Objective To establish the mice model of immunological tolerance,and investigate the significance of haploidentical allogeneic lymphocytes infusion in induction of graft versus host disease and graft versus tumor in mice.Methods Sixty-four BALB/C female mice were randomly divided into 4 groups with 16 mice in each group.Control group:no special treatment was given after inoculation of tumor cells at the 4th day (CT26 colorectal cancer cell lines with mixture of 1 × 107/mL tumor cells suspension was inoculated to the right subcutaneous axillary of mice) ; Chemotherapy group:chemotherapy was applied at the 7th day after inoculation of tumor cells at the 4th day; DLI group:tumor cells were inoculated at the 4th day,and then haploid donor cells were infused at the 13th,15th and 17th day; Chemotherapy + DLI group:tumor cells were inoculated at the 4th day,chemotherapy was applied at the 7th day,and haploid donor cells were infused at the 13th,15th and 17th day.The pretreatment scheme included haploidentical allogeneic lymphocyte + ring ling amide + haploidentical allogeneic lymphocyte,and the chemotherapy regimen included peritoneal infusion of cyclophosphamide at the 3rd day after inoculation of tumor cells in mice.The time from the first day after vaccination to the day of death of mice and the mass of the tumors were detected to calculate the tumor inhibition rate.The clinical indexes of GVHD were observed,and clinical evaluation was made.The numbers of T lymphocytes in peripheral blood were detected by flow cytometry.Three mice were sacrificed in each group at the 15th day to make the tissue specimens,and they were observed under light microscope after HE staining.All data were analyzed using the analysis of variance or LSD-t test.Results The symptoms of GVHD of mice in the chemotherapy + DLI group were milder than those in other groups.The GVHD scores of the control group,chemotherapy group and the chemotherapy + DLI group were 2.3 ±0.6,1.5 ± 1.1,6.7 ±0.9 and 3.4 ±0.5,respectively,with significant difference between the 4 groups (F =148.68,P ＜ 0.05).The tumor masses of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were (3.40 ± 0.20) g,(0.80 ± 0.10) g,(2.20 ± 0.20) g and (0.50 ± 0.30) g,respectively,with significant difference between the 4 groups (F =149.17,P ＜ 0.05).The tumor inhibition rates of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 0,77％ ± 9％,35％ ± 3％,85％ ± 44％.The levels of CD3 + of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 52.3％ ± 2.9％,44.8％ ± 3.1％,62.9％ ± 3.5％,65.9％ ± 3.3％,respectively,with significant difference between the 4 groups (F =28.04,P ＜ 0.05).The levels of CD3 + CD4 + of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 32.1％ ± 2.6％,27.1％ ± 1.1％,42.6％ ± 1.8％ and 41.7％ ± 2.4％,respectively,with significant difference between the 4 groups (F =40.29,P ＜ 0.05).The levels of CD3 + CD8 + of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 22.7％ ± 2.2％,20.7％ ± 1.8％,26.7％ ± 0.8 ％ and 26.1％ ± 0.7％,respectively,with significant difference between the 4 groups (F =10.74,P ＜ 0.05).The levels of CD3 + CD4 + CD25 + of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 8.7％ ±0.6％,6.6％ ±0.6％,11.2％ ±0.4％ and 13.3％ ± 0.7％,respectively,with significant difference between the 4 groups (F =82.88,P ＜ 0.05).Necrosis and bleeding of the tumor tissues were observed in all the 4 groups.Necrosis,shrinking of the tumor cells,inflammatory infiltration were observed in the DLI group and the chemotherapy + DLI group.Proliferation of lymphoid follicles was observed in the chemotherapy + DLI group.The survival time of mice in the control group,chemotherapy group,DLI group,chemotherapy + DLI group were (16.8 ± 2.5) days,(26.3 ± 2.9) days,(23.4 ± 2.5) days and (33.7 ± 4.6) days,respectively,with significant difference between the 4 groups (F =46.45,P ＜ 0.05).Conclusions (1) Pretreatment can induce specific immune tolerance in mice.(2) Haploidentical allogeneic lymphocyte infusion and chemotherapy have synergistic effects,joint application of haploidentical allogeneic lymphocyte infusion and chemotherapy can inhibit the proliferation of tumor cells and prolong the survival time of mice.(3) Chemotherapy can reduce the GVHD of haploidentical allogeneic lymphocyte infusion and enhance the GVT.(4) CD3 + CD4 + CD25 + T lymphocytes play important roles in decreasing GVHD.

Objective To investigate the intraoperative intraperitoneal chemotherapy security with raltitrexed in advanced colorectal cancer surgical operation.Methods Sixty patients with colorectal cancer undergone surgery were randomly divided into trial group (n =30) and control group (n =30) according to the random number table method.The trial group was given surgical operation plus with intraperitoneal chemotherapy with raltitrexed.The control group was given surgical operation plus with intraperitoneal saline perfusion.Theroutine blood test,liver and kidney functions,toxic side effects and complications in two groups before and after surgery were investigated.Results The white blood cells in trial group before and after surgery was (6.36 ± 2.63) × 109/L vs.(8.20 ± 2.08) × 109/L,with statistically significant difference (t =3.06,P ＜0.05).The ratio of absolute neutrophil count in trial group before and after surgery was 65.17％ ± 10.36％ vs.72.21％ ± 10.53％ (t =3.22,P ＜ 0.05).The platelets in trial group before and after surgery was (261.03 ±84.74) × 109/L vs.(228.47 ± 58.69) × 109/L (t =2.07,P ＜ 0.05).The white blood cells,the ratio of absolute neutrophil count and the platelets after surgery had no statistically significant differeuces between the two groups (P ＞0.05).The trial group had higher 1,2 level vomiting (60.00％ vs.23.33％;x2 =8.30,P ＜ 0.05),and nausea (30.00％ vs.6.67％;x2 =5.46,P ＜ 0.05) incidence rates,but there was no statistically significant difference in other toxic side effects (P ＞ 0.05).The major complications post operation included intestinal obstruction,incision infection,abdominal cavity bleeding,and anastomotic fistula.There were equivalent complications in two groups (6.67％ vs.3.33％,x2 =0.35,P ＞0.05;10.00％ vs.6.67％,x2 =0.22,P＞0.05;0 vs.0;3.33％ vs.0,P＞0.05).Conclusion For patients with advanced colorectal cancer,intraoperative intraperitoneal chemotherapy with raltitrexed is safe and feasible,and the adverse reactions can be tolerated without increasing postoperative complications.

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 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 phosphatidylinositol 3-kinase/serine-threonine kinase(PI3K/Akt) signaling pathway in mitochondrial fission in endotoxin-challenged alveolar type Ⅱ epithelial cells of rats.Methods Rat alveolar type Ⅱ epithelial cells CCL-149 were seeded in 6-well plates at a density of 2×105 cells/ml.CCL-149 cells were divided into 6 groups (n =10 each) using a random number table:control group (group C),lipopolysaccharide (LPS) group (group L),LPS plus CO-releasing molecule-2 (CORM-2) group (group L+CO),LPS plus PI3K inhibitor LY294002 group (group L+LY),LPS plus iCORM-2 group (group L+iCO) and LPS plus dimethyl sulfoxide (DMSO) group (group L+D).CCL149 cells were stimulated with 10 μg/ml LPS for 24 h in L,L+CO,L+LY,L+iCO and L+D groups.CORM-2 100 μmol,LY294002 25 μg and iCORM-2 100 μmol were added at 1 h before stimulation with LPS in L+CO,L+LY and LPS+iC0 groups,respectively.In group L+D,0.1％ DMSO 100 μmol was added at 1 h before stimulation with LPS.After the end of incubation,the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture medium were determined by enzyme-linked immunosorbent assay,and the expression of phosphorylated-Akt (p-Akt),heme oxygenase-1 (HO-1),dynamin-related protein 1 (Drpl) and fissionl (Fisl) was detected by Western blot.Results Compared with group C,IL-6 and TNF-α concentrations in the culture medium were significantly increased,and the expression ofp-Akt,HO-1,Drp1and FIS1 was up-regulated in L,L+CO,L+LY,L+iCO and L+D groups (P＜0.05).Compared with group L,IL-6 and TNF-α concentrations in the culture medium were significantly decreased,the expression of p-Akt and HO-1 was up-regulated,and the expression of Drp1 and Fis1 was down-regulated in group L+CO,and IL-6 and TNF-α concentrations in the culture medium were significantly increased,the expression of p-Akt and HO-l was down-regulated,and the expression of Drpl and Fisl was up-regulated in group L+LY (P＜0.05).Conclusion Activation of PI3K/Akt signaling pathway can inhibit mitochondrial fission in endotoxin-challenged alveolar type Ⅱ epithelial cells of rats.