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.

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.

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.

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.

Inhibition of MALT1 Alleviates Spinal Ischemia/Reperfusion Injury-Induced Neuroinflammation by Modulating Glial Endoplasmic Reticulum Stress in Rats.

PURPOSE: Glial activation and the disorders of cytokine secretion induced by endoplasmic reticulum stress (ERS) are crucial pathogenic processes in establishing ischemia/reperfusion (I/R) injury of the brain and spinal cord. This present study aimed to investigate the effects of mucous-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) on spinal cord ischemia/reperfusion (SCI/R) injury via regulating glial ERS. METHODS: SCI/R was induced by thoracic aorta occlusion-reperfusion in rats. The MALT1-specific inhibitor MI-2 or human recombinant MALT1 protein (hrMALT1) was administrated for three consecutive days after the surgery. Immunofluorescent staining was used to detect the localization of MALT1 and ERS profiles in activated astrocyte and microglia of spinal cord. The ultrastructure of endoplasmic reticulum (ER) was examined by transmission electron microscopy. Blood-spinal cord barrier (BSCB) disruption and noninflammatory status were assessed. The neuron loss and demyelination in the spinal cord were monitored, and the hindlimb motor function was evaluated in SCI/R rats. RESULTS: Intraperitoneally postoperative MI-2 treatment down-regulated phos-NF-κB (p65) and Bip (ERS marker protein) expression in the spinal cord after SCI/R in rats. Intraperitoneal injection MI-2 attenuated the swelling/dilation of ER of the glia in SCI/R rats. Furthermore, MI-2 attenuated I/R-induced Evans blue (EB) leakage and microglia M1 polarization in spinal cord, implying a role for MALT1 in the BSCB destruction and neuroinflammation after SCI/R in rats. Furthermore, intrathecal injection of hrMALT1 aggravated the fragmentation of neuron, loss of neurofibrils and demyelination caused by I/R, while 4-PBA, an ERS inhibitor, co-treatment with hrMALT1 reversed these effects in SCI/R rats. hrMALT1 administration aggravated the motor deficit index (MDI) scoring, while 4-PBA co-treatment improved SCI/R-induced motor deficits in rats. CONCLUSION: Inhibition of MALT1 alleviates SCI/R injury-induced neuroinflammation by modulating glial endoplasmic reticulum stress in rats.

Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2.

Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ∼65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

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.

TLR4/MyD88/NF-κB Signaling in the Rostral Ventrolateral Medulla Is Involved in the Depressor Effect of Candesartan in Stress-Induced Hypertensive Rats.

This study aimed to investigate whether the proinflammatory and pressor effects of endogenous angiotensin II (AngII) are mediated by binding to the AngII type 1 receptor (AT1R) and subsequently activating central Toll-like receptor 4 (TLR4) in the rostral ventrolateral medulla (RVLM) of stress-induced hypertensive rats (SIHR). The stress-induced hypertension (SIH) model was established by random electric foot shocks combined with noise stimulation. Mean arterial pressure, heart rate, plasma norepinephrine, and RVLM AngII and TLR4 increased in a time-dependent manner in SIHR. Pro-inflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß)), myeloid differentiation factor 88 (MyD88), and nuclear factor (NF)-κB also increased, while anti-inflammatory cytokine IL-10 decreased in the RVLM of SIHR. These changes were attenuated by 14-day intracerebroventricular (ICV) infusion of VIPER (a TLR4 inhibitor) or candesartan (an AT1R antagonist). Both TLR4 and AT1R were expressed in the neurons and microglia in the RVLM of SIHR. Candesartan attenuated the expression of TLR4 in the RVLM of SIHR. This study demonstrated that endogenous AngII may activate AT1R to upregulate TLR4/MyD88/NF-κB signaling and subsequently trigger an inflammatory response in the RVLM of SIHR, which in turn enhanced sympathetic activity and increased blood pressure.

ß-arrestin 2 quenches TLR signaling to facilitate the immune evasion of EPEC.

The protein translocated intimin receptor (Tir) from enteropathogenic Escherichia coli shares sequence similarity with the host cellular immunoreceptor tyrosine-based inhibition motifs (ITIMs). The ITIMs of Tir are required for Tir-mediated immune inhibition and evasion of host immune responses. However, the underlying molecular mechanism by which Tir regulates immune inhibition remains unclear. Here we demonstrated that ß-arrestin 2, which is involved in the G-protein-coupled receptor (GPCR) signal pathway, interacted with Tir in an ITIM-dependent manner. For the molecular mechanism, we found that ß-arrestin 2 enhanced the recruitment of SHP-1 to Tir. The recruited SHP-1 inhibited K63-linked ubiquitination of TRAF6 by dephosphorylating TRAF6 at Tyr288, and inhibited K63-linked ubiquitination and phosphorylation of TAK1 by dephosphorylating TAK1 at Tyr206, which cut off the downstream signal transduction and subsequent cytokine production. Moreover, the inhibitory effect of Tir on immune responses was diminished in ß-arrestin 2-deficient mice and macrophages. These findings suggest that ß-arrestin 2 is a key regulator in Tir-mediated immune evasion, which could serve as a new therapeutic target for bacterial infectious diseases.

Asthmatic Augmentation of Airway Vagal Activity Involves Decreased Central Expression and Activity of CD73 in Rats.

The severity of asthma is closely related to the intensity of airway vagal activity; however, it is unclear how airway vagal activity is centrally augmented in asthma. Here we report that in an asthma model of male Sprague-Dawley rats, the expression and activity of ecto-5'-nucleotidase (CD73) were decreased in airway vagal centers, ATP concentration in cerebral spinal fluid was increased, and the inhibitory and excitatory airway vagal responses to intracisternally injected ATP (5 µmol) and CD73 inhibitor AMPCP (5 µmol), respectively, were attenuated. In airway vagal preganglionic neurons (AVPNs) identified in medullary slices of neonatal Sprague-Dawley rats, AMPCP (100 µmol·L-1) caused excitatory effects, as are shown in patch-clamp by depolarization, increased neuronal discharge, and facilitated spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, exogenous ATP (100 µmol·L-1, 1 mmol·L-1) primarily caused inhibitory effects, which are similar to those induced by exogenous adenosine (100 µmol·L-1). Adenosine A1 receptor antagonist CPT (5 µmol·L-1) blocked the inhibition of sEPSCs induced by 100 µmol·L-1 exogenous ATP and that by 100 µmol·L-1 exogenous adenosine, whereas 50 µmol·L-1 CPT converted the inhibition of sEPSCs induced by 1 mmol·L-1 ATP to facilitation that was blocked by addition of P2X receptor antagonist PPADS (20 µmol·L-1). These results demonstrate that in rat, the sEPSCs of AVPNs are facilitated by extracellular ATP via activation of P2X receptors and inhibited by extracellular adenosine via activation of A1 receptors; in experimental asthma, decreased CD73 expression and activity in airway vagal centers contribute to the augmentation of airway vagal activity through imbalanced ATP/ADO modulation of AVPNs.

Urinary bladder organ hypertrophy is partially regulated by Akt1-mediated protein synthesis pathway.

AIMS: The present study aims to investigate the role of Akt in the regulation of urinary bladder organ hypertrophy caused by partial bladder outlet obstruction (pBOO). MAIN METHODS: Male rats were surgically induced for pBOO. Real-time PCR and western blot were used to examine the levels of mRNA and protein. A phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was used to inhibit the activity of endogenous Akt. KEY FINDINGS: The urinary bladder developed hypertrophy at 2 weeks of pBOO. The protein but not mRNA levels of type I collagen and α-smooth muscle actin (αSMA) were increased in pBOO bladder when compared to sham control. The phosphorylation (activation) levels of Akt1 (p-Ser473), mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and 4E-BP1 were also increased in pBOO bladder. LY294002 treatment reduced the phosphorylation levels of Akt1 and 4E-BP1, and the protein levels of type I collagen and αSMA in pBOO bladder. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) were increased in pBOO bladder, and PCNA up-regulation occurred in urothelial not muscular layer. LY294002 treatment had no effect on the mRNA and protein levels of PCNA in pBOO bladder. LY294002 treatment partially reduced the bladder weight caused by pBOO. SIGNIFICANCE: pBOO-induced urinary bladder hypertrophy is attributable to fibrosis, smooth muscle cellular hypertrophy, and urothelium cell hyper-proliferation. Akt1-mediated protein synthesis in pBOO bladder contributes to type I collagen and αSMA but not PCNA up-regulation. Target of Akt1 is necessary but not sufficient in treatment of urinary bladder hypertrophy following pBOO.

The protective effects of electro-acupuncture in thoracic surgery on trauma stressed rats involve the rostral ventrolateral medulla and supraoptic nucleus.

The present study was designed to explore whether the rostral ventrolateral medulla (RVLM) and supraoptic nucleus (SON) were involved in the protective effects of electro-acupuncture (EA) in thoracic surgery on trauma-stressed rats. The rats were randomly divided into a non-stressed group (Control), surgical trauma-stressed group (Trauma), and Neiguan EA applied on the surgical trauma-stressed group (Trauma+EA-PC 6). RVLM neuron discharge was observed by using an in vivo electrophysiological method, and micro-dialysis combining high-performance liquid chromatography with fluorometric detection (HPLC-FD) was used to assess expression of amino acids in the RVLM. Immunohistochemical methods were used to assess c-Fos expression in SON neurons. The trauma of surgical stress was shown to dramatically increase the discharge frequency of RVLM neurons and promote the release of glutamate and taurine in the RVLM. The expression of c-Fos was also significantly increased in the SON of traumatized rats. EA application at Neiguan acupoints significantly suppressed trauma-induced increase of discharge frequency of the RVLM neurons, almost completely suppressed the trauma-induced increase of glutamate release but only very slightly reduced the trauma-enhanced taurine release, and inhibited the increase of c-Fos expression in these SON neurons of traumatized rats. These results indicate that Neiguan EA may improve cardiac function by modulating neurons in the RVLM and the SON in surgically traumatized rats. The taurine-mediated negative feedback may be involved in the protective effect of EA on cardiac function.

Emodin ameliorates bleomycin-induced pulmonary fibrosis in rats by suppressing epithelial-mesenchymal transition and fibroblast activation.

Aberrant activation of TGF-ß1 is frequently encountered and promotes epithelial-mesenchymal transition (EMT) and fibroblast activation in pulmonary fibrosis. The present study investigated whether emodin mediates its effect via suppressing TGF-ß1-induced EMT and fibroblast activation in bleomycin (BLM)-induced pulmonary fibrosis in rats. Here, we found that emodin induced apoptosis and inhibited cellular proliferation, migration and differentiation in TGF-ß1-stimulated human embryonic lung fibroblasts (HELFs). Emodin suppressed TGF-ß1-induced EMT in a dose- and time-dependent manner in alveolar epithelial A549 cells. Emodin also inhibited TGF-ß1-induced Smad2, Smad3 and Erk1/2 activation, suggesting that Smad2/3 and Erk1/2 inactivation mediated the emodin-induced effects on TGF-ß1-induced EMT. Additionally, we provided in vivo evidence suggesting that emodin apparently alleviated BLM-induced pulmonary fibrosis and improved pulmonary function by inhibiting TGF-ß1 signaling and subsequently repressing EMT, fibroblast activation and extracellular matrix (ECM) deposition. Taken together, our data suggest that emodin mediates its effects mainly via inhibition of EMT and fibroblast activation and thus has a potential for the treatment of pulmonary fibrosis.

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.

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.

Microglial P2X7 receptor in the hypothalamic paraventricular nuclei contributes to sympathoexcitatory responses in acute myocardial infarction rat.

Several pieces of evidence indicate that the microglial P2X7 receptor (P2X7R) regulate cardiovascular activities. We explored the possible roles of microglial P2X7R in the PVN mediated sympathoexcitatory responses in acute myocardial infarction (AMI) rat. Sprague-Dawley rats underwent coronary artery ligation to induce AMI. The rats received intraperitoneal administration of the P2X7R antagonist Brilliant Blue-G (BBG, 25 or 50 mg kg(-1), once a day for 5 days) prior to myocardial ischemia. Other rats received bilateral microinjection of P2X7R-siRNA (0.015 or 0.03 nmol 0.1µl per side, once a day for 2 days) targeting P2X7R mRNA into the PVN prior to myocardial ischemia. First, we examined the ATP levels and protein expression P2X7R in the PVN in different ischemia time groups, and we found that the change of P2X7R was positive correlated with the ATP levels in a time-dependent manner. The double-immunofluorescence evidence showed that P2X7R was mainly co-localizated with the microglial marker Iba-1 in the PVN. Second, gene knockdown of P2X7R with P2X7-siRNA or inhibition of P2X7R with BBG reduce the mRNA and protein expression of IL-1ß and TNF-α in the PVN of AMI rat. Third, microinjected P2X7-siRNA also suppressed the up-regulation of P2X7R, oxytocin and vasopressin in the PVN of AMI rats. Fourth, P2X7-siRNA and BBG also attenuated the renal sympathetic nerve activity (RSNA) in the AMI rats. Our results indicate that microglial P2X7R activation in PVN mediating the production of proinflammatory cytokines that activate oxytocinergic and vasopressinergic neuron, which augmented the RSNA in the AMI rat.

Suppression of the PI3K pathway in vivo reduces cystitis-induced bladder hypertrophy and restores bladder capacity examined by magnetic resonance imaging.

This study utilized magnetic resonance imaging (MRI) to monitor the real-time status of the urinary bladder in normal and diseased states following cyclophosphamide (CYP)-induced cystitis, and also examined the role of the phosphoinositide 3-kinase (PI3K) pathway in the regulation of urinary bladder hypertrophy in vivo. Our results showed that under MRI visualization the urinary bladder wall was significantly thickened at 8 h and 48 h post CYP injection. The intravesical volume of the urinary bladder was also markedly reduced. Treatment of the cystitis animals with a specific PI3K inhibitor LY294002 reduced cystitis-induced bladder wall thickening and enlarged the intravesical volumes. To confirm the MRI results, we performed H&E stain postmortem and examined the levels of type I collagen by real-time PCR and western blot. Inhibition of the PI3K in vivo reduced the levels of type I collagen mRNA and protein in the urinary bladder ultimately attenuating cystitis-induced bladder hypertrophy. The bladder mass calculated according to MRI data was consistent to the bladder weight measured ex vivo under each drug treatment. MRI results also showed that the urinary bladder from animals with cystitis demonstrated high magnetic signal intensity indicating considerable inflammation of the urinary bladder when compared to normal animals. This was confirmed by examination of the pro-inflammatory factors showing that interleukin (IL)-1α, IL-6 and tumor necrosis factor (TNF)α levels in the urinary bladder were increased with cystitis. Our results suggest that MRI can be a useful technique in tracing bladder anatomy and examining bladder hypertrophy in vivo during disease development and the PI3K pathway has a critical role in regulating bladder hypertrophy during cystitis.