Objective To evaluate effects of carbon monoxide (CO) on lipopolysaccharide (LPS) induced damage and possible mechanism in rat alveolar macrophages. Methods Rat alveolar macrophages were cultured in DMEM containing 10%fetal bovine serum with 5%CO2 at 37℃in Heraeus sepatech. The cells were divided into four groups using random number table (n=10): control group (group C),CO group, LPS group and LPS+CO group. The CO release molecule-2 (CORM-2) 100 μmol/L was added into CO group,LPS 10 mg/L was added into LPS group, cells were pretreated with CORM-2 100μmol/L for 1 h then LPS 10 mg/L was added into LPS+CO group, the same amount of PBS was added to group C. Proliferation was measured by MTT assay. Apoptosis and mitochondrial membrane potential were detected with flow cytometer. The content of ATP was tested by ATP content kit. Drp1 mRNA was measured by RT-PCR, and Drp1 expression was determined by Western blot assay. Results Compared with group C, the cell vitality, content of ATP and mitochondrial membrane potential were decreased in LPS group and LPS+CO group,and cell apoptosis rate, Drp1 mRNA and protein expression were increased (P<0.05). There were no significant changes were found in CO group. Compared with LPS group, the cell vitality, content of ATP and mitochondrial membrane potential were increased in LPS+CO group,and the cell apoptosis rate, Drp1 mRNA and protein expression were decreased (P<0.05). Conclusion Carbon monoxide can alleviate LPS-induced damage in rat alveolar macrophages, which is related with down-regulation of Drp1 and amelioration of mitochondrial function.

Objective To study the clinical course and clinicopathological features of para-aortic lymph node metastases in patients with gallbladder cancer.Methods Forty-two patients with gallbladder cancer who underwent radical resection combined with para-aortic lymphadenectomy at the Mianyang Hospital of Traditional Chinese Medicine from January 2001 to December 2013 were retrospectively studied.The survival rates of the para-aortic lymph node metastasis group were compared with the negative para-aortic lymph node group of patients.Para-aortic lymph node metastasis as well as clinical features were correlated with survival.Results No one died within the perioperative period.The total complication rate was 24.0％,and there was no significant difference between the positive para-aortic lymph node group and the negative group (P ＞0.05).The rate of para-aortic lymph node metastasis on histopathology was 21.4％ (9/42),which was positively correlated with tumor depth of invasion and negatively correlated with the degree of differentiation (P ＜ 0.01).The 1-,2-,and 3-year survival rates of the positive para-aortic lymph node group were significantly inferior to the negative group (P ＜ 0.05).Conclusions Dissection of para-aortic lymph nodes in patients with gallbladder cancer was safe and feasible.Lymphadenectomy did not improve the longterm survival rates of patients with para-aortic lymph node involvement metastases.The extent of lymph node dissection for gallbladder cancer should be decided by intraoperative biopsy.

Objective To investigate the protective effects of recombinant human erythropoietin (rHuEPO) on caecal ligation and puncture (CLP)-induced acute kidney injury (AKI).Methods A total of 260 healthy male Sprague-Dawley rats (250-300 g) were randomly divided into 6 groups:normal control group,sham group,CLP model group,the large dose rHuEPO (5000 U/kg)group,the middle dose rHuEPO (1000 U/kg) group,and the small dose rHuEPO (500 U/kg) group.The rat models of sepsis were established by CLP.In treatment groups,rats were treated with rHuEPO through caudalis injection after CLP surgery.Each group was divided into 2-,6-,12-,24-,36-hour subgroups with 10 rats.Rats were sacrificed and the tissue samples including kidney and blood samples were collected.The kidney function,plasma cytokines [interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α)],kidney injury moleclue 1 (KIM-1) and inducible nitric oxide synthase (iNOS)were measured.Cytokines were determined by ELISA method.The expression of nuclear factor-kappaB (NF-κB) protein in kidneys were detected by immnunohistochemistry method.Pathological changes of kidney tissues were observed by light and transmission electron microscopy for cytokine content and apoptosis.Results Compared with CLP model group,renal function,the levels of TNF-α,IL-6,KIM-1 and iNOS in serum,the expression of NF-κB,significantly decresed in large dose rHuEPO group (all P ＜ 0.05).rHuEPO also lessened the histological changes in large dose group.rHuEPO did not lessen the histological changes in others.Conclusion rHuEPO can inhibit the levels of TNF-α,IL-6 and iNOS in serum,thus modify the inflammatory response and provide protective effects against acute kidney injury induced by sepsis.

Objective To investigate the effects of CORM-2 via p38 mitogeu-activated protein kinase (p38MAPK) signaling pathway on the expression of the mitochondrial fission protein 1 (Fisl) in lipopolysaccharide (LPS)-induced mouse pulmonary macrophages.Methods The rat subculture alveolar macrophages were seeded on 96 well plates with 2 × 105/ml densities.After 24 hours of culture,it was divided into 4 groups by random number table method:normal control group (group C),group LPS (group L),CO releasing agent CORM-2 + LPS group (group LC),p38MAPK inhibitor SB203580 + CORM-2 + LPS group (group LCS).When the cells were incubated for 24 hours,the mitochondrial MDA content and SOD activity were determined by ELISA kit,the levels of HO-1、mitochondrial fission protein Fis1 and p38 were determined by Western blot,the expressions of HO-1 and mitochondrial fission protein Fis1 were detected by RT-PCR.Results Compared with the C group,the levels of MDA [(2.43 ±0.12) vs.(3.59 ±0.07)],HO-1 [(1.31±0.27) vs.(1.65±0.41)],Fis1 [(1.27±0.23) vs.(1.65±0.41)] andp38 [(1.01 ±0.24) vs.(1.36 ±0.17)] in group L were increased,and the activity of SOD [(81.7 ± 1.62) vs.(54.7 ± 1.62)] was decreased (P ＜ 0.05);Compared with the group L,the MDA content [(3.59 ± 0.07) vs.(3.08 ±0.52)] and the level of Fis1 [(2.01 ±0.35) vs.(1.48 ±0.39)] in group LC were down-regulated,and the levels of SOD [(54.7 ± 1.62) vs.(67.4 ± 1.32)]、and the expressions of HO-1 [(1.65±0.41)vs.(2.25±0.18)] andp38 [(1.36±0.17) vs.(1.78±0.23)] wereup-regulated (P ＜0.05).Compared with the group LC,the MDA content [(3.08 ±0.52) vs.(4.16 ±0.19)] and the expression of Fis1 [(1.48 ±0.39) vs.(1.96 ±0.31)] in group LCS were increased,and the level of SOD [(67.4±1.32)vs.(45.9±1.52)]、and the expressions of HO-1 [(2.25±0.18)vs.(1.78± 0.19)] and p38 [(1.78 ±0.23) vs.(1.12 ±0.29)] were decreased (P ＜0.05).Conclusions HO-1/CO system inhibits the expression of Fis1 in LPS-induced lung macrophages,which may be regulated by p38MAPK signaling pathway.

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 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 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.

Objective To evaluate the role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor E2-related factor 2 (Nrf2) signaling pathway in endotoxic shock-induced acute lung injury (ALI) in rabbits.Methods Thirty healthy male New Zealand white rabbits, aged 2 months, weighing 1.5-2.0 kg, were randomly divided into 3 groups (n =10 each) using a random number table: control group (group C), ALI group, and wortmannin group (group W).In group W, wortmannin 0.6 mg/kg (in 0.08 ml/kg dimethyl sulfoxide) was injected via the auricular vein, while the equal volume of normal saline was given in C and ALI groups.And 30 min later, lipopolysaccharide (LPS) 5 mg/kg (in 2 ml of normal saline) was injected via the auricular vein in ALI and W groups, while the equal volume of normal saline was given in group C.The rabbits were sacrificed at 6 h after LPS or normal saline injection.The lung was immediately removed for microscopic examination and for determination of wet/dry lung weight ratio (W/D ratio), expression of phosphorylated Akt (p-Akt) , Nrf2 and heme oxygenase-1 (HO-1) (by Western blot), and expression of Nrf2 and HO-1 mRNA (using fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction).The pathological changes of the lung were scored.Results Compared with group C, the lung injury scores and W/D ratio were significantly increased, and the expression of p-Akt, Nrf2 protein and mRNA, and HO-1 protein and mRNA was up-regulated in ALI and W groups (P＜0.05).Compared with group ALI, the lung injury scores and W/D ratio were significantly increased,and the expression of p-Akt, Nrf2 protein and mRNA, and HO-1 protein and mRNA was up-regulated in group W (P＜0.05).Conclusion Activation of PI3K/Akt/Nrf2 pathway is the regulatory mechanism of the body adapting to the development of endotoxic shock-induced ALI in rabbits.