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Objective:To evaluate the effect of losartan on acute kidney injury (AKI) and the relationship with mitochondrial fusion-fission in septic mice.Methods:One hundred and twenty-eight SPF male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=32 each) using a random number table method: sham operation group (Sham group), sham operation+ losartan group (Sham+ LOS group), sepsis-associated AKI group (SA-AKI group), and sepsis-associated AKI+ losartan group (SA-AKI+ LOS group). Sepsis was induced by cecal ligation and puncture in anesthetized mice. Sham+ LOS group and SA-AKI+ LOS group received intraperitoneal injection of losartan 5 mg/kg, once a day, for 3 consecutive days, starting from 3 days before sham operation or developing the model. The equal volume of solvent was given instead in Sham group and SA-AKI group. Twenty mice were randomly selected to observe the survival 7 days after surgery. At 24 h after sham operation or establishing the model, serum blood urea nitrogen (BUN) and creatinine (Cr) concentrations were determined by the colorimetric method, and serum concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and high-mobility group box 1 protein (HMGB1) were measured using enzyme-linked immunosorbent assay. Renal tissues were obtained for microscopic examination of pathological changes which were scored and for determination of mitochondrial membrane potential (using JC-1 method) and expression of dynamin-related protein 1 (Drp1) and mitofusin-2 (Mfn2) (using Western blot). Results:Compared with Sham group, the survival rate was significantly decreased, the serum BUN, Cr, TNF-α, IL-6 and HMGB1 concentrations and renal tubular injury score were increased, the ATP content and MMP were decreased, the expression of Drp1 was up-regulated, the expression of Mfn2 was down-regulated ( P<0.05), and pathological changes were found in renal tissues in SA-AKI group and SA-AKI+ LOS group. Compared with SA-AKI group, the survival rate was significantly increased, serum concentrations of BUN, Cr, TNF-α, IL-6 and HMGB1 and renal tubular injury score were decreased, the ATP content and MMP were increased, the expression of Drp1 was down-regulated, the expression of Mfn2 was up-regulated ( P<0.05), and the pathological changes of renal tissues were significantly attenuated in SA-AKI+ LOS group. Conclusions:Losartan can alleviate AKI in septic mice, and the mechanism may be related to promoting mitochondrial fusion and inhibiting mitochondrial fission.
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Objective:To investigate whether propofol can cause injury to hippocampal mitochondria in neonatal rats and the regulation of excitatory amino acid receptor AMPA receptor.Methods:Forty-eight Sprague-Dawley (SD) rats aged 7 days were randomly divided into control group, propofol group, propofol+AMPA receptor agonist AMPA group (propofol+AMPA group) and propofol+AMPA receptor inhibitor CNQX group (propofol+CNQX group), with 12 rats in each group. The rats in the propofol groups were intraperitoneally injected with 30 mg/kg propofol, while in control group with 3 mg/kg normal saline. Each group was given 1/2 of the first dose every 20 minutes after the first administration, three times a day, for three consecutive days. The rats in the propofol+AMPA group and the propofol+CNQX group were injected with 1 g/L AMPA or CNQX 5 μL through left ventricle after the first administration. Three days after administration, the rats were sacrificed to obtain brain tissue. Western blotting was used to determine the expression of AMPA receptor glutamate receptors (GluR1, GluR2) subunit totally (T) and on membrane (M) in hippocampus. The expression of dynamin-related protein-1 (DRP-1) and phosphorylated-DRP-1 (p-DRP-1) and mitofusin 2 (Mfn2) related to mitochondrial fission and fusion were determined. The adenosine triphosphate (ATP) content and ATPase activity were determined.Results:Compared with the control group, GluR1 expression and its M/T ratio were significantly increased after treatment of propofol, GluR2 expression and its M/T ratio were significantly decreased, the ATP content and ATP-related enzyme activity were decreased significantly, while the expression of DRP-1 and its phosphorylation was significantly increased, and the expression of Mfn2 was significantly decreased. The changes indicated that repeated intraperitoneal injection of 30 mg/kg propofol leading to the injury of mitochondria in neural cells. Compared with the propofol group, the GluR1 expression and its M/T ratio further increased after AMPA agonist administration [T-GluR1 protein (T-GluR1/β-actin): 2.41±0.29 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 1.18±0.15 vs. 0.79±0.09, M/T ratio: 0.78±0.12 vs. 0.46±0.08, all P < 0.01], GluR2 expression was significantly increased [T-GluR2 protein (T-GluR2/β-actin): 0.65±0.13 vs. 0.30±0.14, P < 0.01; M-GluR2 protein (M-GluR2/β-actin): 0.17±0.05 vs. 0.13±0.07, P > 0.05], but its M/T ratio was further decreased (0.27±0.10 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was further decreased, and the ATP content was further decreased (μmol/g: 0.32±0.07 vs. 0.70±0.10, P < 0.01). Mitochondria DRP-1 expression and its phosphorylation were further increased [DRP-1 protein (DRP-1/GAPDH): 2.75±0.36 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.99±0.14 vs. 0.76±0.15, both P < 0.05], and Mfn2 expression was further decreased (Mfn2/GAPDH: 0.23±0.12 vs. 0.54±0.12, P < 0.05). This indicated that the AMPA agonist increased the expression of the AMPA receptor GluR1 subunit on the cell membrane and shifted the GluR2 into the cell, thus increasing the mitochondrial injury caused by propofol. Compared with the propofol group, the GluR1 expression and its M/T ratio decreased significantly after AMPA inhibitor administration [T-GluR1 protein (T-GluR1/β-actin): 0.99±0.14 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 0.21±0.07 vs. 0.79±0.09, M/T ratio: 0.21±0.07 vs. 0.46±0.08, all P < 0.01], the change of GluR2 expression was not significant, but its M/T ratio was significantly increased (0.59±0.09 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was increased significantly, and the ATP content was increased significantly (μmol/g: 0.87±0.12 vs. 0.70±0.10, P < 0.05). Mitochondria DRP-1 expression and its phosphorylation were significantly decreased [DRP-1 protein (DRP-1/GAPDH): 1.18±0.17 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.37±0.10 vs. 0.76±0.10, both P < 0.05], and Mfn2 expression was significantly increased (Mfn2/GAPDH: 0.78±0.10 vs. 0.54±0.12, P < 0.05). This indicated that AMPA inhibitor promoted the movement to the cell membrane of GluR2 subunits meanwhile inhibited the expression of GluR1 subunits, thus alleviating the injury of mitochondrial caused by propofol in the brain. Conclusions:Repeated intraperitoneal injection of 30 mg/kg propofol for 3 days can increase the expression of GluR1 subunits of AMPA receptor in 7-day neonatal rats hippocampus mainly distributing in the cell membrane, decrease the expression of GluR2 subunits moving into the cell, thus causing injury of mitochondrial function and dynamics, which can be aggravated by AMPA receptor agonist and alleviated by AMPA receptor inhibitors.
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Objective:To evaluate the role of the autophagy in hydrogen-induced reduction of myocardial injury in septic mice.Methods:A total of 192 clean-grade healthy male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 6 groups ( n=32 each) using a random number table method: sham operation group (group Sham), sham operation plus hydrogen group (group Sham+ H 2), sepsis group (group S), sepsis plus hydrogen group (group S+ H 2), sepsis plus bafilomycin A1 group (group S+ BafA1) and sepsis plus hydrogen plus bafilomycin A1 group (group S+ H 2+ BafA1). Sepsis was produced by cecal ligation and puncture (CLP) after anesthesia. The mice inhaled 2% hydrogen for 1 h starting from 1 and 6 h after operation in group Sham+ H 2, group S+ H 2 and group S+ H 2+ BafA1. Bafilomycin A1 1 mg/kg was intraperitoneally injected at 1 h after operation in S+ BafA1 and S+ H 2+ BafA1 groups. Twenty mice in each group were selected to record the 7-day survival rates after operation. Then the mice were sacrificed at 24 h after operation to observe the pathological changes of myocardial tissues which were scored and detect the serum cardiac troponin I (cTnI) concentration (by enzyme-linked immunosorbent assay) and determine the level of microtubule-associated protein 1 light chain 3 B (LC3B) and P62 (by Western blot). LC3Ⅱ/LC3Ⅰratio was calculated. Results:Compared with group Sham, the 7-day survival rate after operation was significantly decreased, the serum cTnI concentrations and pathological scores of myocardial tissues were increased, the expression of P62 was up-regulated ( P<0.05), no significant change was found in LC3Ⅱ/LC3Ⅰratio ( P>0.05), and no significant change was found in the parameters mentioned above in group Sham+ H 2 ( P>0.05). Compared with group S, the 7-day survival rate after operation was significantly increased, the serum cTnI concentrations and pathological scores of myocardial tissues were decreased, LC3Ⅱ/LC3Ⅰratio was increased, and the expression of P62 was down-regulated in group S+ H 2, and LC3Ⅱ/LC3Ⅰratio was significantly decreased, and the expression of P62 was up-regulated in group S+ BafA1 ( P<0.05). Compared with group S+ H 2, the 7-day survival rate was significantly decreased, the serum cTnI concentrations and pathological scores of myocardial tissues were increased, LC3Ⅱ/LC3Ⅰratio was decreased, and the expression of P62 was up-regulated in group Sham+ H 2 ( P<0.05). Conclusions:The mechanism by which hydrogen alleviates myocardial damage may be related to promoting autophagy in septic mice.
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Objective:To evaluate the effects of inhaling high concentration hydrogen on myocardial injury and mitochondrial biogenesis in septic mice.Methods:One hundred and twenty-eight clean-grade healthy male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=32 each) using a random number table method: sham operation group (group Sham), sham operation + hydrogen group (group Sham+ H), sepsis group (group Sep), and sepsis+ hydrogen group (group Sep+ H). The sepsis model was developed by cecal ligation and puncture in anesthetized animals. In Sham+ H and Sep+ H groups, 67% H 2 was inhaled for 1 h starting from 1 and 6 h after operation, respectively. Twenty mice in each group were randomly selected to observe the survival conditions at 7 days after operation. Blood samples were taken from the remaining mice at 24 h after operation for determination of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cardiac troponin I (cTnI) and creatine kinase isoenzyme (CK-MB) (by enzyme-linked immunosorbent assay), for examination of the pathological changes of myocardial tissues (by HE staining), and for determination of the mitochondrial membrane potential (MMP) (by fluorescence spectrophotometry), ATP content (by luciferase assay), and expression of myocardial peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 2 (NRF2) and mitochondrial transcription factor A (TFAM) (by Western blot). Results:Compared with Sham group, the survival rate was significantly decreased, the serum concentrations of TNF-α, IL-1β, cTnI and CK-MB and pathological score were increased, the MMP and content of ATP in myocardial mitochondria were decreased, and the expression of PGC-1α, NRF2 and TFAM in myocardial tissues was down-regulated in Sep group ( P<0.05), and no significant change was found in the parameters mentioned above in Sham+ H group ( P>0.05). Compared with group Sep, the survival rate was significantly increased, the serum concentrations of TNF-α, IL-1β, cTnI and CK-MB and pathological score were decreased, the MMP and content of ATP in myocardial mitochondria were increased, and the expression of PGC-1α, NRF2 and TFAM in myocardial tissues was up-regulated in group Sep+ H ( P<0.05). Conclusions:Inhaling high concentration hydrogen can attenuate sepsis-induced myocardial injury in mice, and the mechanism may be related to promotion of mitochondrial biosynthesis and improvement in mitochondrial function.
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Objective:To evaluate the effects of inhalation of high-concentration hydrogen on acute kidney injury (AKI) and mitochondrial dynamics in septic mice.Methods:One hundred and twenty-eight male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=32 each) using a random number table method: sham operation group (group Sham), sham operation + hydrogen group (group Sham+ H), sepsis AKI group, and sepsis AKI+ hydrogen group (group S-AKI+ H). A mouse model of sepsis-induced AKI was developed by cecal ligation and puncture in anesthetized animals. In Sham+ H and S-AKI+ H groups, 67% H 2+ 33% O 2 was inhaled for 1 h starting from 1 and 6 h after sham operation or developing the model, respectively. Twenty mice were selected to observe the survival at 7 days after developing the model. At 24 h after developing the model, blood samples were collected for determination of serum BUN and Cr concentrations (by colorimetric analysis), and renal tissues were obtained for determination of the contents of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and high mobility group protein B1 (HMGB1) (by enzyme-linked immunosorbent assay), activities of superoxide dismutase (SOD) and catalase (CAT) (by spectrophotometry) and expression of dynamin-related protein 1 (Drp1) and mitofusin 2 (Mfn2) (by Western blot). The damage to the renal tubules was scored after HE staining. Results:Compared with Sham group, the survival rate was significantly decreased, the serum BUN and Cr concentrations, renal tubular damage score and contents of TNF-α, IL-1β and HMGB1 were increased, the activities of SOD and CAT were decreased, the expression of Drp1 was up-regulated, and the expression of Mfn2 was down-regulated in S-AKI group ( P<0.05), and no significant change was found in the parameters mentioned above in Sham+ H group ( P>0.05). Compared with S-AKI group, the survival rate was significantly increased, the serum BUN and Cr concentrations, renal tubular injury score and contents of TNF-α, IL-1β and HMGB1 were decreased, the activities of SOD and CAT were increased, the expression of Drp1 was down-regulated, and the expression of Mfn2 was up-regulated in S-AKI+ H group ( P<0.05). Conclusions:Inhalation of high-concentration hydrogen can alleviate AKI in septic mice, and the mechanism may be related to inhibition of renal mitochondrial fission and promotion of mitochondrial fusion.
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Objective:To evaluate the effect of high concentration hydrogen on myocardial injury and expression of mammalian STE20-like protein kinases 1 (Mst1) in septic mice.Methods:One hundred and five clean-grade healthy male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 3 groups ( n=35 each) using a random number table method: sham operation group (group Sham), sepsis group (group SEP), and sepsis+ high concentration hydrogen group (group SEP+ HCH). The model of sepsis-induced myocardial injury was developed by cecal ligation and perforation in anesthetized mice. In SEP+ HCH group, high concentration hydrogen (66.7%) was inhaled for 1 h starting from 1 and 6 h after the successful preparation of the model. Twenty mice were taken from each group to observe the survival rate at day 7 after operation. Blood samples from the medial canthus were collected after deep anesthesia at 24 h after surgery for determination of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations (by enzyme-linked immunosorbent assay). Then the animals were sacrificed, and the heart tissues were taken for examination of the pathological results (with a light microscope) which were scored and for determination of apoptosis in myocardial cells (by TUNEL) and expression of Mst1, dynamic-related protein 1 (Drp1) and mitofusin 2 (Mfn2) in myocardium (by Western blot). Results:Compared with group Sham, the survival rate at 7 days after operation, pathological score, apoptosis index and concentrations of TNF-α and IL-6 were significantly increased, Mst1 and Drp1 expression was up-regulated, and Mfn2 expression was down-regulated in group SEP ( P<0.05). Compared with group SEP, the survival rate at 7 days after operation, pathological score, apoptosis index and concentrations of TNF-α and IL-6 were significantly decreased, Mst1 and Drp1 expression was down-regulated, and Mfn2 expression was up-regulated in group SEP+ HCH ( P<0.05). Conclusions:Inhalation of high-concentration hydrogen can attenuate the myocardial injury in septic mice, and the mechanism may be related to down-regulation of Mst1 expression, improvement in mitochondrial dynamics, and inhibition of apoptosis in myocardial cells.
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Objective:To evaluate the relationship between B-cell lymphoma/adenovirus E1B19 kDa-interacting protein 3-like protein (BNIP3L)/adenovirus E1B-interacting protein and mitochondrial dysfunction in the hippocampus of mice with sepsis-associated encephalopathy (SAE).Methods:One hundred and eighty C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=45 each) using a random number table method: control group (C group), sham operation group (Sham group), SAE group, and SAE+ BNIP3L agonist carfilzomib group (SC group). The sepsis model was developed by cecal ligation and puncture (CLP) in anesthetized animals. In SC group, carfilzomib 2 mg/kg was intraperitoneally injected at 2 h after CLP. Twenty mice in each group were selected, and the survival at 7 days after operation was recorded. Eight surviving mice in each group were selected at 1 week after CLP for Morris water maze test. The remaining mice were sacrificed at 24 h after surgery, and the hippocampal tissues were harvested for determination of the expression of BNIP3L (by immunofluorescence) and BNIP3L in mitochondrial protein (by Western blot) and for microscopic examination of the morphological structure of mitochondria. The mitochondrial ATP content was measured by fluorescein-fluorescence enzyme luminescence method, and the mitochondrial membrane potential (MMP) was measured by fluorescence spectrophotometry. Results:Compared with C and Sham groups, the survival rate was significantly decreased, the escape latency was prolonged, the time of staying at the original platform quadrant was shortened, and the number of crossing the original platform region was decreased, the expression of BNIP3L in the hippocampal mitochondria was down-regulated, the MMP and content of mitochondrial ATP were decreased ( P<0.05), the intensity of fluorescence of BNIP3L in the hippocampus was decreased, and the damage to mitochondrial ultrastructure was marked in SAE group. Compared with SAE group, the survival rate was significantly increased, the escape latency was shortened, the time of staying at the original platform quadrant was prolonged, and the number of crossing the original platform region was increased, the expression of BNIP3L in the hippocampal mitochondria was up-regulated, the MMP and content of mitochondrial ATP were increased ( P<0.05), the intensity of fluorescence of BNIP3L in the hippocampus was decreased, and the damage to mitochondrial ultrastructure was attenuated in SC group. Conclusions:BNIP3L-mediated mitochondrial dysfunction may be involved in the mechanism of SAE developed in mice.
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Objective:To evaluate the effect of sleep fragmentation on postoperative cognitive dysfunction (POCD) and hippocampal glutaminergic metabolism in aged mice anesthetized with isoflurane.Methods:Forty healthy SPF-grade male C57BL/6J mice, aged 18 months, weighing 20-30 g, were divided into 4 groups ( n= 10 each) by the random number table method: normal control group (group C), sleep fragmentation group (group SF), isoflurane anesthesia/surgery group (group I/S), and sleep fragmentation plus isoflurane anesthesia/surgery group (group SF+ I/S). Group C did not received any treatment. Group SF received sleep fragmentation for 24 h. The right carotid artery exposure was performed under isoflurane anesthesia in group I/S. Group SF+ I/S received isoflurane anesthesia/right carotid artery exposure at 24 h after sleep fragmentation. The metabolic levels of glutamate (Glu), glutamine (Gln), Glu/Gln complex (Glx), and N-acetylaspartate (NAA) and their ratio to creatine (Cr) were measured by in vivo 9.4T hydrogen proton magnetic resonance spectroscopy at 2 h after anaesthesia. Y maze and Morris water maze tests were used to evaluate the cognitive function at 1-7 days after surgery. The mice were sacrificed after the behavioral testing, brain tissues were immediately obtained, and the number of Nissl bodies and density of dendritic spines in the hippocampal CA1 region were measured by Nissl staining and Golgi staining, respectively. Results:Compared with group C, the percentage of exploration time and shuttle times at the novel arm were significantly decreased, the number of crossing the original platform was decreased, the time of stay at the target quadrant was shortened, the ratios of Glu/Cr, Gln/Cr and Glx/Cr in the hippocampal CA1 region were increased, and the ratio of NAA/Cr was decreased, and the number of Nissl bodies and density of dendritic spines were decreased in SF, I/S and SF+ I/S groups ( P<0.05). Compared with group SF and group I/S, the percentage of exploration time and shuttle times at the novel arm were significantly decreased, the number of crossing the original platform was decreased, the time of stay at the target quadrant was shortened, the ratios of Glu/Cr and Glx/Cr in hippocampal CA1 region was increased, the ratio of NAA/Cr was decreased, and the number of Nissl bodies and density of dendritic spines were decreased in group SF+ I/S ( P<0.05). Conclusions:Sleep fragmentation exacerbates POCD in aged mice anesthetized with isoflurane, and the mechanism is related to nerve injury induced by abnormality in hippocampal glutaminergic metabolism excitability.
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Objective:To evaluate the effect of gender on anesthetic potency of ciprofol for gastroscopy when combined with fentanyl.Methods:American Society of Anesthesiologists Physical Status classification Ⅰ or Ⅱ patients, aged 18-50 yr, with body mass index of 18-25 kg/m 2, undergoing elective gastroscopy with intravenous anesthesia, were divided into 2 groups according to gender: male group (M group) and female group (F group). After fentanyl 1.5 μg/kg was intravenously injected, ciprofol was given by the Dixon′s up-and-down method, with the initial dose of 0.4 mg/kg followed by dose increment/decrement of 0.04 mg/kg. The ED 50 and 95% confidence interval of ciprofol for gastroscopy anesthesia were calculated by the probit regression analysis. Results:The ED 50 (95% confidence interval) of ciprofol for gastroscopy was 0.33 (0.32-0.34) mg/kg in F group and 0.27 (0.26-0.28) mg/kg in M patients when combined with fentanyl 1.5 μg/kg. There was no significant difference between the two groups ( P>0.05). Conclusions:There is no significant gender difference in the anesthetic potency of ciprofol for gastroscopy (ED 50: female 0.33 mg/kg, male 0.27 mg/kg) when combined with fentanyl (1.5 μg/kg).
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Objective:To identify the differentially expressed long-chain non-coding RNA(lncRNA) and mRNA using ribonucleic acid sequencing(RNA-seq), and construct a competing endogenous RNA(ceRNA) regulatory network in mice with sepsis-associated encephalopathy.Methods:Ten clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 2 groups( n=5 each) using a random number table method: sham operation group(group Sham) and sepsis group(group Sepsis). Sepsis was induced by cecal ligation and puncture(CLP) in group Sepsis, while group Sham only underwent laparotomy without CLP. Morris water maze test and contextual fear conditioning test were performed to detect the cognitive function on 1 day before CLP and 3 days after CLP. Three mice were randomly sacrificed in group Sham, and 3 mice with the worst results in the cognitive function test were sacrificed in group Sepsis. The hippocampal tissues were obtained for RNA-seq via the BGISEQ-500 platform, and the differentially expressed mRNA and lncRNA were identified. The differentially expressed mRNAs and lncRNAs were visualized and analyzed by Dr. Tom platform provided by Shenzhen BGI Technology Service Co., Ltd., and the ceRNA regulatory network was constructed using the online visualization tool Cytoscape software. Results:Compared with group Sham, the escape latency was significantly prolonged, and the percentage of time of staying at the target quadrants and percentage of time spent freezing were decreased in group Sepsis( P<0.05). A total of 62 differentially expressed lncRNAs were obtained from RNA-seq, of which the expression of 45 lncRNAs was up-regulated and the expression of 17 lncRNAs was down-regulated.There were 282 differentially expressed mRNAs identified from RNA-seq, of which the expression of 173 mRNAs was up-regulated, and the expression of 109 mRNAs was down-regulated.Gene Ontology enrichment analysis revealed that the differentially expressed mRNAs were involved in biological processes such as memory, learning or memory, inflammatory responses, regulation of aging-related behavioral decline, and regulation of synaptic plasticity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that differentially expressed mRNAs were enriched in IL-17 signaling pathway, TNF signaling pathway, NF-κB signaling pathway and etc. KDA analysis was performed on the differentially expressed mRNAs to identify the key driver genes, and the results showed that Ch25h, Il6ra, Lcn2, Sgk1, Nr4a3, Osm, Saa3, Ccl7, Sqle, Dhcr24 were the key SAE genes.A competing endogenous RNA regulatory network was successfully constructed based on 9 lncRNAs, 28 mRNAs and 134 miRNAs in the hippocampus of mice with SAE. Conclusions:The results of RNA-seq find that 10 mRNAs including Ch25h, Il6ra, Lcn2, Sgk1, Nr4a3, Osm, Saa3, Ccl7, Sqle, Dhcr24 and lncRNAs such as Rian, Gm35874 and Gm34347 are key genes regulating SAE in mice. Meanwhile, a ceRNA regulatory network based on lncRNA-miRNA-mRNA is successfully constructed in the hippocampus of mice with SAE.
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Objective:To evaluate the effect of hydrogen-rich saline (HRS) on mitochondrial biogenesis and dynamics in hippocampus of mice with sepsis-associated encephalopathy (SAE).Methods:One hundred and twenty-eight male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=32 each) using a random number table method: sham operation group (Sham group), sham operation plus HRS group (Sham+ HRS group), SAE group and SAE plus HRS group.Sepsis was developed by cecal ligation and puncture (CLP) in anesthetized mice.HRS 10 ml/kg was intraperitoneally injected at 1 and 6 h after CLP in Sham+ HRS and SAE+ HRS groups.Twenty mice were randomly selected from each group to record the 7-day survival after operation.The working memory of the mice was observed by Y-maze test on days 3, 5 and 7 after CLP.The hippocampal tissues were obtained at 24 h after CLP for determination of the content of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and high-mobility group box 1 protein (HMGB1) (by enzyme-linked immunosorbent assay), activities of superoxide dismutase (SOD) and catalase (CAT) (by spectrophotometry), and expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), nuclear respiratory factor 2 (NRF2), mitochondrial transcription factor A (Tfam), dynamin-related protein 1 (Drp1) and mitochondrial fusion protein mitofusin 2 (Mfn2) (by Western blot). Results:Compared with group Sham, the postoperative 7-day survival rate was significantly decreased, the time spent in novel arm was shortened, the contents of TNF-α, IL-6 and HMGB1 were increased, the activities of SOD and CAT were decreased, the expression of PGC-1α, NRF2 and Tfam was up-regulated, the expression of Drp1 was up-regulated, and the expression of Mfn2 was down-regulated in group SAE ( P<0.05). Compared with group SAE, the postoperative 7-day survival rate was significantly increased, the time spent in novel arm was prolonged, the contents of TNF-α, IL-6 and HMGB1 were decreased, the activities of SOD and CAT were increased, the expression of PGC-1α, NRF2 and Tfam was up-regulated, the expression of Drp1 was down-regulated, and the expression of Mfn2 was up-regulated in group SAE+ HRS ( P<0.05). Conclusions:The mechanism by which HRS alleviates SAE may be related to promotion of mitochondrial biogenesis, regulation of dynamics, and reduction of oxidative stress in hippocampus of mice.
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Objective:To evaluate the effects of endothelial progenitor cell (EPC)-derived exosomes on neuronal injury induced by oxygen-glucose deprivation and restoration (OGD/R).Methods:HT22 neurons of mice were cultured and divided into 3 groups ( n=30 each) using a random number table method: control group (C group), OGD/R group and OGD/R plus EPC-derived exosome group (OGD/R+ EXO group). Cells in group C were cultured in normal atmosphere.In group OGD/R, the cells were exposed to 94%N 2-1%O 2-5%CO 2 for 6 h in glucose- and serum-free DMEM medium, followed by 24 h restoration of O 2 and glucose in the normal medium.In group OGD/R+ EXO, 20 μg/ml EPC-derived exosomes were added to the culture medium at 24 h before developing the model.EPCs were identified by immunofluorescence staining.Exosomes were identified by Western blot, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Cell viability was measured by CCK-8 assay, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by enzyme-linked immunosorbent assay.The neuronal apoptosis was detected by TUNEL staining, and the apoptosis rate was calculated.The expression of Bax, Bcl-2, caspase-3, and cleaved caspase-3 was determined by Western blot, and cleaved caspase-3/caspase-3 ratio was calculated. Results:The cultured cells were EPCs, and EPC-derived exosomes were successfully extracted.Compared with group C, the cell viability was significantly decreased, the content of MDA was increased, the activity of SOD was decreased, the apoptosis rate was increased, the expression of Bax was up-regulated, the expression of Bcl-2 was down-regulated, and the ratio of cleaved-caspase-3/caspase-3 was increased in group OGD/R and group OGD/R+ EXO ( P<0.05). Compared with group OGD/R, the cell viability was significantly increased, the content of MDA was decreased, the activity of SOD was increased, the apoptosis rate was decreased, the expression of Bax was down-regulated, the expression of Bcl-2 was up-regulated, and the ratio of cleaved-caspase-3/caspase-3 was decreased in group OGD/R+ EXO ( P<0.05). Conclusions:EPC-derived exosomes can reduce OGD/R-induced neuronal injury, which is related to inhibition of oxidative stress and neuronal apoptosis.
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Objective:To identify the key genes for neuropathic pain in rats.Methods:The genomic data of spinal cord tissues of rats (GSE18803) were downloaded from the Gene Expression Database at the American Center for Biotechnology Information to identify differentially expressed genes associated with neuropathic pain, and key genes were obtained by further analysis of the protein-protein interaction networks.Single-cell localization and expression of the key genes were analyzed by the Tabula Muris database.Results:The protein-protein interaction networks identified 10 hub genes, including Tyrobp, Clec4a3, C1qc, Ptprc, Laptm5, Csf1r, C1qa, C1qb, Fcgr3a, Cd53. Cd53, Laptm5 and Ptprc were mainly expressed in macrophages, B cells, NK cells, monocytes and granulocytes. Clec4a3 and Csf1r were mainly expressed in monocytes, Fcgr3a in monocytes and granulocytes, and Tyrobp in macrophages, monocytes, granulocytes, and pluripotent progenitor cells. Conclusions:Ten target genes associated with neuropathic pain are identified using bioinformatics, and their distribution and expression in immune inflammatory cells are obtained through comprehensive analysis.
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Objective:To evaluate the effect of high-concentration hydrogen inhalation on sepsis-associated encephalopathy (SAE) in mice.Methods:Healthy male ICR mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=50 each) using the random number table method: sham operation group (Sham group), SAE group, sham operation plus high-concentration hydrogen group (Sham+ H 2 group), and SAE plus high-concentration hydrogen group (SAE+ H 2 group). SAE model was prepared by cecal ligation and puncture (CLP) in anesthetized animals.At 1 and 6 h after operation, Sham+ H 2 and SAE+ H 2 groups inhaled the mixture of hydrogen and oxygen (67% hydrogen-33% oxygen) for 1 h, and Sham and SAE groups inhaled the mixture of nitrogen and oxygen (67% nitrogen-33% oxygen) for 1 h. The postoperative 7-day survival rate was recorded.Cognitive function was assessed by Y maze test at days 3, 5 and 7 after operation.The mice were sacrificed at 24 h after operation, and hippocampal tissues were obtained for microscopic examination of the pathological changes of neurons in hippocampal CA1 region (with a light microscope) and for determination of normal neuron count, contents of tumor necrosis factor-ɑ (TNF-α) and high mobility group box-1 (HMGB1) (by enzyme-linked immunosorbent assay), mitochondrial membrane potential (MMP) (by fluorescence spectrophotometry) and content of mitochondrial ATP (by fluorescein-fluorescent enzyme luminescence method). Results:Compared with Sham group, the 7-day survival rate after operation, percentage of spontaneous alternation at each time point after operation, and the number of normal neurons were significantly decreased, the contents of TNF-ɑ and HMGB1 were increased, and the contents of ATP and MMP were decreased in SAE and SAE+ H 2 groups ( P<0.05), and no significant change was found in Sham+ H 2 group ( P>0.05). Compared with SAE group, the 7-day survival rate after operation, percentage of spontaneous alternation at each time point after operation, and the number of normal neurons were significantly increased, the contents of TNF-ɑ and HMGB1 were decreased, and the contents of ATP and MMP were increased in SAE+ H 2 group ( P<0.05). Conclusions:High-concentration hydrogen inhalation can reduce SAE, and the mechanism may be related to reduction of hippocampal inflammatory responses and improvement in mitochondrial function in mice.
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Objective:To evaluate the effect of hydrogen on activation of A1 astrocytes in the hippocampus of mice with sepsis-associated encephalopathy (SAE).Methods:A total of 164 clean-grade healthy male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=41 each) using a random number table method: sham operation group (group Sham), sham operation plus hydrogen group (group Sham+ H 2), group SAE and SAE plus hydrogen group (group SAE+ H 2). The SAE model was established by cecal ligation and perforation.Group Sham+ H 2 and group SAE+ H 2 inhaled 2% hydrogen starting from 1 and 6 h after operation, respectively.Twenty mice in each group were selected to observe the 7-day survival rate after operation.The remaining mice were sacrificed at 12 h after operation, and brain tissues were removed for examination of the pathological changes in hippocampal CA1 region (with a light microscope) and for determination of the apoptosis in neurons (by TUNEL), co-expression of hippocampal glial fibrillary acidic protein (GFAP) and complement C3 (by immunofluorescence staining), expression of A1 astrocyte marker C3 (by Western blot), and contents of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and high-mobility group box 1 protein (HMGB1) (by enzyme-linked immunosorbent assay). The abnormal cell ratio and apoptosis rate were calculated.Six mice in each group were selected at 7 days after operation to perform Y-Maze paradigm. Results:Compared with group Sham, the 7-day survival rate after operation was significantly decreased, the abnormal cell ratio and apoptosis rate of hippocampal neurons were increased, the contents of TNF-α, IL-6 and HMGB1 were increased, the expression of C3 was up-regulated, the number of cells coexpressing GFAP and C3 was increased, the exploration time spent in the novel arm in Y-Maze paradigm was shortened, and the preference index was decreased in group SAE ( P<0.05). Compared with group SAE, the 7-day survival rate after operation was significantly increased, the abnormal cell ratio and apoptosis rate of hippocampal neurons were decreased, the contents of TNF-α, IL-6 and HMGB1 were decreased, the expression of C3 was down-regulated, the number of cells coexpressing GFAP and C3 was decreased, the exploration time spent in the novel arm in Y-Maze paradigm was prolonged, and the preference index was increased in group SAE+ H 2 ( P<0.05). There was no significant difference in each parameter mentioned above between Sham group and Sham+ H 2 group ( P>0.05). Conclusion:The mechanism by which hydrogen improves SAE may be related to inhibiting activation of A1 type astrocytes in mice.
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Objective:To evaluate the effects of propofol on α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor expression in the hippocampus of neonatal rats.Methods:Eighty-four clean-grade healthy Sprague-Dawley rats of either sex, aged 7 days, weighing 14-18 g, were divided into 2 groups ( n=42 each) using a random number table method: control group (group C) and propofol group (group P). Propofol 30 mg/kg was intraperitoneally injected in group P, fat emulsion 3 mg/kg was intraperitoneally injected in group C, 1/2 of the initial dose was given at a 20 min interval, 3 times in total, for 3 consecutive days.The arterial blood samples were taken for blood gas analysis after administration on 1st day.The rats were sacrificed at 3, 7 and 28 days after the last administration of propofol, and the bilateral hippocampus was obtained for detection of the expression of AMPA receptors containing GluR1, GluR2 and GluR3 subunits in total and membrane protein (by Western blot), and the ratio of membrane protein to total protein (M/T) was calculated.The concentrations of free calcium ion were measured.The learning and memory ability was evaluated by Morris water maze test on 28 days after the last administration. Results:Compared with group C, the expression of AMPA receptor containing GluR1 subunit in total and membrane protein was significantly up-regulated, M/T was increased, the expression of AMPA receptor containing GluR2 subunit in total and membrane protein was down-regulated, and M/T was decreased at each time point ( P<0.05), no significant change was found in the expression of AMPA receptor containing GluR3 subunits ( P>0.05), the concentrations of free calcium ion in hippocampal cells were increased, and the escape latency was prolonged, the number of crossing the original platform was decreased, and the time of staying at the target quadrant was shortened at 2-4 days of training in group P ( P<0.05). Conclusion:The mechanism by which propofol reduces cognitive function is related to up-regulation of the expression of AMPA receptors containing GluR1 subunit in the hippocampus and down-regulation of the expression of AMPA receptors containing GluR2 subunits, which increases the concentration of free calcium ions in nerve cells of neonatal rats.
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Objective:To evaluate the effect of hydrogen on mitochondrial dynamics in hippocampus of mice with sepsis-associated encephalopathy (SAE).Methods:A total of 224 clean-grade healthy male C57 mice, weighing 20-25g, aged 6-8 weeks, were divided into 4 groups ( n=56 each) using a random number table method: sham operation group (group Sham), sham operation + hydrogen group (group Sham+ H 2), SAE group and SAE + hydrogen group (group SAE+ H 2). Sepsis was produced by cecum ligation and puncture (CLP). Sham+ H 2and SAE+ H 2 groups inhaled 2% hydrogen for 1 h starting from 1 and 6 h after CLP, respectively.The postoperative 7-day survival rate was recorded.Brain tissues were obtained at 24 h after operation for examination of the pathological changes in hippocampal CA1 region (with a light microscope) and for determination of mitochondrial membrane potential (MMP) (by fluorescence spectrophotometry) and ATP content (by a bioluminescence assay) in hippocampal tissues.At 6, 12 and 24 h after operation, hippocampal mitochondria were isolated for determination of the expression of dynamin-related protein 1 (Drp1) and the mitochondrial fusion proteins mitofusin 2 (Mfn2) (by Western blot). Results:Compared with group Sham, the postoperative 7-day survival rate was significantly decreased, the contents of MMP and ATP were decreased, the expression of Drp1 was up-regulated, and the expression of Mfn2 was down-regulated( P<0.05), the pathological changes were aggravated in hippocampal CA1 region in SAE and SAE+ H 2 groups, and no significant change was found in the parameters mentioned above in group Sham+ H 2 ( P>0.05). Compared with group SAE, the postoperative 7-day survival rate was significantly increased, the contents of MMP and ATP were increased, the expression of Drp1 was down-regulated, and the expression of Mfn2 was up-regulated( P<0.05), the pathological changes were attenuated in hippocampal CA1 region in group SAE+ H 2. Conclusion:The mechanism by which hydrogen improves mitochondrial function is probably associated with promoting mitochondrial fusion and inhibiting mitochondrial fission in hippocampus of mice with SAE.
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Objective:To evaluate the role of hemopexin (HPX) in cerebral ischemia-reperfusion (I/R) injury in rats.Methods:One hundred and twenty healthy male Sprague-Dawley rats, aged 7-8 weeks, weighing 250-280 g, were divided into sham operation group (S group, n=36), cerebral I/R group (I/R group, n=36), vehicle group (V group, n=24), and HPX group ( n=24). The model of cerebral I/R injury was established by 120 min middle cerebral artery occlusion followed by reperfusion in anesthetized rats.At 6, 12 and 24 h of reperfusion, 4 rats in S group and I/R group were sacrificed, and the ischemic penumbra of the ipsilateral cerebral cortex was obtained to detect the expression of HPX by Western blot.In I/R, V and HPX groups, 0.9% normal saline 10 μl, 0.1% NaN 3 10 μl, and 1.86 mg/ml HPX 10 μl were injected into the lateral ventricle, respectively, immediately after reperfusion.Eight rats in each group were selected, and neurological deficit was scored at 1-7 days of reperfusion.Eight rats in each group were sacrificed at 1 and 7 days of reperfusion, brains were removed, and brain tissues were obtained for measurement of infarct size, and the percentage of infarct size was calculated. Results:Compared with S group, the expression of HPX in cerebral ischemic penumbra was significantly up-regulated at 24 h of reperfusion in I/R group, and the neurological deficit scores were significantly decreased at 1-7 days of reperfusion, and the percentage of cerebral infarct size was increased at 1 and 7 days of reperfusion in I/R, V and HPX groups ( P<0.05). Compared with I/R group, the neurological deficit scores were significantly increased at 1-7 days of reperfusion, and the percentage of cerebral infarct size was decreased at 1 and 7 days of reperfusion in HPX group ( P<0.05), and no significant change was found in the above indicators in V and I/R groups ( P>0.05). Conclusion:Up-regulation of HPX expression is the endogenous protective mechanism of cerebral I/R injury in rats.
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Objective:To evaluate the effect of hydrogen on the immunosuppressive status of septic rats.Methods:SPF healthy adult male Sprague-Dawley rats, aged 7-8 weeks, weighing 220-260 g, were studied.This study was performed in two parts.Part Ⅰ The rats were divided into 2 groups: sepsis group (Sep group, n=36) and sham operation group (Sham group, n=12). The model of sepsis was established by cecal ligation puncture in anesthetized rats.The histocompatibility DR antigen (HLA-DR)/CD14 + monocyte level in peripheral blood was detected by flow cytometry immediately after CLP and at 1, 2, 3 and 4 days after CLP.The establishment of sepsis-induced immunosuppression model was considered successful when the levels of HLA-DR/CD14 + monocyte in peripheral blood were <30%.Part Ⅱ Twelve rats with sepsis-induced immunosuppression were randomly selected and divided into 2 groups ( n=6 each) by a random number table method: sepsis immunosuppression group (Sep-IS group), sepsis immunosuppression plus hydrogen treatment group (Sep-IS+ H group). Another 12 rats were selected and divided into 2 groups ( n=6 each) by a random number table method: sham operation group (Sham group) and sham operation plus hydrogen group (Sham+ H group). In Sep-IS+ H group, 67% hydrogen was inhaled for 1 h starting from the time point immediately after successful establishment of sepsis-induced immunosuppression and from 6 h after establishment, and 67% hydrogen was inhaled for 1 h at the corresponding time points in Sham+ H group.The levels of helper T lymphocytes 17 (Th17 cells), regulatory T lymphocytes (Treg cells) and HLA-DR/CD14 + monocyte in peripheral blood were determined by flow cytometry immediately after the end of hydrogen inhalation mentioned above (T 0, T 1) and at 12 h after establishing the model (T 2). Results:Part Ⅰ Compared with Sham group, the levels of HLA-DR/CD14 + monocyte in peripheral blood were significantly decreased at 1-4 days after CLP in Sep group ( P<0.05). Part Ⅱ Compared with Sham group, the level of HLA-DR/CD14 + monocytes in peripheral blood was significantly decreased, and the levels of Treg and Th17 cells were increased at each time point in Sep-IS and Sep-IS+ H groups ( P<0.05), and no significant change was found in the parameters mentioned above in Sham+ H group ( P>0.05). Compared with Sep-IS group, the level of HLA-DR/CD14 + monocytes in peripheral blood was significantly increased at T 1, 2, the levels of Th17 cells were increased at T 2, and the levels of Treg cells were decreased at T 1, 2 in Sep-IS+ H group ( P<0.05). Conclusion:Hydrogen can improve the immunosuppressive state of septic rats.
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Objective:To evaluate the effects of vitamin K 2 on sevoflurane-induced cognitive decline in aged mice. Methods:A total of 72 SPF healthy female C57BL/6J mice, aged 12 months, weighing 20-25 g, were divided into 4 groups ( n=18 each) using a random number table method: control+ corn oil group (group Con+ Oil), sevoflurane+ corn oil group (group Sevo+ Oil), control+ vitamin K 2 group (group Con+ K 2) and sevoflurane+ vitamin K 2 group (group Sevo+ K 2). The mice in Sevo+ Oil and Sevo+ K 2 groups were anesthetized with 2.5% sevoflurane+ 33% oxygen for 2 h. The mice in Con+ Oil and Con+ K 2 groups were treated with 33% oxygen only.The animals in Con+ Oil and Sevo+ Oil groups were intraperitoneally injected with corn oil 100 μl at 30 min before oxygen or sevoflurane inhalation.Vitamin K 2 (dissolved in corn oil, concentration 1 mg/ml) 100 mg/kg was injected intraperitoneally in Con+ K 2 and Sevo+ K 2 groups.At 24 h after sevoflurane inhalation, 8 mice from each group were randomly selected and sacrificed, and the hippocampal tissues were removed for determination of activity of ATPase, contents of interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay) and the expression of AT8 and PHF1 (by Western blot). The remaining 10 mice in each group received standardized feeding, and the cognitive function was assessed using Y-maze at 1, 3, 5, 7 and 14 days after sevoflurane inhalation. Results:Compared with group Con+ Oil, the contents of IL-1β, IL-6 and TNF-α were significantly increased, expression of AT8 and PHF1 were up-regulated, activity of ATPase was decreased, and spontaneous alternation percentage was decreased at 1, 3, 5, 7 and 14 days after sevoflurane inhalation in group Sevo+ Oil ( P<0.05). Compared with group Sevo+ Oil, the contents of IL-1β, IL-6 and TNF-α were significantly decreased, expression of AT8 and PHF1 were down-regulated, activity of ATPase was increased, and spontaneous alternation percentage was increased at 1, 3, 5, 7 and 14 days in group Sevo+ K 2 ( P<0.05). There was no significant difference in the above indicators between group Con+ K 2 and group Sevo+ K 2 ( P>0.05). Conclusion:Vitamin K 2 can improve sevoflurane-induced cognitive decline in aged mice, the mechanism is related to increasing activity of ATPase and inhibiting the up-regulation of AT8 and PHF1 expression in hippocampus.