Objective:To evaluate the role of glucagon-like peptide-1 receptor (GLP-1R) signaling pathway in sevoflurane postconditioning-induced attenuation of myocardial ischemia-reperfusion (I/R) injury in rats.Methods:Eighty SPF healthy adult male Sprague-Dawley rats, aged 8-10 weeks, weighing 300-340 g, were divided into 4 groups ( n=20 each) by a random number table method: sham operation group (group S), myocardial I/R group (group I/R), myocardial I/R plus sevoflurane postconditioning group (group ISP), and myocardial I/R plus sevoflurane postconditioning plus GLP-1R antagonist group (group ISPE). The myocardial I/R injury model was developed by ligating the left anterior descending branch of the coronary artery for 40 min followed by 2-h reperfusion in anesthetized rats.In group ISP, the rats inhaled 2.4% sevoflurane for 15 min starting from the beginning of reperfusion.In group ISPE, GLP-1R antagonist Exendin9-39 50 μg/kg (in 1 ml 0.9% normal saline) was intraperitoneally injected once a day from 28 days before development of the model, the last intraperitoneal injection was completed at 40 min before inhalation of sevoflurane, and the other treatments were the same as those previously described in group ISP.Blood samples from the abdominal aorta were collected immediately after reperfusion to determine the serum levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). Then the rats were sacrificed, and the hearts were obtained for microscopic examination of the histopathological changes of myocardial tissues (by HE staining) and the ultrastructure of cardiomyocytes (with a transmission electron microscope) for determination of the myocardial infarct size (TTC staining), expression of GLP-1R in myocardium (by immunohistochemical staining), expression of GLP-1R, cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), cAMP response element-binding protein (CREB), phospho-CREB (p-CREB), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated x protein (Bax) in myocardium (by Western blot). The ratios of p-CREB/CREB and Bcl-2/Bax were calculated. Results:Compared with group S, the serum levels of CK-MB and LDH and percentage of myocardial infarct size were significantly increased, the expression of GLP-1R was up-regulated, the expression of cAMP and PKA was down-regulated, and the p-CREB/CREB ratio and Bcl-2/Bax ratio were decreased in group I/R ( P<0.05). Compared with group I/R, the serum levels of CK-MB and LDH and percentage of myocardial infarct size were significantly decreased, the expression of GLP-1R, cAMP and PKA was up-regulated, and p-CREB/CREB ratio and Bcl-2/Bax ratio were increased in group ISP ( P<0.05). Compared with group ISP, the serum levels of CK-MB and LDH and percentage of myocardial infarct size were significantly increased, the expression of GLP-1R, cAMP and PKA was down-regulated, and the p-CREB/CREB ratio and Bcl-2/Bax ratio were decreased in group ISPE ( P<0.05). Conclusions:Sevoflurane postconditioning can attenuate myocardial I/R injury by activation of GLP-1R signal pathway and inhibition of cardiomyocyte apoptosis in rats.

Objective:To investigate the role of Sestrin2 overexpression in regulating mitochondrial fission and its mechanism in human neuroblastoma SH-SY5Y cell model of glucose and oxygen deprivation/recovery (OGD/R). Methods:(1) SH-SY5Y cells were divided into normal control group, OGD/R group, Vector group, and Sestrin2 overexpression group; Sestrin2 overexpression or empty vector stable cell lines in the Sestrin2 overexpression group and Vector group were constructed by lentivirus infection; cells in the later 3 groups were subjected to oxygen-glucose deprivation (OGD) for 4 h followed by restoration of O 2 supply for 18 h. The cell survival rate was detected by cell counting kit (CCK)-8 assay. The protein levels of Sestrin2, dynamin-related protein 1 (Drp1), mitochondrial fission protein 1 (Fis1), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Kelch-like ECH-related protein 1 (Keap1) in the cytoplasm and nuclear factor E2-related factor (Nrf2) in the nucleus were detected by Western blotting. The mitochondria ultrastructure was observed by transmission electron microscope. The Nrf2 nuclear translocation was detected by immunofluorescence staining. (2) Cell lines with Sestrin2 overexpression were divided into Sestrin2 overexpression group, Brusatol+ Sestrin2 overexpression group, and DMSO+ Sestrin2 overexpression group. Cells in the Brusatol+ Sestrin2 overexpression group were pretreated with normal medium containing Brusatol (Keap1/Nrf2 pathway inhibitor, final concentration: 100 nmol/L) for 4 h before OGD/R; cells in the DMSO+ Sestrin2 group were pretreated with normal medium containing DMSO (final volume fraction: 0.1%) for 4 h before OGD/R. Cells in these groups were then subjected to OGD for 4 h followed by restoration of O 2 supply for 18 h. The protein levels of Drp1, Fis1, Keap1 in the cytoplasm, and Nrf2 in the nucleus were measured by Western blotting. Results:(1) As compared with those in the OGD/R group, cells in the Sestrin2 overexpression group had significantly increased survival rate (61.33%±1.15% vs. 81.00%±3.00%), significantly up-regulated Bcl-2/Bax ratio (0.467±0.006 vs. 0.880±0.010), significantly decreased Drp1, Fis1 and cytoplasmic Keap1 protein levels (1.089±0.033 vs. 0.865±0.014; 0.829±0.009 vs. 0.350±0.007; 0.967±0.017 vs. 0.881±0.024), and significantly up-regulated nuclear Nrf2 protein level (0.627±0.025 vs. 0.957±0.015, P<0.05). The mitochondrial structure in the Sestrin2 overexpression group under electron microscope was more complete than that in the OGD/R group, and obvious nuclear translocation of Nrf2 was noted. (2) As compared with the Sestrin2 overexpression group, Brusatol+ Sestrin2 overexpression group had significantly decreased nuclear Nrf2 protein level (0.920±0.013 vs. 0.627±0.035), and statistically increased Drp1 and Fis1 protein levels (0.994±0.020 vs. 1.084±0.005; 0.728±0.010 vs. 0.906±0.022, P<0.05). Conclusion:Sestrin2 overexpression could suppress mitochondrial fission, reduce cell apoptosis, and attenuate OGD/R injury of SH-SY5Y cells by activating Keap1/Nrf2 pathway via down-regulating cytoplasmic Keap1 protein level and promoting Nrf2 nuclear translocation.

1 临床资料 患者,马某某,女,43 岁,因"声嘶半年余"就诊,门诊行电子喉镜检查示:右侧声带近前联合处暗红色新生物,肿物表面光滑,声带活动良好. 于2018年5月2 日以"声带息肉"收入青岛大学附属医院耳鼻咽喉科. 入院后采集病史告知哮喘病史,未规律服药治疗. 查体:肥胖体型,颈粗,下颌短小. 相关辅助检查无明显异常. 于2018 年5月4日行全麻下支撑喉镜下声带息肉切除术. 手术过程顺利,撤镜时发现右侧腭舌弓下方轻度撕裂,给予压迫止血,血止术毕.

Objective To observe the influence of flumazenil on plasma orexin-A level during the recovery period of anesthesia in patients with general anesthesia of propofol combined with sufentanil and explore whether the effect of promoting anesthesia recovery of flumazenil was related to orexin-A.Methods Forty patients undergoing laparoscopic hysteromyoma decollement or pan hysteromyoma under general anesthesia with ASA classification Ⅰ-Ⅱ were selected.The patients were randomly divided into two groups (n =20 each):flumazenil group (F group) and normal saline group (N group).After general anesthesia,propofol sedation was given to maintain bispectral index (BIS) at the level of 55 to 65.After operation,intravenous injection of 0.008 mg/kg flumazenil (F group) or the same dose of normal saline (N group) was respectively given.Drawing 3.0 ml of radial artery blood at the time points of T0 (before medication of flumazenil or normal saline),T1 (5 min after medication),T2 (15 min after medication) and T3 (30 min after medication),the levels of plasma orexin-A were detected (by ELISA methods) after centrifugation.The patients' eyes open time in calling and the time pulling out tracheal intubation were recorded.Results There were significant differences in mean artery pressure(MAP),heart rate (HR) and BIS between T0 time point and T1,T2,T3 time points in both two groups(all P ＜ 0.05).There were significant differences in MAP and HR at T1 and T2 time points between F group and N group(all P ＜ 0.05).There were significant differences in BIS values at T1,T2 and T3 time points between two groups (all P ＜ 0.05).There were significant differences in orexin-A levels between T0 time point and T1,T2,T3 time points in both two groups(all P ＜ 0.05).There were significant differences in orexin-A levels at T1,T2 and T3 time points between two groups(all P ＜ 0.05).There were significant differences in eyes open time in calling and time pulling out tracheal intubation between two groups (all P ＜ 0.05).There was no significant difference in postoperative complication rate (P ＞ 0.05).Conclusion Flumazenil can increase the plasma orexin-A level and shorten the anesthesia recovery time for the patients under general anesthesia with propofol combined with sufentanil.

目的:探究术中给予不同剂量右美托嘧啶对于预防痣切除术患者单次腰麻术后寒战的效果.方法:选取我院2016年1月至2016年8月收治的痔疮患者需行痔切除术者150例,随机数表法分为A、B、C3组.三组患者均使用0.75％罗哌卡因施行单次腰麻,A组患者麻醉同时静脉微泵注射0.9％生理盐水,B组患者采用0.3ug·kg-1右美托嘧啶微量泵注射,C组患者采用0.5ug·kg-1右美托嘧啶微量泵注射,三组患者注射时间均为10min.观察患者静脉泵用药前及用药后10min、30min、60min及90min后的生命体征变化,包括体温(T)、心率(HR)、呼吸频率(RR)、平均动脉压(MAP)、氧饱和度(SpO2)、寒战Wrench分级以及OAA/S评分变化.结果:三组患者静脉用药前后各时间点T、RR、SpO2比较无统计学意义(P>0.05);三组患者注药前HR和MAP比较无统计学意义(P>0.05),用药后B组及C组患者各时间点的HR及MAP均低于A组患者(P<0.05).三组患者中寒战发生率以A组最高,B组及C组患者寒战发生率低于A组患者(P<0.05);B组及C组患者间寒战发生率比较无统计学意义(P>0.05).B组及C组患者的镇静率高于A组患者(P<0.05),B组及C组患者间镇静率比较无统计学意义(P>0.05).结论:右美托咪定可降低痔疮手术接受单次腰麻的患者手术期间寒战的发生率,减轻术后焦虑程度,且无明显不良反应.

目的 为缺血性脑损伤模型的制备及研究建立一种更好的体外培养海马神经元的方法.方法 实验组取出生24 h内的Wistar新生鼠海马,用木瓜酶和DNA酶消化,以1×108/L的密度接种于含体积分数0.20胎牛血清的DMEM-F12培养液中,24 h后更换无血清培养液,每隔3d半量换液.对照组采用传统的培养方法培养细胞.培养期间用倒置显微镜观察细胞形态及数量变化.采用免疫荧光法测定神经元特异性烯醇化酶表达,判断海马神经元培养是否成功.结果实验组的海马神经元培养12h,可见大部分细胞贴壁;培养24 h,大部分细胞可见3～4个突起,突起长度为(25.0±2.5)μm;培养3d,神经元胞体增大,可见明显光晕;之后神经元分化逐渐成熟,胞体透亮,呈锥形或多极形,光晕明显,神经元之间的突起联系更加紧密,形成密集的神经细胞网络;培养13 d后,神经元细胞开始退化、变性,胞体萎缩,神经细胞网络开始老化.随着培养天数的增加,神经元可出现少量凋亡,但实验组培养1、3、5、8d时的神经元数量明显高于对照组(t=11.5～49.5,P＜0.05).免疫荧光法鉴定显示,实验组神经元细胞阳性率为(93.53±1.67)％.结论用上述改良方法培养得到的海马神经元细胞生长状态良好,可用于后续实验研究.

目的:线粒体通透性转换孔通透性改变是导致缺血再灌注损伤的原因,线粒体功能的致命性改变最终引起细胞凋亡,本研究旨在观察线粒体通透性转换孔(mitochondrial permeability transition pore,MPTP)在缺血再灌注及缺血预处理脑保护中的作用;方法:将体外培养8天的海马神经元细胞分为五组,正常对照组(A组),缺血再灌注组(B组),缺血预处理+缺血再灌注组(C组),苍术苷+缺血再灌注组(D组),缺血预处理+苍术苷+缺血再灌注组(E组).使用流式细胞术检测各组细胞凋亡率,罗丹明123染色流式细胞术检测线粒体膜电位,Western-blot检测Bcl-2,Bax的表达.结果:与A组比较,其余四组线粒体膜电位均降低,神经元凋亡率升高(P＜0.05);与B组比较,C组线粒体膜电位升高,神经元凋亡率升高,Bcl-2表达上调,Bax表达下调(P＜0.05);与C组比较,E组粒体膜电位降低,神经元凋亡率升高,Bcl-2表达下调,Bax表达上调(P＜0.05).结论:我们在细胞及分子生物学水平对MPTP及缺血预处理的研究后发现,缺血预处理能有效减轻海马神经元缺血再灌注损伤,抑制缺血再灌注后神经细胞凋亡,其机制与抑制MPTP的开放有关.

Objective To investigate whether mitochondrial calcium uniporte (MCU) can regulate the mitochondrial permeability transition pore(mPTP) in primary hippocampal neurons. Methods The primary hippocampal hippocampal neurons were randomly divided into seven groups according to the random number table:control group (n=6):without any prior administration,Spermine(Sper) group(n=6):gave 30 μmol/L Sper before the test,Ru360 group(n=6):gave 10 μmol/L Ru360 30 min before the test,Ciclosporin A(CsA) group(n=6):gave 0.2 μmol/L CsA 30 min before the test,Atractyloside(Atr) group(n=6):gave 400 μmol/L Atr before the test,Ru360+Atr group(n=6):gave 10 μmol/L Ru360 30 min before the test and then gave 400 μmol/L Atr before the test,CsA+Sper group (n=6):gave 0.2 μmol/L CsA 30 min before the test and then gave 30 μmol/L Sper before the test.200 μmol/L CaCl2 were added to each group at the time of the test to establish the model of calcium overload.Then monitored the mitochondrial calcein AM fluorescence intensity continuously over time for 6 min under the laser confocal microscope. Results The mitochondrial calcein AM fluorescence intensity of the control group reduced rapidly (0.073±0.011),compared with the control group,the mitochondrial calcein AM fluorescence intensity of the Ru360 group (0.779±0.015) and CsA group (0.923±0.011) reduced slowly (P＜0.05).Compared with the Sper group (0.023±0.006),the mitochondrial calcein AM fluorescence intensity of the CsA+Sper group (0.713±0.032) reduced slowly (P＜0.05).The mitochondrial calcein AM fluorescence intensity of the Ru360+Atr group (0.643±0.087) was slower than the Atr group (0.047±0.015) (P＜0.05),the mitochondrial calcein AM fluorescence intensity of the Ru360 group (0.779±0.015) reduced slower than the Sper group (0.023±0.006) (P＜0.05). Conclusions Mitochondrial calcium uniporter could regulate the mPTP in hippocampal neurons in vitro.

Objective To investigate whether diazoxide preconditioning protects cerebrum against i(s)chemia reperfusion injury via mitochondrial calcium uniporter(MCU)or not.Methods The ischemia reperfusion model was performed by thread occlusion of middle cerebral artery for 2 h,and then 24 h reperfusion.Fifty-two rats were randomly divided into four groups:group A (sham-operated group),group B(ischemia/reperfusion group),group C(diazoxide group),group D(diazozide-spermine group).After 24 h reperfusion,neuropsychological score,the mitochondrial calcium content([Ca2+]m),,the activity of superoxide dismutase (SOD)and glutathione peroxidase(GSH-Px),the content of malondialdehyde(MDA)and nitric oxide(NO)in ischemic hemisphere were observed in above groups respectively.Results Compared with group B,diazoxide preconditioning in group C improved neuropsychological score(13.1 ±0.8,P＜0.01),improved the activity of SOD(143 ±-18)U/mgprot and GSH-Px(84 ±8)U/mgprot(P＜0.05),and decreased the content of MDA(0.799 ±0.092)nmol/mgprot and NO(0.216±0.138)nmol/mgprot(P＜0.05)and[Ca2+]m (293±7)nmol/mgprot(P＜0.01).Spermine in group D attenuated the beneficial effects of diazoxide preconditioning in group C.Compared with group C,spermine attenuated neuropsychological score(8.0±0.7,P＜0.01)and the activity of SOD(128±7)U/mgprot and GSH-Px(73 ±6)U/mgprot(P＜0.05),and improved[Ca2+]m(322±10)nmol/mgprot(P＜0.01)and the content of MDA (0.955±0.141)nmol/mgprot and NO(0.575±0.292)nmol/mgprot(P＜0.05).Conclusions The effects of diazoxide preconditioning against cerebral ischemia reperfusion injury by attenuating[Ca2+]m and peroxidation injury in rats may involve in the suppression of MCU during reperfusion.