Cervical Vagus Nerve Stimulation Improves Neurologic Outcome After Cardiac Arrest in Mice by Attenuating Oxidative Stress and Excessive Autophagy.

BACKGROUND: Cerebral ischemia and reperfusion (I/R) induces oxidative stress and activates autophagy, leading to brain injury and neurologic deficits. Cervical vagus nerve stimulation (VNS) increases cerebral blood flow (CBF). In this study, we investigate the effect of VNS-induced CBF increase on neurologic outcomes after cardiac arrest (CA). MATERIALS AND METHODS: A total of 40 male C57Bl/6 mice were subjected to ten minutes of asphyxia CA and randomized to vagus nerve isolation (VNI) or VNS treatment group. Eight mice received sham surgery and VNI. Immediately after resuscitation, 20 minutes of electrical stimulation (1 mA, 1 ms, and 10 Hz) was started in the VNS group. Electrocardiogram, blood pressure, and CBF were monitored. Neurologic and histologic outcomes were evaluated at 72 hours. Oxidative stress and autophagy were assessed at 3 hours and 24 hours after CA. RESULTS: Baseline characteristics were not different among groups. VNS mice had better behavioral performance (ie, open field, rotarod, and neurologic score) and less neuronal death (p < 0.05, vs VNI) in the hippocampus. CBF was significantly increased in VNS-treated mice at 20 minutes after return of spontaneous circulation (ROSC) (p < 0.05). Furthermore, levels of 8-hydroxy-2'-deoxyguanosine in the blood and autophagy-related proteins (ie, LC-3Ⅱ/Ⅰ, Beclin-1, and p62) in the brain were significantly decreased in VNS mice. Aconitase activity was also reduced, and the p-mTOR/mTOR ratio was increased in VNS mice. CONCLUSIONS: Oxidative stress induced by global brain I/R following CA/ROSC leads to early excessive autophagy and impaired autophagic flux. VNS promoted CBF recovery, ameliorating these changes. Neurologic and histologic outcomes were also improved.

The role of DJ-1/Nrf2 pathway in the pathogenesis of diabetic nephropathy in rats.

Diabetic nephropathy (DN) is one of the most common chronic complications of diabetes, which is associated with an increased oxidative stress induced by hyperglycemia and alterations in DJ-1/NF-E2-related factor-2 (Nrf2) pathway. In the present study, we investigated the role and the proper time nodes of DJ-1/Nrf2 pathway in the pathogenesis of DN. Diabetes mellitus (DM) model of rats was induced by intraperitoneal injection of streptozotocin (STZ) on male Sprague-Dawley (SD) rats. Then, the diabetic rats were divided into 4, 8 and 12 weeks groups. As early at 4 weeks of diabetes, renal histologic evaluation score, cystatin C (Cys C), ß2-microglobulin (ß2-MG) and malondialdehyde (MDA) levels were increased, and total antioxidative capacity (T-AOC) level was decreased as compared with that in the control group. The protein expressions of DJ-1, NF-E2-related factor-2 (Nrf2) and heme oxygenase-1 (HO-1) were upregulated compared with the control group from 4 weeks and further increased with the progression of DM. The protein expressions of DJ-1, Nrf2 and HO-1 in renal tissues have good line correlations with renal histologic evaluation score, respectively. Taken together, these results suggested that the activation of DJ-1/Nrf2 pathway was involved in the pathogenesis of diabetic nephropathy in rats.

Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation.

Most cardiac arrest (CA) survivors experience varying degrees of neurologic deficits. To understand the mechanisms that underpin CA-induced brain injury and, subsequently, develop effective treatments, experimental CA research is essential. To this end, a few mouse CA models have been established. In most of these models, the mice are placed in the supine position in order to perform chest compression for cardiopulmonary resuscitation (CPR). However, this resuscitation procedure makes the real-time imaging/monitoring of brain physiology during CA and resuscitation challenging. To obtain such critical knowledge, the present protocol presents a mouse asphyxia CA model that does not require the chest compression CPR step. This model allows for the study of dynamic changes in blood flow, vascular structure, electrical potentials, and brain tissue oxygen from the pre-CA baseline to early post-CA reperfusion. Importantly, this model applies to aged mice. Thus, this mouse CA model is expected to be a critical tool for deciphering the impact of CA on brain physiology.

Intracarotid Infusion of Redox-Active Manganese Porphyrin, MnTnBuOE-2-PyP5+, following Reperfusion Improves Long-Term, 28-Day Post-Stroke Outcomes in Rats.

Endovascular mechanical thrombectomy, combined with a tissue plasminogen activator (t-PA), is efficacious as a standard care for qualifying ischemic stroke patients. However, > 50% of thrombectomy patients still have poor outcomes. Manganese porphyrins, commonly known as mimics of superoxide dismutases, are potent redox-active catalytic compounds that decrease oxidative/nitrosative stress and in turn decrease inflammatory responses, mitigating therefore the secondary injury of the ischemic brain. This study investigates the effect of intracarotid MnTnBuOE-2-PyP5+ (BMX-001) administration on long-term, 28-day post-stroke recovery in a clinically relevant setting. The 90 min of transient middle cerebral artery occlusion was performed in young, aged, male, female, and spontaneous hypertension rats. All physiological parameters, including blood pressure, blood gas, glucose, and temperature, were well controlled during ischemia. Either BMX-001 or a vehicle solution was infused through the carotid artery immediately after the removal of filament, mimicking endovascular thrombectomy, and was followed by 7 days of subcutaneous injection. Neurologic deficits and infarct volume were assessed at 28 days in a blinded manner. The effects of BMX-001 on the carotid arterial wall and blood-brain barrier permeability and its interaction with t-PA were assessed in normal rats. There were no intra-group differences in physiological variables. BMX-001-treated stroke rats regained body weight earlier, performed better in behavioral tests, and had smaller brain infarct size compared to the vehicle-treated group. No vascular wall damage and blood-brain barrier permeability changes were detected after the BMX-001 infusion. There was no drug interaction between BMX-001 and t-PA. Intracarotid BMX-001 infusion was safe, and it significantly improved stroke outcomes in rats. These findings indicate that BMX-001 is a candidate drug as an adjunct treatment for thrombectomy procedure to further improve the neurologic outcomes of thrombectomy patients. This study warrants further clinical investigation of BMX-001 as a new stroke therapy.

H2O2-Driven Anticancer Activity of Mn Porphyrins and the Underlying Molecular Pathways.

Mn(III) ortho-N-alkyl- and N-alkoxyalkyl porphyrins (MnPs) were initially developed as superoxide dismutase (SOD) mimics. These compounds were later shown to react with numerous reactive species (such as ONOO-, H2O2, H2S, CO3 •-, ascorbate, and GSH). Moreover, the ability of MnPs to oxidatively modify activities of numerous proteins has emerged as their major mechanism of action both in normal and in cancer cells. Among those proteins are transcription factors (NF-κB and Nrf2), mitogen-activated protein kinases, MAPKs, antiapoptotic bcl-2, and endogenous antioxidative defenses. The lead Mn porphyrins, namely, MnTE-2-PyP5+ (BMX-010, AEOL10113), MnTnBuOE-2-PyP5+ (BMX-001), and MnTnHex-2-PyP5+, were tested in numerous injuries of normal tissue and cellular and animal cancer models. The wealth of the data led to the progression of MnTnBuOE-2-PyP5+ into four Phase II clinical trials on glioma, head and neck cancer, anal cancer, and multiple brain metastases, while MnTE-2-PyP5+ is in Phase II clinical trial on atopic dermatitis and itch.

Penehyclidine hydrochloride exerts protective effects in rats with acute lung injury via the Fas/FasL signaling pathway.

Acute lung injury (ALI) is a critical syndrome that is associated with high morbidity and mortality rates. The activation of the Fas/Fas ligand (FasL) signaling pathway may be an important pathophysiological mechanism during ALI development. Penehyclidine hydrochloride (PHC) has been revealed to exhibit anti-apoptotic properties and may attenuate the observed systemic inflammatory response. The present study was performed to elucidate the molecular mechanism of PHC in the regulation of the Fas/FasL signaling pathway in rats with ALI. An ALI rat model was constructed by inducing blunt chest trauma and hemorrhagic shock (T/HS), with PHC administration prior to or following T/HS. At 6 h following T/HS, blood samples and lung tissues were collected. Western blotting, arterial blood gas analysis, ELISA, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and biochemical indicator analysis were performed to determine the degree of lung injury and the key signaling pathways associated with lung damage. The results indicated that the administration of PHC following T/HS effectively attenuates lung injury by improving pulmonary oxygenation, decreasing histopathological damage, decreasing polymorphonuclear neutrophil count and decreasing Fas, FasL, caspase-8, caspase-3, tumor necrosis factor-α, interleukin (IL)-6 and IL-1ß expression. The results indicated that PHC exhibits anti-apoptotic functions and exerts protective effects in ALI rats induced by T/HS, which may be attributed to the inhibition of the Fas/FasL signaling pathway.

Penehyclidine hydrochloride alleviates lipopolysaccharide­induced acute lung injury in rats: Potential role of caveolin­1 expression upregulation.

The aim of the present study was to examine the protective effect of caveolin­1 (Cav­1) in the penehyclidine hydrochloride (PHC)­based inhibition of lipopolysaccharide (LPS)­induced acute lung injury (ALI) in vivo and in vitro, in addition to the potential underlying mechanisms. In vivo, an ALI rat model was established via intratracheal administration of LPS (5 mg/kg), and PHC (2 mg/kg) was administered 30 min following LPS treatment. In vitro, the Cav­1 gene was knocked down by small interfering (si)RNA in J774A.1 cells. Cells were incubated with LPS (1 µg/ml) for 2 h, and subsequently incubated with PHC (2 µg/ml) for an additional 2 h. Lung injury was assessed by lung histology and the ratio of polymorphonuclear leukocytes (PMNs) to total cells was assessed in bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) activity, BALF protein content and lung wet/dry (W/D) ratio. The levels of pro­inflammatory factors, including tumor necrosis factor­α (TNF­α), interleukin (IL)­6 and IL­1ß, in the sera of rats and cell culture supernatant were determined by ELISA. The protein expression levels of Cav­1, toll­like receptor 4 (TLR4), phosphorylated (p)­p38 mitogen activated protein kinases (p38 MAPKs) and nuclear factor kappa­light­chain­enhancer of activated B cells transcription factor p65 subunit (NF­κB p65) in lung tissues and J774A.1 cells were analyzed by western blot analysis. The results indicated that PHC effectively alleviated lung injury by decreasing neutrophil infiltration and protein concentration in BALF, and the lung W/D ratio and MPO activity and pro­inflammatory cytokine production induced by LPS. Furthermore, PHC significantly decreased the degrees of histopathological changes and pulmonary dysfunction. In vitro, treatment with PHC inhibited pro­inflammatory cytokine levels and MPO activity in LPS­stimulated J774A.1 cells. However, the results in the J774A.1 cells with Cav­1 gene knockdown were contrary. In addition, PHC decreased TLR4, p­p38 MAPKs and nuclear NF­κB p65 expression levels and upregulated the expression level of Cav­1, in vivo and in vitro. These data demonstrated that PHC exhibited a protective effect against LPS­induced ALI in rats and LPS­stimulated J774A.1 cells, which may be due to the inhibition of p38 MAPKs phosphorylation and TLR4/NF­κB signaling pathway by Cav­1 upregulation.

Effects of Dezocine-Remifentanil Intravenous Anaesthesia on Perioperative Signs, Serum TNF-&aipha; and IL-6 in Liver Cancer Patients undergoing Radiofrequency Ablation.

OBJECTIVE: To analyse the impact of dezocine-remifentanil intravenous anaesthesia on perioperative signs, serum tumour necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in liver cancer patients undergoing radiofrequency ablation (RFA). STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Renmin Hospital of Wuhan University, Wuhan, China, from January 2017 to February 2018. METHODOLOGY: Eighty patients with small hepatocellular carcinoma (SHCC) were selected as the research object. They were divided into Group A and Group B with the random number table method, with 40 cases in each group. Group A were given dezocine-remifentanil intravenous anaesthesia and Group B were given midazolam-remifentanil intravenous anaesthesia. Patients' situations in the surgery were compared between the two groups. Changes in heart rate (HR), mean arterial pressure (MAP) and blood oxygen saturation (SpO2) were recorded before the surgery (T0), at 5 minutes after the RFA (T1) and at the end of the RFA (T2). Levels of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on the 12 day after the RFA were compared between the two groups. RESULTS: The wake-up time in Group A was shorter than Group B (p<0.001), and the VAS pain score in Group A was lower than Group B (p<0.001). At T1, the MAP in Group A was higher than Group B (p<0.001). There was no significant difference in MAP between the two groups at T0 and T2 (p=0.881, 0.696, respectively). At T1 and T2, the HR in Group A was lower than Group B (all p<0.001). There was no significant difference in HR between the two groups at T0 (p=0.684). There was no significant difference in SpO2 between the two groups at T0, T1 and T2 (p=0.654, 0.884 and 0.798, respectively). On the 1st day after the RFA, the level of TNF-α, IL-6 in Group A were lower than those of Group B (all p<0.001). There was no significant difference in the incidence of intraoperative complications between the two groups (p=0.644). CONCLUSION: Compared with midazolam-remifentanil intravenous anaesthesia, the dezocine-remifentanil method has a better analgesic effect, shorter wake-up time, and can effectively regulate the expression of inflammatory cytokines TNF-α and IL-6. However, the effect of remifentanil on the respiratory function is dose-dependent. Therefore, respiratory cycle monitoring and management should be strengthened during the surgery.

Mechanism of myocardial ischemia/reperfusion-induced acute kidney injury through DJ-1/Nrf2 pathway in diabetic rats.

The objective of the present study was to investigate acute kidney injury (AKI) induced by myocardial ischemia/reperfusion (MIR) in diabetic rats and elucidate its underlying mechanism. A rat model of MIR was established by left anterior descending coronary artery occlusion for 30 min, followed by reperfusion for 2 h. Rats were randomly divided into four groups: i) Sham group, ii) sham + MIR group, iii) diabetic group and iv) diabetes + MIR group. Myocardial injury was detected by plasma creatine kinase isoenzyme MB and lactate dehydrogenase assays. AKI induced by MIR in diabetic rats was characterized by increases in cystatin C and ß2-microglobulin levels. Oxidative stress injury was accompanied by an increase of malondialdehyde levels and a decrease of total antioxidative capacity in the renal tissues. Immunohistochemistry and western blot analysis demonstrated that the expression of DJ-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) were significantly increased in the diabetes + MIR group compared with that in the sham + MIR and diabetic groups. Taken together, these results suggested that AKI induced by MIR in diabetic rats may be associated with activation of the DJ-1/Nrf2 pathway.

Protective effects of SOCS3 overexpression in high glucose­induced lung epithelial cell injury through the JAK2/STAT3 pathway.

Previous studies have suggested that the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway is involved in hyperglycemia­induced lung injury. The present study aimed to investigate the roles of suppressor of cytokine signaling3 (SOCS3) in the regulation of JAK2/STAT3 activation following high glucose (HG) treatment in A549 human pulmonary epithelial cells. Cell viability was evaluated using Cell Counting Kit-8 and lactate dehydrogenase assays. HG­induced inflammatory injury in A549 cells was assessed through the evaluation of interleukin­6 (IL­6) and tumor necrosis factor­α (TNF­α) levels using ELISA. The protein expression levels of SOCS3, JAK2, STAT3, phosphorylated (p)­JAK2 and p­STAT3 were determined using western blot analysis. Cellular viability was significantly decreased, whereas IL­6 and TNF­α levels were significantly increased, following HG stimulation of A549 cells. In addition, the protein levels of SOCS3, p­JAK2 and p­STAT3 were significantly increased in HG­treated cells. Treatment with the JAK2/STAT3 inhibitor tyrphostin AG490, or SOCS3 overexpression, appeared to prevent the HG­induced alterations in protein expression. Furthermore, cellular viability was enhanced, whereas the levels of proinflammatory cytokines were suppressed. These finding suggested the involvement of the SOCS3/JAK2/STAT3 signaling pathway in HG­induced responses in lung cells. Therefore, it may be hypothesized that the inhibition of the JAK2/STAT3 pathway through SOCS3 overexpression may prevent hyperglycemia­induced lung injury, and may have therapeutic potential for the treatment of patients with diabetic lung injury.

Protective effect of ginsenoside Rb1 against intestinal ischemia-reperfusion induced acute renal injury in mice.

Ginsenoside Rb1 (RB1), the most clinically effective constituent of ginseng, possesses a variety of biological activities. The objectives of this study were to investigate the protective effects of RB1 and its underlying mechanism on renal injury induced by intestinal ischemia-reperfusion (IIR) in mice. RB1 was administered prior to inducing IIR achieved by occluding the superior mesenteric artery for 45 min followed by 120 min of reperfusion. All-trans-retinoic acid (ATRA) was used as an inhibitor of NF-E2-related factor-2 (Nrf2) signaling. Adult male C57BL/6J mice were randomly divided into six groups: (1) sham group, (2) IIR group, (3) RB1 group, (4) sham + ATRA group, (5) IIR + ATRA group, and (6) RB1 + ATRA group. Intestinal histology and pathological injury score were observed. Intestinal mucosal injury was also evaluated by measuring serum diamine oxidase (DAO). Renal injury induced by IIR was characterized by increased levels of histological severity score, blood urea nitrogen (BUN), serum creatinine (Scr) and neutrophil gelatinase-associated lipocalin (NGAL), which was accompanied with elevated renal TUNEL-positive cells and the Bcl-2/Bax expression ratio. RB1 significantly reduced renal injury and apoptosis as compared with IIR group, which was reversed by ATRA treatment. Immunohistochemistry and Western blot analysis demonstrated that RB1 significantly upregulated the protein expression of heme oxygenase-1 (HO-1) and Nrf2, which were attenuated by ATRA treatment. Taken together, these results suggest that the protective effects of RB1 pretreatment against renal injury induced by IIR are associated with activation of the Nrf2/ anti-oxidant response element (ARE) pathway.