1.Role of p38MAPK signaling pathway in electroacupuncture-induced reduction of ALI in rabbits with endotoxic shock: the relationship with Nrf2
Xuesong GAO ; Lirong GONG ; Jianbo YU ; Jia SHI ; Shu'an DONG ; Lili WU ; Yuan ZHANG
Chinese Journal of Anesthesiology 2015;35(4):481-485
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.
2.Role of PKCα in electroacupuncture-induced reduction of acute kidney injury induced by endotoxic shock in rabbits: the relationship with Nrf2/HO-1 pathway
Xinshun CAO ; Jia SHI ; Jianbo YU ; Yuan ZHANG ; Shu'an DONG ; Lili WU ;
Chinese Journal of Anesthesiology 2015;35(6):727-731
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.
3.Effect of lipopolysaccharide on viability of rat alveolar macrophages
Wei LIU ; Dan WANG ; Jianbo YU ; Lirong GONG ; Yuan ZHANG ; Shu'an DONG ; Qiang FU
Chinese Journal of Anesthesiology 2015;35(10):1261-1263
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.
4.Effect of electroacupuncture on endoplasmic reticulum stress in lung tissues of rats with acute lung injury induced by endotoxin
Changkun LI ; Jia SHI ; Lirong GONG ; Shu'an DONG ; Yuan ZHANG ; Dan WANG ; Jianbo YU
Chinese Journal of Anesthesiology 2016;36(10):1266-1269
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.
5.Role of PI3K/Akt signaling pathway in carbon monoxide-induced up-regulation of mitofusin-1 expression in endotoxin-challenged rat alveolar macrophages
Zhen LI ; Jia SHI ; Jianbo YU ; Dan WANG ; Shu'an DONG ; Lirong GONG ; Yuan ZHANG
Chinese Journal of Anesthesiology 2017;37(1):112-115
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.
6.Effects of heme oxygenase-1/carbon monoxide pathway on the mitochondrial fusion in rat alveolar epithelial type Ⅱ cells stimulated by lipopolysaccharide
Haojuan JIA ; Jia SHI ; Shu'an DONG ; Yuan ZHANG ; Jianbo YU
Chinese Critical Care Medicine 2018;30(3):209-213
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.
7.Effects of electroacupuncture on postoperative acute lung injury in patients with acute abdomen complicated with abdominal infection
Lina WU ; Shu'an DONG ; Junjian LIU ; Jia SHI ; Lili WU ; Jianbo YU
Chinese Journal of Anesthesiology 2019;39(2):235-238
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.
8.Role of PI3K/Akt signaling pathway in mitochondrial fission in endotoxin-challenged alveolar type Ⅱ epithelial cells of rats
Hanbing CAO ; Jia SHI ; Lirong GONG ; Yuan ZHANG ; Shu'an DONG ; Jianbo YU
Chinese Journal of Anesthesiology 2018;38(5):618-621
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.
9.Role of PI3K/Akt signaling pathway in carbon monoxide-induced up-regulation of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells of rats
Manying HUANG ; Jia SHI ; Yuan ZHANG ; Lili WU ; Lirong GONG ; Shu'an DONG ; Jianbo YU
Chinese Journal of Anesthesiology 2018;38(6):728-731
Objective To evaluate the role of 1-phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/Akt) signaling pathway in carbon monoxide (CO)-induced up-regulation of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells of rats.Methods The rat alveolar epithelial cells were cultured in a 5% CO2 cell culture incubator at 37 ℃ with F12K complete medium containing 10% fetal bovine serum and 1% green chain double antibody,and divided into 10 groups (n=5 each) according to the random number table method:control group (C group),endotoxin group (L group),lipopolysaccharide (LPS) plus exogenous CO-releasing agent CO-releasing-molecule-2 (CORM-2) group (L+CO group),LPS plus PI3K inhibitor LY294002 group (L+LY group),LPS plus CORM-2 plus LY294002 group (L+ CO+LY group),LPS plus inactive CO releasing agent iCORM-2 group (L+iCO group),LPS plus dimethyl sulfoxide (DMSO) group (L+D group),CORM-2 group (CO group),LY294002 group (LY group) and CORM-2 plus LY294002 group (CO+LY group).LPS 10 μg/ml was added to the culture medium in group L.CORM-2 100 μmol/L was added to the culture medium and 1 h later 10 μg/ml LPS was added in L+CO group.LY294002 25 μmol/L was added to the medium,and 1 h later LPS 10 μg/ml was added in L+LY group.In L+CO+LY group,25 μmol/L LY294002 was added to the culture medium,CORM-2 100 μmol/L was added 1 h later,and then LPS 10 μg/ml was added 1 h later.iCORM 100 μmol/L was added to the culture medium and 1 h later LPS 10 μg/ml was added in L+iCO group.In L+D group,the equal concentration of DMSO was added to the culture medium and 1 h later LPS 1 μg/ml was added.CORM-2 100 μmol/L was added to the culture medium in CO group.LY294002 25 μmol/L was added to culture medium in LY group.LY294002 25 μmol/L was added to the culture medium,and 1 h later CORM-2 100 μmol/L was added in CO+LY group.Cells were harvested after 24 h of incubation for measurement of the malondialdehyde (MDA) content,superoxide dismutase (SOD) activity and expression of heme oxygenase-1 (HO-1),phosphorylated Akt (p-Akt),mitochondrial fusion proteins mitofusion 1 (Mfn1),Mfn2 and optic atrophy 1 (OPA1) by Western blot.Results Compared with group C,the MDA content was significantly increased and SOD activity was decreased in L,L+CO,L+LY,L+CO+LY,L+iCO,L+D groups,and the expression of HO-1,Mfn1,Mfn2,OPA1 protein and p-Akt was significantly up-regulated in group L (P<0.05).Compared with group L,MDA content was significantly decreased and SOD activity was increased in group L+CO,MDA content was significantly increased and SOD activity was decreased in group L+LY,the expression of HO-1,Mfn1,Mfn2,OPA1 protein and p-Akt was significantly up-regulated in group L+ CO,and the expression of HO-1,Mfn1,Mfn2,OPA1 protein and p-Akt was significantly down-regulated in group L+LY (P<0.05).Compared with group L+CO,the MDA content was significantly increased,SOD activity was decreased,and the expression of HO-1,Mfn1,Mfn2,OPA1 and p-Akt was down-regulated in group L+CO+LY (P<0.05).Conclusion The mechanism by which CO up-regulates the expression of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells is related to activating PI3K/Akt signaling pathway in rats.
10.Role of α7nAChR in electroacupuncture-induced reduction of acute lung injury induced by endotoxin in rats: the relationship with JAK2/STAT3 signaling pathway
Lirong GONG ; Jia SHI ; Yuan ZHANG ; Shu'an DONG ; Jianbo YU
Chinese Journal of Anesthesiology 2018;38(6):739-742
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.