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.

Objective To evaluate the role of protein kinase Ca (PKCα) in electroacupuncture (EA)-induced reduction of acute kidney injury (AKI) induced by endotoxic shock,and the relationship with nuclear factor E2-related factor 2/heme oxygenase-1 Nrf2/HO-1 pathway in rabbits.Methods Eighty heahhy male New Zealand white rabbits,aged 2 months,weighing 1.5-2.0 kg,were randomly divided into 8 groups (n=10 each) using a random number table:sham operation group (group S),group AKI,specific PKCα inhibitor chelerythrine + AKI group (group CHA),chelerythrine group (group Che),dimethyl sulfoxide (DMSO) group (group D),EA at acupoints + AKI group (group EA),EA at non-acupoints + AKI group (group SEA),and EA at acupoints + chelerythrine + AKI group (group CEA).Bilateral 30 min EA (disperse-dense wave,wave length 0.2-0.6 ms,frequency 2/15 Hz,intensity 1-2 mA) stimulation of Zusanli and Shenshu acupoints was performed once a day for 4 days before establishment of the model and during the process of establishment of the model in EA and CEA groups.In group SEA,EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Shenshu with the same parameters.The animals were anesthetized with iv 20％ urethane 5 ml/kg,tracheostomized and kept spontaneous breathing.Lipopolysaccharide 5 mg/kg (in 2 ml of normal saline) was injected via the auricular vein to establish the model of endotoxic shock-induced AKI in AKI,CHA,EA,SEA and CEA groups,while the equal volume of normal saline was given in S,Che and D groups.At 30 min before establishment of the model,chelerythrine 5 mg/kg (in 0.5 ml of 1％ DMSO) was injected intravenously in CHA and CEA groups,the equal volume of chelerythrine was given in Che group,while the equal volume of DMSO was given in group D.At 6 h after lipopolysaccharide or normal saline injection,blood samples were taken from the internal carotid artery for determination of serum urea nitrogen (BUN) and creatinine (Cr) concentrations.The rabbits were then sacrificed by exsanguinations.The kidney specimens were removed for microscopic examination of pathologic changes which were scored and for determination of superoxide dismutase (SOD) activities,malondialdehyde (MDA) contents,and expression of PKCα protein and HO-1 protein,and expression of Nrf2 in nucleoprotein and total protein.Results Compared with group S,the serum BUN and Cr concentrations were significantly increased,MDA contents were increased,the activities of SOD were decreased,the kidney injury scores were increased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was up-regulated in AKI,CHA,EA,SEA and CEA groups.Compared with group AKI,the serum BUN and Cr concentrations were significantly decreased,MDA contents were decreased,the activities of SOD were increased,the kidney injury scores were decreased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was up-regulated in group EA,and the serum BUN and Cr concentrations were significantly increased,MDA contents were increased,the activities of SOD were decreased,the kidney injury scores were increased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was down-regulated in CHA and CEA groups.The serum BUN and Cr concentrations were significantly higher,MDA contents were higher,the activities of SOD were lower,the kidney injury scores were higher,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was lower in group CEA than in group EA,and in CHA group than in CEA group.Conclusion PKCα mediates reduction of endotoxic shock-induced AKI by EA of Zusanli and Shenshu acupoints in rabbits,and the mechanism may be related to activation of Nrf2/HO-1.

Objective To evaluate the effect of lipopolysaccharide (LPS) on the viability of rat alveolar macrophages.Methods The rat alveolar macrophages were seeded in 96-well plate at a density of 4× 104/ml.After being cultured for 24 h, the cells were randomly divided into 6 groups (n =5 each) using a random number table : control group (group C), LPS 0.1 μg/ml group (group LPS0.1), LPS 1.0 μg/ml group (group LPS1.0), LPS 10.0 μg/ml group (group LPS10), LPS 5.0 μg/ml group (group LPS50), and LPS 100.0 μg/ml group (group LPS100).Phosphate buffer solution was added to the culture medium in group C, and LPS with the final concentrations of 0.1, 1.0, 10, 50.0 and 100.0 μg/ml were added to the culture medium in LPS0.1, LPS1.0, LPS10, LPS50, and LPS100 groups, respectively.At 6, 12, 24 and 48 h after addition of PBS or LPS, the cell viability was measured by methyl thiazolyl tetrazolium assay.Results Compared with group C, the viability of alveolar macrophages was significantly increased at 6 and 12 h after addition of LPS in the other five groups , and was decreased at 24 and 48 h after addition of LPS in groups LPS50and LPS100 (P＜0.05), and no significant change was found in LPS0.1, LPSL0 and LPS10 groups (P＞0.05).Conclusion Incubation with LPS 0.1-100.0 μg/ml for less than 12 h can enhance the viability of rat alveolar macrophages;incubation with LPS with the concentration ≥ 50.0 μg/ml for more than 24 h can decrease the cell viability.

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.

Objective To evaluate the role of phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/Akt) signaling pathway in carbon monoxide (CO)-induced up-regulation of the mitofusin-1 (Mfn1) expression in endotoxin-challenged rat alveolar macrophages.Methods Alveolar macrophages obtained from the rats aged 12-20 weeks were subcuhured and seeded in 96 well plates at a density of 4× 104 cells/ml.After being cultured for 24 h,the cells were divided into 4 groups (n=10 each) using a random number table:control group (group C),endotoxin group (group L),lipopolysaccharide (LPS) +CO-releasing molecule-2 (CORM-2) group (group L+C) and LPS+CORM-2+PI3K inhibitor LY294002 group (group L+C+LY).Cells were cultured normally in group C.Cells were stimulated by using LPS 10 μg/ml in L,L+C and L+C+LY groups.In group L+C,CORM-2 100 μmol was given at 1 h before stimulation with LPS.In group L+C+LY,LY294002 20 μg and CORM-2 100 μ mol were given at 1.5 and 1.0 h before stimulation with LPS,respectively.The cells were continuously incubated for 24 h after the end of treatment.The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in the supernatant were determined by enzyme-linked immunosorbent assay.The expression of PI3K,phosphorylated Akt (p-Akt) and Mfn1 in cells was measured by real-time polymerase chain reaction and Western blot.Results Compared with group C,the concentration of TNF-α was significantly increased,and the IL-10 concentration was decreased in L,L+C and L+C+LY groups (P＜0.05).Compared with group L,the concentration of IL-10 was significantly increased,the TNF-α concentration was decreased,and the expression of PI3K,p-Akt and Mfn1 was up-regulated in group L+C (P＜0.05).Compared with group L+C,the concentration of IL-10 was significantly decreased,the TNF-α concentration was increased,and the expression of PI3K,p-Akt and Mfn1 was down-regulated in group L+C+LY (P＜0.05).Conclusion PI3K/Akt signaling pathway is involved in CO-induced up-regulation of Mfn1 expression in endotoxin-challenged rat alveolar macrophages.

Objective To investigate the effects of heme oxygenase-1/carbon monoxide (HO-1/CO) pathway on mitochondrial fusion in rat alveolar epithelial type Ⅱ cells (AECⅡ) stimulated by lipopolysaccharide (LPS). Methods Once the cultured in vitro rat AECⅡcells line RLE-6TN reached confluency of 85%, they were subcultured and randomly divided into seven groups (n = 5 each). RLE-6TN cells were routinely cultured in control group. The cells in LPS group was stimulated with 10 mg/L LPS to reproduce the model of endotoxin challenge in AECⅡ cells. The cells in carbon monoxide-releasing molecule-2 (CORM-2, in vitro CO release agent) + LPS group (CL group) and Hemin (HO-1 inducer) + LPS group (HL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin for 1 hour, respectively, followed by 10 mg/L LPS stimulation. The cells in zinc protoporphyrin-Ⅸ (ZnPP-Ⅸ, HO-1 inhibitor) + LPS group (ZL group) was pretreated with 10 μmol/L ZnPP-Ⅸ for 0.5 hour followed by 10 mg/L LPS stimulation. The cells in CORM-2 + ZnPP-Ⅸ + LPS group (CZL group) and Hemin + ZnPP-Ⅸ + LPS group (HZL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin respectively for 1 hour, and other treatments were similar to those previously described in ZL group. At 24 hours after LPS stimulation, interleukin-6 (IL-6)and tumor necrosis factor-α (TNF-α) in the supernatant were determined by enzyme linked immunosorbent assay (ELISA), the protein expressions of HO-1, mitochondrial fusion related proteins 1 and 2 (Mfn1, Mfn2) and optic atrophy 1 (OPA1) were determined by Western Blot. Results Compared with control group, IL-6 and TNF-α contents in the supernatant were increased, HO-1 protein expression was up-regulated, Mfn1, Mfn2 and OPA1 protein expressions were down-regulated in all treatment groups. Compared with LPS group, IL-6 and TNF-α contents were significantly decreased after CORM-2 or Hemin pretreatment [IL-6 (ng/L): 48.6±3.7, 48.4±3.1 vs. 58.7±2.5; TNF-α (ng/L):40.7±5.3, 39.4±4.3 vs. 51.8±5.1], the protein expressions of HO-1, Mfn1, Mfn2 and OPA1 were significantly up-regulated (HO-1 protein: 0.873±0.051, 0.839±0.061 vs. 0.671±0.044; Mfn1 protein: 0.673±0.037, 0.654±0.025 vs. 0.568±0.021; Mfn2 protein: 0.676±0.044, 0.683±0.035 vs. 0.571±0.043; OPA1 protein: 0.648±0.031, 0.632±0.031 vs. 0.554±0.032; all P < 0.05); while opposite effects were found after ZnPP-Ⅸ preincubation, and there were significant differences in IL-6 and TNF-α contents and protein expressions of HO-1, Mfn1, Mfn2 and OPA1 as compared with those of LPS group [IL-6 (ng/L): 69.8±5.1 vs. 58.7±2.5, TNF-α (ng/L): 61.9±3.3 vs. 51.8±5.1, HO-1 protein: 0.545±0.023 vs. 0.671±0.044, Mfn1 protein: 0.406±0.051 vs. 0.568±0.021, Mfn2 protein:0.393±0.051 vs. 0.571±0.043, OPA1 protein: 0.372±0.050 vs. 0.554±0.032; all P < 0.05]. There were no significant differences in the parameters mentioned above between HL group and CL group, as well as among LPS, CZL and HZL groups. Conclusion HO-1/CO pathway promotes mitochondrial fusion and alleviates inflammatory response in LPS-induced rat AECⅡ cells.

Objective To evaluate the effects of electroacupuncture (EA) on postoperative acute lung injury (ALI) in patients with acute abdomen complicated with abdominal infection.Methods Ninety patients of both sexes with acute abdomen complicated with abdominal infection,with body mass index of 18-30 kg/m2,aged 35-64 yr,of American Society of Anesthesiologists physical status Ⅰ or Ⅱ,scheduled for elective abdominal surgery with general anesthesia,were divided into 3 groups (n =30 each) using a random number table method:control group (group C),EA at acupoint group (group E) and EA at non-acupoint group (group N).Anesthesia was induced with midazolam,propofol,cis-atracurium and sufentanil.The patients were mechanically ventilated after tracheal intubation.Anesthesia was maintained with inhalation of sevoflurane,Ⅳ infusion of propofol and cis-atracurium and intermittent Ⅳ boluses of sufentanil.At 15 min of anesthesia induction,2 h after skin incision and 2,12 and 24 h after tracheal intubation,Hegu,Zusanli and Neiguan acupoints were stimulated for 15 min every time in group E,and the points 1 cm lateral to the acupoints of Hegu,Zusanli and Neiguan were stimulated for 15 min every time in group N,with disperse-dense waves,frequency 2/15 Hz,wave length 0.2-0.6 ms,at a voltage of 1-2 mA.Arterial blood samples were collected at 15 min before induction (T1) and 24 and 48 h after operation (T2 and T3) for blood gas analysis,oxygenation index was calculated,and the development of ALI (oxygenation index ＜ 300 mmHg) was recorded at T2 and T3.The concentrations of club cell protein 16 (CC16),surfactant protein D (SP-D),soluble receptor for advanced glycation end products (sRAGE),interleukin-1 (IL-1),IL-10,and tumor necrosis factor-alpha (TNF-α) in serum were detected by enzyme-linked immunosorbent assay.Results Compared with the baseline at T1,the serum concentrations of CC16,SP-D,sRAGE,IL-1 and TNF-α were significantly increased,and the serum IL-10 concentrations were decreased at T2 in C,E and N groups (P＜0.05).Compared with group C,the serum concentrations of CC16,SP-D,sRAGE,IL-1 and TNF-α were significantly decreased,and the serum IL-10 concentrations were increased at T2 and T3,and AI was increased at T3 in group E (P＜0.05).There was no significant difference in the incidence of ALI at T2 and T3 among three groups (P＞0.05).Conclusion Although EA does not decrease the occurrence of ALI,it mitigates the degree of ALI to some extent in the patients with acute abdomen complicated with abdominal infection.

Objective To evaluate the relationship between p38 mitogen-activated protein kinase (p38MAPK) signaling pathway and lipopolysaccharide (LPS)-induced mitochondrial fission in alveolar epithelial cells using an in vitro experiment.Methods The cultured alveolar epithelial cells were subcultured and seeded in 96-well plates at the density of 2 × 105 cells/ml (200 μl/well).The cells were divided into 4 groups (n=10 each) when cell confluence reached 80％ using a random number table method:control group (group C),LPS group,LPS+SB203580 group (group LPS+SB) and LPS+dimethyl sulfoxide (DMSO) group.Cells were incubated with LPS 10 μg/ml for 24 h in group LPS.Cells were incubated with p38MAPK inhibitor SB203580 10 μmol (dissolved in DMSO) for 1 h and then with LPS 10 μg/ml for 24 h in group LPS+SB.Cells were incubated with the equal volume of DMSO for 1 h and then with LPS 10 μg/ml for 24 h in group LPS+DMSO.Malonaldehyde (MDA) content and superoxide dismutase (SOD) activity were measured.The expression of phosphorylated p38MAPK (p-p38MAPK),heme oxygenase-1 (HO-1),dynamin-related protein 1 (DRP1) and fission protein 1 (FIS1) was determined 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,HO-1,FIS1 and DRP1 was up-regulated in LPS,LPS+SB and LPS+ DMSO groups (P＜0.05).Compared with group LPS,the MDA content was significantly increased,the SOD activity was decreased,the expression of p-p38MAPK and HO-1 was down-regulated,the expression of FIS1 and DRP1 was up-regulated in group LPS+SB (P＜0.05),and no significant change was found in the parameters mentioned above in group LPS+DMSO (P＞0.05).Conclusion The p38MAPK signaling pathway activation can up-regulate the expression of HO-1,thus reducing LPS-induced mitochondrial fission in alveolar epithelial cells.

Objective To evaluate the role of protein kinase Cα (PKCoα)/heme oxygenase-1 (HO-1) signaling pathway in lipopolysaccharide (LPS)-caused damage to type Ⅱ alveolar epithelial cells of rats and the relationship with mitochondrial fusion.Methods Type Ⅱ alveolar epithelial cells were seeded in 96-well plates at a density of 2× 105 cells/ml and divided into 5 groups (n =40 each) using a random number table method:control group (group C),Go6976 group (group G),LPS group (group L),LPS plus PKCα inhibitor Go6976 group (group LG) and LPS plus dimethyl sulfoxide (DMSO) group (group LD).Group LG and group LD were pretreated with 5 μmol/L Go6976 and the equal volume of 0.1％ DMSO,respectively,for 30 min,lipopolysaccharide (LPS) 10 μg/ml was then given to establish the model of type Ⅱ alveolar epithelial cell damage in L,LG and LD groups,Go6976 5 μmol/L was added in group G,and the equal volume of phosphate buffer solution was added in group C.The cells were collected after 24 h of incubation for measurement of malondialdehyde (MDA) and reactive oxygen species (ROS) contents,superoxide dismutase (SOD) activity and expression of PKCα,HO-1,mitochondrial fusion-related proteins 1 and 2 (Mfn1,Mfn2),optic atrophy 1 (OPA1) protein and mRNA (by fluorescent quantitative polymerase chain reaction or Western blot).Results Compared with group C,MDA and ROS contents were significantly increased,the SOD activity was decreased,the expression of PKCα and HO-1 protein and mRNA was up-regulated,and the expression of Mfn1,Mfn2 and OPA1 protein and mRNA was downregulated in L,LG and LD groups (P＜0.05),and no significant change was found in the parameters mentioned above in group G (P＞0.05).Compared with group L,MDA and ROS contents were significantly increased,the SOD activity was decreased,and the expression of PKCα,HO-1,Mfn1,Mfn2 and OPA1 protein and mRNA was down-regulated in group LG (P＜0.05),and no significant change was found in the parameters mentioned above in group LD (P＞0.05).Conclusion Activation of PKCα/HO-1 signaling pathway is the endogenous protective mechanism of LPS-caused damage to type Ⅱ alveolar epithelial cells,which may be related to promoting mitochondrial fusion in rats.

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.