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Objective:To evaluate the relationship between silent information regulator 2 homologue 3 (SIRT3) and mitochondrial function in mice with endotoxin-induced lung injury.Methods:Twenty clean-grade healthy adult male wild C57BL/6 (SIRT3 + /+ ) mice, 20 SIRT3 knockout (SIRT3 -/-) mice, weighing 20-25 g, aged 6-8 weeks, were studied.SIRT3 + /+ mice and SIRT3 -/- mice were divided into 4 groups ( n=5 each) according to the random number table method: blank control group (group C, group SIRT3 -/-C), endotoxin-induced lung injury group (group L, group SIRT3 -/-L), endotoxin-induced lung injury plus resveratrol group (group L+ R, group SIRT3 -/-L+ R), and resveratrol group (group R, group SIRT3 -/-R). Resveratrol 15 mg/kg was intraperitoneally injected once a day for 7 consecutive days in L+ R, R, SIRT3 -/-L+ R and SIRT3 -/-R groups, while the equal volume of normal saline was injected in the rest groups.Lipopolysaccharid 15 mg/kg was injected via the tail vein to develop a mouse model of endotoxin-induced lung injury at 30 min after resveratrol injection on 7th day, in L+ R and SIRT3 -/-L+ R groups and at the corresponding time points in L and SIRT3 -/-L groups, while the equal volume of normal saline was injected in the other groups.Blood samples were collected from the orbital venous plexus at 12 h after injection of normal saline or lipopolysaccharid for determination of serum total oxidation state (TOS) and total antioxidant state (TAS) levels by the xylenol orange method and ABTS colorimetric method, and the oxidative stress index (OSI) was calculated.After the mice were sacrificed, the lung tissues were taken for microscopic examination of the pathological changes which were scored and for determination of the mitochondrial membrane potential (MMP) (by JC-1 method), cellular oxygen consumption rate (OCR) (by the specific fluorescent probe method), and expression of SIRT3 (by Western blot). Results:Compared with group C or group SIRT3 -/-C, the lung injury score, serum TOS concentration and OSI were significantly increased, TAS concentration, MMP and OCR were decreased, and SIRT3 expression was down-regulated in L, L+ R, SIRT3 -/-L and SIRT3 -/-L+ R groups ( P<0.05). Compared with group L, the lung injury score, serum TOS concentration and OSI were significantly decreased, TAS concentration, MMP and OCR were increased, and SIRT3 expression was up-regulated in group L+ R, and lung injury score, serum TOS concentration and OSI were significantly increased, TAS concentration, MMP and OCR were decreased, and SIRT3 expression was down-regulated in group SIRT3 -/-L ( P<0.05). Compared with group L+ R, the lung injury score, serum TOS concentration and OSI were significantly increased, the TAS concentration, MMP and OCR were decreased, and the expression of SIRT3 was down-regulated in group SIRT3 -/- L+ R ( P<0.05). There was no significant difference in the indicators mentioned above between group SIRT3 -/-L+ R and group SIRT3 -/-L ( P>0.05). Conclusions:Down-regulation of SIRT3 expression can lead to impaired mitochondrial function, which is involved in the pathophysiological mechanism of endotoxin-induced lung injury.
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Objective:To evaluate the role of nicotinamide adenine dinucleotide (NAD + )-mediated deacetylation activity of silent information regulator 1 (SIRT1) in endotoxin-induced acute lung injury (ALI) in mice. Methods:Twenty-five SPF clean-grade healthy male C57BL/6 mice including 10 wild-type (WT) and 15 NMNAT1 conditional-knockout (KO) mice, aged 6-8 weeks, weighing 20-25 g, were selected.The WT mice were divided into 2 groups ( n=5 each) using a random number table method: control group (group WT+ C) and ALI group (group WT+ ALI). The KO mice were divided into 3 groups ( n=5 each) using a random number table method: control group (group KO+ C), ALI group (group KO+ ALI) and ALI plus NAD + precursor substances nicotinamide mononucleotide (NMN) group (KO+ LPS+ NMN group). ALI was produced with lipopolysaccharide (LPS) 15 mg/kg injected intravenously.NMN 500 mg/kg was intraperitoneally injected at 1 h before injection of LPS in KO+ ALI+ NMN group, while the equal volume of normal saline was given instead in control group.Blood samples were collected from the abdominal aorta at 12 h after LPS or normal saline injection for blood gas analysis, and the animals were then sacrificed and the lung tissues were removed for microscopic examination of pathologic changes which were scored and for determination of wet/dry weight ratio (W/D ratio), and interleukin-6 (IL-6), IL-1β and tumor necrosis factor-alpha (TNF-α) contents (by enzyme-linked immunosorbent assay)and content of NAD + (using a spectrophotometer) and levels of SIRT1, acetylated nuclear factor kappaB (Ac-NF-κB), acetylated p53 (Ac-p53), acetylated FoxO1 (Ac-FoxO1) and acetylated PGC1α (Ac-PGC1α) (by Western blot). Results:Compared with group C, pH value and PaO 2 were significantly decreased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β, TNF-α and NAD + were increased, expression of SIRT1 was up-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was down-regulated in group ALI ( P<0.05). Compared with group WT+ ALI, pH value and PaO 2 were significantly decreased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β and TNF-α were increased, NAD + content was decreased, expression of SIRT1 was down-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was up-regulated in group KO+ ALI ( P<0.05). Compared with group KO+ ALI, pH value and PaO 2 were significantly increased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β and TNF-α were decreased, NAD + content was increased, expression of SIRT1 was up-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was down-regulated in group KO+ ALI+ NMN ( P<0.05). Conclusion:The enhanced NAD + -mediated deacetylation activity of SIRT1 is involved in the endogenous protective mechanism in mice with endotoxin-induced ALI.
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Objective:To evaluate the effect of electroacupuncture (EA) pretreatment on oxidative stress response of hippocampus in rats with sepsis-associated encephalopathy (SAE) and the relationship with nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (Nrf2/HO-1) signaling pathway.Methods:A total of 64 healthy adult male Sprague-Dawley rats, weighing 250-300 g, were divided into 4 groups ( n=16 each) using a random number table method: sham operation group (Sham group), SAE group, SAE+ EA group and SAE plus sham EA group (SAE+ SEA group). In SAE+ EA group, Baihui, Quchi and Zusanli acupoints were stimulated for 30 min once a day for 5 consecutive days.Sepsis was induced by cecal ligation and puncture immediately after the end of the last EA.At 1 and 7 days after establishment of the model, the hippocampal malondialdehyde (MDA) and superoxide dismutase (SOD) levels were detected by enzyme-linked immunosorbent assay, the expression of hippocampal Nrf2 mRNA was detected using real-time reverse transcription polymerase chain reaction, and the expression of Nrf2 and HO-1 was determined by Western blot.At 3-7 days after establishment of the model, cognitive function was assessed using the Morris water maze test, and the escape latency and the target quadrant exploration time were recorded. Results:Compared with Sham group, the content of MDA was significantly increased and the activity of SOD was decreased at 1 and 7 days after establishment of the model, the expression of Nrf2 protein and mRNA and HO-1 was down-regulated at day 7 after establishment of the model, the escape latency was prolonged, and the target quadrant exploration time was shortened in SAE group ( P<0.05). Compared with SAE group, the content of MDA was significantly decreased and the activity of SOD was increased at 1 and 7 days after establishment of the model, the expression of Nrf2 protein and mRNA and HO-1 was up-regulated at day 7 after establishment of the model, the escape latency was shortened, and the target quadrant exploration time was prolonged in group SAE+ EA ( P<0.05), and no significant change was found in the parameters mentioned above in group SAE+ SEA ( P>0.05). Conclusion:EA pretreatment can reduce oxidative stress response of hippocampus in rats with SAE, and the mechanism may be related to activating Nrf2/HO-1 signaling pathway.
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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 effect of electroacupuncture (EA) on pyroptosis in renal tubular epithelial cells of rats with acute kidney injury (AKI) induced by endotoxin.Methods:Twenty-four healthy clean-grade Sprague-Dawley rats of either gender, aged 6-8 weeks, weighing 160-182 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), group AKI, EA plus AKI group (group EA), sham EA at non-acupoint plus AKI group (group SEA). The model of endotoxemia was established by intraperitoneally injecting 10 mg/kg lipopolysaccharide.Bilateral 30 min EA stimulation of Zusanli and Shenyu (according to atlas of animal acupoint) was performed starting from 5 days before establishing the model (once a day) and at 30 min before lipopolysaccharide administration on the day of establishing the model, with disperse-dense waves, frequency of 15 Hz, and the needle was kept until 6 h after injection of LPS in group EA.EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Shenyu in group SEA.At 6 h after LPS injection, blood was taken from the heart, and the concentrations of serum blood urea nitrogen (BUN) and creatinine (Cr) were detected by an automatic biochemical analyzer, and the serum concentrations of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) by enzyme-linked immunosorbent assay.The rats were then sacrificed, and the left renal cortex was obtained for determination of pyroptosis rate of renal tubular epithelial cells (by TUNEL). The right renal cortex was obtained to detect the expression of caspase-1 and IL-1β by Western blot, and the expression of caspase-1 mRNA and IL-1β mRNA was detected by real-time polymerase chain reaction. Results:Compared with group C, the concentrations of BUN, Cr, NGAL, KIM-1, TNF-α, and IL-6 were significantly increased, the pyroptosis rate of renal tubular epithelial cells was increased, the expression of caspase-1 and IL-1β protein and mRNA in the renal cortex was up-regulated in group AKI ( P<0.05). Compared with group AKI, the concentrations of BUN, Cr, NGAL, KIM-1, TNF-α, and IL-6 were significantly decreased, the pyroptosis rate of renal tubular epithelial cells was decreased, the expression of caspase-1 and IL-1β protein and mRNA in the renal cortex was down-regulated in group SEA ( P>0.05). Conclusion:The mechanism by which EA reduces AKI may be related to inhibiting pyroptosis in renal tubular epithelial cells of rats.
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Objective:To investigate the effect of electroacupuncture (EA) on synaptic damage to hippocampal neurons in rats with sepsis-associated encephalopathy (SAE).Methods:A total of 48 healthy adult male Sprague-Dawley rats, weighing 250-300 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (Sham group), SAE group, SAE+ EA group and SAE+ sham EA group (SAE+ SEA group). SAE was induced by cecal ligation and puncture in anesthetized rats.Baihui, Quchi and Zusanli acupoints were stimulated with constant voltage (2/15 Hz) and disperse-dense waves for 30 min once a day for 10 consecutive days, and the stimulation intensity was defined as less than 1.5 mA causing slight muscle contraction at 2 days before operation in group SAE+ EA.In group SAE+ SEA, stimulating electrodes were placed at the points 5 mm lateral to the corresponding acupoints, but no electrical stimulation was applied.On day 14 after operation, the rats were sacrificed, and hippocampal tissues were obtained and stained with haematoxylin and eosin for examination of the pathological changes in the hippocampal CA1 region, for determination of the expression of synaptophysin (SYN) and postsynaptic density protein 95 (PSD-95) (by Western blot), and for calculation of dendritic spine density of neurons in the hippocampal CA1 area (using Golgi staining) and pyramidal neurons counts. Results:Compared with Sham group, the expression of SYN and PSD-95 in hippocampus was significantly decreased, and the basal and apical dendrite spine density of neurons in hippocampal CA1 area was decreased in SAE group, the expression of PSD-95 was decreased, and the apical dendrite spine density of neurons in the hippocampal CA1 area was increased in SAE+ EA group, and the pyramidal neuron counts in the hippocampal CA1 area were reduced in SAE, SAE+ EA and SAE+ SEA groups ( P<0.05). Compared with group SAE, the expression of SYN and PSD-95 was significantly up-regulated, the basal and apical dendrite spine density of neurons in the hippocampal CA1 area was increased and the pyramidal neuron counts were increased in group SAE+ EA ( P<0.05), the pathological damage of hippocampal CA1 area was alleviated and no significant change was found in the parameters mentioned above in group SAE+ SEA ( P>0.05). Compared with group SAE+ EA, the expression of SYN and PSD-95 was down-regulated, the basal and apical dendrite spine density of neurons in the hippocampal CA1 area was decreased, and the pyramidal neuron counts were reduced in SAE+ SEA group ( P<0.05). Conclusion:The mechanism by which EA alleviates SAE may be related to reducing synaptic damage to hippocampal neurons in rats.
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Objective:To evaluate the role of heme oxygenase-1 (HO-1) in electroacupuncture (EA)-induced alleviation of cognitive dysfunction in mice with sepsis-associated encephalopathy (SAE) and the relationship with mitochondrial fusion-division balance.Methods:Thirty clean-grade male C57BL/6 mice (24 wide-type mice and 6 HO-1 knockout mice), aged 6-8 weeks, weighing 18-22 g, were studied.Twenty-four wide-type mice were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), SAE group, SAE plus EA group (group SAE+ EA), and SAE plus sham EA group (group SAE+ SEA). HO-1 knockout mice in which EA intervention was performed after establishing SAE model served as SAE plus EA plus HO-1 knockout group (group SAE + EA+ H). Sepsis was induced by intraperitoneally injecting lipopolysaccharide 15 mg/kg.EA of Zusanli and Baihui acupoints lasting 30 min was performed after intraperitoneal injection of lipopolysaccharide once a day for 5 consecutive days in SAE+ EA and SAE+ EA+ H groups.Cognitive function was assessed using Morris water maze test before stimulation every day.The mice were sacrificed, and hippocampal tissues were removed for detection of the expression of mitofusin 2 (Mfn2), optic atrophy 1 (OPA1) and mitochondrial dynamin-related protein 1 (Drp1) by Western blot. Results:Compared with group C, the expression of Mfn2 and OPA1 was significantly down-regulated, the escape latency was prolonged, and the time spent in the target quadrant was shorted in SAE, SAE+ SEA and SAE+ EA+ H groups, and the expression of Drp1 was significantly up-regulated in SAE, SAE+ EA, SAE+ SEA and SAE+ EA+ H groups ( P<0.05). Compared with group SAE, the expression of Mfn2 and OPA1 was significantly up-regulated, the expression of Drp1 was down-regulated, the escape latency was shortened, and the time spent in the target quadrant was prolonged in group SAE+ EA ( P<0.05), and no significant change was found in the parameters mentioned above in group SAE+ SEA ( P>0.05). Compared with group SAE+ EA, the expression of Mfn2 and OPA1 was significantly down-regulated, the expression of Drp1 was up-regulated, the escape latency was prolonged, and the time spent in the target quadrant was shortened in SAE+ SEA and SAE+ EA+ H groups ( P<0.05). Conclusion:HO-1 is involved in EA-induced alleviation of cognitive dysfunction in mice with SAE, and the mechanism may be related to the regulation of mitochondrial mitochondrial fusion-division balance.
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Objective:To evaluate the relationship between changes in Golgi apparatus morphological structure and endotoxin-induced acute lung injury (ALI) in mice.Methods:Twenty healthy male C57BL/6J mice, weighing 18-20 g, aged 6-8 weeks, were divided into 2 groups ( n=10 each) using a random number table method: sham operation group (group Sham) and endotoxin-induced ALI group (group ALI). Lipopolysaccharide (LPS) 10 mg/kg was injected intravenously in group ALI, while the equal volume of normal saline 0.5 ml was given instead in group Sham.The animals were sacrificed at 12 h after LPS injection and the lung tissues were taken for detection of the content of reactive oxygen species (ROS) and wet to dry weight ratio (W/D ratio), for observation of the pathological changes (using HE staining) and Golgi apparatus morphological structure (with a transmission electron microscope) and for determination of expression of Golgi matrix protein 130 (GM130), Golgin97 and mannosidase alpha class II member 1 (MAN2A1) and its mRNA (by Western blot and quantitative polymerase chain reaction). Results:Compared with group Sham, ROS content and the W/D ratio in lung tissues were significantly increased, GM130, MAN2A1, Golgin97 protein and its mRNA expression were down-regulate ( P<0.01), the pathological changes of lung tissues were accentuated, the Golgi cisternae was swollen, and Golgi fragments were dispersed in the cytoplasm in group ALI. Conclusion:The mechanism of endotoxin-induced ALI may be related to the changes in Golgi apparatus morphological structure.
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Objective:To evaluate the effect of transcutaneous electrical acupoint stimulation (TEAS) on postoperative gastrointestinal function in patients undergoing laparoscopic non-gastrointestinal hand surgery.Methods:Two hundred and sixty-seven patients, regardless of gender, aged 18-64 yr, of American Society of Anesthesiology physical status Ⅰ-Ⅲ, scheduled for elective laparoscopic cholecystectomy, laparoscopic bile duct exploration, and laparoscopic ovarian lesion resection under general anesthesia, were divided into 2 groups using a random number table method: control group (C group, n=147) and TEAS group ( n=120). From 30 min before induction of anesthesia to the end of operation, transcutaneous electrical stimulation of bilateral Zusanli and Hegu acupoints was performed at 30 min before induction of general anesthesia and maintained until the end of surgery with disperse-dense waves at frequency of 15-20 Hz and the maximum tolerated intensity.Combined intravenous-inhalational anesthesia was used in both groups.The time to first flatus and defecation was recorded.The occurrence of nausea and vomiting and abdominal distension within 24 h after surgery were recorded.The quantitative score of traditional Chinese medicine symptom of gastrointestinal function and Hamilton Anxiety Scale score were performed after surgery.The plasma motilin and gastrin levels were measured by enzyme-linked immunosorbent assay at 30 min before operation and 12 h after operation. Results:Compared with group C, the time to first flatus and defecation was significantly shortened, the incidence of postoperative nausea and vomiting and abdominal distension was decreased, the quantitative score of the traditional Chinese medicine and Hamilton Anxiety Scale score were decreased after operation, and the plasma motilin and gastrin levels were increased at 12 h after surgery in group TEAS ( P<0.05). Conclusion:TEAS can promote the recovery of gastrointestinal function after laparoscopic non-gastrointestinal surgery in the patients.
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Objective:To evaluate the role of melatonin in electroacupuncture (EA)-induced reduction of lung injury induced by limb ischemia-reperfusion (I/R) in rabbits.Methods:Fifty clean-grade healthy male New Zealand white rabbits, weighing 2.0-2.5 kg, aged 3 months, were divided into 5 groups ( n=10 each) using a random number table method: sham operation group (group Sham), limb I/R group (group IR), EA group, sham EA group (group SEA) and EA plus melatonin receptor antagonist luzindele group (group EA+ L). The model of limb I/R injury was established by clamping the femoral artery for 3 h followed by 4-h reperfusion in anesthetized animals.In group EA and group EA + L, bilateral Zusanli and Feishu acupoints (4-6 mm depth) were stimulated with constant voltage (2/15 Hz, l-2 mA, disperse-dense waves) for 30 min once a day during 1-7 days before establishing the model and during establishment of the model.EA was performed at the points (3 mm depth) 0.5 cm lateral to the acupoints of Zusanli and Feishu instead in group SEA.Luzinole 30 mg/kg was intraperitoneally injected at 30 min before establishing the model in group EA+ L.Blood samples from the right internal jugular vein were collected before ischemia (T 0), at 3 h of ischemia (T 1) and 4 h of reperfusion (T 2) for determination of the serum melatonin concentrations by enzyme-linked immunosorbent assay.Bronchoalveolar lavage fluid (BALF) was collected at 4 h of reperfusion for measurement of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 concentrations (by enzyme-linked immunosorbent assay), superoxide dismutase (SOD) activity (by xanthine oxidase method), and malondialdehyde (MDA) concentration (by thiobarbituric acid method). Then the rabbits were sacrificed, and the lung tissues were taken for determination of wet to dry weight ratio (W/D ratio) and for microscopic examination of the pathological changes (with a light microscope) which were scored and ultrastructure (with a transmission electron microscope). The number of mitochondria and relative cross-sectional area of mitochondria were calculated. Results:Compared with group Sham, lung injury scores and W/D ratio were significantly increased, the number of mitochondria was decreased, the relative cross-sectional area of mitochondria was increased, levels of TNF-α, IL-1β, IL-6 and MDA in BALF were increased, and activities of SOD in BALF were decreased in the other four groups, and the serum melatonin concentration was decreased at T 1 and T 2 in group I/R and increased at T 0 in EA and EA+ L groups ( P<0.05). Compared with group IR, the lung injury score and W/D ratio were significantly decreased, the number of mitochondria was increased, the relative cross-sectional area of mitochondria was decreased, levels of TNF-α, IL-1β, IL-6 and MDA in BALF were decreased, and activities of SOD in BALF were increased in group EA, the serum melatonin concentration was increased at each time point in EA and EA+ L groups ( P<0.05), and no significant change was found in the parameters mentioned above in group SEA ( P>0.05). Compared with group EA, lung injury scores and W/D ratio were significantly increased, the number of mitochondria was decreased, the relative cross-sectional area of mitochondria was increased, levels of TNF-α, IL-1β, IL-6 and MDA in BALF were increased, and activities of SOD in BALF were decreased in SEA and EA+ L groups, and the serum melatonin concentration was decreased at each time point in group SEA ( P<0.05). Conclusion:EA can reduce lung injury induced by limb I/R by increasing serum melatonin level in rabbits.
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Objective:To evaluate the role of heme oxygenase-1 (HO-1)-induced endogenous protection in lipopolysaccharide (LPS)-caused apoptosis in rat alveolar macrophages and the relationship with endoplasmic reticulum stress.Methods:Alveolar macrophages of rats were randomized into 4 groups ( n=32 each) using a random number table method: control group (group C), LPS group (group L), Con siRNA group and HO-1 siRNA group. Cells were cultured normally in group C, and 10 μg/ml LPS was added to the culture medium in the other three groups. Con siRNA and HO-1 siRNA transfection was performed at 48 h before adding LPS in Con siRNA and HO-1 siRNA groups. At 24 h of treatment with LPS, MTT method was used to measure the cell viability, flow cytometry was used to determine the cell apoptosis rate, and Western blot was used to detect the expression of glucose regulatory protein 78 (GRP78), phosphorylated kinase receptor-like endoplasmic reticulum kinase (p-PERK), CCAAT/enhancer-binding protein homologous protein (CHOP), phosphorylated type I endoplasmic reticulum transmembrane protein kinase (p-IRE-1), phosphorylated stress-activated protein kinase (p-JNK) and caspase-12. Results:Compared with group C, the cell viability was significantly decreased, cell apoptosis rate was increased, and the expression of HO-1, GRP78, CHOP, p-PERK, p-IRE-1, p-JNK and caspase-12 was up-regulated in the other three groups ( P<0.05). Compared with group L, the cell viability was significantly decreased, cell apoptosis rate was increased, and the expression of HO-1 was down-regulated, and the expression of GRP78, CHOP, p-PERK, p-IRE-1, p-JNK and caspase-12 was up-regulated in group HO-1 siRNA ( P<0.05), and no significant change was found in each parameter in group Con siRNA ( P>0.05). Compared with group Con siRNA, the cell viability was significantly decreased, cell apoptosis rate was increased, and the expression of HO-1 was down-regulated, and the expression of GRP78, CHOP, p-PERK, p-IRE-1, p-JNK and caspase-12 was up-regulated in group HO-1 siRNA ( P<0.05). Conclusion:The mechanism of HO-1-induced endogenous protection is related to inhibiting endoplasmic reticulum stress and then reducing LPS-induced apoptosis in alveolar macrophages of rats.
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Objective:To evaluate the relationship between melatonin receptors and mitochondrial fission proteins and to clarify the mechanism of melatonin alleviating lipopolysaccharide(LPS)-induced damage to type Ⅱ alveolar epithelial cells of rats.Methods:The rat type Ⅱalveolar epithelial cells were seeded in 6-well plates at a density of 2×10 5 cells/ml and divided into 5 groups ( n=10 each) using a random number table method: control group (C group), LPS group (L group), LPS plus melatonin group (LM group), LPS plus melatonin receptor blocking group (LL group), and LPS plus melatonin plus melatonin receptor blocker group (LML group). The model of LPS-induced damage to cells was established by incubating with LPS 10 μg/ml for 24 h. Melatonin 0.1 mmol/L and/or melatonin receptor blocker luzindole 0.2 μmol/L was added in LM group, LL group and LML group.The concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay after the end of incubation.The mitochondrial respiratory control rate (RCR) was measured by GENMED purified mitochondrial RCR quantitative detection kit in each group.Western blot was used to detect the expression of dynamin-related protein 1 (Drp1) and mitochondrial adaptor fission 1 (Fis1). Results:Compared with C group, the concentrations of TNF-α and IL-6 in culture medium were significantly increased, RCR was decreased, and the expression of Drp1 and Fis1 was up-regulated in L, LM, LL and LML groups ( P<0.05). Compared with L group, the concentrations of TNF-α and IL-6 in culture medium were significantly decreased, RCR was increased, and the expression of Drp1 and Fis1 was down-regulated in LM group ( P<0.05), and no significant change was found in parameters mentioned above in LL group ( P>0.05). Compared with LM group, the concentrations of TNF-α and IL-6 in culture medium were significantly increased, RCR was decreased, and the expression of Drp1 and Fis1 was up-regulated in LML group ( P<0.05). Conclusion:The mechanism by which melatonin attenuates LPS-induced damage to type Ⅱ alveolar epithelial cells is related to activating melatonin receptors and inhibiting the expression of mitochondrial fission proteins in rats.
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Objective:To evaluate the role of hippocampal hemeoxygenase-1 (HO-1) in electroacupuncture (EA)-induced reduction of lipopolysaccharide (LPS)-induced brain injury in mice.Methods:Twenty-four healthy adult C57BL/6J mice of both sexes, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), LPS-induced brain injury group (LPS group), LPS plus EA group, and LPS plus EA plus HO-1 inhibitor zinc protoporphyria (ZnPP) group (LPS+ EA+ ZnPP group). A virus carrying calcium ion fluorescent probes was injected into and an optical fiber was implanted into the hippocampal CA1 region to record changes in the calcium fluorescence signals.Three weeks later, Baihui, Quchi and Zusanli acupoints were stimulated with constant voltage (2/15 Hz) and disperse-dense waves for 30 min once a day for 5 consecutive days, and the stimulation intensity was defined as less than 1 mA causing slight muscle contraction.ZnPP 50 μmol/kg was intraperitoneally injected at 12 h before each stimulation in LPS+ EA+ ZnPP group, and the equal volume of normal saline was given instead in the other groups.After the end of EA stimulation on the last day, LPS 5 mg/kg was intraperitoneally injected to induce brain injury.Open field tests were performed at 1 day after LPS injection to record the number of rearing and amplitude of neuronal calcium signals during rearing.Novel object recognition tests were conducted at 3 days after LPS injection, and the exploration index and amplitude of neuronal calcium signals while exploring novel objects were recorded.The mice were sacrificed after the end of behavioral testing, and the brain tissues were obtained and stained by Nissl, and the neurons in the hippocampal CA1 region were counted. Results:Compared with group C, the number of rearing and amplitude of calcium signals in neurons in the hippocampal CA1 region during rearing were significantly decreased, the exploration index and amplitude of calcium signals in neurons in the hippocampal CA1 region while exploring novel objects were decreased, and the neuron counts were reduced in LPS, LPS+ EA and LPS+ EA+ ZnPP groups ( P<0.05 or 0.01). Compared with group LPS, the number of rearing and amplitude of calcium signals in neurons in the hippocampal CA1 region during rearing were significantly increased, and the exploration index and amplitude of calcium signals in neurons in the hippocampal CA1 region while exploring novel objects were increased in group LPS+ EA ( P<0.05), and no significant change was found in the parameters mentioned above in group LPS+ EA+ ZnPP ( P>0.05). Compared with group LPS+ EA, the number of rearing and amplitude of calcium signals in neurons in the hippocampal CA1 region during rearing were significantly decreased, and the exploration index and amplitude of calcium signals in neurons in the hippocampal CA1 region while exploring novel objects were decreased in group LPS+ EA+ ZnPP ( P<0.05). Conclusion:The mechanism by which EA reduces LPS-induced brain injury is related to the activation of the endogenous protective mechanism HO-1 in mice.
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Objective:To evaluate the relationship between heme oxygenase-1/carbon monoxide (HO-1/CO) signaling pathway and mitochondrial fission in lipopolysaccharide (LPS)-challenged alveolar type Ⅱ epithelial cells (AECⅡ) of rats.Methods:The cultured AECⅡ were subcultured and inoculated in 96-well plates (about 2 × 10 5 cells/ml) and divided into 7 groups ( n=12 each) using a random number table method: blank control group (group C), LPS group (group L), carbon monoxide release molecule-2 (CORM -2) + LPS group (group CL), HO-1 inducer hemin+ LPS group (group HL), HO-1 inhibitor ZnPP-Ⅸ+ LPS group (group ZL), CORM-2+ ZnPP-Ⅸ+ LPS group (group CZL) and hemin+ ZnPP-Ⅸ+ LPS group (group HZL). In group C, the cells were continuously incubated and received no treatment.Cells were incubated with LPS 10 μg/ml in group LPS. In CL, HL and ZL groups, cells were incubated with 100 μmol/L CORM-2 for 1 h, with 20 μmol/L hemin for 1 h and with 10 μmol/L ZnPP- Ⅸ for 0.5 h, respectively, and then the cells were incubated with 10 μg/ml LPS.In group CZL, the cells were incubated with 100 μmol/L CORM-2 for 1 h, then with 10 μmol/L ZnPP-Ⅸ for 0.5 h, and finally with 10 μg/ml LPS.In group HZL, the cells were incubated with 20 μmol/L hemin for 1 h, then with 10 μmol/L ZnPP-Ⅸ for 0.5 h, and finally with 10 μg/ml LPS.Cell viability was evaluated by methyl thiazolyl tetrazolium assay at 48 h of culture or incubation with LPS.The expression of HO-1, dynamin-related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1) was determined using Western blot. Results:Compared with group C, the cell viability was significantly decreased, and the expression of HO-1, Drp1 and Fis1 was up-regulated in other six groups ( P<0.05). Compared with group L, the cell viability was significantly increased, the expression of HO-1 was up-regulated, and the expression of Drp1 and Fis1 was down-regulated in CL and HL groups, the cell viability was decreased, HO-1 expression was down-regulated and the expression of Drp1 and Fis1 was up-regulated in group ZL ( P<0.05), and no significant change was found in the parameters mentioned above in CZL and HZL groups ( P>0.05). Compared with CL and HL groups, the cell viability was significantly decreased, HO-1 expression was down-regulated, and the expression of Drp1 and Fis1 was up-regulated in group ZL ( P<0.05). Conclusion:HO-1/CO signaling pathway can inhibit mitochondrial fission, which exerts endogenous protection when LPS induces injury to AEC Ⅱ in 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 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 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.
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Objective To investigate the relationship between the mechanism underlying hydrogeninduced improvement of intestinal barrier function and RhoA-mDia1 signaling pathway in septic mice.Methods Sixty male C57BL/6 mice,weighing 20-25 g,aged 6-8 weeks,were divided into 3 groups (n =20 each) using a random number table method:control group (group C),sepsis group (group S),and sepsis plus H2 group (group SH).Sepsis was produced by cecal ligation and puncture (CLP) in mice anesthetized with chloral hydrate.Mice in group SH inhaled air containing 2％ hydrogen for 1 h starting from 1 and 6 h after CLP.At 24 h after CLP,blood samples were obtained by cardiac puncture to measure the activity of serum diamine oxidase (DAO) and to count colony-forming unit (CFU) after bacterial culture,the small intestinal samples were obtained for microscopic examination of the ultrastructure of intestinal epithelial cells (with a transmission electron mnicroscope) and for measurement of the expression and distribution of tight junction protein ZO-1 in intestinal epithelial cells (by immunofluorescence) and expression of RhoA,expression and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) and expression of mDia1 (by Western blot).The p-MYPT1/MYPT1 ratio was calculated.Results Compared with group C,the serum DAO activity and whole blood CFU counts were significantly increased,the expression of RhoA was up-regulated,and the expression of ZO-1 was down-regulated in S and SH groups,the p-MYPT1/MYPT1 ratio was significantly increased,and the expression of mDia1 was down-regulated in group S,and the p-MYPT1/MYPT1 ratio was significantly decreased,and the expression of mDia1 was up-regulated in group SH (P＜0.05).Compared with group S,the serum DAO activity,whole blood CFU counts and p-MYPT1/MYPT1 ratio were significantly decreased,the expression of RhoA was down-regulated,the expression of ZO-1 and mDia1 was up-regulated (P＜0.05),and the ultrastructure changes of intestinal tissues were significantly improved in group SH.Conclusion The mechanism by which hydrogen improves intestinal barrier function is related to inhibiting RhoA activity and increasing mDia1 activity in intestinal epithelial cells of septic mice.