Role and intervention of PAD4 in NETs in acute respiratory distress syndrome.

BACKGROUND: Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. Acute respiratory distress syndrome (ARDS) is a common sepsis-associated injury that can increase postoperative mortality but the mechanism is still unclear. MAIN TEXT: The role of neutrophils in the pathophysiology of sepsis was deeply challenged after the discovery of NETosis, a process resulting in neutrophil extracellular traps (NETs) release. NETs can support thrombin generation and the concept of immunothrombosis has emerged as a new innate response to infection. Immunothrombosis leads to thrombosis in microvessels and supports immune cells together with specific thrombus-related molecules. ARDS is a common sepsis-associated organ injury. Immunothrombosis participates in thrombosis in pulmonary capillaries. Intervention regarding immunothrombosis in ARDS is a key scientific problem. PAD4 is the key enzyme regulating the NET skeleton protein histone H3 to citrulline histone to form NETs in immune thrombosis. This review summarizes NETosis and immunohaemostasis, ARDS and therapeutic opportunities targeting PAD4 via PAD4 inhibitors and lncRNAs potentially, providing future therapies. CONCLUSIONS: We identified and summarized the fundamental definition of ARDS and the concept of immune thrombosis and its composition. NETs activation has become particularly relevant in the formation of immune thrombosis. The taskforce highlighted the intervention targets of PAD4, including noncoding RNAs, potentially providing future therapeutic targets to confront the high postoperative mortality of ARDS.

Effects of preoperative oral enzyme-hydrolyzed rice flour solution on gastric emptying and insulin resistance in patients undergoing laparoscopic cholecystectomy: a prospective randomized controlled trial.

BACKGROUND: The effect of preoperative oral carbohydrates (POC) on insulin resistance (IR) of laparoscopic cholecystectomy (LC) remains debatable. Enzyme-hydrolyzed rice flour (EHR) is a kind of water-soluble micromolecular carbohydrates. This study aimed to investigate the impact of preoperative oral EHR solution on gastric emptying and IR in patients undergoing LC. METHODS: Patients (n = 100) undergoing LC were divided into oral-water group (group C) or oral-EHR solution (group E) randomly (n = 50 each), and the patients drank 300 ml water or EHR solution 2-3 h before surgery respectively. Gastric emptying which was quantized by gastric volume (GV) from antrum ultrasonography, IR indicators, subjective comfort indicators, handgrip strength, postoperative recovery indexes, and complications were recorded. RESULTS: There were no differences in GV between the two groups before oral administration (V0), immediately after oral administration (V1) and before anesthesia induction(V2). The GV at V2 (GV2) reduced to the level of V0 (GV0) in the two groups. Fasting glucose (FG), fasting insulin (FINS) and Homa-IR in the two groups increased at postoperative day 1 (Pos 1d) compared with those at preoperative day 1(Pre 1d). Homa-IS and Homa-ß in the two groups decreased at Pos 1d compared with those at Pre 1d. FG, FINS and Homa-IR in group E were lower than those in group C at Pos 1d, and Homa-IS and Homa-ß were higher in group E than those in group C at Pos 1d. Subjective comfort indictors (hunger, fatigue and anxiety) in group E were lower than those in group C at preoperative 15 min (Pre 15 min) and postoperative 1 h (Pos 1 h). Handgrip strength in group E was raised compared with that in group C at Pre 15 min, Pos 1 h and Pos 1d. There was a lower incidence of nausea and earlier exhaust time in group E. CONCLUSION: Oral 300 ml EHR solution 2-3 h before LC surgery did not increase the occurrence of reflux and aspiration during anesthesia induction with a normal gastric emptying, ameliorated postoperative IR, improved subjective comfort, and promoted postoperative gastrointestinal function recovery. TRIAL REGISTRATION: Prospectively registered at the China Clinical Trial Registry, registration number: ChiCTR2000039939, date of registration:14/11/2020.

Relationship Between mTOR Signaling Activation and Postoperative Neurocognitive Disorder in Aged Rats.

BACKGROUND: Although incidence rates of postoperative neurocognitive disorder (PND) in aged individuals following noncardiac major surgery are rising as individuals are living longer, the mechanism of PND remains poorly understood. We wondered if mammalian target of rapamycin (mTOR) signaling might be associated with PND since mTOR controls some essential intracellular events. OBJECTIVE: To investigate whether surgery activates the mTOR signaling pathway in aged rats, leading to PND, and whether the mTOR inhibitor, rapamycin, can be used to alleviate PND. METHODS: We randomly assigned aged rats to four groups: normal control (C), isoflurane (I), surgery (S), and rapamycin (R). Then, we anesthetized Groups I, S, and R, following which, Groups S and R underwent a splenectomy. After surgery, Group R was administered rapamycin. We used the Morris water maze to test the rats' spatial learning and memory after surgery. RESULTS: In Group S, escape latency (ie, the time to find the platform) was markedly higher, and the ratio of swimming time in the target quadrant was lower, compared to the other groups. In Group R, escape latency was markedly lower as compared with Group S, and the ratio of swimming time in the target quadrant was higher. CONCLUSIONS: Our results indicate that an altered mTOR signaling pathway after a splenectomy causes PND in aged rats, which can be alleviated by rapamycin.

[Effects of stellate ganglion block on AMP-ativated protein kinase and astrocyte in hippocampal neurons in postoperative aged rats].

OBJECTIVE: To investigate the effects of stellate ganglion block on AMP-activated protein kinase and astrocyte in hippocampal neurons in postoperative aged rats. METHODS: A total of 96 male Sprague-Dawley rats aged 18-20 months, weighing 450-550 g, were randomly divided into 4 groups (n = 24): Control group (Group I); Surgery group (Group II); Normal saline + surgery group (Group III); SGB+ surgery group (Group IV). SGB with 0.25% bupivacaine 0.15 ml and operation was conducted after 15 min in groups II, III and IV. The eight rats were randomly sacrificed in each group postoperative 1, 3, and 5 d. The escape latency and swimming distance were recorded by morris water maze. The expression of AMPKmRNA,AMPK and phosphorylated AMPK(p-AMPK) was measured by RT-PCR and Western blot. The expression of GFAP was measured by immunohistochemisty. RESULTS: Compared with I group, the escape latency and swimming distance were significantly prolonged and the expression of AMPK mRNA, AMPK and p-AMPK was up-regulated and the expression of GFAP in astrocyte was significatively increased in groups II, III and IV. Compared with II and III group, the escape latency and swimming distance were significantly shorten and the expression of AMPK mRNA, AMPK and p-AMPK was down-regulated and the expression of GFAP in astrocyte was significatively decreased in group IV. CONCLUSION: SGB can improve the postoperative cognitive function, and the mechanism may be associated with down-regulating the expression of AMPK and restraining the activation of the astrocyte.

Peroxiredoxin 1 alleviates oxygen-glucose deprivation/ reoxygenation injury in N2a cells via suppressing the JNK/caspase-3 pathway.

Objectives: Cerebral ischemia/reperfusion (I/R) injury inevitably aggravates the initial cerebral tissue damage following a stroke. Peroxiredoxin 1 (Prdx1) is a representative protein of the endogenous antioxidant enzyme family that regulates several reactive oxygen species (ROS)-dependent signaling pathways, whereas the JNK/caspase-3 proapoptotic pathway has a prominent role during cerebral I/R injury. This study aimed to examine the potential mechanism of Prdx1 in Neuro 2A (N2a) cells following oxygen-glucose deprivation and reoxygenation (OGD/R) injury. Materials and Methods: N2a cells were exposed to OGD/R to simulate cerebral I/R injury. Prdx1 siRNA transfection and the JNK inhibitor (SP600125) were used to interfere with their relative expressions. CCK-8 assay, flow cytometry, and lactate dehydrogenase (LDH) assay were employed to determine the viability and apoptosis of N2a cells. The intracellular ROS content was assessed using ROS Assay Kit. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were conducted to detect the expression levels of Prdx1, JNK, phosphorylated JNK (p-JNK), and cleaved caspase-3. Results: Firstly, Prdx1, p-JNK, and cleaved caspase-3 expression were significantly induced in OGD/R-exposed N2a cells. Secondly, the knockdown of Prdx1 inhibited cell viability and increased apoptosis rate, expression of p-JNK, and cleaved caspase-3 expression. Thirdly, SP600125 inhibited the JNK/caspase-3 signaling pathway and mitigated cell injury following OGD/R. Finally, SP600125 partially reversed Prdx1 down-regulation-mediated cleaved caspase-3 activation and OGD/R damage in N2a cells. Conclusion: Prdx1 alleviates the injury to N2a cells induced by OGD/R via suppressing JNK/caspase-3 pathway, showing promise as a potential therapeutic for cerebral I/R injury.

[Effect of M1 microglia-derived exosomal microRNA-20a-5p on neuronal injury after oxygen-glucose deprivation and restoration injury].

OBJECTIVE: To investigate the effect of M1 microglia-derived exosomes (M1-exo) on neuronal injury after oxygen-glucose deprivation and restoration, and to explore its mechanism. METHODS: The mouse microglia BV2 cells grown in logarithmic growth phase were added with 100 µg/L liposolysaccharide (LPS) and 20 µg/L interferon-γ (IFN-γ) to induce the polarization of microglia into M1 phenotype. M1 microglia were identified by Western blotting, quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence. The supernatant of M1 microglia was collected, and exosomes were extracted by ExoQuick-TCTM kit. The morphology of exosomes were observed by transmission electron microscope and nanoparticle tracking analysis (NTA), and the expression of characteristic proteins CD9 and CD63 of exosomes were detected by Western blotting. The well-growing mouse neuroblastoma N2a cells were divided into six groups: the cells in group C were conventionally-cultured; and the cells in group O were subjected to oxygen-glucose deprivation for 3 hours followed by restoration of oxygen-glucose supply 24 hours to establish the model of oxygen-glucose deprivation and restoration injury; and the N2a cells in group E were co-cultured with M1-exo 24 hours after oxygen-glucose deprivation 3 hours; NC group, M group and I group constructed negative control, overexpression and knockdown of microRNA-20a-5p (miR-20a-5p) M1-exo, respectively. The succession of transfection was detected by qPCR and N2a cells in group NC, group M and group I were co-cultured with such transfected M1-exo for 24 hours after oxygen-glucose deprivation 3 hours. Cell viability were detected by cell counting kit-8 (CCK-8) assay, cell apoptosis were detected by flow cytometry, and the expression of miR-20a-5p were detected by qPCR. RESULTS: Compared with M0 microglia, the fluorescence intensity and mRNA and protein expressions of CD32 and inducible nitric oxide synthase (iNOS), specific markers of M1 microglia, were increased [CD32 (fluorescence intensity): 36.919±1.541 vs. 3.533±0.351, CD32 mRNA (2-ΔΔCt): 4.887±0.031 vs. 1.003±0.012, CD32/ß-actin: 2.663±0.219 vs. 1.000±0.028; iNOS (fluorescence intensity): 29.513±1.197 vs. 7.933±0.378, iNOS mRNA (2-ΔΔCt): 4.829±0.177 vs. 1.000±0.016, iNOS/ß-actin: 1.991±0.035 vs. 1.000±0.045; all P < 0.01], indicating M1 microglia were successfully activated. Under electron microscopy, M1-exo had round or oval vesicular bodies with obvious membranous structures, with diameters ranging from 100 nm. Western blotting showed that the exosomes expressed specific CD63 and CD9 proteins. Compared with group C, the cell viability was decreased, the apoptosis rate and the expression of miR-20a-5p were significantly increased in group O [cell viability (A value): 0.540±0.032 vs. 1.001±0.014, apoptosis rate: (19.857±0.910)% vs. (13.508±0.460)%, miR-20a-5p (2-ΔΔCt): 5.508±0.291 vs. 1.033±0.101, all P < 0.01]. Compared with O group, cell viability was decreased, apoptosis rate and the expression of miR-20a-5p were increased in group E [cell viability (A value): 0.412±0.029 vs. 0.540±0.032, apoptosis rate: (31.802±0.647)% vs. (19.857±0.910)%, miR-20a-5p (2-ΔΔCt): 8.912±0.183 vs. 5.508±0.291, all P < 0.01], indicating that M1 microglia-derived exosomes further aggravated the damage of N2a cells after oxygen-glucose deprivation and restoration. Compared with group E, cell viability was decreased, apoptosis rate and the expression of miR-20a-5p were increased in group M [cell viability (A value): 0.311±0.028 vs. 0.412±0.029, apoptosis rate: (36.343±0.761)% vs. (31.802±0.647)%, miR-20a-5p (2-ΔΔCt): 32.348±0.348 vs. 8.912±0.183, all P < 0.01]; and the cell viability was increased, apoptosis rate and the expression of miR-20a-5p were decreased in group I [cell viability (A value): 0.498±0.017 vs. 0.412±0.029, apoptosis rate: (26.437±0.793)% vs. (31.802±0.647)%, miR-20a-5p (2-ΔΔCt): 6.875±0.219 vs. 8.912±0.183, all P < 0.01]. There was no significant difference in cell viability, apoptosis rate and the expression of miR-20a-5p between group E and group NC. CONCLUSIONS: M1 microglia-derived exosomes aggravate the injury of neurons after oxygen and glucose deprivation and reoxygenation, which may be related to miR-20a-5p carried by M1-exo.

Selective-cerebral-hypothermia-induced neuroprotection against-focal cerebral ischemia/reperfusion injury is associated with an increase in SUMO2/3 conjugation.

Selective cerebral hypothermia is considered an effective treatment for neuronal injury after stroke and avoids the complications of general hypothermia. Several recent studies hanve suggested that SUMO2/3 conjugation occurs following cerebral ischemia/reperfusion (I/R) injury. However, the relationship between the cerebral protective effect of selective cerebral hypothermia and SUMO2/3 conjugation remains unclear. In this study, we investigated the effect of selective cerebral hypothermia on SUMO2/3 conjugation during focal cerebral I/R injury. A total of 140 Sprague-Dawley rats were divided into four groups. In the sham group, only the carotid artery was exposed. The endoluminal filament technique was used to induce middle cerebral artery occlusion in the other three groups. After 2 h of occlusion, the filaments were slowly removed to allow blood reperfusion in the I/R group. In the hypothermia (HT) group and normothermia (NT) group, normal saline at 4 °C and 37 °C, respectively , was perfused through the carotid artery, followed by the restoration of blood flow. The results of the modified neurological severity score (mNSS), 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining demonstrated that selective cerebral hypothermia significantly decreased I/R-induced neuronal injury (mNSS, n = 8, 24 h, HT (5.88 ± 2.36) vs. I/R (8.63 ± 3.38), P < 0.05. 48 h, HT (5.75 ± 2.25) vs. I/R (8.5 ± 2.88), P < 0.05. Cerebral infarct volume percentages, n = 5, HT (18.71 ± 2.13) vs. I/R (41.52 ± 2.90), P < 0.01. Cell apoptosis rate, n = 5, 24 h, HT (21.28 ± 2.61) vs. I/R (43.72 ± 4.30), P < 0.05. 48 h, HT (20.50 ± 2.53) vs. I/R (38.94 ± 2.93), P < 0.05). The expression of Ubc9 and conjugated SUMO2/3 proteins was increased at 24 and 48 h after reperfusion in the 3 non-sham groups, and hypothermia further upregulated the expression of Ubc9 and conjugated SUMO2/3 proteins in the HT group. The expression of SENP3 was increased in the NT group and I/R group, while it was decreased in the HT group at 24 and 48 h after reperfusion (Relative quantities, n = 5, Ubc9, 24 h, HT (2.44 ± 0.22) vs. I/R (1.55 ± 0.39), P < 0.05. 48 h, HT (2.69 ± 0.16) vs. I/R (2.25 ± 0.33), P < 0.05. SENP3, 24 h, HT (0.47 ± 0.15) vs. I/R (2.18 ± 0.43), P < 0.05. 48 h, HT (0.72 ± 0.06) vs. I/R (1.51 ± 0.19), P < 0.05. conjugated SUMO2/3 proteins, 24 h, HT (2.84 ± 0.24) vs. I/R (2.51 ± 0.20), P < 0.05. 48 h, HT (2.73 ± 0.13) vs. I/R (2.44 ± 0.13), P < 0.05). Further analysis showed that the variation in SENP3 expression was more obvious than that in Ubc9 under hypothermia intervention in the HT group. These findings suggest that selective cerebral hypothermia could increase SUMO2/3 modification mainly via down-regulating the expression of SENP3, and then exert neuroprotective effects in rats with cerebral I/R injury.

Selective brain hypothermia-induced neuroprotection against focal cerebral ischemia/reperfusion injury is associated with Fis1 inhibition.

Selective brain hypothermia is considered an effective treatment for neuronal injury after stroke, and avoids the complications of general hypothermia. However, the mechanisms by which selective brain hypothermia affects mitochondrial fission remain unknown. In this study, we investigated the effect of selective brain hypothermia on the expression of fission 1 (Fis1) protein, a key factor in the mitochondrial fission system, during focal cerebral ischemia/reperfusion injury. Sprague-Dawley rats were divided into four groups. In the sham group, the carotid arteries were exposed only. In the other three groups, middle cerebral artery occlusion was performed using the intraluminal filament technique. After 2 hours of occlusion, the filament was slowly removed to allow blood reperfusion in the ischemia/reperfusion group. Saline, at 4°C and 37°C, were perfused through the carotid artery in the hypothermia and normothermia groups, respectively, followed by restoration of blood flow. Neurological function was assessed with the Zea Longa 5-point scoring method. Cerebral infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride staining, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Fis1 and cytosolic cytochrome c levels were assessed by western blot assay. Fis1 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction. Mitochondrial ultrastructure was evaluated by transmission electron microscopy. Compared with the sham group, apoptosis, Fis1 protein and mRNA expression and cytosolic cytochrome c levels in the cortical ischemic penumbra and cerebral infarct volume were increased after reperfusion in the other three groups. These changes caused by cerebral ischemia/reperfusion were inhibited in the hypothermia group compared with the normothermia group. These findings show that selective brain hypothermia inhibits Fis1 expression and reduces apoptosis, thereby ameliorating focal cerebral ischemia/reperfusion injury in rats. Experiments were authorized by the Ethics Committee of Qingdao Municipal Hospital of China (approval No. 2019008).

Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1.

Electroacupuncture preconditioning at acupoint Baihui (GV20) can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 (Drp1) can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 (depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day). Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

[Impacts of electroacupucnture at Zusanli (ST 36) and Feishu (BL 13) on the postoperative inflammatory reaction and pulmonary complications in senile patients after radical resection of pulmonary carcinoma].

OBJECTIVE: To observe the impacts of electroacupuncture (EA) at Zusanli (ST 36) and Feishu (BL 13) applied 30 min before the operation till the end of the operation on the postoperative inflammatory reaction and pulmonary complications in the senile patients after radical resection of pulmonary carcinoma. METHODS: Eighty senile patients of pulmonary carcinoma were selected and randomized into an observation group and a control group, 40 cases in each one. In the observation group, EA stimulation at Zusanli (ST 36) and Feishu (BL 13) was used 30 min before the operation till the end of the operation. In the control group, electric stimulation was not used. Separately, before operation (T1, basic state), 12 h after operation (T2) and 24 h after operation (T3), blood sample was collected from the central vein. The concentrations of plasma tumor necrosis factor-ɑ (TNF-ɑ) and interleukin-10 (IL-10) were detected. Additionally, the radial arterial blood sample was collected at the above time points; oxygen partial pressure (PaO2) was determined; pulmonary alveoli-arterial partial pressure of oxygen (PA-aDO2) and oxygenation index (OI) were calculated. The pulmonary complication in the two days after operation was recorded. RESULTS: Compared with the control group, in the observation group, at T2 and T3, TNF-ɑ concentration and PA-aDO2 were lower (all P<0.05); plasma IL-10 concentration and OI were higher (all P<0.05). In the observation group, the incidences of postoperative pneumonia and acute pulmonary injury were lower than those in the control group (both P<0.05). CONCLUSIONS: EA reduces the postoperative inflammatory reaction in the senile patients with radical resection of pulmonary carcinoma and decreases the postoperative pulmonary complicattizen.

Hypothermia-induced ischemic tolerance is associated with Drp1 inhibition in cerebral ischemia-reperfusion injury of mice.

Excessive mitochondrial fission activation has been implicated in cerebral ischemia-reperfusion (IR) injury. Hypothermia is effective in preventing cerebral ischemic damage. However, effects of hypothermia on ischemia-induced mitochondrial fission activation is not well known. Therefore, the aim of this study was to investigate whether hypothermia protect the brain by inhibiting mitochondrial fission-related proteins activation following cerebral IR injury. Adult male C57BL/6 mice were subjected to transient forebrain ischemia induced by 15min of bilateral common carotid artery occlusion (BCCAO). Mice were divided into three groups (n=48 each): Hypothermia (HT) group, with mild hypothermia (32-34°C) for 4h; Normothermia (NT) group, similarly as HT group except for cooling; Sham group, with vessels exposed but without occlusion or cooling. Hematoxylin and eosin (HE), Nissl staining, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and behavioral testing (n=6 each) demonstrated that hypothermia significantly decreased ischemia-induced neuronal injury. The expressions of Dynamin related protein 1 (Drp1) and Cytochrome C (Cyto C) (n=6 each) in mice hippocampus were measured at 3, 6, 24, and 72h of reperfusion. IR injury significantly increased expressions of total Drp1, phosphorylated Drp1 (P-Drp1 S616) and Cyto C under normothermia. However, mild hypothermia inhibited Drp1 activation and Cyto C cytosolic release, preserved neural cells integrity and reduced neuronal necrosis and apoptosis. These findings indicated that mild hypothermia-induced neuroprotective effects against ischemia-reperfusion injury is associated with suppressing mitochondrial fission-related proteins activation and apoptosis execution.

[Impacts on oxidative stress in the patients with cardiac valve replacement treated with electroacupuncture at Neiguan (PC 6)].

OBJECTIVE: To observe the myocardial protective effect of electroacupuncture (EA) at Neiguan (PC 6) in the patients with valve replacement via extracorporeal circulation. METHODS: Fifty patients of rheumatic cardiac disease planned for valve replacement were graded as II or III level according to America Society of Anesthesiologists (ASA) and were randomized into an observation group and a control group, 25 cases in each one. The same anesthesia and valve replacement via extracorporeal circulation were adopted in the patients of the two groups. In the observation group, 30 min before operation, EA was used to stimulate bilateral Neiguan (PC 6) till the end of operation. The venous blood was collected at 5 time points separately, named before aorta blockage (T1), 15 min after aorta open (T2), 30 min after aorta open (T3), 6 h after opening (T4) and 24 h after opening (T5). The concentrations of malondial dehyde (MDA), superoxide dismutase (SOD) and cardiac troponin 1 (cTnI) were determined in serum. The heart re-beating and the total dosage of vasoactive drugs after operation were recorded. RESULTS: Compared with those before aorta blockage, MDA and cTnI at each time point of aorta open were all apparently increased in the patients of the two groups (all P<0. 05), and SOD was reduced apparently (P<0. 05). Compared with the control group, at the time points from T3 to T5 , MDA and cTnL were lower apparently in the observation group as compared with those in the control group (all P<0. 05) and SOD was higher than that in the control group (P<0. 05). The dosage of vasoactive drugs was reduced apparently (P<. 05). CONCLUSION: EA at Neiguan (PC 6) alleviates oxidative stress injury and has the protective effect on ischemic reperfusion myocardium.

Electroacupuncture preconditioning attenuates ischemic brain injury by activation of the adenosine monophosphate-activated protein kinase signaling pathway.

Electroacupuncture has therapeutic effects on ischemic brain injury, but its mechanism is still poorly understood. In this study, mice were stimulated by electroacupuncture at the Baihui (GV20) acupoint for 30 minutes at 1 mA and 2/15 Hz for 5 consecutive days. A cerebral ischemia model was established by ligating the bilateral common carotid artery for 15 minutes. At 72 hours after injury, neuronal injury in the mouse hippocampus had lessened, and the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-positive cells reduced after electroacupuncture treatment. Moreover, expression of adenosine monophosphate-activated protein kinase α (AMPKα) and phosphorylated AMPKα was up-regulated. Intraperitoneal injection of the AMPK antagonist, compound C, suppressed this phenomenon. Our findings suggest that electroacupuncture preconditioning alleviates ischemic brain injury via AMPK activation.

[Effect of electroacupuncture pretreatment on apoptotic neurons and expression of GRP 78 and GADD 153 in the hippocampus in rats with global cerebral ischemia/reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) pretreatment on the number of survival neurons and the expression of glucose regulated protein 78 (GRP 78) and growth arrest and DNA damage-inducible gene 153 (GADD 153) in the hippocampus in rats with global cerebral ischemia /reperfusion injury (CI/R), so as to study its underlying mechanism in neuroprotective action. METHODS: SD male rats were randomly divided into 3 groups (n =48 each):sham operation,Cl/R model and EA pretreatment group. Global CI/R model was induced by 4-vessel occlusion (bilateral vertebral artery cauterization and bilateral carotid artery ligation for 5 min, followed by reperfusion). Before modeling, EA pretreatment of "Baihui" (GV 20) and "Dazhui" (GV 14, 2 Hz/15 Hz, 1 mA) was given to rats of the EA pretreatment group for 30 min, once daily for 5 days. At 6, 12, 24 and 48 h after CI/R, the hippocampus tissues of rats in different subgroups were separately sampled to be stained with H. E. method for detecting the number of the survived neurons, stained with TUNEL method for assaying the apoptotic neurons in the CA 1 region, and processed with Western blot (WB) for assaying the expression of GRP 78 and GADD 153 proteins. RESULTS: Compared with the sham group, the number of hippocampal survival neurons was significantly decreased at the time-points of 12 h, 24 h and 48 h after CI/R in the model group (P<0. 05) and was apparently increased by EA pretreatment at 24 h and 48 h (P<0. 05). The number of the apoptotic neurons in the hippocampal CA 1 region at the time-points of 6 h, 12 h, 24 h and 48 h after CI/R was significantly bigger in the model group than in the sham group (P<0. 05), and was obviously decreased at 12 h, 24 h, 48 h after CI/R in the EA pretreatment group (P<0.05). WB detection showed that the expression levels of hippocampal GRP 78 and GADD 153 proteins at the four time-points after CI/R were significantly higher in the model group than in the sham group (P<0. 05). Compared with the model group, hippocampal GRP 78 protein expression levels at the 4 time-points were further markedly up-regulated (P<0.05), while GADD 153 protein expression levels at the 4 time-points were significantly suppressed in the EA pretreatment group (P<0.05). CONCLUSION: EA pretreatment can effectively suppress the number of hippocampal apoptotic neurons and increase survival rate of neurons in CI/R rats, which may be closely associated with its effects in up-regulating the expression of GRP 78 protein and down-regulating the expression of GADD 153 protein in the hippocampus.

[Effect of electroacupuncture pretreatment on expression of glucose-regulated protein 78 in hippocampus of rats with cerebral ischemia/reperfusion].

OBJECTIVE: To explore the effects and action mechanism of electroacupuncture (EA) pretreatment on rats with transient cerebral ischemia/reperfusion. METHODS: A total of 144 healthy SD male rats were randomly divided into a sham operation group (group S), an ischemia/reperfusion group (group I/R) and an EA pretreatment group (group EA), 48 rats in each one. The model of cerebral ischemia/reperfusion was established by using 4-vessel occlusion method in the group I/R; after 5 min of cerebral ischemia, the reperfusion was performed. The group EA was treated with EA at "Dazhui" (GV 14) and "Baihui" (GV 20) 5 days before model establishment, 30 min per time, once a day. In group S, bilateral foramen alares were exposed without burning on the vertebral arteries, and bilateral common carotid arteries were unfolded and not occluded. The rats in the group I/R and group EA were sacrificed 6 h, 12 h, 24 h and 48 h after reperfusion and those in the group S were sacrificed at corresponding time to collect hippocampus example. The Western-blot method was used to measure the expression of glucose-regulated protein 78 (GRP 78), and HE staining method was used to count the number of surviving neurons, and TUNEL method was used to measure the number of apoptotic neurons. RESULTS: Compare with the group S, the number of surviving neurons in hippocampus was reduced at each reperfusion time point and the number of apoptotic neurons was increased (all P<0.05) in the group I/R and the group EA; the expression of GRP 78 at each reperfusion time point in group I/R and group EA was increased (P<0.05). Compared with the group I/R, the number of surviving neurons in hippocampus was increased at each reperfusion time point and the number of apoptotic neurons was reduced in the group EA (P<0.05); the expression of GRP 78 at each reperfusion time point was further increased (P<0.05). CONCLUSION: The electroacupuncture pretreatment has obvious cerebral protection on rats with ischemia/reperfusion, which is related with further increasing the expression of GRP 78 in ischemia area, leading to relieved endoplasmic reticulum stress.

Hypothermia protects the brain from transient global ischemia/reperfusion by attenuating endoplasmic reticulum response-induced apoptosis through CHOP.

Endoplasmic reticulum (ER) stress has been implicated in the pathology of cerebral ischemia. Apoptotic cell death occurs during prolonged period of stress or when the adaptive response fails. Hypothermia blocked the TNF or Fas-mediated extrinsic apoptosis pathway and the mitochondria pathway of apoptosis, however, whether hypothermia can block endoplasmic reticulum mediated apoptosis is never known. This study aimed to elucidate whether hypothermia attenuates brain cerebral ischemia/reperfusion (I/R) damage by suppressing ER stress-induced apoptosis. A 15 min global cerebral ischemia rat model was used in this study. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in hippocampus CA1 were assessed after reperfusion of the brain. The expressions of C/EBP-homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) in ischemic hippocampus CA1 were measured at 6, 12, 24 and 48 h after reperfusion. The results showed that hypothermia significantly attenuated brain I/R injury, as shown by reduction in cell apoptosis, CHOP expression, and increase in GRP78 expression. These results suggest that hypothermia could protect brain from I/R injury by suppressing ER stress-induced apoptosis.