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Objective:To evaluate the role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in sepsis-associated encephalopathy (SAE) and the relationship with pyroptosis in microglia of mice.Methods:Twenty-four SPF healthy male C57BL/6J mice, aged 6-8 weeks, weighing 18-22 g, were divided into 3 groups ( n=6 each) using a random number table method: sham operation group (Sham group), SAE group and SAE plus an NLRP3 inhibitor MCC950 group (SAE+ MCC950 group). The mouse model of SAE was prepared by cecal ligation and puncture after anesthesia. MCC950 20 mg/kg was intraperitoneally injected at 1 h after developing the model in SAE+ MCC950 group, and the equal volume of normal saline was given instead in the other groups. Open field tests were conducted at 1 day after developing the model to record the number of rearing and time spent in the central area. Novel object recognition tests were conducted at 2-3 days after developing the model to record the recognition index. After the behavioral experiment on 3 day after developing the model, mice were sacrificed and hippocampal tissues were collected for determination of the expression of NLRP3 (by Western blot), count of cells co-expressing NLRP3 and microglia-specific ionized calcium-binding adaptor molecule 1 (Iba-1) (by immunofluorescence), activity of caspase-1, and contents of interleukin-1beta(IL-1β) and IL-18 (by enzyme-linked immunosorbent assay). Results:Compared with Sham group, the number of rearing was significantly reduced, the time spent in the central area was shortened, the recognition index was decreased, the expression of NLRP3 was up-regulated, the count of NLRP3 + -Iba-1 + cells was increased, and the activity of caspase-1 and contents of IL-1β and IL-18 were increased in SAE and SAE+ MCC950 groups ( P<0.05). Compared with SAE group, the number of rearing was significantly increased, the time spent in the central area was prolonged, the recognition index was increased, the expression of NLRP3 was down-regulated, the count of NLRP3 + -Iba-1 + cells was decreased, and the activity of caspase-1 and contents of IL-1β and IL-18 were decreased in SAE+ MCC950 group ( P<0.05). Conclusions:NLRP3 is involved in the development of SAE, which may be related to the mediation in microglial pyroptosis in mice.
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The clinical data of patients with severe acute pancreatitis admitted to the Department of Intensive Care Unit in our hospital from January 1, 2016 to December 31, 2020 were retrospectively collected.The patients were divided into electroacupuncture combined with Qingyi decoction treatment group (acupuncture group) and conventional group according to whether the patients received electroacupuncture combined with Qingyi decoction treatment.A prediction model of treatment propensity score was established for paired screening, with 122 cases in each group.The acupoints such as Zusanli, Sanyinjiao, Hegu, Shangjuxu, Xiajuxu, and Taichong were selected, and then electroacupuncture treatment was performed after qi arrival using the manipulation technique, 1 or 2 times per day.Qingyi decoction was injected through the stomach and/or Qingyi decoction was given by coloclysis, 2-4 doses per day.The main outcome was the incidence of acute respiratory distress syndrome (ARDS), and the secondary outcome was the occurrence of complications and outcome of discharge.Compared with conventional group, the incidence of ARDS was significantly decreased, the time of mechanical ventilation was shortened, the incidence of renal dysfunction, score for acute physiology and chronic health score system, sequential organ failure score, and score for the severity of bedside acute pancreatitis were decreased, the rate of surgical intervention was increased, the total length of hospital stay was prolonged, and the fatality rate during hospitalization was reduced in acupuncture group ( P<0.05). The results of subgroup analysis showed that the onset time of disease (<1 week), a history of cardiovascular disease, diabetes mellitus, biliary pancreatitis and alcoholic pancreatitis, high fever, puncture and drainage were influencing factors for ARDS developed in the patients who received electroacupuncture combined with Qingyi decoction for treating severe acute pancreatitis.In conclusion, electroacupuncture combined with Qingyi decoction as an adjuvant treatment for severe acute pancreatitis can reduce acute lung injury, promote recovery, and decrease fatality rate.
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Objective To evaluate the endotoxin-induced endogenous protective mechanism of alveolar type Ⅱ epithelial cells of rats and the relationship with p38 mitogen-activated protein kinase (p38MAPK)-HO-1-mitochondrial fusion signaling pathway.Methods Rat alveolar type Ⅱ epithelial cells were seeded in 6-well plates at a density of 2× 105 cells/ml and divided into 5 groups (n =15 each) using a random number table method:control group (group C),lipopolysaccharide (LPS) group (group L),LPS plus p38MAPK inhibitor SB203580 group (group LS),LPS plus dimethyl sulfoxide group (group LD),and SB203580 group (group S).Cells were conventionally cultured in group C.The model of endotoxin-challenged alveolar type Ⅱ epithelial cells was established by giving LPS 10 μg/ml in L,LS and LD groups.SB203580 10 μmol and 0.1％ dimethyl sulfoxide 100 μμmol were added at 1 h before giving LPS in group LS and group LD,respectively.SB203580 10 μ mol was added to the culture medium in group S.All the cells were incubated for 24 h.The malonaldehyde (MDA) content and superoxide dismutase (SOD) activity in the culture medium were determined by thiobarbituric acid assay and xanthine oxidase method,respectively.The expression of p38MAPK,phosphorylated p38MAPK (p-p38MAPK),hemeoxygenase-1 (HO-1),mitofusin 1 (Mfn1),Mfn2,and optical atrophy-1 (OPA1) was measured by Western blot.Results Compared with group C,the MDA content was significantly increased,the SOD activity was decreased,and the expression of p-p38MAPK and HO-1 was up-regulated,and the expression of Mfn1,Mfn2 and OPA1 was down-regulated in L,LS and LD groups (P＜0.05).Compared with group L,the MDA content was significantly increased,the SOD activity was decreased,and the expression of pp38MAPK,HO-1,Mfn1,Mfn2 and OPA1 was down-regulated in group LS (P＜0.05),and no significant change was found in the indices mentioned above in group LD (P＞0.05).Conclusion The endotoxin-induced endogenous protective mechanism of alveolar type Ⅱ epithelial cells is related to p38MAPK-HO-1-mitochondrial fusion signaling pathway in rats.
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Objective@#To evaluate the role of heme oxygenase-1 (HO-1) on lipopolysaccharide (LPS)-induced activation of NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes in alveolar macrophages of rats.@*Methods@#NR8383 cells of rat alveolar macrophages cultured in vitro were seeded in 96-well plates at a density of 4×104 cells/ml and divided into 4 groups (n=48 each) using a random number table method: control group (group C), group LPS (group L), LPS+ HO-1 siRNA group (group L+ HO-1 siRNA), and LPS+ Con siRNA group (group L+ Con siRNA). The cells were cultured in normal culture atmosphere in group C. NR8383 cells were stimulated with 10 μg/ml LPS in L, L+ HO-1 siRNA and L+ Con siRNA groups.Cells were transfected with HO-1 siRNA at 48 h before stimulation with LPS in group L+ HO-1 siRNA and with Con siRNA at 48 h before stimulation with LPS in group L+ Con siRNA.The cells were collected and incubated for 24 h after stimulation with LPS for measurement of the cell viability, expression of HO-1 and NLRP3 mRNA (by real-time polymerase chain reaction), expression of HO-1, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 (by Western blot), and concentrations of interleukin-1beta (IL-1β) and interleukin-18 (IL-18) in the supernatant (by enzyme-linked immunosorbent assay).@*Results@#Compared with group C, the cell viability was significantly decreased, the expression of HO-1 and NLRP3 protein and mRNA, ASC and caspase-1 was up-regulated, and the IL-1β and IL-18 concentrations were increased in L, L+ HO-1 siRNA and L+ Con siRNA groups (P<0.05). Compared with group L, the cell viability was significantly decreased, the expression of HO-1 protein and mRNA was down-regulated, and the expression of NLRP3 protein and mRNA, ASC and caspase-1 was up-regulated, and the IL-1β and IL-18 concentrations were increased in group L+ HO-1 siRNA (P<0.05), and no significant change was found in the parameters mentioned above in group L+ Con siRNA (P>0.05).@*Conclusion@#HO-1 is involved in LPS-induced activation of NLRP3 inflammasomes in alveolar macrophages of rats.
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Objective@#To evaluate the effect of dexmedetomidine on mitochondrial dynamics in mice with endotoxin-induced acute lung injury (ALI).@*Methods@#Thirty clean-grade healthy adult male C57BL/6 mice, weighing 20-25 g, aged 2 months, were divided into 3 groups (n=10 each) using a random number table method: control group (group C), endotoxin-induced ALI group (group LPS) and endotoxin-induced ALI plus dexmedetomidine group (group LPS+ DEX). In LPS and LPS+ DEX groups, lipopolysaccharide (LPS) 10 mg/kg was injected via the caudal vein to establish the model of endotoxin-induced ALI.In group LPS+ DEX, dexmedetomidine 50 μg/kg was intraperitoneally injected at 30 min before injection of LPS, while the equal volume of normal saline was given instead in C and LPS groups.The mice were sacrificed at 6 h after LPS administration, and lung tissues were obtained for examination of the pathological changes (with a light microscope) which were scored and for determination of the level of reactive oxygen species (ROS) and expression of mitochondrial fusion proteins mitofusin 1 (Mfn1), Mfn2, optic atrophy 1 (OPA1), dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1)(using Western blot).@*Results@#Compared with group C, the lung injury scores and ROS level in lung tissues were significantly increased, the expression of Mfn1, Mfn2 and OPA1 was down-regulated, and the expression of Drp1 and Fis1 was up-regulated in LPS and LPS+ DEX groups (P<0.05). Compared with group LPS, the lung injury scores and ROS level in lung tissues were significantly decreased, the expression of Mfn1, Mfn2 and OPA1 was up-regulated, and the expression of Drp1 and Fis1 was down-regulated in group LPS+ DEX (P<0.05).@*Conclusion@#Dexmedetomidine can reduce endotoxin-induced ALI through maintaining the mitochondrial fusion-fission balance in mice.
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Objective To evaluate the effect of dexmedetomidine on mitochondrial dynamics in mice with endotoxin-induced acute lung injury (ALI).Methods Thirty clean-grade healthy adult male C57BL/6 mice,weighing 20-25 g,aged 2 months,were divided into 3 groups (n=10 each) using a random number table method:control group (group C),endotoxin-induced ALI group (group LPS) and endotoxin-induced ALI plus dexmedetomidine group (group LPS+DEX).In LPS and LPS+DEX groups,lipopolysaccharide (LPS) 10 mg/kg was injected via the caudal vein to establish the model of endotoxin-induced ALI.In group LPS+DEX,dexmedetomidine 50 μg/kg was intraperitoneally injected at 30 min before injection of LPS,while the equal volume of normal saline was given instead in C and LPS groups.The mice were sacrificed at 6 h after LPS administration,and lung tissues were obtained for examination of the pathological changes (with a light microscope) which were scored and for determination of the level of reactive oxygen species (ROS) and expression of mitochondrial fusion proteins mitofusin 1 (Mfn1),Mfn2,optic atrophy 1 (OPA1),dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1) (using Western blot).Results Compared with group C,the lung injury scores and ROS level in lung tissues were significantly increased,the expression of Mfn1,Mfn2 and OPA1 was down-regulated,and the expression of Drp1 and Fis1 was up-regulated in LPS and LPS+DEX groups (P＜0.05).Compared with group LPS,the lung injury scores and ROS level in lung tissues were significantly decreased,the expression of Mfn1,Mfn2 and OPA1 was up-regulated,and the expression of Drp1 and Fis1 was down-regulated in group LPS+DEX (P＜ 0.05).Conclusion Dexmedetomidine can reduce endotoxin-induced ALI through maintaining the mitochondrial fusion-fission balance in mice.
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Objective To evaluate the role of heme oxygenase-1 (HO-1) on lipopolysaccharide (LPS)-induced activation of NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes in alveolar macrophages of rats.Methods NR8383 cells of rat alveolar macrophages cultured in vitro were seeded in 96-well plates at a density of 4× 104 cells/ml and divided into 4 groups (n =48 each) using a random number table method:control group (group C),group LPS (group L),LPS+HO-1 siRNA group (group L+HO-1 siRNA),and LPS+Con siRNA group (group L+Con siRNA).The cells were cultured in normal culture atmosphere in group C.NR8383 cells were stimulated with 10 μg/ml LPS in L,L+HO-1 siRNA and L+Con siRNA groups.Cells were transfected with HO-1 siRNA at 48 h before stimulation with LPS in group L+HO-1 siRNA and with Con siRNA at 48 h before stimulation with LPS in group L+Con siRNA.The ceils were collected and incubated for 24 h after stimulation with LPS for measurement of the cell viability,expression of HO-1 and NLRP3 mRNA (by real-time polymerase chain reaction),expression of HO-1,NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)and caspase-1 (by Western blot),and concentrations of interleukin-1beta (IL-1β) and interleukin-18 (IL-18) in the supernatant (by enzyme-linked immunosorbent assay).Results Compared with group C,the cell viability was significantly decreased,the expression of HO-1 and NLRP3 protein and mRNA,ASC and caspase-1 was up-regulated,and the IL-1β and IL-18 concentrations were increased in L,L+HO-1 siRNA and L+Con siRNA groups (P＜0.05).Compared with group L,the cell viability was significantly decreased,the expression of HO-1 protein and mRNA was down-regulated,and the expression of NLRP3 protein and mRNA,ASC and caspase-1 was up-regulated,and the IL-1β and IL-18 concentrations were increased in group L+HO-1 siRNA (P＜0.05),and no significant change was found in the parameters mentioned above in group L+Con siRNA (P＞0.05).Conclusion HO-1 is involved in LPS-induced activation of NLRP3 inflammasomes in alveolar macrophages of rats.
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Objective To evaluate the role of endogenous heme oxygenase-1/carbon monoxide ( HO-1/CO) signaling pathway in endoplasmic reticulum stress during endotoxin-induced acute lung injury ( ALI) in rats. Methods Forty healthy clean-grade male Sprague-Dawley rats, aged 8 weeks, weighing 190-210 g, were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), ALI group, ALI plus ZnPP-IX group (group AZ), and ALI plus vehicle sodium bicarbonate group ( group AV) . ALI was induced by intravenously injecting lipopolysaccharide 5 mg/kg in anesthetized rats. At 30 min before establishing the model, ZnPP-IX 10μmol/kg (diluted to 1 ml in 50 mmol/L sodium bicarbonate) was intraperitoneally injected in group AZ, and 50 mmol/L sodium bicarbonate 1 ml was intra-peritoneally injected in group AV. After injecting lipopolysaccharide for 6 h, blood samples were collected from the common carotid artery for determination of plasma CO concentration, the rats were then sacrificed, and lungs were removed for microscopic examination of the pathological changes which were scored and for determination of CO level, wet to dry weight ratio ( W/D ratio) , cell apoptosis ( by TUNEL) , and expres-sion of heme oxygenase-1 ( HO-1) , glucose-regulated protein 78 ( GRP78) , phosphorylated protein kinase R-like endoplasmie reticulum kinase (p-PERK), phosphorylated eukaryotic translation initiation factor 2 alpha ( p-elF2 ) , CCAAT/enhancer-binding protein homologous protein ( CHOP ) and caspase-12 in lung tissues ( by Western blot) . Apoptosis index ( AI) was calculated. Results Compared with group C, the lung injury scores, W/D ratio, AI and CO levels in plasma and lung tissues were significantly increased, and the expression of HO-1, GRP78, p-PERK, p-elF2, CHOP and caspase-12 was up-regulated in the other three groups ( P＜0. 05) . Compared with group ALI, lung injury scores, W/D ratio and AI were sig-nificantly increased, CO levels in plasma and lung tissues were decreased, the expression of HO-1 was down-regulated, and the expression of GRP78, p-PERK, p-elF2, CHOP and caspase-12 was up-regula-ted in group AZ (P＜0. 05), and no significant change was found in the parameters mentioned above in group AV ( P＞0. 05) . Conclusion HO-1/CO signaling pathway produces endogenous protection possibly through inhibiting endoplasmic reticulum stress during endotoxin-induced ALI in rats.