Activation of adenosine A2B receptor alleviates myocardial ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress and restoring autophagy flux.

Myocardial ischemia-reperfusion injury (MIRI) poses a significant threat to patients with coronary heart disease. Adenosine A2A receptors have been known as a protective role in MIRI by regulating autophagy, so we assumed that activation of adenosine A2B receptor (A2BAR) might exert a similar effect during MIRI and underlying mechanism be related to proteostasis maintenance as well. In situ hearts were subjected to 30 min of ischemia and 120 min of reperfusion (IR), while invitro cardiomyocytes from neonatal rats experienced 6 h of oxygen-glucose deprivation followed by 12 h of reoxygenation (OGDR). Initially, we observed that post-ischemia-reperfusion induced autophagy flux blockade and ERS both in vivo and in vitro, evident through the increased expression of p62, LC3II, and BIP, which indicated the deteriorated proteostasis. We used a selective A2BAR agonist, Bay 60-6583, to explore the positive effects of A2BAR on cardiomyocytes and found that A2BAR activation rescued damaged cardiac function and morphological changes in the IR group and improved frail cell viability in the OGDR group. The A2BAR agonist also alleviated the blockage of autophagic flux, coupled with augmented ERS in the IR/OGDR group, which was reassured by using an autophagy inhibitor chloroquine (CQ) and ERS inhibitor (4-PBA) in vitro. Additionally, considering cAMP/PKA as a well-known downstream effector of A2BAR, we utilized H89, a selective PKA inhibitor. We observed that the positive efficacy of Bay 60-6583 was inhibited by H89. Collectively, our findings demonstrate that the A2BAR/cAMP/PKA signaling pathway exerts a protective role in MIRI by mitigating impaired autophagic flux and excessive ERS.

Optimum cycles of induction chemotherapy in concurrent chemo-radiotherapy management of unresectable stage III non-small cell lung cancer: Results from a single institutional database.

Induction chemotherapy (IC) prior to concurrent chemo-radiotherapy is the recommended treatment for unresectable stage III non-small cell lung cancer (NSCLC). However, the optimum number of IC cycles for improved survival outcomes is still not known. Here, we assessed the efficacy of 2 or more cycles of IC for unresectable stage III NSCLC patients from our hospital. Data on unresectable stage III NSCLC patients treated with IC + concurrent chemo-radiotherapy at our hospital between 2018 and 2022 were retrieved and analyzed, and survival outcomes compared between IC = 2 and IC > 2 patients. Univariate and multivariate Cox regression, and Chi-square or Fisher exact test were used to assess prognosis and acute toxicity profiles. One hundred twenty-six patients were recruited; 90 for IC = 2 and 36 for IC > 2. Median follow-up time was 26 months [IQR 16-38]. Three-year overall survival was not statistically significant between the 2 groups (77.8% vs 75.0%, P = .453). Distant metastasis free survival, loco-regional recurrence free survival and progression free survival were also not significant, (90.0% vs 86.1%, P = .068), 97.8% vs 97.2%, P = .056), and (73.3% vs 66.7%, P = .446) respectively. Univariate and multivariate Cox regression analysis revealed smoking, T_stage, N_stage, and IC_regimen as independent prognostic factor for overall survival, while drinking and T_stage were risk factors for progression free survival. In summary, 2 cycles of platinum-based IC was effective for stage III unresectable NSCLC and adding more than 2 cycles did not offer extra survival benefits.

Adenosine 2 receptor regulates autophagy and apoptosis to alleviate ischemia reperfusion injury in type 2 diabetes via IRE-1 signaling.

PURPOSE: This study aimed to determine the effect and mechanism of action of adenosine 2 receptor (A2R) activation on myocardial ischemia reperfusion injury (MIRI) under diabetic conditions. METHODS: MIRI type 2 diabetic rats and H9C2 cardiomyocytes were treated with A2R agonist and then subjected to hypoxia for 6 h and reoxygenation for 18 h. Myocardial damage, and infarct size were determined by cardiac ultrasound. Indicators of cardiomyocyte injury, creatine kinase-MB and cardiac troponin I were detected by Enzyme Linked Immunosorbent Assay. Endoplasmic reticulum stress (ERS) was determined through measuring the expression levels of ERS related genes GRP78, p-IRE1/IRE1, and p-JNKJNK. The mechanism of A2R cardio protection in MIRI through regulating ERS induced autophagy was determined by investigating the ER resident protein IRE-1. The ER-stress inducer Tunicamycin, and the IRE-1 inhibitor STF in combination with the A2R agonist NECA were used, and the cellular responses were assessed through autophagy proteins expression Beclin-1, p62, LC3 and apoptosis. RESULTS: NECA improved left ventricular function post MIRI, limited myocardial infarct size, reduced myocardial damage, decreased cardiomyocytes apoptosis, and attenuated ERS induced autophagy through regulating the IRE-XBP1s-CHOP pathway. These actions resulted into overall protection of the myocardium against MIRI. CONCLUSION: In summary, A2R activation by NECA prior to ischemia attenuates apoptosis, reduces ERS induced autophagy and restores left ventricular function. This protective effect occurs through regulating the IRE1-XBPs-CHOP related mechanisms. NECA is thus a potential target for the treatment of MIRI in patient with type 2 diabetes.

Identification of the hub susceptibility genes and related common transcription factors in the skeletal muscle of Type 2 Diabetes Mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) and its related complications contribute to the high morbidity and mortality in worldwide. Skeletal muscle insulin resistance plays a critical role in the onset of T2DM due to the decreasing in the insulin-stimulated glucose uptake. T2DM is associated not only with the inherited factors but also with the noninherited factors. However, the susceptibility genes related with the two factors and the transcription factors (TF) regulating the susceptibility genes in skeletal muscle, which aggravate the development of T2DM were still ill-defined. METHODS: In the present study, the expression profiles by the array of GSE25462 were retrieved from the GEO database. GEO2R was performed to validate the susceptibility differentially expressed genes (SDEG) in skeletal muscle of T2DM. Gene Ontology (GO) analysis and The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted via The Database for Annotation, Visualization, and Integrated Discovery (DAVID). A Protein-Protein Interaction (PPI) network was performed with the STRING. RESULTS: With the performance of GEO2R, 229 SDEGs in skeletal muscle of T2DM were identified. The biological processes (BP) of SDEGs was enriched in the cellular response to UV-B most significantly. KEGG pathway analysis revealed that the SDEGs were most significantly enriched in glycosaminoglycan degradation. 5 hub susceptibility genes (GPR84, CALCB, GCG, PTGDR, GNG8) in the skeletal muscle of T2DM were identified. Eventually, the common transcription factors regulating the hub susceptibility genes were identified by means of the online tool PROMO. CONCLUSIONS: Five hub susceptibility genes (GPR84, CALCB, GCG, PTGDR, GNG8) in the skeletal muscle of T2DM and the common transcription factors were identified. The outputs would provide new clues on the novel potential targets and the therapeutic strategies for treating T2DM and its related diseases.

Adenosine A2a Receptor Regulates Autophagy Flux and Apoptosis to Alleviate Ischemia-Reperfusion Injury via the cAMP/PKA Signaling Pathway.

Exploring effective methods to lessen myocardial ischemia-reperfusion injury still has positive significance. The adenosine A2a receptor (A2aR) has played a crucial part in cardiac ischemia-reperfusion injury. Previous studies revealed that the adenosine A2a receptor regulated autophagy, but the specific mechanism in myocardial ischemia-reperfusion injury was still unclear. We established an ischemia-reperfusion model (30 min of ischemia and 2 h of reperfusion) in vivo and a model with oxygen-glucose deprivation for 6 h and reoxygenation for 18 h (OGDR) in vitro. The ischemia-reperfusion injury resulted in prolonged QTc interval, left ventricular systolic dysfunction, and myocardial infarction. In vitro model, we found that the OGDR-induced autophagosomes and apoptosis caused myocardial cell death, as evidenced by a significant increase in the generation of lactate dehydrogenase and creatine kinase-MB. Furthermore, overactivated autophagy with rapamycin showed an anti-apoptotic effect. The interaction between autophagy and apoptosis in myocardial ischemia-reperfusion injury was complex and variable. We discovered that the activation of adenosine A2a receptor could promote the expression of Bcl-2 to inhibit the levels of Beclin-1 and LC3II. The number of autophagosomes exceeded that of autolysosomes under OGDR, but the result reversed after A2aR activation. Activated A2aR with its agonist CGS21680 before reperfusion saved cellular survival through anti-apoptosis and anti-autophagy effect, thus improving ventricular contraction disorders, and visibly reducing myocardial infarction size. The myocardial protection of adenosine A2a receptor after ischemia may involve the cAMP-PKA signaling pathway and the interaction of Bcl-2-Beclin-1.

HDAC6 inhibitor ACY-1215 improves neuropathic pain and its comorbidities in rats of peripheral nerve injury by regulating neuroinflammation.

The fact that neuropathic pain (NP) has no effective therapy and is frequently accompanied by psychiatric comorbidities is well established. Aberrant neuroinflammation plays an important role in the development and maintenance of NP. HDAC6 inhibitors have been demonstrated to ameliorate mechanical allodynia brought on by chemotherapy and peripheral nerve damage. However, its pharmacological mechanisms and its effects on NP-related mental disorders have not been fully elucidated. The present study was dedicated to exploring the effects of ACY-1215 (a specific HDAC6 inhibitor) on neuroinflammation and behavioral abnormalities associated with NP. In this work, spinal nerve ligation (SNL) was performed as an NP model on rats. Mechanical allodynia, cognitive impairment, and depressive-like behavior caused by SNL were attenuated by continuous intraperitoneal injection of ACY-1215. Moreover, ACY-1215 administration suppressed SNL-induced neuroinflammatory responses (including microgliosis, the elevation of pro-inflammatory factors IL-1ß and TNF-α) in ligation of the ipsilateral spinal dorsal horn (iSDH), hippocampus (HPC) and prefrontal cortex (PFC). Mechanistically, MyD88-dependent pro-inflammatory pathways (MyD88/NF-κB and MyD88/ERK) were activated in the iSDH following SNL and were inhibited by ACY-1215. Moreover, ACY-1215 enhanced the acetylation modification of MyD88 and inhibited the SNL-induced elevation of MyD88 without affecting its transcription in the iSDH. These findings suggest that pharmacological inhibition of HDAC6 can ameliorate NP and its psychiatric complications through modulating neuroinflammation, in part by blocking the MyD88-mediated pro-inflammatory pathways. The possible mechanism is that ACY-1215 prevents the elevation of MyD88 reactivity by increasing its acetylation level. Notably, neither SNL nor ACY-1215 significantly altered MyD88 expression in HPC and PFC, indicating differentiated pro-inflammatory mechanisms in the supraspinal neural regions.

Puerarin Attenuates Complete Freund's Adjuvant-Induced Trigeminal Neuralgia and Inflammation in a Mouse Model via Sirt1-Mediated TGF-ß1/Smad3 Inhibition.

BACKGROUND: Puerarin, an active compound of radix puerariae, is a major compound used in Chinese herbal medicines and it has been well known for its pharmacological effects, including antioxidant, anti­inflammatory, neuroprotective and cardioprotective properties. The aim of the present study was to determine the role of puerarin (Pue) in complete Freund's adjuvant (CFA)-induced trigeminal neuralgia (TN) and the effects of this compound on Sirt1 activity and on the progression of CFA-induced TN. METHODS: Mice were injected with CFA on the unilateral face to induce TN. A cell model of inflammation-associated TN was established by interleukin-1ß (IL-1ß; 10 ng/mL) and tumor necrosis factor-α (TNF-α; 50 ng/mL) stimulation of neurons. Reverse transcription-quantitative PCR and Western blot analyses were performed to analyze mRNA and protein expression levels in trigeminal ganglion and nerve cells. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was used to determine nerve cell apoptosis following IL-1ß/TNF-α or Pue treatment. RESULTS: Pue is a conceivable Sirtuin1 (Sirt1) activator used for the prevention of trigeminal nerve injury that attenuates CFA-induced TN and inflammatory cytokine-evoked overactivation of neuronal inflammation and apoptosis. Treatment of mice with inflammatory cytokines induced upregulation of cleaved caspase-3 protein expression, which was neutralized by Pue supplementation. Both in vivo and in vitro experiments led to the conclusion that Pue modulated Sirt1 activation and repressed transforming growth factor-ß1 (TGF-ß1) protein expression and drosophila mothers against decapentaplegic homolog3 (Smad3) phosphorylation in order to exert neuroprotection. CONCLUSION: The findings suggested that Pue functioned as a potential Sirt1 activator to improve neuroinflammation-induced TN and neuronal apoptosis via the suppression of TGF-ß1/Smad3 activity. The pharmacological activity of Pue provides a new perspective for the effective prevention and treatment of TN.

Exercise-Linked Irisin Prevents Mortality and Enhances Cognition in a Mice Model of Cerebral Ischemia by Regulating Klotho Expression.

Irisin, which can be released in the hippocampus after physical exercise, is demonstrated to have beneficial effects on neurovascular diseases. This study investigated the impact of exercise linked-irisin on mortality and cognition in a mice model of cerebral ischemia and further explored its underlying mechanism. The cerebrospinal concentrations of irisin and klotho from ischemic stroke patients were measured with an enzyme-linked immunosorbent assay (ELISA). The cognitive function of mice was evaluated by a series of behavioural experiments. The expressions of klotho, MnSOD, and FOXO3a in the hippocampus of mice were detected by Western blot. Superoxide production in the brain tissue of mice was evaluated with the dihydroethidium (DHE) dying. The results demonstrated that stroke patients showed a positive correlation between their CSF irisin concentration and klotho concentration. In addition, when mice subjected to cerebral ischemia, their cognitive function was impaired, the protein expressions of klotho, MnSOD, and FOXO3a downregulated, and the production of reactive oxygen species (ROS) increased compared with the sham group. After pretreatment with exogenous irisin, improved cognitive impairment, upregulated protein expressions of klotho, MnSOD, and FOXO3a, and reduced ROS generation were observed in mice with MCAO. However, the neuroprotective effects of irisin compromised with the evidence of severe cognitive impairment, decreased protein expressions of MnSOD and FOXO3a, and increased ROS production in klotho knockout mice. Thus, our results indicated that exercise-linked irisin could prevent mortality and improve cognitive impairment after cerebral ischemia by regulating klotho expression.

Valproic acid mitigates spinal nerve ligation-induced neuropathic pain in rats by modulating microglial function and inhibiting neuroinflammatory response.

Spinal inflammation is a pathophysiological state of neuropathic pain (NP). The subsequent microglial activation and neuroinflammatory response are contributing factors for long-lasting behavioral hypersensitivity. Valproic acid (VPA), a histone deacetylase inhibitor, has promising anti-inflammatory and neuroprotective properties for clinical use in the treatment of neurological disorders. However, the underlying mechanisms of its effects on NP have not been determined. This study aimed to clarify the possible mechanisms by which VPA alleviates NP in rat models induced by spinal nerve ligation (SNL). Intraperitoneal injection of VPA (300 mg/kg) efficiently attenuated mechanical allodynia in rats with NP. VPA exerted anti-inflammatory effects by downregulating proinflammatory cytokines (tumor necrosis factor-α, cytokines interleukin-1ß, cytokines interleukin-6; TNF-α, IL-1ß, and IL-6) and upregulating anti-inflammatory cytokines (transforming growth factor-ß, cytokines interleukin-10, cytokines interleukin-4; TGF-ß, IL-10 and IL-4). Additionally, VPA suppressed spinal microgliosis and promoted the polarization of microglia towards the M2 phenotype to further ameliorate spinal neuroinflammation. VPA also exerted neuroprotective effects by decreasing spinal cell apoptosis. The anti-inflammatory and neuroprotective effects may have depended on changes in nuclear histone deacetylase 3 (HDAC3) expression following VPA treatment. Moreover, VPA treatment inhibited nuclear factor-κB (NF-κB) p65 nuclear expression and upregulated acetylated the signal transducer and activator of transcription 1 (STAT1). In addition, VPA suppressed SNL-induced phosphorylation of Janus Kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Taken together, our results demonstrate that VPA is a promising anti-inflammatory agent suitable for NP therapy that regulates microglial function and suppresses spinal neuroinflammation via the STAT1/NF-κB and JAK2/STAT3 signaling pathways.

Psychological Status Among Anesthesiologists and Operating Room Nurses During the Outbreak Period of COVID-19 in Wuhan, China.

Background: Coronavirus Disease 2019 (COVID-19) caused by a novel strain of coronavirus (SARS-CoV-2) posed a major threat to public health. Anesthesiologists and operating room (OR) nurses are at high risk of occupational exposure to SARS-CoV-2 and developing COVID-19. We conducted a single-center survey to investigate the psychological status and perceived social support among operation room (OR) medical staffs during the outbreak of Coronavirus Disease 2019 (COVID-19). Methods: A total of 197 OR medical staffs were enrolled in the survey. The authors performed a cohort study during the period of Wuhan lockdown and then conducted a longitudinal follow-up after lifting of lockdown. The Patient Health Questionaire-9 (PHQ-9) was used to assess for depression and Generalized Anxiety Disorder-7 (GAD-7) for anxiety. The Multidimensional Scale of Perceived Social Support (MSPSS) was used to assess perceived social support. We compared the psychological status of OR medical staffs before and after lifting of Wuhan lockdown. Results: During the period of city lockdown, 177 (89.8%) had close contact with confirmed COVID-19 cases. The prevalence of depression and anxiety in OR medical staffs was 41.6 and 43.1% under Wuhan lockdown, while 13.2 and 15.7% after lifting of lockdown (P = 0.002, P = 0.004). Logistic regression analysis showed that being female, living in suburb areas, shortage of protective equipment and close contact with COVID-19 patients were associated with a higher risk of depression and anxiety. Perceived social support was negatively correlated with depression and anxiety severity in the OR medical staffs (P < 0.05). Conclusions: OR medical staffs exhibited high incidence of anxiety and depression faced with the high risk of exposure to COVID-19 patients. More social support and social recognition for anesthesiologists and OR nurses might potentially help them relieve their psychological pressure.

Effect of Nalmefene on Delayed Neurocognitive Recovery in Elderly Patients Undergoing Video-assisted Thoracic Surgery with One Lung Ventilation.

The intravenous use of nalmefene has been found to exert neuroprotective effect in patients with severe traumatic brain injury and acute cerebral infarction; nonetheless, it is unknown whether nalmefene alleviates delayed neurocognitive recovery. Our purpose of the current research was to clarify the impact of nalmefene on delayed neurocognitive recovery in aged patients experiencing video-assisted thoracic surgery (VATS) with intraoperative use of one lung ventilation (OLV). The present study involved 120 patients undergoing selective VATS, randomized to accept low-dose nalmefene (N1 group, n=40), high-dose nalmefene (N2 group, n=40) or equal volume of physiologic saline (control group, n=40). A battery of neuropsychological tests were used to estimate cognitive function 1 day before surgery (t0) and 10 days after surgery or before discharge (t1). Regional cerebral oxygen saturation (rSO2) was detected 5 min before induction (t0), 5 min after induction (t1), 15 and 60 min after onset of OLV (t2 and t3), and 15 min after termination of OLV (t4). The plasma values of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and adiponectin (ADP) were also detected prior to induction of anesthesia (T0), 1 h, 2 h and 6 h after surgery (T1, T2, T3). On t1, delayed neurocognitive recovery occurred in 5/40 (12.5%) patients of N1 group, in 5/40 (12.5%) patients of N2 group and in 13/40 (32.5%) patients of control group (P0.05). There were no statistical differences in rSO2 among three groups at different time points. At T1, T2 and T3, IL-1ß, IL-6 and TNF-α values significantly increased and ADP value significantly decreased (P0.05) in control group. In contrast, at T1, T2 and T3, IL-1ß, IL-6 and TNF-α values decreased and ADP value decreased less in N1 and N2 groups (P0.05). At T1, T2 and T3, IL-1ß, IL-6 and TNF-α concentrations presented a trend of N2 group N1 group control group and ADP presented a trend of N2 groupN1 groupcontrol group (P0.05). The result of our present research supports the hypothesis that the perioperative intravenous treatment with nalmefene to VATS with OLV ameliorates postoperative cognitive function and decreases the incidence of delayed neurocognitive recovery, most likely by suppression of inflammatory responses.

Direct Peritoneal Resuscitation with Pyruvate Protects the Spinal Cord and Induces Autophagy via Regulating PHD2 in a Rat Model of Spinal Cord Ischemia-Reperfusion Injury.

Direct peritoneal resuscitation with pyruvate (Pyr-PDS) has emerged as an interesting candidate to alleviate injury in diverse organs, while the potential mechanism has yet to be fully elucidated. To explore the effect of autophagy in the spinal cord ischemia-reperfusion (SCIR) injury and the underlying mechanism, we established a model of SCIR in vivo and in vitro. In vivo, male SD rats underwent aortic occlusion for 60 min and then followed by intraperitoneally infused with 20 mL of pyruvate or normal saline for 30 min, and the spinal cords were removed for analysis after 48 h of reperfusion. The functional and morphological results showed that Pyr-PDS alleviated SCIR injury; meanwhile, the expression of autophagy-related genes and transmission electron microscopy displayed autophagy was activated by SCIR injury, and Pyr-PDS treatment could further upregulate the degree of autophagy which plays a protective part in the SCIR injury, while there is no significant difference after treatment with saline. In addition, SCIR injury inhibited expression of PHD2, which results to activate its downstream HIF-1α/BNIP3 pathway to promote autophagy. In the Pyr-PDS, the results revealed PHD2 was further inhibited compared to the SCIR group, which could further activate the HIF-1α/BNIP3 signaling pathway. Additionally, oxygen-glucose deprivation and reoxygenation were applied to SH-SY5Y cells to mimic anoxic conditions in vitro, and the expression of autophagy-related genes, PHD2, and its downstream HIF-1α/BNIP3 pathway showed the same trend as the results in vivo. Besides, IOX2, a specific inhibitor of PHD2 was also treated to SH-SY5Y cells during reoxygenation, in which the result is as same as the pyruvate group. Then, we observed the expression of autophagy-related genes and the HIF-1α signal pathway in the process of reoxygenation; the results showed that as the reoxygenation goes, the expression of the HIF-1α signal pathway and degree of autophagy came to decrease gradually, while treated with pyruvate could maintain autophagy high and stable through keeping PHD2 at a lower level during reoxygenation, and the latter was observed downregulated during reoxygenation process from 0 to 24 hours in a time-effect way. The above results indicated that direct peritoneal resuscitation with pyruvate showed effective protection to ischemia-reperfusion of the spinal cord through activating autophagy via acting on PHD2 and its downstream HIF-1α/BNIP3 pathway.

Effects of irisin on the dysfunction of blood-brain barrier in rats after focal cerebral ischemia/reperfusion.

OBJECTIVE: To investigate whether irisin could protect against blood-brain barrier (BBB) dysfunction following focal cerebral ischemia/reperfusion in rats. METHODS AND MATERIALS: Seventy-two adult male Sprague Dawley rats weighing 280-320 g were randomly divided into three groups: sham operation group (S), focal cerebral ischemia/reperfusion group (FC), and irisin group (IR). Focal cerebral ischemia was induced by improved thread occlusion of right middle cerebral artery (MCAO) for 2 hr followed by reperfusion for 24 hr in rats. After 24 hr of reperfusion, the neurological evaluation was performed by the method of Longa's score. The histopathological changes were observed by HE staining. The brain water content was determined by detecting the wet weight and dry weight. The BBB permeability was assessed by fluorescence spectrophotometer and fluorescence microscopy for Evans blue (EB) extravasation. The activity and expression of matrix metalloproteinase-9 (MMP-9) in different groups were detected by immunohistochemical staining, Western blot, and gel gelatin zymography. RESULTS: After MCAO, the neurological deficit scores, the infarct volume, the brain water content, and the EB content were higher in the FC group than those in the S group (p < .05). While after irisin treatment, these indicators mentioned above were lower than those in the IR group (p < .05). Moreover, the protein expression of MMP-9 in the cortex increased significantly after MCAO, while irisin treatment could decrease the protein expression of MMP-9 in the cortex (p < .05). CONCLUSION: Our data suggest that irisin can attenuate brain damage both morphologically and functionally and protect BBB from disruption after focal cerebral ischemia/reperfusion, which is highly associated with the inhibition of the expression and activity of MMP-9 in the brain tissue.

Quercetin improves blood-brain barrier dysfunction in rats with cerebral ischemia reperfusion via Wnt signaling pathway.

Reperfusion therapy after cerebral ischemia often leads to reperfusion injury which may cause brain edema and blood-brain barrier (BBB) dysfunction. As a natural bioflavonoid, quercetin may exert protective effects on BBB dysfunction. This study aimed to investigate effects of quercetin in a rat model of global cerebral ischemia reperfusion (I/R) injury and explore the potential mechanism. Male rats were randomly divided into 4 groups: sham group, I/R group, quercetin-treated group (25 µmol/kg twice daily for 3 consecutive days before I/R), and quercetin/DKK-1-treated group. Global cerebral I/R was induced by bilateral common carotid artery occlusion combined with hypotension for 20 min and reperfusion for 24 h. Neurological function was scored, and then rats were sacrificed. The brain was harvested for HE staining, NeuN staining, and detection of brain water content. The BBB structure and permeability were examined by transmission electron microscopy and Evans blue extravasation, respectively. The protein expression of MMP-9, ZO-1, Claudin-5, ß-catenin, and GSK-3ß, and the mRNA expression of Axin and LEF1 were detected in either the absence or presence of Wnt/ß-catenin inhibitor DKK-1. Results showed that quercetin reduced brain edema and BBB leakage, and improved BBB dysfunction. Quercetin could increase the expression of ZO-1, Claudin-5, ß-catenin, and LEF1, and decrease the expression of MMP-9, GSK-3ß and Axin. And all these protective effects of quercetin could be reversed by DKK-1. Thus, quercetin can alleviate BBB dysfunction after global cerebral I/R in rats and the mechanism may be related to the activation of canonical Wnt/ß-catenin signaling pathway.

Neuroprotective effects of irisin against cerebral ischemia/ reperfusion injury via Notch signaling pathway.

Irisin, a 112-amino acid peptide induced with exercise in mice and human is thought to have correlation with the short-term outcomes of patients in ischemic stroke. In the present study, the neuroprotective effects of irisin were evaluated in vivo and in vitro and its underlying mechanism was also explored. The global cerebral ischemia/reperfusion (I/R) model was established by bilateral common carotid artery occlusion for 20 min and reperfusion for 24 h in mice and oxygen-glucose deprivation/reperfusion in HT22 cells. Neurological function was scored and then the mice were sacrificed. The brains were harvested for HE staining and detection of brain water content (BWC). The percentage of neuronal apoptosis was evaluated by TUNEL and flow cytometry analysis. The mRNA expression of TNF-α and IL-1ß was detected by RT-PCR analysis. The Notch intracellular domain (NICD) was detected by double immunofluorescence staining and western blot, and the protein expression of Notch1 and Hes 1 was detected by western blot. It was observed that irisin could alleviate morphological damage and improve neurological function after global cerebral I/R injury in mice. The apoptosis of hippocampal neurons reduced in the presence of irisin in vivo and in vitro. Additionally irisin could downregulate the expression of IL-1ß and TNF-α and upregulate the expression of NICD, Notch1 and Hes 1 in vitro and in vivo. After the application of γ-secretase inhibitor DAPT, all the morphological, neurological and biochemical changes were reversed. Taken together, these results suggest that irisin could regulate the Notch signaling pathway that leads to the alleviation of transient global cerebral I/R injury.

Serum levels of irisin predict short-term outcomes in ischemic stroke.

OBJECTIVE: Irisin is a 112-amino acid peptide found in rat and human skeletal muscle after exercise. Previous studies had suggested that higher circulating irisin levels were associated with an increased risk of vascular atherosclerosis and cardiovascular diseases. In this study, we determined irisin levels in serum, and investigated their associations with functional outcomes in a 3-month follow-up study in Chinese patients with first-ever acute ischemic stroke (AIS). METHODS: From September 2015 to December 2016, consecutive first-ever AIS patients admitted to the Department of Emergency of our hospital were identified. Serum irisin levels were measured at admission. Functional impairment was evaluated at discharge using the modified Rankin scale. The levels of irisin were expressed as median and interquartile ranges [IQR]. RESULTS: The irisin level was obtained in 324 patients (97.6%) with a median value of 291.2 ng/ml (IQR: 214.1-404.2 ng/ml). There were significantly negative correlations between levels of irisin and NHISS (r = -0.272; P < 0.001) and BMI (r = -0.193; P = 0.003). A poor functional outcome was found in 99 patients (30.6%; 95%CI: 25.5-35.6%). The poor functional outcome distribution across the irisin quartiles ranged between 51.9% (first quartile: Q1) to 12.4% (fourth quartile: Q4). In a multivariate model using the Q1 of irisin vs. Q2-4 together with the clinical variables, the marker displayed prognostic information and increased risk of poor outcomes by 94% (OR for Q1, 1.94 [95% CI, 1.19-3.42]; P = 0.018) and mortality 66% (OR for Q1, 1.66 [95% CI, 1.11-3.07]; P = 0.009). In addition, a model containing known risk factors plus irisin compared with a model containing known risk factors without irisin showed a greater discriminatory ability to predict poor outcomes (P = 0.01) and mortality (P = 0.02). CONCLUSIONS: A low serum irisin level is a predictor of poor early functional outcome in ischemic stroke patients. The underlying mechanisms of these associations remain to be investigated.

Suberoylanilide Hydroxamic Acid Triggers Autophagy by Influencing the mTOR Pathway in the Spinal Dorsal Horn in a Rat Neuropathic Pain Model.

Histone acetylation levels can be upregulated by treating cells with histone deacetylase inhibitors (HDACIs), which can induce autophagy. Autophagy flux in the spinal cord of rats following the left fifth lumber spinal nerve ligation (SNL) is involved in the progression of neuropathic pain. Suberoylanilide hydroxamic acid (SAHA), one of the HDACIs can interfere with the epigenetic process of histone acetylation, which has been shown to ease neuropathic pain. Recent research suggest that SAHA can stimulate autophagy via the mammalian target of rapamycin (mTOR) pathway in some types of cancer cells. However, little is known about the role of SAHA and autophagy in neuropathic pain after nerve injury. In the present study, we aim to investigate autophagy flux and the role of the mTOR pathway on spinal cells autophagy activation in neuropathic pain induced by SNL in rats that received SAHA treatment. Autophagy-related proteins and mTOR or its active form were assessed by using western blot, immunohistochemistry, double immunofluorescence staining and transmission electron microscopy (TEM). We found that SAHA decreased the paw mechanical withdrawal threshold (PMWT) of the lower compared with SNL. Autophagy flux was mainly disrupted in the astrocytes and neuronal cells of the spinal cord dorsal horn on postsurgical day 28 and was reversed by daily intrathecal injection of SAHA (n = 100 nmol/day or n = 200 nmol/day). SAHA also decreased mTOR and phosphorylated mTOR (p-mTOR) expression, especially p-mTOR expression in astrocytes and neuronal cells of the spinal dorsal horn. These results suggest that SAHA attenuates neuropathic pain and contributes to autophagy flux in astrocytes and neuronal cells of the spinal dorsal horn via the mTOR signaling pathway.

Flurbiprofen axetil attenuates cerebral ischemia/reperfusion injury by reducing inflammation in a rat model of transient global cerebral ischemia/reperfusion.

Ischemic stroke has been ranked as the second cause of death in patients worldwide. Inflammation which is activated during cerebral ischemia/reperfusion (I/R) is an important mechanism leading to brain injury. The present study aimed to investigate the effect of flurbiprofen axetil on cerebral I/R injury and the role of inflammation in this process. Rats were subjected to sham operation or global cerebral I/R with or without flurbiprofen axetil (5 or 10 mg/kg). Global cerebral ischemia was achieved by occlusion of bilateral common carotid arteries combined with hypotension for 20 min followed by reperfusion for 72 h. Then the neurological deficit score, hippocampal cell apoptosis, levels of aquaporin (AQP) 4, AQP9, intercellular cell adhesion molecule-1 (ICAM-1), nuclear factor-κB (NF-κB), tumor necrosis factor (TNF-α), interleukin-1 ß (IL-1ß), thromboxane B2 (TXB2), and 6-keto-PGI1α were assessed. After reperfusion, neurological deficit score was significantly increased accompanied by severe neuronal damage (exacerbated morphological deficit, increased terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL)-positive cells and cleaved caspase-3 protein expression in hippocampal CA1 region). Cerebral I/R injury also enhanced expressions of TNF-α, IL-1ß, NF-κB, AQP4 and AQP9 as well as TXB2 and TXB2/6-keto-PGI1α. All these changes were reversed by pretreatment with flurbiprofen axetil. Flurbiprofen axetil protects the brain from cerebral I/R injury through reducing inflammation and brain edema.

Restenosis Inhibition and Re-differentiation of TGFß/Smad3-activated Smooth Muscle Cells by Resveratrol.

To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFß) and its signaling protein Smad3, known contributors to IH. TGFß/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFß/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies - a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.

HO-1 Protects against Hypoxia/Reoxygenation-Induced Mitochondrial Dysfunction in H9c2 Cardiomyocytes.

BACKGROUND: Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial's structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism. METHODS AND RESULTS: The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed. CONCLUSIONS: During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect.