1.Effect of etomidate post-conditioning on mPTP in rat cortical neurons subjected to oxygen-glucose deprivation and restoration: the relationship with Robo receptors
Rui LI ; Limin ZHANG ; Xingliao LUO ; Wenbo SUN
Chinese Journal of Anesthesiology 2016;36(2):253-256
Objective To evaluate the role of etomidate post-conditioning on mitochondrial permeability transition pore (mPTP) in the rat cortical neurons subjected to oxygen-glucose deprivation and restoration (OGD/R) and the relationship with Robo receptors.Methods The cortical neurons obtained from Sprague-Dawley rats (< 24 h after birth) were cultured in vitro and seeded in 6-well plates (2 ml/well).The neurons were divided into 4 groups (n=24 each) using a random number table:control group (group C),OGD/R group,etomidate post-conditioning group (group E),and etomidate post-conditioning + Robo receptor blocker group (group ER).The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h.In E and ER groups,etomidate was added to the culture medium with the final concentration of 6 μmol/L immediately after onset of O2-glucose supply.In group ER,Robo blocker RoboN was added to the culture medium with the final concentration of 1 μg/ml at 6 h before O2-glucose deprivation.The neuronal apoptosis was detected using Hoechst/PI double staining,the viability of neurons was measured by MTT assay,and the amount of lactic dehydrogenase (LDH) released was measured using colorimetric method.The mitochondria were extracted,and mitochondrial permeability transition pore (mPTP) opening was detected.Results Compared with group C,the apoptosis rate,amount of LDH released,and mPTP opening were significantly increased,and the cell survival rate was decreased in OGD/R,E and ER groups (P<0.05).Compared with group OGD/R,the apoptosis rate,amount of LDH released,and mPTP opening were significantly decreased,and the cell survival rate was increased in group E,and the apoptosis and amount of LDH released were significantly decreased,and the cell survival rate was increased in group ER (P<0.05).Compared with group E,the apoptosis rate,amount of LDH released,and mPTP opening were significantly increased,and the cell survival rate was decreased in group ER (P<0.05).Conclusion Etomidate post-conditioning mitigates OGD/R-induced damage to the cortical neurons through activating Robo receptors and inhibiting mPTP opening in rats.
2.Role of mito-KATP channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose deprivation and restoration-induced pyroptosis in primary rat cardiomyocytes
Limin ZHANG ; Dongxue ZHANG ; Wenbo SUN ; Rui LI ; Xingliao LUO
Chinese Journal of Anesthesiology 2017;37(7):883-887
Objective To evaluate the role of mitochondrial ATP-seusitive potassium (mito-KATP) channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose dcprivation and restoration (OGD/R)-induced pyroptosis in primary rat cardiomyocytes.Methods Cardiomyocytes of newborn Sprague-Dawley rats (<48 h after birth) were cultured in vitro and seeded in 6-well dishes (2 cm in diameter)or in 96-well plates.The cells were divided into 6 groups (n =15 each) using a random number table:control group (group C),OGD/R group (group O),sevoflurane postconditioning group (group Sev),sevoflurane postconditioning plus 5-hydroxydecanoate (5-HD) group (group SH),5-HD group (group H) and OGD/R plus 5-HD group (group HO).The cardiomyocytes were subjected to oxygen-glucose deprivation for 4 h followed by restoration of oxygen-glucose supply for 24 h.After oxygen-glucose restoration,the cardiomyocytes in the culture media were exposed to 2% sevoflurane for 1 h to perform sevoflurane postconditioning.At 1 h before oxygen-glucose deprivation,a specific mito-KATP channel blocker 5-HD 100 μmol/L was added to the culture media.Cardiomyocytes were cultured in normal culture atmosphere in group C.Cardiomyocytes were collected at 24 h of oxygen-glucose restoration.Cell pyroptosis was detected by double flow cytometry AlexaFour488 (caspase-1 FLICA staining) and TMR red (DNA staining) staining.The pyroptosis rate was calculated.The cell survival rate was measured by methyl thiazolyl tetrazolium assay.The content of reactive oxygen species (ROS) in mitochondria was determined by 2',7'-dichlorofluorescin diacetate assay.The mitochondrial membrane potential (MMP) was measured by using JC-I fluorescent probe.The expression of interleukin-1beta (IL-1β) was determined by Western blot.Results Compared with group C,the pyroptosis rate and ROS content were significantly increased,the cell survival rate and MMP were decreased,and the expression of IL-1β was up-regulated in group O (P<0.05).Compared with group O,the pyroptosis rate and ROS content were significantly decreased,the cell survival rate and MMP were increased,and the expression of IL-1β was down-regulated in group Sev (P<0.05).Compared with group Sev,the pyroptosis rate and ROS content were significantly increased,the cell survival rate and M MP were decreased,and the expression of IL-1β was up-regulated in group SH (P<0.05).Compared with group SH,the pyroptosis rate and ROS content were significantly increased,the cell survival rate and MMP were decreased,and the expression of IL-1β was up-regulated in group H O (P<0.05).Conclusion The mechanism by which sevoflurane postconditioning inhibits OGD/R-induced pyroptosis in primary rat cardiomyocytes is probably associated with increasing mito-KATP channel opening.
3.Role of ERK signaling pathway in inhibition of OGD/R-induced apoptosis in rat cortical neurons by sevoflurane: the relationship with mPTP
Chunling WU ; Limin ZHANG ; Dongxue ZHANG ; Wenbo SUN ; Rui LI ; Xingliao LUO
Chinese Journal of Anesthesiology 2016;36(4):452-455
Objective To evaluate the role of extracellular signal-regulated kinase (ERK) signaling pathway in inhibition of oxygen-glucose deprivation and restoration (OGD/R)-induced apoptosis in rat cortical neurons by sevoflurane and the relationship with mitochondrial permeability transition pore (mPTP).Methods The rat cortical neurons were cultured in vitro and seeded in 6-well or 12-well culture plates.The neurons were randomly divided into 4 groups (n =18 each) using a random number table:control group (group C);OGD/R group (group O);sevoflurane group (group OS);sevoflurane + ERK1/2 inhibitor PD98059 group (group OSP).The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h in group O.The neurons were incubated with 2% sevoflurane for 2 h after OGD/R in group OS.OGD/R was performed at 1 h after ERK1/2 inhibitor PD98059 30 μmol/L was added,and the neurons were incubated with 2% sevoflurane for 2 h after OGD/R in group OSP.At 24 h of restoration of O2-glucose supply,the expression of phosphorylated ERK1/2 (p-ERK1/2) in neurons was measured by Western blot,the neuronal apoptosis was detected using Annexin V-FITC/PI double staining combined with flow cytometry,and the opening of mPTP was determined through measuring the optical density at 540 nm.The apoptosis rate was calculated.Results Compared with group C,the expression of p-ERK1/2 in neurons was significantly up-regulated,and the apoptosis rate and mPTP opening were significantly increased in group O (P<0.05).Compared with group O,the expression of p-ERK1/2 in neurons was significantly up-regulated,and the apoptosis rate and mPTP opening were significantly decreased in group OS (P<0.05).Compared with group OS,the expression of p-ERK1/2 in neurons was significantly down-regulated,and the apoptosis rate and mPTP opening were significantly increased in group OSP (P<0.05).Conclusion The mechanism by which sevoflurane inhibits OGD/R-induced apoptosis in rat cortical neurons is related to inhibition of mPTP opening after activation of ERK signaling pathway.
4.Risk factors for postoperative short-term cognitive dysfunction in patients undergoing carotid endar-terectomy
Limin ZHANG ; Yanchao PENG ; Rui LI ; Mingli WANG ; Yali MA ; Xingliao LUO
Chinese Journal of Anesthesiology 2019;39(6):676-679
Objective To identify the risk factors for postoperative short-term cognitive dysfunction ( STCD) in the patients undergoing carotid endarterectomy. Methods A total of 158 patients with carotid artery stenosis, aged≥18 yr, without preoperative cognitive dysfunction, undergoing carotid endarterecto-my from January 1, 2014 to May 30, 2017 in our hospital, were selected. Factors including age, sex, height, weight, education, presurgical complications ( hypertension, type Ⅱ diabetes mellitus, myocar-dial infarction and brain infarction) , degree of internal carotid artery stenosis, preoperative mean arterial pressure ( MAP ) , amount of dexmedetomidine, duration of operation, time of carotid artery occlusion, MAP during occlusion period, intraoperative volume of fluid infused ( crystalloid solution and colloid solu-tion), urine volume, blood loss, significant change in electroencephalogram (EEG) and visual analogue scale scores at 24 h after surgery were collected. Mini-Mental State Examination was used to evaluate the cognitive function at 24 h after surgery. The patients were divided into STCD group and non-STCD group ac-cording to whether or not patients developed postoperative STCD. The risk factors of which P values were less than 0. 05 would enter the bivariate logistic regression analysis to stratify the risk factors for postopera-tive STCD. Results Forty-nine patients developed postoperative STCD ( 31. 0%) . The intraoperative a-mount of colloid solution infused<5 ml/kg, increase in MAP<20% of the baseline value during the occlu-sion period, and significant change in EEG during the occlusion period were independent risk factors for postoperative STCD ( P<0. 05) . Conclusion The intraoperative amount of colloid solution infused<5 ml/kg, increase in MAP<20% of the baseline value during the occlusion period, and significant change in EEG during the occlusion period are independent risk factors for postoperative STCD in the patients undergo-ing carotid endarterectomy.