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Objective:To evaluate the role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in sepsis-associated encephalopathy (SAE) and the relationship with pyroptosis in microglia of mice.Methods:Twenty-four SPF healthy male C57BL/6J mice, aged 6-8 weeks, weighing 18-22 g, were divided into 3 groups ( n=6 each) using a random number table method: sham operation group (Sham group), SAE group and SAE plus an NLRP3 inhibitor MCC950 group (SAE+ MCC950 group). The mouse model of SAE was prepared by cecal ligation and puncture after anesthesia. MCC950 20 mg/kg was intraperitoneally injected at 1 h after developing the model in SAE+ MCC950 group, and the equal volume of normal saline was given instead in the other groups. Open field tests were conducted at 1 day after developing the model to record the number of rearing and time spent in the central area. Novel object recognition tests were conducted at 2-3 days after developing the model to record the recognition index. After the behavioral experiment on 3 day after developing the model, mice were sacrificed and hippocampal tissues were collected for determination of the expression of NLRP3 (by Western blot), count of cells co-expressing NLRP3 and microglia-specific ionized calcium-binding adaptor molecule 1 (Iba-1) (by immunofluorescence), activity of caspase-1, and contents of interleukin-1beta(IL-1β) and IL-18 (by enzyme-linked immunosorbent assay). Results:Compared with Sham group, the number of rearing was significantly reduced, the time spent in the central area was shortened, the recognition index was decreased, the expression of NLRP3 was up-regulated, the count of NLRP3 + -Iba-1 + cells was increased, and the activity of caspase-1 and contents of IL-1β and IL-18 were increased in SAE and SAE+ MCC950 groups ( P<0.05). Compared with SAE group, the number of rearing was significantly increased, the time spent in the central area was prolonged, the recognition index was increased, the expression of NLRP3 was down-regulated, the count of NLRP3 + -Iba-1 + cells was decreased, and the activity of caspase-1 and contents of IL-1β and IL-18 were decreased in SAE+ MCC950 group ( P<0.05). Conclusions:NLRP3 is involved in the development of SAE, which may be related to the mediation in microglial pyroptosis in mice.
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Objective:To evaluate the role of small ubiquitin-associated modifier (SUMO) E3 ligase (PIAS)-regulated SUMOylation of peroxisome proliferator-activated receptor γ (PPARγ) in the endogenous protective mechanism against endotoxin-induced acute lung injury (ALI) in mice.Methods:Experiment Ⅰ Twenty-four clean-grade wild type male C57BL/6 mice, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (C group), ALI group, ALI+ PPARγ inducer TZD group (ALI+ T group) and ALI+ TZD+ SUMOylation inhibitor anacardic acid group (ALI+ T+ A group). Lipopolysaccharide (LPS) 15 mg/kg was injected into the tail vein to develop the ALI model. In ALI+ T+ A group, anacardic acid 5 mg/kg was intraperitoneally injected at 1 h before LPS administration. In ALI+ T group and ALI+ T+ A group, TZD 50 mg/kg was intraperitoneally injected at 30 min before LPS administration. The mice were sacrificed at 12 h after LPS administration, and the lung tissues were obtained to examine the pathological changes which were scored and to determine the wet/dry (W/D) weight ratio, and expression of PIAS1, PIAS2, PIAS3 and PIASy protein and mRNA (by Western blot or polymerase chain reaction). Experiment Ⅱ Mouse alveolar macrophages (MH-S cells) were cultured in vitro and divided into 4 groups ( n=5 each) using a random number table method: control group (C group), LPS group, LPS+ PIAS2 siRNA group (L+ P group) and LPS+ Con siRNA group (L+ C group). Cells were routinely cultured in group C. Cells were stimulated with 10 μg/ml LPS to develop the model of endotoxin challenge. PIAS2 siRNA 50 nmol/L and Con siRNA 50 nmol/L were transfected at 48 h before LPS was added in L+ P group and L+ C group, respectively. The cells were collected at 24 h of incubation with LPS to determine the cell viability, levels of M1 and M2 alveolar macrophages (by flow cytometry), expression of PIAS2 and PPARγ (by Western blot), co-expression of PPARγ-SUMO1 (by immunoprecipitation) and expression of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) mRNA (by polymerase chain reaction). The ratio of M1/M2 was calculated. Results:Experiment Ⅰ Compared with C group, the lung injury scores and W/D ratio were significantly increased, and the expression of PIAS2 protein and mRNA was up-regulated in the other three groups ( P<0.05). Compared with ALI group, the lung injury scores and W/D ratio were significantly decreased, and the expression of PIAS2 protein and mRNA was up-regulated in ALI+ T group and ALI+ T+ A group ( P<0.05). Compared with ALI+ T group, the lung injury scores and W/D ratio were significantly increased, and the expression of PIAS2 protein and mRNA was down-regulated in ALI+ T+ A group ( P<0.05). There was no significant difference in the expression of PIAS1, PIAS3 and PIASy protein and mRNA in lung tissues among the four groups ( P>0.05). Experiment Ⅱ Compared with C group, the cell viability was significantly decreased, the expression of PPARγ and co-expression of PPARγ-SUMO1 was up-regulated, the levels of M1 and M2 macrophages and M1/M2 ratio were increased, the expression of TNF-α mRNA was up-regulated, and the expression of IL-10 mRNA was down-regulated in the other three groups, and PIAS2 expression was significantly up-regulated in L group and L+ C group ( P<0.05). Compared with L group, the cell viability was significantly decreased, the expression of PIAS2 and PPARγ and PPARγ-SUMO1 co-expression were down-regulated, the M1 macrophage level and M1/M2 ratio were increased, TNF-α mRNA expression was up-regulated, and the expression of IL-10 mRNA was down-regulated in L+ P group ( P<0.05), and no significant change was found in the parameters mentioned above in L+ C group ( P>0.05). Compared with L+ C group, the cell viability was significantly decreased, the expression of PIAS2 and PPARγ and co-expression of PPARγ-SUMO1 were down-regulated, the level of M1 alveolar macrophages and M1/M2 ratio were increased, the expression of TNF-α mRNA was down-regulated, and the expression of IL-10 mRNA was up-regulated in L+ P group ( P<0.05). Conclusions:PIAS2-regulated SUMOylation of PPARγ is the endogenous protective mechanism against endotoxin-induced ALI in mice, which may be related to inhibition of macrophage polarization into M1 type and alleviation of inflammatory responses.
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Objective:To evaluate the effect of electroacupuncture (EA) on Golgi apparatus stress in the rats with endotoxin-induced acute lung injury (ALI).Methods:Twenty clean-grade male Sprague-Dawley rats, aged 2 months, weighing 160-185 g, were divided into 4 groups ( n=5 each) according to a random number table method: control group (C group), endotoxin group (LPS group), EA plus endotoxin group (EA+ LPS group), and sham EA plus endotoxin group (SEA+ LPS group).The model of endotoxin-induced ALI was developed by intravenous injection of lipopolysaccharide (LPS) 5 mg/kg in anesthetized animals.Bilateral Zusanli (ST36) and Neiguan (PC6) acupoints were stimulated with an electric stimulator for 30 min once a day at 1-4 days before and during model preparation in group EA+ LPS.In group SEA+ LPS, acupuncture needles were inserted to the surface of ST36 and PC6 acupoints with no current stimulation, and the other parameters were the same as those previously described in group EA+ LPS.Blood samples were collected from the abdominal aorta at 6 h after development of the model for measurement of concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum by enzyme-linked immunosorbent assay.The animals were sacrificed and lungs were removed for microscopic examination of the pathological changes of lung tissues (with a light microscope) and morphological changes of Golgi apparatus (with a transmission electron microscope) and for determination of wet to dry lung weight (W/D) ratio, cell apoptosis index (by TUNEL), activity of superoxide dismutase (SOD) (by WST-1 method), content of malondialdehyde (MDA) (by TBA method), and expression of Golgi matrix protein 130 (GM130), Golgin-84 and Golgi phosphoprotein 3 (GOLPH3) protein and mRNA in lung tissues (by Western blot or real-time polymerase chain reaction). Results:Compared with group C, the lung injury score, W/D ratio, cell apoptosis index, serum IL-6 and TNF-α concentrations and MDA content were significantly increased, SOD activity was decreased, the expression of GM130 and Golgin-84 protein and mRNA was down-regulated, the expression of GOLPH3 protein and mRNA was up-regulated ( P<0.05), and Golgi apparatus was swollen and vacuolated in the other three groups.Compared with group LPS, lung injury score, W/D ratio, cell apoptosis index, serum IL-6 and TNF-α concentrations and MDA content were significantly decreased, SOD activity was increased, the expression of GM130 and Golgin-84 protein and mRNA was up-regulated, the expression of GOLPH3 protein and mRNA was down-regulated ( P<0.05), and swelling and vacuolization of Golgi apparatus were reduced in group EA+ LPS, and no significant change was found in the parameters mentioned above in group SEA+ LPS ( P>0.05). Conclusions:The mechanism by which EA reduces endotoxin-induced ALI may be related to inhibition of Golgi apparatus stress in lung tissues of rats.
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Objective:To evaluate the role of nicotinamide adenine dinucleotide (NAD + )-mediated deacetylation activity of silent information regulator 1 (SIRT1) in endotoxin-induced acute lung injury (ALI) in mice. Methods:Twenty-five SPF clean-grade healthy male C57BL/6 mice including 10 wild-type (WT) and 15 NMNAT1 conditional-knockout (KO) mice, aged 6-8 weeks, weighing 20-25 g, were selected.The WT mice were divided into 2 groups ( n=5 each) using a random number table method: control group (group WT+ C) and ALI group (group WT+ ALI). The KO mice were divided into 3 groups ( n=5 each) using a random number table method: control group (group KO+ C), ALI group (group KO+ ALI) and ALI plus NAD + precursor substances nicotinamide mononucleotide (NMN) group (KO+ LPS+ NMN group). ALI was produced with lipopolysaccharide (LPS) 15 mg/kg injected intravenously.NMN 500 mg/kg was intraperitoneally injected at 1 h before injection of LPS in KO+ ALI+ NMN group, while the equal volume of normal saline was given instead in control group.Blood samples were collected from the abdominal aorta at 12 h after LPS or normal saline injection for blood gas analysis, and the animals were then sacrificed and the lung tissues were removed for microscopic examination of pathologic changes which were scored and for determination of wet/dry weight ratio (W/D ratio), and interleukin-6 (IL-6), IL-1β and tumor necrosis factor-alpha (TNF-α) contents (by enzyme-linked immunosorbent assay)and content of NAD + (using a spectrophotometer) and levels of SIRT1, acetylated nuclear factor kappaB (Ac-NF-κB), acetylated p53 (Ac-p53), acetylated FoxO1 (Ac-FoxO1) and acetylated PGC1α (Ac-PGC1α) (by Western blot). Results:Compared with group C, pH value and PaO 2 were significantly decreased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β, TNF-α and NAD + were increased, expression of SIRT1 was up-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was down-regulated in group ALI ( P<0.05). Compared with group WT+ ALI, pH value and PaO 2 were significantly decreased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β and TNF-α were increased, NAD + content was decreased, expression of SIRT1 was down-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was up-regulated in group KO+ ALI ( P<0.05). Compared with group KO+ ALI, pH value and PaO 2 were significantly increased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β and TNF-α were decreased, NAD + content was increased, expression of SIRT1 was up-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was down-regulated in group KO+ ALI+ NMN ( P<0.05). Conclusion:The enhanced NAD + -mediated deacetylation activity of SIRT1 is involved in the endogenous protective mechanism in mice with endotoxin-induced ALI.
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Objective:To evaluate the relationship between heme oxygenase-1/carbon monoxide (HO-1/CO) signaling pathway and mitochondrial fission in lipopolysaccharide (LPS)-challenged alveolar type Ⅱ epithelial cells (AECⅡ) of rats.Methods:The cultured AECⅡ were subcultured and inoculated in 96-well plates (about 2 × 10 5 cells/ml) and divided into 7 groups ( n=12 each) using a random number table method: blank control group (group C), LPS group (group L), carbon monoxide release molecule-2 (CORM -2) + LPS group (group CL), HO-1 inducer hemin+ LPS group (group HL), HO-1 inhibitor ZnPP-Ⅸ+ LPS group (group ZL), CORM-2+ ZnPP-Ⅸ+ LPS group (group CZL) and hemin+ ZnPP-Ⅸ+ LPS group (group HZL). In group C, the cells were continuously incubated and received no treatment.Cells were incubated with LPS 10 μg/ml in group LPS. In CL, HL and ZL groups, cells were incubated with 100 μmol/L CORM-2 for 1 h, with 20 μmol/L hemin for 1 h and with 10 μmol/L ZnPP- Ⅸ for 0.5 h, respectively, and then the cells were incubated with 10 μg/ml LPS.In group CZL, the cells were incubated with 100 μmol/L CORM-2 for 1 h, then with 10 μmol/L ZnPP-Ⅸ for 0.5 h, and finally with 10 μg/ml LPS.In group HZL, the cells were incubated with 20 μmol/L hemin for 1 h, then with 10 μmol/L ZnPP-Ⅸ for 0.5 h, and finally with 10 μg/ml LPS.Cell viability was evaluated by methyl thiazolyl tetrazolium assay at 48 h of culture or incubation with LPS.The expression of HO-1, dynamin-related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1) was determined using Western blot. Results:Compared with group C, the cell viability was significantly decreased, and the expression of HO-1, Drp1 and Fis1 was up-regulated in other six groups ( P<0.05). Compared with group L, the cell viability was significantly increased, the expression of HO-1 was up-regulated, and the expression of Drp1 and Fis1 was down-regulated in CL and HL groups, the cell viability was decreased, HO-1 expression was down-regulated and the expression of Drp1 and Fis1 was up-regulated in group ZL ( P<0.05), and no significant change was found in the parameters mentioned above in CZL and HZL groups ( P>0.05). Compared with CL and HL groups, the cell viability was significantly decreased, HO-1 expression was down-regulated, and the expression of Drp1 and Fis1 was up-regulated in group ZL ( P<0.05). Conclusion:HO-1/CO signaling pathway can inhibit mitochondrial fission, which exerts endogenous protection when LPS induces injury to AEC Ⅱ in rats.
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Objective:To evaluate the role of melatonin in electroacupuncture (EA)-induced reduction of lung injury induced by limb ischemia-reperfusion (I/R) in rabbits.Methods:Fifty clean-grade healthy male New Zealand white rabbits, weighing 2.0-2.5 kg, aged 3 months, were divided into 5 groups ( n=10 each) using a random number table method: sham operation group (group Sham), limb I/R group (group IR), EA group, sham EA group (group SEA) and EA plus melatonin receptor antagonist luzindele group (group EA+ L). The model of limb I/R injury was established by clamping the femoral artery for 3 h followed by 4-h reperfusion in anesthetized animals.In group EA and group EA + L, bilateral Zusanli and Feishu acupoints (4-6 mm depth) were stimulated with constant voltage (2/15 Hz, l-2 mA, disperse-dense waves) for 30 min once a day during 1-7 days before establishing the model and during establishment of the model.EA was performed at the points (3 mm depth) 0.5 cm lateral to the acupoints of Zusanli and Feishu instead in group SEA.Luzinole 30 mg/kg was intraperitoneally injected at 30 min before establishing the model in group EA+ L.Blood samples from the right internal jugular vein were collected before ischemia (T 0), at 3 h of ischemia (T 1) and 4 h of reperfusion (T 2) for determination of the serum melatonin concentrations by enzyme-linked immunosorbent assay.Bronchoalveolar lavage fluid (BALF) was collected at 4 h of reperfusion for measurement of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 concentrations (by enzyme-linked immunosorbent assay), superoxide dismutase (SOD) activity (by xanthine oxidase method), and malondialdehyde (MDA) concentration (by thiobarbituric acid method). Then the rabbits were sacrificed, and the lung tissues were taken for determination of wet to dry weight ratio (W/D ratio) and for microscopic examination of the pathological changes (with a light microscope) which were scored and ultrastructure (with a transmission electron microscope). The number of mitochondria and relative cross-sectional area of mitochondria were calculated. Results:Compared with group Sham, lung injury scores and W/D ratio were significantly increased, the number of mitochondria was decreased, the relative cross-sectional area of mitochondria was increased, levels of TNF-α, IL-1β, IL-6 and MDA in BALF were increased, and activities of SOD in BALF were decreased in the other four groups, and the serum melatonin concentration was decreased at T 1 and T 2 in group I/R and increased at T 0 in EA and EA+ L groups ( P<0.05). Compared with group IR, the lung injury score and W/D ratio were significantly decreased, the number of mitochondria was increased, the relative cross-sectional area of mitochondria was decreased, levels of TNF-α, IL-1β, IL-6 and MDA in BALF were decreased, and activities of SOD in BALF were increased in group EA, the serum melatonin concentration was increased at each time point in EA and EA+ L groups ( P<0.05), and no significant change was found in the parameters mentioned above in group SEA ( P>0.05). Compared with group EA, lung injury scores and W/D ratio were significantly increased, the number of mitochondria was decreased, the relative cross-sectional area of mitochondria was increased, levels of TNF-α, IL-1β, IL-6 and MDA in BALF were increased, and activities of SOD in BALF were decreased in SEA and EA+ L groups, and the serum melatonin concentration was decreased at each time point in group SEA ( P<0.05). Conclusion:EA can reduce lung injury induced by limb I/R by increasing serum melatonin level in rabbits.
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Objective:To evaluate the relationship between melatonin receptors and mitochondrial fission proteins and to clarify the mechanism of melatonin alleviating lipopolysaccharide(LPS)-induced damage to type Ⅱ alveolar epithelial cells of rats.Methods:The rat type Ⅱalveolar epithelial cells were seeded in 6-well plates at a density of 2×10 5 cells/ml and divided into 5 groups ( n=10 each) using a random number table method: control group (C group), LPS group (L group), LPS plus melatonin group (LM group), LPS plus melatonin receptor blocking group (LL group), and LPS plus melatonin plus melatonin receptor blocker group (LML group). The model of LPS-induced damage to cells was established by incubating with LPS 10 μg/ml for 24 h. Melatonin 0.1 mmol/L and/or melatonin receptor blocker luzindole 0.2 μmol/L was added in LM group, LL group and LML group.The concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay after the end of incubation.The mitochondrial respiratory control rate (RCR) was measured by GENMED purified mitochondrial RCR quantitative detection kit in each group.Western blot was used to detect the expression of dynamin-related protein 1 (Drp1) and mitochondrial adaptor fission 1 (Fis1). Results:Compared with C group, the concentrations of TNF-α and IL-6 in culture medium were significantly increased, RCR was decreased, and the expression of Drp1 and Fis1 was up-regulated in L, LM, LL and LML groups ( P<0.05). Compared with L group, the concentrations of TNF-α and IL-6 in culture medium were significantly decreased, RCR was increased, and the expression of Drp1 and Fis1 was down-regulated in LM group ( P<0.05), and no significant change was found in parameters mentioned above in LL group ( P>0.05). Compared with LM group, the concentrations of TNF-α and IL-6 in culture medium were significantly increased, RCR was decreased, and the expression of Drp1 and Fis1 was up-regulated in LML group ( P<0.05). Conclusion:The mechanism by which melatonin attenuates LPS-induced damage to type Ⅱ alveolar epithelial cells is related to activating melatonin receptors and inhibiting the expression of mitochondrial fission proteins in rats.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Objective To evaluate the role of α7 nicotinic acetylcholine receptor (α7nAChR) in electroacupuncture (EA)-induced reduction of acute lung injury (ALI) induced by endotoxin and the relationship with Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) signaling pathway in rats.Methods Sixty clean-grade healthy male Sprague-Dawley rats,aged 8 weeks,weighing 200-220 g,were divided into 6 groups (n=10 each) using a random number table method:control group (group C),endotoxin-induced ALI group (group ALI),EA at acupoints plus ALI group (group EA),specific α7nAChR antagonist α-bungarotoxin (α-BGT) plus ALI group (group BA),EA at acupoints plus α-BGT plus ALI group (group EBA),and EA at non-acupoints plus ALI group (group SEA).ALI was induced by intravenously injecting lipopolysaccharide 5 mg/kg.EA stimulation of bilateral Zusanli and Neiguan acupoints was performed (frequency of disperse-dense wave 2/15 Hz,wave length 0.2-0.5 ms,intensity 1-2 mA) once a day (time for stimulation 9:00 to 10:00,30 min per time) at 1-4 days before establishing the model in EA and EAB groups.EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Neiguan with the same parameters of stimulation in group SEA.o-BGT 1 μg/kg was intraperitoneally injected at 30 min before establishing the model in BA and EBA groups.Rats were sacrificed at 6 h after injecting lipopolysaccharide,and lungs were removed for microscopic examination of the pathological changes which were scored and for determination of wet to dry weight ratio (W/D ratio),contents of tumor necrosis factor-alpha (TNF-α),interleukin-lbeta (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay),and expression of α7nAChR,phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) (by Western blot).Results Compared with group C,the lung injury scores,W/D ratio and contents of TNF-α,IL-1β and IL-6 were significantly increased,and the expression of α7nAChR,p-JAK2 and p-STAT3 was up-regulated in the other five groups (P＜0.05).Compared with group ALI,the lung injury scores,W/D ratio and contents of TNF-α,IL-1β and IL-6 were significantly decreased,and the expression of α7nAChR,p-JAK2 and p-STAT3 was up-regulated in group EA,the lung injury scores,W/D ratio and contents of TNF-α,IL-1β and IL-6 were significantly increased,and the expression of o7nAChR,p-JAK2 and p-STAT3 was down-regulated in group BA (P＜0.05),and no significant change was found in the parameters mentioned above in group SEA (P＞0.05).Compared with group EA,the lung injury scores,W/D ratio and contents of TNF-α,IL-1β and IL-6 were significantly increased,and the expression of o7nAChR,p-JAK2 and p-STAT3 was down-regulated in group EBA (P＜0.05).Conclusion Up-regulated expression of α7nAChR activates JAK2/STAT3 signaling pathway,which is involved in EA-induced reduction of ALI in rats.
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Objective To evaluate the 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.
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Objective To evaluate the efficacy of self?made breathing circuit joint for intermittent positive pressure ventilation ( IPPV) in patients with central airway obstruction undergoing interventional fi?beroptic bronchoscopy ( FOB) . Methods Sixty?two patients of both sexes with central airway obstruction requiring tracheal intubation, aged 60-80 yr, with body mass index of 20-26 kg∕m2 , of American Society of Anesthesiologists physical status Ⅲ or Ⅳ and Medical Research Council dyspnea scale grade Ⅲ or Ⅳ, undergoing interventional FOB under general anesthesia, were divided into 2 groups ( n=31 each) using a random number table:high frequency jet ventilation ( HFJV) group and IPPV group. The patients were tra?cheally intubated after induction of general anesthesia. The self?made breathing circuit joint was connected, then the anesthesia machine was connected to perform IPPV, and the ventilator settings were adjusted to maintain the end?tidal pressure of carbon dioxide 35-45 mmHg in group IPPV, and HFJV was used in group HFJV. Before induction ( baseline) , at 10, 20, 30 and 40 min after start of operation, and at the end of operation, arterial blood samples were collected for blood gas analysis, the pH value, arterial oxy?gen partial pressure, and arterial carbon dioxide partial pressure were recorded. The development of hyper?capnia was recorded. Results Hyoxemia was not found in the two groups. The incidence of hypercapnia was 74%, and in addition the incidence of severe hypercapnia was 10% in group HFJV. The incidence of hypercapnia was 16%, and all the patients presented with permissive hypercapnia in group IPPV. Com?pared with group HFJV, the incidence of hypercapnia was significantly decreased, and the pH value and arterial oxygen partial pressure were increased, and arterial carbon dioxide partial pressure was decreased from 10 min after start of operation to the end of operation in group IPPV (P<0.05). Conclusion The self?made breathing circuit joint provides better efficacy than HFJV when used for IPPV in the patients with central airway obstruction undergoing interventional FOB.
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Objective To evaluate the effect of electroacupuncture on endoplasmic reticulum stress in lung tissues of rats with acute lung injury (ALI) induced by endotoxin.Methods Forty healthy pathogen-free male Sprague-Dawley rats,aged 8 weeks,weighing 180-210 g,were divided into 4 groups (n=10 each) using a random number table:control group (group C),ALI group,electroacupuncture group (group E),and electroacupuncture at non-acupoint group (group NE).Lipopolysaccharide 5 mg/kg (in 0.5 ml normal saline) was injected intravenously to establish the model of endotoxin-induced ALI.Bilateral 30 min electroacupuncture stimulation of Zusanli and Neiguan acupoints was performed with the dispersedense wave (frequency 2/15 Hz,wave length 0.2-0.6 ms,intensity 1-2 mA) once a day (time for stimulation 9:30-10:30) for 4 consecutive days before and during establishment of the model in group E.Electroacupuncture was performed with the same parameters at the points 0.5 cm lateral to the acupoints of Zusanli and Neiguan in group NE.At 6 h after lipopolysaccharide injection,the rats were sacrificed,and lungs were removed for microscopic examination and for determination of wet to dry weight ratio (W/D ratio),apoptosis in alveolar epithelial cells and expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12 in lung tissues (by Western blot).The pathological changes of lungs were scored.Apoptosis index (AI) was calculated.Results Compared with group C,lung injury scores,W/D ratio and AI were significantly increased,and the expression of GRP78,CHOP and caspase-12 in lung tissues was up-regulated in the other three groups (P＜0.05).Compared with group ALI,lung injury scores,W/D ratio and AI were significantly decreased,and the expression of GRP78,CHOP and caspase-12 in lung tissues was down-regulated in group E (P＜0.05),and no significant change was found in the paramneters mentioned above in group NE (P＞0.05).Conclusion The mechanism by which electroacupuncture attenuates endotoxin-induced ALI is related to inhibition of endoplasmic reticulum stress and reduction of apoptosis in alveolar epithelial cells in rats.
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Objective To evaluate the role of p38MAPK signaling pathway in electroacupuncture (EA)-induced reduction of acute lung injury (ALI) in rabbits with endotoxic shock and the relationship with nuclear factor E2-related factor 2 (Nrf2).Methods Seventy healthy male New Zealand white rabbits,aged 2 months,weighing 1.5-2.5 kg,were randomly divided into 7 groups (n=10 each) using a random number table:control group (group C),endotoxin-induced ALI group (group A),p38MAPK inhibitor SB203580 group (group SB),ALI + SB203580 group (group A-SB),ALI + EA group (A-EA group),ALI + EA at non-acupoint group (A-NEA group) and ALI + EA at acupoints+ SB203580 group (A-EA-SB group).The rabbits were anesthetized with urethane and tracheostomized and kept spontaneous breathing.Right common carotid artery was cannulated for mean arterial pressure monitoring.The auricular vein was cannulated for drug administration.Bilateral 30 min EA (wave length 0.2-0.6 ms,frequency 2/100 Hz,intensity ≤ 1-2 mA) stimulation of Zusanli and Feishu was performed once a day for 4 days before establishment of the model and during establishment of the model in A-EA and A-EA-SB groups.In group A-NEA,EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Feishu according to the method previously described in group EA.In A,A-SB,A-EA,A-NEA and A-EA-SB groups,ALI was induced by endotoxin (5 mg/kg) injection,while the equal volume of normal saline was given in C and SB groups.After establishment of the model,SB203580 5 μmol/kg was injected intravenously in SB,A-SB and A-EA-SB groups,the equal volume of normal saline was given in group C,and the equal volume of dehydrated alcohol was given in the other groups.At 6 h after endotoxin or normal saline administration,arterial blood samples were collected for blood gas analysis.The rabbits were then sacrificed,and lungs were removed for microscopic examination and for determination of malondialdehyde (MDA) content,superoxide dismutase (SOD) activity,and expression of phosphor-p38MAPK (p-p38MAPK) and Nrf2 in lung tissues.The pathological changes of lungs were scored.Wet to dry lung weight ratio (W/D ratio) was calculated.Results Compared to group C,the pathological scores,W/D ratio,MDA content,and expression of pp38MAPK and Nrf2 were significantly increased,and SOD activities were decreased in A,A-SB,A-EA,ANEA and A-EA-SB groups.Compared to group A,the pathological scores,W/D ratio and MDA content were significantly decreased,and SOD activities and expression of p-p38MAPK and Nrf2 were increased in A-EA group.Compared to group A-EA,the pathological scores,W/D ratio and MDA content were significantly increased,and SOD activities and expression of p-p38MAPK and Nrf2 were significantly decreased in group A-EA-SB.Conclusion p38MAPK signaling pathway mediates EA-induced reduction of ALI in rabbits with endotoxic shock,and up-regulated expression of Nrf2 is involved in the mechanism.
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Objective To 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.