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Objective:To investigate the effect of electroacupuncture on calcium homeostasis in hippocampal neurons of mice with sepsis-associated encephalopathy (SAE).Methods:Twenty-four healthy male C57BL/6J mice, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (Sham group), SAE group, SAE plus electroacupuncture group (SAE+ EA group), and SAE plus sham electroacupuncture group (SAE+ SEA group). The virus carrying calcium ion (Ca 2+ ) fluorescent probes was injected and then an optical fiber was implanted into the hippocampal CA1 area to record the fluorescence signals of Ca 2+ . SAE was induced by cecal ligation and puncture in anesthetized mice at 3 weeks after administration. Starting from 3 days before surgery, Baihui and bilateral Quchi and bilateral Zusanli acupoints were stimulated for 30 min per day for 7 consecutive days in SAE+ EA group. In SAE+ SEA group, electroacupuncture was performed at the points 0.2 mm lateral to the corresponding acupoints without electrical stimulation. Open field tests were conducted at 5 days after surgery to record the number of rearing and changes in related Ca 2+ signals in hippocampal CA1 neurons. Novel object recognition tests were conducted at 6-7 days after surgery to record the recognition time and changes in related Ca 2+ signals in hippocampal CA1 neurons. Mice were sacrificed after the end of behavioral testing on 7 days after surgery, and brain tissues ipsilateral to the optical fiber implant were obtained and the fluorescence intensity of Ca 2+ in the hippocampal CA1 neurons was acquired using a fluorescent microscope. Results:Compared with Sham group, the number of rearing and amplitudes of related Ca 2+ signals in hippocampal CA1 neurons while rearing were significantly decreased in SAE group and SAE+ SEA group ( P<0.05), and no statistically significant changes were found in the parameters mentioned above in SAE+ EA group ( P>0.05), and the recognition index and amplitudes of related Ca 2+ signals while recognizing were significantly deceased, and the fluorescence intensity of Ca 2+ in hippocampal CA1 neurons was increased in SAE, SAE+ EA and SAE+ SEA groups ( P<0.05). Compared with SAE group and SAE+ SEA group, the number of rearing and amplitudes of related Ca 2+ signals in hippocampal CA1 neurons while rearing were significantly increased, the recognition index and amplitudes of related Ca 2+ signals in hippocampal CA1 neurons while recognizing were increased, and the fluorescence intensity of Ca 2+ in hippocampal CA1 neurons was decreased in SAE+ EA group ( P<0.05). There were no statistically significant differences in the parameters mentioned above between SAE group and SAE+ SEA group ( P>0.05). Conclusions:The mechanism by which electroacupuncture alleviates SAE may be related to regulation of Ca 2+ homeostasis in hippocampal neurons of mice.
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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 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 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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Objective:To evaluate the effect of electroacupuncture (EA) on lung injury caused by extremity ischemia-reperfusion.Methods:Forty-five American Society of Anesthesilogists physical status ⅠorⅡpatients, aged 20-60 yr, with body mass index of 18-28 kg/m 2, undergoing unilateral lower extremity operation requiring tourniquet with neuraxial anesthesia were divided into 3 groups ( n=15 each) using a random number table method: control group (C group), EA group and EA at non-acupoint group (group N). Bilateral acupoints Feshu and Zusanli were stimulated with disperse-dense waves, frequency 2/15 Hz, the current intensity the maximum current that patients could tolerant until the end of surgery in group EA.EA was performed at the points 1 cm lateral to the acupoints of Feshu and Zusanli in group N. Before anesthesia (T 1) and at 10, 30 and 60 min after tourniquet loosening (T 2-4), blood samples were collected from the radial artery for blood gas analysis, the partial pressure of arterial oxygen(PaO 2) and arterial carbon dioxide partial pressure (PaCO 2) were recorded, alveolar-arterial oxygen partial pressure difference (P A-aDO 2), oxygenation index (OI) and respiratory index (RI) were calculated, the malondialdehyde (MDA) content was measured by thiobarbituric acid method, the concentration of serum nitric oxide (NO) was determined by nitrate reductase method, and the concentrations of serum endothelin-1 (ET-1) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay. Results:Compared with the baseline at T 1, OI and RI were significantly decreased, P A-aO 2 was increased, and serum MDA, IL-6, ET-1 and NO levels were increased at T 2-4 in three groups ( P<0.05). Compared with group C, OI was significantly increased, P A-aO 2 and RI were decreased, serum MDA, IL-6, ET-1 and NO levels were decreased at T 2-4 in group EA ( P<0.05). Conclusion:EA can reduce lung injury caused by extremity ischemia-reperfusion, and the mechanism may be related to maintaining NO/ET-1 balance.
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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 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 α7 nicotinic acetylcholine receptor (α7nAChR) in electroacupuncture (EA)-induced reduction of acute lung injury (ALI) induced by endotoxin and the relationship with Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) signaling pathway in rats.Methods Sixty clean-grade healthy male Sprague-Dawley rats,aged 8 weeks,weighing 200-220 g,were divided into 6 groups (n=10 each) using a random number table method:control group (group C),endotoxin-induced ALI group (group ALI),EA at acupoints plus ALI group (group EA),specific α7nAChR antagonist α-bungarotoxin (α-BGT) plus ALI group (group BA),EA at acupoints plus α-BGT plus ALI group (group EBA),and EA at non-acupoints plus ALI group (group SEA).ALI was induced by intravenously injecting lipopolysaccharide 5 mg/kg.EA stimulation of bilateral Zusanli and Neiguan acupoints was performed (frequency of disperse-dense wave 2/15 Hz,wave length 0.2-0.5 ms,intensity 1-2 mA) once a day (time for stimulation 9:00 to 10:00,30 min per time) at 1-4 days before establishing the model in EA and EAB groups.EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Neiguan with the same parameters of stimulation in group SEA.o-BGT 1 μg/kg was intraperitoneally injected at 30 min before establishing the model in BA and EBA groups.Rats were sacrificed at 6 h after injecting lipopolysaccharide,and lungs were removed for microscopic examination of the pathological changes which were scored and for determination of wet to dry weight ratio (W/D ratio),contents of tumor necrosis factor-alpha (TNF-α),interleukin-lbeta (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay),and expression of α7nAChR,phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) (by Western blot).Results Compared with group C,the lung injury scores,W/D ratio and contents of TNF-α,IL-1β and IL-6 were significantly increased,and the expression of α7nAChR,p-JAK2 and p-STAT3 was up-regulated in the other five groups (P＜0.05).Compared with group ALI,the lung injury scores,W/D ratio and contents of TNF-α,IL-1β and IL-6 were significantly decreased,and the expression of α7nAChR,p-JAK2 and p-STAT3 was up-regulated in group EA,the lung injury scores,W/D ratio and contents of TNF-α,IL-1β and IL-6 were significantly increased,and the expression of o7nAChR,p-JAK2 and p-STAT3 was down-regulated in group BA (P＜0.05),and no significant change was found in the parameters mentioned above in group SEA (P＞0.05).Compared with group EA,the lung injury scores,W/D ratio and contents of TNF-α,IL-1β and IL-6 were significantly increased,and the expression of o7nAChR,p-JAK2 and p-STAT3 was down-regulated in group EBA (P＜0.05).Conclusion Up-regulated expression of α7nAChR activates JAK2/STAT3 signaling pathway,which is involved in EA-induced reduction of ALI in rats.
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Objective To evaluate the effect of electroacupuncture on endoplasmic reticulum stress in lung tissues of rats with acute lung injury (ALI) induced by endotoxin.Methods Forty healthy pathogen-free male Sprague-Dawley rats,aged 8 weeks,weighing 180-210 g,were divided into 4 groups (n=10 each) using a random number table:control group (group C),ALI group,electroacupuncture group (group E),and electroacupuncture at non-acupoint group (group NE).Lipopolysaccharide 5 mg/kg (in 0.5 ml normal saline) was injected intravenously to establish the model of endotoxin-induced ALI.Bilateral 30 min electroacupuncture stimulation of Zusanli and Neiguan acupoints was performed with the dispersedense wave (frequency 2/15 Hz,wave length 0.2-0.6 ms,intensity 1-2 mA) once a day (time for stimulation 9:30-10:30) for 4 consecutive days before and during establishment of the model in group E.Electroacupuncture was performed with the same parameters at the points 0.5 cm lateral to the acupoints of Zusanli and Neiguan in group NE.At 6 h after lipopolysaccharide injection,the rats were sacrificed,and lungs were removed for microscopic examination and for determination of wet to dry weight ratio (W/D ratio),apoptosis in alveolar epithelial cells and expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12 in lung tissues (by Western blot).The pathological changes of lungs were scored.Apoptosis index (AI) was calculated.Results Compared with group C,lung injury scores,W/D ratio and AI were significantly increased,and the expression of GRP78,CHOP and caspase-12 in lung tissues was up-regulated in the other three groups (P＜0.05).Compared with group ALI,lung injury scores,W/D ratio and AI were significantly decreased,and the expression of GRP78,CHOP and caspase-12 in lung tissues was down-regulated in group E (P＜0.05),and no significant change was found in the paramneters mentioned above in group NE (P＞0.05).Conclusion The mechanism by which electroacupuncture attenuates endotoxin-induced ALI is related to inhibition of endoplasmic reticulum stress and reduction of apoptosis in alveolar epithelial cells in rats.
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Objective To evaluate the role of p38MAPK signaling pathway in electroacupuncture (EA)-induced reduction of acute lung injury (ALI) in rabbits with endotoxic shock and the relationship with nuclear factor E2-related factor 2 (Nrf2).Methods Seventy healthy male New Zealand white rabbits,aged 2 months,weighing 1.5-2.5 kg,were randomly divided into 7 groups (n=10 each) using a random number table:control group (group C),endotoxin-induced ALI group (group A),p38MAPK inhibitor SB203580 group (group SB),ALI + SB203580 group (group A-SB),ALI + EA group (A-EA group),ALI + EA at non-acupoint group (A-NEA group) and ALI + EA at acupoints+ SB203580 group (A-EA-SB group).The rabbits were anesthetized with urethane and tracheostomized and kept spontaneous breathing.Right common carotid artery was cannulated for mean arterial pressure monitoring.The auricular vein was cannulated for drug administration.Bilateral 30 min EA (wave length 0.2-0.6 ms,frequency 2/100 Hz,intensity ≤ 1-2 mA) stimulation of Zusanli and Feishu was performed once a day for 4 days before establishment of the model and during establishment of the model in A-EA and A-EA-SB groups.In group A-NEA,EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Feishu according to the method previously described in group EA.In A,A-SB,A-EA,A-NEA and A-EA-SB groups,ALI was induced by endotoxin (5 mg/kg) injection,while the equal volume of normal saline was given in C and SB groups.After establishment of the model,SB203580 5 μmol/kg was injected intravenously in SB,A-SB and A-EA-SB groups,the equal volume of normal saline was given in group C,and the equal volume of dehydrated alcohol was given in the other groups.At 6 h after endotoxin or normal saline administration,arterial blood samples were collected for blood gas analysis.The rabbits were then sacrificed,and lungs were removed for microscopic examination and for determination of malondialdehyde (MDA) content,superoxide dismutase (SOD) activity,and expression of phosphor-p38MAPK (p-p38MAPK) and Nrf2 in lung tissues.The pathological changes of lungs were scored.Wet to dry lung weight ratio (W/D ratio) was calculated.Results Compared to group C,the pathological scores,W/D ratio,MDA content,and expression of pp38MAPK and Nrf2 were significantly increased,and SOD activities were decreased in A,A-SB,A-EA,ANEA and A-EA-SB groups.Compared to group A,the pathological scores,W/D ratio and MDA content were significantly decreased,and SOD activities and expression of p-p38MAPK and Nrf2 were increased in A-EA group.Compared to group A-EA,the pathological scores,W/D ratio and MDA content were significantly increased,and SOD activities and expression of p-p38MAPK and Nrf2 were significantly decreased in group A-EA-SB.Conclusion p38MAPK signaling pathway mediates EA-induced reduction of ALI in rabbits with endotoxic shock,and up-regulated expression of Nrf2 is involved in the mechanism.
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Objective To investigate the effect of electro-acupuncture (EA) on nuclear factor E2-related factor 2 (Nrf2) expression in the renal tissues of rabbits with endotoxic shock-induced acute kidney injury (AKI) and the relationship with p38 mitogen-activated protein kinase (p38MAPK) signaling pathway.Methods Seventy male New Zealand white rabbits,weighing 1.5-2.0 kg,aged 2 months,were randomized into 7 groups (n =10 each) using a random number table:normal control group (C group),endotoxic shock-induced AKI group (AKI group),EA + endotoxic shock-induced AKI group (EA group),non-acupoints + endotoxic shock-induced AKI group (SA group),EA + endotoxic shock-induced AKI + specific p38MAPK blocker SB203580 group (EAS group),SB203580 group (S group),and ethanol group (A group).EA (intensity 1-2 mA,frequency 2/100 Hz,wave length 0.2-0.6 ms) of Zusanli and Shenyu lasting for 15 min was performed once a day for 5 consecutive days in EA and EAS groups.In SA group,EA was performed at the points 0.5 cm lateral to the acupoints of bilateral Zusanli and Shenyu using the parameters of EA mentioned above.At 24 h after the last EA,endotoxic shock-induced AKI was induced by injection of lipopolysaccharide (LPS) 5 mg/kg (in 2 ml normal saline) in AKI,EA,SA and EAS groups,while the equal volume of normal saline was given in the other groups.At 30 min before the model was established,5/μmol/kg SB203580 (in 0.5 ml ethanol) was injected intravenously in EAS and S groups,while ethanol 0.5 ml was given in A group and the equal volume of normal saline was given in the other groups.Blood samples were obtained at 6 h after administration of LPS or normal saline for determination of serum urea nitrogen (BUN) and creatinine (Cr) concentrations.The animals were sacrificed and kidney specimens were obtained for microscopic examination of pathological changes which were scored and for measurement of Nrf2 protein expression and phosphorylation of p38MAPK (by Western blot) and Nrf2 mRNA expression (using fluorescent quantitative PCR).Results Compared with C group,the pathological score and serum BUN and Cr concentrations were significantly increased,and Nrf2 mRNA and protein expression was up-regulated in AKI,EA,SA and EAS groups,the phosphorylation of p38MAPK was increased in AKI,EA and SA groups,and no significant changes were found in the parameters mentioned above in S and A groups.Compared with AKI group,the pathological score and serum BUN and Cr concentrations were significantly decreased,and Nrf2 mRNA and protein expression was up-regulated in EA and EAS groups,the phosphorylation of p38MAPK was increased in EA group,the phosphorylation of p38MAPK was decreased in EAS group,and no significant changes were found in the parameters mentioned above in SA group.Compared with EA group,the pathological score and serum BUN and Cr concentrations were significantly increased,Nrf2 mRNA and protein expression was down-regulated,and the phosphorylation of p38MAPK was decreased in EAS group.Conclusion The mechanism by which EA mitigates endotoxic shock-induced AKI may be related to activation of p38MAPK signaling pathway and up-regulation of Nrf2 expression in renal tissues of rabbits.
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Objective To evaluate the role of activator protein-1 (AP-1 ) in electro-acupuncture (EA )-induced up-regulation of heme oxygenase-1 (HO-1 ) expression in lung tissues in a rabbit model of endotoxic shock .Methods Seventy healthy male New Zealand white rabbits ,aged 2 months ,weighing 1.5-2.0 kg ,were randomly divided into 7 groups ( n=10 each ) using a random number table :normal control group (group C ) , endotoxic shock-induced acute lung injury (ALI ) group (group ALI ) , EA + ALI group (group EL ) , non-acupoint+EA+ ALI group (group NEL ) , curcumin (HO-1 inhibitor ) group (group Cur ) , dimethyl sulfoxide group (group D ) ,and EA+ALI+curcumin group (group ELC ) .Bilateral 15 min EA stimulation of Zusanli and Feishu (according to atlas of animals acupoints ) was performed (frequency 15Hz ) once a day for 5 consecutive days before lipopolysaccharide (LPS ) administration in EL and ELC groups ,while in group NEL ,EA stimulation was performed at non-acupoints located 0.5 cm lateral to Zusanli and Feishu acupoints with the same parameters . The animals were anesthetized with urethane and tracheostomized .The animals kept spontaneous breathing .Right internal carotid artery was cannulated for blood pressure monitoring . Ear vein was cannulated for drug administration .At 5 days after EA stimulation ,curcumin 25 mg/kg (in 0.5 ml of 0.1% dimethyl sulfoxide ) was injected in Cur and ELC groups ,0.1% dimethyl sulfoxide 0.5 ml was injected in D group ,and the equal volume of normal saline was given in the other groups .LPS 5 mg/kg (in 2 ml of 0.9% normal saline ) was injected intravenously at 30 min after administration in ALI ,EL ,NEL and ELC groups ,while the equal volume of normal saline was given in the other three groups .Endotoxic shock was confirmed by decrease in mean arterial pressure to 75% of the baseline value within 2 h after LPS injection .Blood samples were collected from the common carotid artery at 6 h after LPS or normal saline administration and the rabbits were then sacrificed .The lungs were removed for microscopic examination and the pathological changes were scored .The wet/dry lung weight ratio (W/D ratio ) was calculated .The malondialdehyde (MDA ) content and superoxide dismutase (SOD ) activity in lung tissues were detected ,and the expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun in lung tissues was determined by Western blot .Results Compared with group C ,the pathological score ,W/D ratio and MDA content were significantly increased ,and the SOD activity was decreased in ALI ,EL ,NEL and ELC groups ,the expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun was up-regulated in groups ALI ,EL and NEL ( P0.05) .There was no significant difference in the parameters mentioned above between Cur and D groups ( P>0.05 ) .Compared with group ALI ,the pathological score ,W/D ratio and MDA content were significantly decreased ,and SOD activity was increased ,and the expression of HO-1 mRNA and HO-1 was up-regulated in EL and ELC groups ( P0.05) .The pathological score ,W/D ratio and MDA content were significantly higher ,and the SOD activity and expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun were lower in group ELC than in group EL ( P<0.05) .Conclusion EA up-regulates HO-1 expression through activating AP-1 during endotoxic shock-induced ALI in rabbits ,thus protecting the lung .
ABSTRACT
Heme oxygenase (HO)-1 has been reported to play a great role in attenuating lung injury during endotoxic shock in our previous research. Although electro-acupuncture has been explored to reduce oxidative stress and decrease inflammatory reaction in animals with endotoxic shock, the mechanism of this effect is still unclear. The aim of this study was to determine whether HO-1 is involved in the effect of electro-acupuncture on the injured lung during endotoxic shock in rabbits. Sixty New England white rabbits were randomly divided into groups C, Z, ES, EA, AP, and EAZ. Before inducing endotoxic shock, group ES received no electro-acupuncture, while group EA received electro-acupuncture at ST36 (zusanli) and BL13 (feishu) acupoints on both sides for five days and group AP received electro-acupuncture (EA) stimulation at a non-acupoint. Groups ES, AP, EA, and EAZ received LPS to replicate the experimental model of injured lung induced by endotoxic shock, and electro-acupuncture was performed throughout the procedure with the same parameter. Groups EAZ and Z received the HO-1 inhibitor, ZnPP-IX, intraperitoneally. The animals were sacrificed by blood-letting at 6 h after LPS administration. The blood samples were collected for serum examination, and the lungs were removed for pathology examination, detection of alveolaer epithelial cell apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL assay), determination of wet to dry ratio, measurement of Evans blue (EB) contents, and determination of HO-1protein and mRNA expression. According to the results, EA at ST36 and BL13 could increase the expression of HO-1. At the same time, index of quantitative assessment (IQA) score and the number of TUNEL-positive cells decreased, while electro-acupuncture at the other points did not exert this effect, and pretreatment with ZnPP-IX in group EAZ suppressed the efficacy of electro-acupuncture preconditioning. In summary, electro-acupuncture stimulation at ST36 and BL13, while not the non-acupoint, could attenuate the lung injury during the endotoxic shock, and this effect was due to increased expression of HO-1.
Subject(s)
Acupuncture Therapy , Acute Lung Injury/therapy , Endotoxins/pharmacology , Heme Oxygenase-1/metabolism , Shock, Septic/chemically induced , Acupuncture Therapy/methods , Acute Lung Injury/etiology , Animals , Disease Models, Animal , Electric Stimulation/methods , Heme Oxygenase-1/antagonists & inhibitors , Male , Malondialdehyde/blood , Oxidative Stress/drug effects , RabbitsABSTRACT
Objective To evaluate the effect of electro-acupuncture (EA) at Zusanli and Feishu on endotoxin shock-induced acute lung injury in rabbits.Methods Sixty healthy male New Zealand white rabbits aged 2 months weighing 1.5-2.0 kg were randomly divided into 6 groups (n =10 each):group sham operation (group S); group zinc protoporphyrin-Ⅸ (ZnPP-Ⅸ) (group Z); group lipopolysaccharide (LPS) (group L); group LPS + EA (group EL) ; group LPS + sham EA (group SEL) and group LPS + EA + ZnPP-Ⅸ (group ELZ).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 400 mg/kg and tracheostomized.The animals kept spontaneous breathing.Right internal carotid artery was cannulated for BP monitoring.Ear vein was cannulated for drug administration.LPS 5 mg/kg was injected iv in groups L,EL,SEL,ELZ.Endotoxin shock was confirmed by decrease in BP by 20 ％ of the baseline value and PaO2/FiO2 ≤ 300.ZnPP-Ⅸ (heme oxygenase (HO-1 ) inhibitor)10μmol/kg was injected intraperitoneal at 2 h after LPS injection in groups Z and ELZ.Bilateral 15 min EA stimulation of Zusanli and Feishu ( according to atlas of animal acu-points) was performed once a day for 5 days before LPS administration in groups EL and ELZ.The animals were sacrificed by blood-letting at 6 h after LPS administration.The lungs were removed for microscopic examination (0 =no injury,4 =most severe injury),detection of alveolar epithelial cell apoptosis (by TUNEL) and determination of HO-1 protein and mRNA expression.Results LPS significantly increased lung injury scores,alveolar epithelial cell apoptosis index (the number of apoptotic cells/total cells) and HO-1 protein and mRNA expression.EA significantly attenuated lung injury and alveolar epithelial cell apoptosis induced by LPS and further increased the expression of HO-1 protein and mRNA in group EL as compared with group L.The protective effects of EA was counteracted by ZnPP- Ⅸ in group ELZ.Conclusion EA at Zusanli and Feishu can attenuate endotoxin shock-induced lung injury by up-regulation of HO-1 expression and inhibiting alveolar epithelial cell apoptosis in the lung.