrolateral medulla in stress-induced hypertension rats.

BACKGROUND: Neuroinflammation plays hypertensive roles in the uninjured autonomic nuclei of the central nervous system, while its mechanisms remain unclear. The present study is to investigate the effect of neuroinflammation on autophagy in the neurons of the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone reside. METHODS: Stress-induced hypertension (SIH) was induced by electric foot-shock stressors with noise interventions in rats. Systolic blood pressure (SBP) and the power density of the low frequency (LF) component of the SAP spectrum were measured to reflect sympathetic vasomotor activity. Microglia activation and pro-inflammatory cytokines (PICs (IL-1ß, TNF-α)) expression in the RVLM were measured by immunoblotting and immunostaining. Autophagy and autophagic vacuoles (AVs) were examined by autophagic marker (LC3 and p62) expression and transmission electron microscopy (TEM) image, respectively. Autophagy flux was evaluated by RFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors transfected into the RVLM. Tissue levels of glutamate, gamma aminobutyric acid (GABA), and plasma levels of norepinephrine (NE) were measured by using high-performance liquid chromatography (HPLC) with electrochemical detection. The effects of the cisterna magna infused minocycline, a microglia activation inhibitor, on the abovementioned parameters were analyzed. RESULTS: SIH rats showed increased SBP, plasma NE accompanied by an increase in LF component of the SBP spectrum. Microglia activation and PICs expression was increased in SIH rats. TEM demonstrated that stress led to the accumulation of AVs in the RVLM of SIH rats. In addition to the Tf-LC3 assay, the concurrent increased level of LC3-II and p62 suggested the impairment of autophagic flux in SIH rats. To the contrary, minocycline facilitated autophagic flux and induced a hypotensive effect with attenuated microglia activation and decreased PICs in the RVLM of SIH rats. Furthermore, SIH rats showed higher levels of glutamate and lower level of GABA in the RVLM, while minocycline attenuated the decrease in GABA and the increase in glutamate of SIH rats. CONCLUSIONS: Collectively, we concluded that the neuroinflammation might impair autophagic flux and induced neural excitotoxicity in the RVLM neurons following SIH, which is involved in the development of SIH.

Prognostic Value of Real Time Myocardial Contrast Echocardiography after Percutaneous Coronary Intervention.

Real time myocardial contrast echocardiography (RTMCE) is a cost-effective and simple method to quantify coronary flow reserve (CFR). We aimed to determine the value of RTMCE to predict cardiac events after percutaneous coronary intervention (PCI). We have studied myocardial blood volume (A), velocity (ß), flow indexes (MBF, A × ß), and vasodilator reserve (stress-to-rest ratios) in 36 patients with acute coronary syndrome (ACS) who underwent PCI. CFR (MBF at stress/MBF at rest) was calculated for each patient. Perfusion scores were used for visual interpretation by MCE and correlation with TIMI flow grade. In qualitative RTMCE assessment, post-PCI visual perfusion scores were higher than pre-PCI (Z = -7.26, P < 0.01). Among 271 arteries with TIMI flow grade 3 post-PCI, 72 (36%) did not reach visual perfusion score 1. The ß- and A × ß-reserve of the abnormal segments supplied by obstructed arteries increased after PCI comparing to pre-PCI values (P < 0.01). Patients with adverse cardiac events had significantly lower ß- and lower A × ß-reserve than patients without adverse cardiac events. In the former group, the CFR was ≥ 1.5 both pre- and post-PCI. CFR estimation by RTMCE can quantify myocardial perfusion in patients with ACS who underwent PCI. The parameters ß-reserve and CFR combined might predict cardiac events on the follow-up.

2-(3-Benzoylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid ameliorates metabolic disorders in high-fat diet-fed mice.

AIM: Sterol-regulatory element binding proteins (SREBPs) are major transcription factors that regulate liver lipid biosynthesis. In this article we reported a novel synthetic compound 2-(3-benzoylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (ZJ001) that inhibited the SREBP-1c pathway, and effectively reduced hepatic lipid accumulation in diet-induced obesity (DIO) mice. METHODS: A luciferase reporter driven by an SRE-containing promoter transfected into HepG2 cells was used to discover the compound. Two approaches were used to evaluate the lipid-lowering effects of ZJ001: (1) diet-induced obesity (DIO) mice that were treated with ZJ001 (15 mg·kg(-1)·d(-1), po) for 7 weeks; and (2) HepG2 cells and primary hepatocytes used as in vitro models. RESULTS: ZJ001 (10, 20 µmol/L) dose-dependently inhibited the activity of SRE-containing promoter. ZJ001 administration ameliorated lipid metabolism and improved glucose tolerance in DIO mice, accompanied by significantly reduced mRNA levels of SREBP-1C and SREBP-2, and their downstream genes. In HepG2 cells and insulin-treated hepatocytes, ZJ001 (10-40 µmol/L) dose-dependently inhibited lipid synthesis, and reduced mRNA levels of SREBP-1C and SREBP-2, and their downstream genes. Furthermore, ZJ001 dose-dependently increased the phosphorylation of AMPK and regulatory-associated protein of mTOR (Raptor), and suppressed the phosphorylation of mTOR in insulin-treated hepatocytes. Moreover, ZJ001 increased the ADP/ATP ratio in insulin-treated hepatocytes. CONCLUSION: ZJ001 exerts multiple beneficial effects in diet-induced obesity mice. Its lipid-lowering effects may result from the suppression of mTORC1, which regulates SREBP-1c transcription. The results suggest that the SREBP-1c pathway may be a potential therapeutic target for the treatment of lipid metabolic disorders.

Hepatic involvement of Langerhans cell histiocytosis in children--imaging findings of computed tomography, magnetic resonance imaging and magnetic resonance cholangiopancreatography.

BACKGROUND: Langerhans cell histiocytosis is a rare disease that occurs mainly in children, and hepatic involvement is generally a poor prognostic factor. OBJECTIVE: To describe CT and MRI findings of hepatic involvement of Langerhans cell histiocytosis in children, especially the abnormal bile duct manifestation on magnetic resonance cholangiopancreatography (MRCP). MATERIALS AND METHODS: Thirteen children (seven boys, six girls; mean age 28.9 months) were diagnosed with disseminated Langerhans cell histiocytosis. They underwent CT (n = 5) or MRI (n = 4), or CT and MRI examinations (n = 4) to evaluate the liver involvement. RESULTS: Periportal abnormalities presented as band-like or nodular lesions on CT and MRI in all 13 children. The hepatic parenchymal lesions were found in the peripheral regions of the liver in seven children, including multiple nodules on MRI (n = 6), and cystic-like lesions on CT and MRI (n = 3). In 11 of the 13 children the dilatations of the bile ducts were observed on CT and MRI. Eight of the 13 children underwent MR cholangiopancreatography, which demonstrated stenoses or segmental stenoses with slight dilatation of the central bile ducts, including the common hepatic duct and its first-order branches. The peripheral bile ducts in these children showed segmental dilatations and stenoses. CONCLUSION: Stenosis of the central bile ducts revealed by MR cholangiopancreatography was the most significant finding of liver involvement in Langerhans cell histiocytosis in children.

In vitro human gut microbiota fermentation of litchi pulp polysaccharides as affected by Lactobacillus pre-treatment.

In this study, litchi polysaccharides were obtained from unfermented or fermented pulp by Lactobacillus fermentum (denoted as LP and LPF, respectively). The differences between LP and LPF in the colonic fermentation characteristics and modulatory of gut microbiota growth and metabolism were investigated with an in vitro fecal fermentation model. Results revealed that the strategies of gut bacteria metabolizing LP and LPF were different and LPF with lower molecular weight (Mw) was readily utilized by bacteria. The monosaccharide utilization sequence of each polysaccharide was Ara > Gla > GalA > GlcA ≈ Glu ≈ Man. Moreover, LPF promoted stronger proliferation of Bifidobacterium, Megamonas, Prevotella, and Bacteroides and higher SCFAs production (especially acetic and butyric acids) than LP. Correlation analysis further revealed that Mw could represent an essential structural feature of polysaccharides associated with its microbiota-regulating effect. Overall, Lactobacillus fermentation pre-treatment of litchi pulp promoted the fermentation characteristics and prebiotic activities of its polysaccharide.

Endothelin-1-mediated Brainstem Glial Activation Produces Asthmatic Airway Vagal Hypertonia Via Enhanced ATP-P2X4 Receptor Signaling in Sprague-Dawley Rats.

The occurrence of major asthma symptoms is largely attributed to airway vagal hypertonia, of which the central mechanisms remain unclear. This study tests the hypotheses that endothelin-1-mediated brainstem glial activation produces asthmatic airway vagal hypertonia via enhanced action of adenosine 5'-triphosphate on neuronal purinergic P2X4 receptors. A rat model of asthma was prepared using ovalbumin. Airway vagal tone was evaluated by the recurrent laryngeal discharge and plethysmographic measurement of pulmonary function. The changes in the brainstem were examined using ELISA, Western blot, luciferin-luciferase, quantitative reverse transcription-polymerase chain reaction, enzyme activity assay and immunofluorescent staining, respectively. The results showed that in the medulla of rats, endothelin receptor type B and P2X4 receptors were primarily expressed in astrocytes and neurons, respectively, and both of which, along with endothelin-1 content, were significantly increased after ovalbumin sensitization. Ovalbumin sensitization significantly increased recurrent laryngeal discharge, which was blocked by acute intracisternal injection of P2X4 receptor antagonist 5-BDBD, knockdown of brainstem P2X4 receptors, and chronic intraperitoneal injection of endothelin receptor type B antagonist BQ788, respectively. Ovalbumin sensitization activated microglia and astrocytes and significantly decreased ecto-5'-nucleotidase activity in the medulla, and all of which, together with the increase of medullary P2X4 receptor expression and decrease of pulmonary function, were reversed by chronic BQ788 treatment. These results demonstrated that in rats, allergic airway challenge activates both microglia and astrocytes in the medulla via enhanced endothelin-1/endothelin receptor type B signaling, which subsequently causes airway vagal hypertonia via augmented adenosine 5'-triphosphate/P2X4 receptor signaling in central neurons of airway vagal reflex.

Loneliness, spiritual well-being, and death perception, as well as their risk factors in urological cancer patients.

Cancer patients commonly suffer from loneliness, poor spiritual status, and fear of death; however, these evaluations are rarely revealed in urological cancer patients. Thus, this study aimed to assess the loneliness, spiritual well-being, and death perception, as well as their risk factors in urological cancer patients. A total of 324 urological (including renal, bladder, and prostate) cancer patients and 100 healthy controls were included. The University of California and Los Angeles loneliness scale (UCLA-LS), functional assessment of chronic illness therapy-spiritual well-being (FACIT-Sp), and death attitude profile-revised (DAP-R) scores were evaluated. The results showed that the UCLA-LS score was higher, but the FACIT-Sp score was lower in urological cancer patients than in healthy controls. According to the DAP-R score, fear of death, death avoidance, and approaching death acceptance were elevated, but neutral acceptance was lower in urological cancer patients than in healthy controls. Among urological cancer patients, the UCLA-LS score was highest but the FACIT-Sp score was lowest in bladder cancer patients; regarding the DAP-R score, fear of death and death avoidance were highest, but approaching death acceptance was lowest in bladder cancer patients. Interestingly, single/divorced/widowed status, bladder cancer diagnosis, higher pathological grade, surgery, systemic treatment, and local treatment were independent factors for higher UCLA-LS score or lower FACIT-Sp score. In conclusion, urological cancer (especially bladder cancer) patients bear increased loneliness and reduced spiritual well-being; they also carry higher fear of death, death avoidance, and approaching death acceptance but lower neutral acceptance of death.

Gated CNN-Transformer Network for Automatic Cardiovascular Diagnosis using 12-lead Electrocardiogram.

12-lead electrocardiogram (ECG) is a widely used method in the diagnosis of cardiovascular disease (CVD). With the increase in the number of CVD patients, the study of accurate automatic diagnosis methods via ECG has become a research hotspot. The use of deep learning-based methods can reduce the influence of human subjectivity and improve the diagnosis accuracy. In this paper, we propose a 12-lead ECG automatic diagnosis method based on channel features and temporal features fusion. Specifically, we design a gated CNN-Transformer network, in which the CNN block is used to extract signal embeddings to reduce data complexity. The dual-branch transformer structure is used to effectively extract channel and temporal features in low-dimensional embeddings, respectively. Finally, the features from the two branches are fused by the gating unit to achieve automatic CVD diagnosis from 12-lead ECG. The proposed end-to-end approach has more competitive performance than other deep learning algorithms, which achieves an overall diagnostic accuracy of 85.3% in the 12-lead ECG dataset of CPSC-2018.

Natural product P57 induces hypothermia through targeting pyridoxal kinase.

Induction of hypothermia during hibernation/torpor enables certain mammals to survive under extreme environmental conditions. However, pharmacological induction of hypothermia in most mammals remains a huge challenge. Here we show that a natural product P57 promptly induces hypothermia and decreases energy expenditure in mice. Mechanistically, P57 inhibits the kinase activity of pyridoxal kinase (PDXK), a key metabolic enzyme of vitamin B6 catalyzing phosphorylation of pyridoxal (PL), resulting in the accumulation of PL in hypothalamus to cause hypothermia. The hypothermia induced by P57 is significantly blunted in the mice with knockout of PDXK in the preoptic area (POA) of hypothalamus. We further found that P57 and PL have consistent effects on gene expression regulation in hypothalamus, and they may activate medial preoptic area (MPA) neurons in POA to induce hypothermia. Taken together, our findings demonstrate that P57 has a potential application in therapeutic hypothermia through regulation of vitamin B6 metabolism and PDXK serves as a previously unknown target of P57 in thermoregulation. In addition, P57 may serve as a chemical probe for exploring the neuron circuitry related to hypothermia state in mice.

Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons.

BACKGROUND: Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated. RESULTS: In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and phospho-CREB, and reverses cystitis-induced increases in micturition frequency. CONCLUSIONS: A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.

Up-regulation of brain-derived neurotrophic factor in primary afferent pathway regulates colon-to-bladder cross-sensitization in rat.

BACKGROUND: In humans, inflammation of either the urinary bladder or the distal colon often results in sensory cross-sensitization between these organs. Limited information is known about the mechanisms underlying this clinical syndrome. Studies with animal models have demonstrated that activation of primary afferent pathways may have a role in mediating viscero-visceral cross-organ sensitization. METHODS: Colonic inflammation was induced by a single dose of tri-nitrobenzene sulfonic acid (TNBS) instilled intracolonically. The histology of the colon and the urinary bladder was examined by hematoxylin and eosin (H&E) stain. The protein expression of transient receptor potential (TRP) ion channel of the vanilloid type 1 (TRPV1) and brain-derived neurotrophic factor (BDNF) were examined by immunohistochemistry and/or western blot. The inter-micturition intervals and the quantity of urine voided were obtained from analysis of cystometrograms. RESULTS: At 3 days post TNBS treatment, the protein level of TRPV1 was increased by 2-fold (p < 0.05) in the inflamed distal colon when examined with western blot. TRPV1 was mainly expressed in the axonal terminals in submucosal area of the distal colon, and was co-localized with the neural marker PGP9.5. In sensory neurons in the dorsal root ganglia (DRG), BDNF expression was augmented by colonic inflammation examined in the L1 DRG, and was expressed in TRPV1 positive neurons. The elevated level of BDNF in L1 DRG by colonic inflammation was blunted by prolonged pre-treatment of the animals with the neurotoxin resiniferatoxin (RTX). Colonic inflammation did not alter either the morphology of the urinary bladder or the expression level of TRPV1 in this viscus. However, colonic inflammation decreased the inter-micturition intervals and decreased the quantities of urine voided. The increased bladder activity by colonic inflammation was attenuated by prolonged intraluminal treatment with RTX or treatment with intrathecal BDNF neutralizing antibody. CONCLUSION: Acute colonic inflammation increases bladder activity without affecting bladder morphology. Primary afferent-mediated BDNF up-regulation in the sensory neurons regulates, at least in part, the bladder activity during colonic inflammation.

Inhibition of cGAS in Paraventricular Nucleus Attenuates Hypertensive Heart Injury Via Regulating Microglial Autophagy.

Neuroinflammation in the cardiovascular center plays a critical role in the progression of hypertensive heart disease. And microglial autophagy is involved in the regulation of neuroinflammation. Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, senses mitochondrial DNA (mtDNA) and regulates autophagy. The detailed mechanisms of central cGAS affects neuroinflammatory response in hypertensive heart disease via regulating autophagy remain unknown. Angiotensin II (Ang II, 1.5 mg·kg-1·12 h-1, 2 weeks) was intraperitoneally injected to induce hypertension in mice. The cGAS-STING pathway was activated in the paraventricular nucleus (PVN) of Ang II-induced hypertensive mice. The contractile dysfunction of heart was alleviated in Ang II-induced hypertensive cGAS-/- mice. To observe the central effects of cGAS on regulating hypertensive heart disease, the RU.521 (a cGAS inhibitor) was intracisternally infused in hypertensive mice. Intracisternal infusion of the RU.521-alleviated myocardial interstitial fibrosis, cardiomyocyte hypertrophy, and the contractile dysfunction in Ang II-induced hypertensive mice. Intracisternal infusion of RU.521 attenuated the microglial activation, neuroinflammation, sympathetic/parasympathetic activity ratio, and lowered blood pressure. The autophagic flux in the PVN cells was blocked, while intracisternal infusion of RU.521 alleviated this effect in the Ang II-induced hypertensive mice. In vitro, it was found that cGAS-STING activation-induced autophagic flux blockage, while when the impaired autophagic flux was facilitated by rapamycin, an autophagy inducer, the microglial M1 polarization was decreased correspondingly. In conclusion, cGAS induces the inflammatory phenotype of microglia via impairing autophagic flux, thereby participating in neuroinflammation, which leads to sympathetic overactivation in hypertension and further caused hypertensive myocardial injury.

The Inhibitory Role of Hydrogen Sulfide in UII-Induced Cardiovascular Effects and the Underlying Signaling Pathways.

Urotensin II (UII) could increase blood pressure and heart rate via increased central reactive oxygen species (ROS) levels. We reported previously that hydrogen sulfide (H2S) exerts an antihypertensive effect by suppressing ROS production. The aim of the current study is to further examine the effects of endogenous and exogenous H2S on UII-induced cardiovascular effects by using an integrated physiology approach. We also use cell culture and molecular biological techniques to explore the inhibitory role of H2S on UII-induced cardiovascular effects. In this study, we found that cystathionine-ß-synthase (CBS), the main H2S synthesizing enzyme in CNS, was expressed in neuronal cells of the rostral ventrolateral medulla (RVLM) area. Cellular distribution of CBS and urotensin II receptor (UT) in SH-SY5Y cells that are confirmed as glutamatergic were identified by immunofluorescent and Western blots assay. In Sprague-Dawley rats, administration of UII into the RVLM resulted in an increase in mean arterial pressure (MAP), heart rate (HR), ROS production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and phosphorylation of p47phox, extracellular signal-regulated protein kinase (ERK)1/2 and p38MAPK, but not stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK). These effects of UII were attenuated by application into the RVLM of endogenous (L-cysteine, SAM) or exogenous (NaHS) H2S. These results were confirmed in SH-SY5Y cells. UII-induced cardiovascular effects were also significantly abolished by pretreatment with microinjection of Tempol, Apocynin, SB203580, or PD98059 into the RVLM. Preincubated SH-SY5Y cells with Apocynin before administration of UII followed by Western blots assay showed that ROS is in the upstream of p38MAPK/ERK1/2. Gao activation assay in SH-SY5Y cells suggested that H2S may exert an inhibitory role on UII-induced cardiovascular effects by inhibiting the activity of Gαo. These results suggest that both endogenous and exogenous H2S attenuate UII-induced cardiovascular effects via Gαo-ROS-p38MAPK/ERK1/2 pathway.

Inhibition of the cGAS-STING Pathway Attenuates Lung Ischemia/Reperfusion Injury via Regulating Endoplasmic Reticulum Stress in Alveolar Epithelial Type II Cells of Rats.

Purpose: Endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis of lung ischemia/reperfusion (I/R) injury. Cyclic GMP-AMP synthase (cGAS) is a cytosol dsDNA sensor, coupling with downstream stimulator of interferon genes (STING) located in the ER, which involves innate immune responses. The aim of our present study was to investigate the effects of cGAS on lung I/R injury via regulating ERS. Methods: We used Sprague-Dawley rats to make the lung I/R model by performing left hilum occlusion-reperfusion surgery. cGAS-specific inhibitor RU.521, STING agonist SR-717, and 4-phenylbutyric acid (4-PBA), the ERS inhibitor, were intraperitoneally administered in rats. Double immunofluorescent staining was applied to detect the colocalization of cGAS or BiP, an ERS protein, with alveolar epithelial type II cells (AECIIs) marker. We used transmission electron microscopy to examine the ultrastructure of ER and mitochondria. Apoptosis and oxidative stress in the lungs were assessed, respectively. The profiles of pulmonary edema and lung tissue injury were evaluated. And the pulmonary ventilation function was measured using a spirometer system. Results: In lung I/R rats, the cGAS-STING pathway was upregulated, which implied they were activated. After cGAS-STING pathway was inhibited or activated in lung I/R rats, the ERS was alleviated after cGAS was inhibited, while when STING was activated after lung I/R, ERS was aggravated in the AECIIs, these results suggested that cGAS-STING pathway might trigger ERS responses. Furthermore, activation of cGAS-STING pathway induced increased apoptosis, inflammation, and oxidative stress via regulating ERS and therefore resulted in pulmonary edema and pathological injury in the lungs of I/R rats. Inhibition of cGAS-STING pathway attenuated ERS, therefore attenuated lung injury and promoted pulmonary ventilation function in I/R rats. Conclusion: Inhibition of the cGAS-STING pathway attenuates lung ischemia/reperfusion injury via alleviating endoplasmic reticulum stress in alveolar epithelial type II cells of rats.

Paraventricular Nucleus P2X7 Receptors Aggravate Acute Myocardial Infarction Injury via ROS-Induced Vasopressin-V1b Activation in Rats.

The present study was designed to investigate the mechanisms by which P2X7 receptors (P2X7Rs) mediate the activation of vasopressinergic neurons thereby increasing sympathetic hyperactivity in the paraventricular nucleus (PVN) of the hypothalamus of rats with acute myocardial ischemia (AMI). The left anterior descending branch of the coronary artery was ligated to induce AMI in rats. The rats were pretreated with BBG (brilliant blue G, a P2X7R antagonist), nelivaptan (a vasopressin V1b receptor antagonist), or diphenyleneiodonium (DPI) [an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor]. Hemodynamic parameters of the heart were monitored. Myocardial injury and cardiomyocyte apoptosis were assessed. In the PVN of AMI rats, P2X7R mediated microglial activation, while reactive oxygen species (ROS) and NADPH oxidase 2 (NOX2) were higher than in the sham group. Intraperitoneal injection of BBG effectively reduced ROS production and vasopressin expression in the PVN of AMI rats. Moreover, both BBG and DPI pretreatment effectively reduced sympathetic hyperactivity and ameliorated AMI injury, as represented by reduced inflammation and apoptosis of cardiomyocytes. Furthermore, microinjection of nelivaptan into the PVN improved cardiac function and reduced the norepinephrine (AE) levels in AMI rats. Collectively, the results suggest that, within the PVN of AMI rats, P2X7R upregulation mediates microglial activation and the overproduction of ROS, which in turn activates vasopressinergic neuron-V1b receptors and sympathetic hyperactivity, hence aggravating myocardial injury in the AMI setting.