Effects of nuclear factor-κB on one lung ventilation-induced lung injury
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Objective To study the changes of nuclear factor-κB(NF-κB) activation and expression of cytokines in lung tissues after one lung ventilation(OLV) in the rabbit models.Methods Twelve white Japanese rabbits were randomly divided into 2 groups with 6 animals each.Two-lung ventilation(TLV) was performed in group TLV for 3 h.OLV was performed for 2 h followed by TLV for 1 h in group OLV.Lung separation was achieved with an artificial double-lumen tube.The lung tissue samples of group OLV were marked as OLVL and OLVR,respectively.NF-κB activity in nuclear protein from lung tissues was measured by electrophoretic mobility shift assay(EMSA),quantity of IκB-α by Western blotting and expression of TNF-α and ICAM-1 mRNA by RT-PCR.Light microscopic evaluations were performed and W/D ratio was determined.Results NF-κB activity,quantity of IκB-α,the expressions of TNF-α and ICAM-1 mRNA were significantly different between group TLV and OLV(P0.01).To compared with OLVR,NF-κB activity of OLVL was significantly increased while quantity of IκB-α was significantly decreased(P0.01).The expressions of TNF-α and ICAM-1 mRNA of OLVL were higher than those of OLVR(P0.05).Conclusion IκB-α degradation and NF-κB activation probably play an important role in lung injury caused by OLV.Cite
Objective
To investigate the effect of dexmedetomidine pretreatment on the expression of caspase-12 in lung tissues undergoing one-lung ventilation(OLV)in rats.
Methods
Thirty male Sprague-Dawley rats, aged 6–8 weeks, weighing 180–220 g, were randomly allocated into 3 groups(n=10 each)using a random number table: two-lung ventilation(TLV)group, OLV group and dexmedetomidine group(Dex group). Bilateral lungs were ventilated for 2 h in group TLV.In OLV and Dex groups, unilateral lung was ventilated for 1.5 h followed by 0.5 h TLV.In group Dex, dexmedetomidine was infused intravenously at a rate of 3.0 μg·kg–1·h–1 over 60 min starting from 60 min prior to OLV.The equal volume of normal saline was given instead of dexmedetomidine in OLV and TLV groups.Peak airway pressure(Ppeak)and mean airway pressure(Paw)were recorded at 45 min of OLV and 15 min of TLV in OLV and Dex groups, and at 15 min of TLV in group TLV.The rats were then sacrificed, and left lungs were removed for microscopic examination of the pathologic changes(using HE staining)and the ultrastructure of lung tissues(with transmission electron microscope)and for determination of wet to dry lung weight ratio(W/D ratio), cell apoptosis in lung tissues(by TUNEL), caspase-12 mRNA expression(using real-time reverse transcriptase-polymerase chain reaction), and caspase-12 expression(by Western blot).
Results
Ppeak and Paw were significantly lower at 15 min of TLV than at 45 min of OLV in OLV and Dex groups(P<0.05). Compared to group TLV, W/D ratio and AI were significantly increased, and the expression of caspase-12 protein and mRNA was up-regulated in OLV and Dex groups(P<0.01). Compared to group OLV, W/D ratio and AI were significantly decreased, and the expression of caspase-12 protein and mRNA was down-regulated in group Dex(P<0.01). The pathologic changes of lung tissues were significantly alleviated in group Dex as compared with group OLV.
Conclusion
The mechanism by which dexmedetomidine pretreatment alleviates acute lung injury caused by OLV is associated with down-regulated expression of caspase-12 and inhibited cell apoptosis in rats.
Key words:
Dexmedetomidine; Respiration, artificial; Respiratory distress syndrome, adult; Caspase 12
Dexmedetomidine
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[Objective] To observe the effects of lidocaine on lung injury and the expression changes of NF-κB by large tidal volume mechanical ventilation on rat lung.[Methods] 36 depuratory-class adult male SD rats were randomly divided into three groups: R group、N group and L group.The tidal volume of N group or L group wes 40 ml/kg;The experiment was terminated atlasted for 3 hours.Respirating air.The animals' BP,HR,ECG and PET CO_2 were monitored,blood gas analysis was detected terminating the experiment.The endotoxin contents in the blood were checked before mechanical ventilation,the rats detected ofwith endotoxin contents were knocked out the experiment to guarantee the experiment would not be influenced by endotoxin.Aseptic techniques were strictly executed throughout the whole experiment.6 rats in each group were injected by EB.Pathomorphological integration and the expression of NFκB mRNA expression in whole lung homogenates was assessed by RT-PCR,the expression of NF-κB protein on were assessed.[Results] There was no significant pathomorphological changes in R group.Compared with R group,lung pathomorphological integrationchanges were more significant,W/D and the content of EB were all elevated(P0.05 or P0.01) in N group and L group;WBC in L group had no change and elevated significantly in N group(P0.01).The expression of NF-κB protein and NF-κB mRNA in lung were significantly higher in N group than in R group(P0.01),but the difference between L group and R group was not significant(P0.05).[Conclusions] The large tidal volume mechanical ventilation on rat lung increased the lungs' permeability and inflammatory response,induced up-regulation of NF-κB expression in rat's lung.Lidocaine inhibited this inflammatory response and up-regulation of NF-κB expression.
Group B
Group A
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Objective To investigate the effect of different one-lung ventilation （OLV） time on TNF-a and IL-8 in rabbit bronchoalveolar lavage fluid, and the surfactant protein-A（SP-A） in lung tissue. Methods 60 male rabbits were randomly divided into group C, group O1, O2, O3, O4 and O5. GroupO1-O5 were established the fight lung ventilation and given l, 2, 3, 4 and 5 hours ventilation. Five rabbits in each group were made execution after completing ventilation, the other five rabbits were executed after feeding 24 h. TNF-a, IL-8 concentrations and SP-A of all rabbits were measured. Results When compared with group C during OLV, cell number in left and right lung of group O3-O5 increased over 4.6d±0.79（P〈0.01 ）, PaCO2 in group O4-O5 increased over 43.6±5.7 （P〈0.01）; TNF-a and IL-8 in left lung of group O3-O5 increased over 1O1.90±18.65（P〈0.01 ）, and TNF-a and IL-8 in right lung of group O4-O5 increased 256.16±30.32（P〈0.01 ）. 24 h later, cell number in left lung was still higher than 7.5±1.9 （P〈 0.01）; IL-8 in left lung of group O5 was still higher than 214.60±76.74 （P〈0.01）. TNF-a and IL-8 in left and fight lung had no difference during OLV and 24 h later（P〉0.05）. When compared with group C, SP-A in left lung of group O1-O3 increased over than 13.84±3.47 （P〈0.01）, and SP-A in right lung of group O1-O5 increased over than 5.51±1.48 （P〈0.01）, but SP-A decreased expression in group O4-O5. SP-A in left and fight lung of group O1-O5 increased expression more than 12.17±4.75 in 24 h later （P〈 0.01 ）. Conclusions Lung inflammatory response increases and SP-A decreases with prolonged OLV; especially when OLV is longer than 3 h, lung injury is difficult to repair even after 24 h. It should be avoided to have long time one-lung ventilation.
Key words:
One-lung ventilation; Inflammatory cytokines; Bronchoalveolar lavage; Surfactant protein-A
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Objective To study the effects of fraction of inspired oxygen on nuclear factor-κB (NF-κB) of lung tissue in rabbit one-lung ventilation(OLV) models.Methods 12 white Japanese rabbits were randomly divided into 2 groups(n=6).Bronchial intubation was performed with artificial double-lumen tube,and right OLV was conducted for 2 h and then tollowed tow-lung ventilation(TLV) for 1 h.Fraction of inspired oxygen was set as 1.0(group A) or 0.6(group B).NF-κB in lung tissue was detected,and arterial blood gases were analyzed before OLV,at 30 min of OLV and 30 min after TLV reversion.Pathology of lung tissue was examined and wet/dry(W/D) of lung weight was measured.Results After TLV restore,the oxygenation index was higher and lower than 300 in group B and group A,respectively.The W/D,the activation and the level of NF-κB in left lung tissue was less in group B than in group A (P＜0.01),and pathological change in left lung tissue was lighter in group B than in group A.Conclusion OLV with 60% oxygen may attenuate lung injury by decreasing the activation and the level of NF-κB in lung tissue.
Key words:
One-lung ventilation; Nuclear factor-κB; Fraction of inspired oxygen; Lung injury; Rabbit
Oxygenation index
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Objective To investigate changes of NF-κB activation in lung tissues and expression of cytokines in homogenate from lung tissues in mechanical ventilation(MV),and endotoxin and MV combined with endotoxin in infant rabbits. Methods Sixty healthy infant rabbits were randomly divided into four groups(n=15 each):combined non ventilation as controls(NMV),MV with large tidal volume(LVMV),endotoxin with no MV(ENMV) and MV cobined with endotoxin (EMV).NF-κB activation in nuclear protein from lung tissues was measured in different groups with EMSA.IκBαin lung tissues was analyzed with Western blot method,while genetic expression and protein quantity of TNF-α and IL-8 in homogenate from lung tissue were analyzed with RT-PCR and ELISA,and the pathological changes were examined. Results The activation of NF-κB reached peak at 2 h after occurrence of tissue injury caused by endotoxin and at 4 h caused by MV.Active strength of NF-κB at different time points after lung injury in EMV was significantly higher than that in the other 2 groups(P0.01).The lowest quantity of IκBα was found at 4 h after lung injury in ENMV,and 6 h in MV.Dropping of IκBα quantity at 2,4,6 h in EMV was very significantly faster than that of in the other 2 groups(P0.01).The expressions of TNF-α and IL-8 mRNA and their protein quantity in lung tissues reached peaks in ENMV and EMV earlier than in LVMV.They were significantly higher at 2,4,6 h after injury in EMV than that in the other 2 groups(P0.01 and P0.05),respectively. Conclusion(Both MV) and endotoxin could enhance NF-κB activation through different ways and thus induce transcription of pro-inflammatory cytokines causing lung injury.The effects of the MV and endotoxin on the body earlier or later promote the activation of NF-κB to be more aggressive by their mutual reaction and accelerate lung injury.
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To explore the role and mechanism of mitophagy in ventilator-induced lung injury (VILI) in rats.Thirty-six adult Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 12): spontaneous breathing group (CON group), normal tidal volume (VT) group (NVT group, VT was 8 mL/kg) and high VT group (HVT group, VT was 40 mL/kg). All rats received endotracheal tube by tracheostomy. The rats in CON group were maintained spontaneous breathing, while those in NVT and HVT groups received mechanical ventilation with corresponding VT. After 4 hours of ventilation, the serum, bronchoalveolar lavage fluid (BALF) and lung tissues were harvested. The lung wet/dry weight (W/D) ratio was assessed, and the histopathology changes were observed by light microscopy, and the ultra structure changes in type II alveolar epithelial cell (AEC II) were observed by electron microscopy. The levels of interleukin (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) in serum and BALF were determined by enzyme linked immunosorbent assay (ELISA). The total protein in BALF was measured by bicinchoninic acid methods, and the infiltrated cells in BALF were counted. The mRNA expressions and protein levels of microtube associated light chain 3B (LC3B), mitochondrial DNA coded cytochrome C oxidase IV (COX-IV) and nuclear factor-KB p65 (NF-KB p65) in lung tissues were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot.The histopathology of lung tissue and the ultra structure of AEC II were normal in CON group and NVT group, and the obvious inflammatory changes and pathological injury were found in HVT group. Compared with CON and NVT groups, the W/D ratio in HVT group was significantly increased (8.53±1.05 vs. 5.12±0.65, 5.57±0.55, both P < 0.05), and total protein, infiltrated cells, and IL-1β, IL-6 and TNF-α in BALF were significantly increased [total protein (g/L): 2.35±0.45 vs. 1.46±0.34, 1.76±0.51; infiltrated cells (×105/mL): 2.05±0.48 vs. 0.40±0.08, 0.60±0.23; IL-1β (ng/L): 119.82±6.56 vs. 76.15±3.32, 79.39±4.44; IL-6 (μg/L): 4.10±0.52 vs. 1.97±0.40, 2.27±0.36; TNF-α (mg/L): 1.49±0.28 vs. 0.43±0.23, 0.61±0.24; all P < 0.05], IL-1β, IL-6 and TNF-α levels in serum were also elevated [IL-1β (ng/L): 127.53±7.10 vs. 79.40±2.80, 82.95±2.25; IL-6 (μg/L): 6.28±0.82 vs. 2.96±0.35, 3.36±0.72; TNF-α (mg/L): 1.59±0.42 vs. 0.53±0.22, 0.78±0.25; all P < 0.05]. The mRNA expressions and protein levels of LC3B, COX-IV and NF-KB p65 in lung tissue of HVT group were significantly higher than those of CON group and NVT group, the mRNA expressions of LC3B-II, COX-IV and NF-KB p65 were (3.52±0.90), (3.76±1.16) and (9.54±2.06) folds of those in CON group, and the protein expressions were (1.76±0.24), (1.65±0.20) and (1.91±0.12) folds of those in CON group, with significantly statistical differences (all P < 0.05). There were no significant differences in the parameters mentioned above between NVT group and CON group.Mitophagy may be associated with VILI resulting from escaped mitochondrial DNA for activation of inflammation.
Histopathology
Bicinchoninic acid assay
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Objective To investigate the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the lung tissue in a rat model of ventilator-induced lung injury. Methods Thirty healthy male SD rats weighing 200-250g were randomly divided into 3 groups( n = 10 each) : group A spontaneous breathing; group B small tidal volume (VT = 7 ml·kg-1, RR = 40 bpm, FiO2=0.21) and group C large tidal volume (VT = 40 ml·kg-1, RR = 20 bpm, FiO2=0.21). The animals were mechanically ventilated for 4 h in group B and C. The lung injury was assessed by measurement of PaO2 /FiO2 . At the end of the experiment the animals were killed and the lungs were removed. The right lung was used for determination of the expression of mRNAs of MMP-2, MMP-9, TIMP-1 and TTMP-2 using RT-PCR and histologic examination. The left lung was weighed and then lavaged. The broncho-alveolar lavage fluid (BALF) was collected for total WBC and neutrophil counts and determination of total protein content and gelatinase activity. After lavage the left lung was heated at 60℃. The W/D lung weight ratio was calculated. Results PaO2/FiO2 was significantly lower after 4h mechanical ventilation in group C than in group A and B. The total WBC count and total protein content in BALF were significantly higher in group C than in group A and B. The W/D ratio of the left lung was significantly higher in group C than in group A and B. Microscopic examination showed that there were marked WBC infiltration and destructive changes of alveolar walls in group C. The levels of gelatinase (MMP-2 and MMP-9) activities in BALF were significantly higher in group C than in group A and B. The expression of the mRNA of MMP-2 and MMP-9 was significantly higher in group C than in group A and B, but there was no significant difference in the expression of the mRNA of TIMP-1 and TIMP-2 between group C and group A and B. Conclusion Large tidal volume ventilation can induce acute lung injury. MMP-2 and MMP-9 play an important role in the development of ventilator induced lung injury and the imbalance between MMPs and TIMPs contributes to the mechanism of ventilator-induced lung injury.
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Objective To investigate the expression and significance of Beclin-1 and nuclear factor-κB (NF-κB) in a rat model of ventilator-induced lung injury (VILI).Methods Thirty healthy SD rats were randomly divided into three groups (each 10 rats):control group (group A,no ventilation),conventional ventilation group (group B,VT=10 ml/kg),and lung injury group (group C,VT =40 ml/kg).After anesthesia and tracheotomy were installed,rats received ventilation with different volumes for 4 h.Thus rats were killed by exsanguinations.The lung wet/dry weight,myeloperoxidase (MPO) activity,and total protein level and counts of white blood cells (WBC) in broneho-alveolar lavage fluid (BALF) were tested.The expression of Beclin-1 and NF-κB was detected by Western blotting.Results After injurious ventilation for 4 h,the level of lung wet/dry weight,total protein level,MPO activity and counts of WBC were 6.13 ± 0.37,(4.12 ±0.29) g/ml,(5.89 ±0.75) × 106/L,and (6.75 ± 1.18) U/g respectively.The average values of Beclin-1 and NF-κB proteins were 1.21 ±0.14 and 0.47 ±0.11 respectively,and there was a negative correlation between the two proteins.Conclusion Tidal volume mechanical ventilation can induce lung tissue damage and autophagy can inhibit the activity of NF-κB to protect lung tissue to avoid VILI in rats.
Key words:
Ventilator-induced lung injury; Autophagy; Nuclear factor-κB
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Objective
To evaluate the effect of Shenfu injection(SF) on ventilator-induced lung injury in rats and its mechanism.
Methods
Forty adult male Wister rats were equally and randomly divided into 4 groups(n=10) using a random number table :control group(group C), normal tidal volume ventilation group(group N), large tidal volume ventilation group(group L), and large tidal volume ventilation+ SF group(group SF). The spontaneous breathing of rats was maintained in group C, while the other rats were tracheostomized and mechanically ventilated for 4 h in group N, group L and group SF. Rats were killed at the end of the ventilation and was lavaged their left lung, then the bronchoalveolar lavage fluid(BALF) was collected for determination of the concentrations of protein, IL-1β, IL-18 and TNF-α. The lung tissues were removed for determination of the wet/dry(W/D) lung weight, and immunohistochemistry was used to detected the expression of NF-κB. The pathological changes of the lungs were determinated by using light microscope and the lung injury scores were also determinated.
Results
Compared with group SF[TNF-α (65±11) ng/L, IL-1β (47±9) ng/L, IL-18 (58±8) ng/L], the protein expression level of IL-1β, IL-18 and TNF-α in BALF were significantly increased in group L[TNF-α (99±7) ng/L, IL-1β (69±7) ng/L, IL-18(86±7) ng/L](P<0.05). W/D(5.0±1.6) in group SF were significantly decreased while W/D (5.5±1.8) in group L (P<0.05). The protein expression level of NF-κB were (0.32 ± 0.28) in group SF while (0.54 ± 0.33) in group L. The protein expression level of NF-κB were significantly decreased in group SF (P<0.05) and the pathological changes were also significantly reduced in group SF.
Conclusions
SF can reduce ventilator-induced lung injury in rats and its mechanism may be related to the inhibition of the activity of NF-κB pathway and reducing the release of inflammatory cytokines in the lungs.
Key words:
Shenfu injection; Mechanical ventilation; Lung injury; Nuclear factor-κB
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OBJECTIVE To evaluate the effect of lung protective ventilation strategy on pulmonary inflammatory response in acute respiratory distress syndrome (ARDS). METHODS The ARDS rabbit model was duplicated by saline alveolar-lavage. The rabbits were divided into six groups: (1) normal control group (N group); (2) ARDS group (M group); (3) low-volume with best end-expiratory pressure (PEEP, A group) group: tidal volume (V(T)) 6 ml/kg, PEEP 2 cm H(2)O greater than the pressure of lower inflection point in pressure-volume curve (P(LIP)); (4) normal-volume with best PEEP group (B group): V(T) 6 ml/kg, and PEEP P(LIP) + 2 cm H(2)O; (5) low-volume with high PEEP group (C group): V(T) 6 ml/kg, and PEEP 15 cm H(2)O; and (6) high-volume with zero PEEP group (D group): V(T) 20 ml/kg. Lung wet/dry weight ratios (W/D) were recorded to evaluate lung injury. After 4 h of ventilation, lung homogenates were prepared to detect nuclear factor-kappaB (NF-kappaB) activity by electrophoretic mobility gel shift assay (EMSA), tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) levels by enzyme-linked immunosorbent assay (ELISA) and their mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Myeloperoxidase (MPO) and malondialdehyde (MDA) in lung homogenates were also assessed. RESULTS After 4 h ventilation, W/D in A group (5.6 +/- 1.1) were significantly lower than those in B group, C group and D group (6.6 +/- 0.8, 6.6 +/- 1.0, 6.9 +/- 1.0, all P < 0.05). But there was no difference between A group and M group (5.8 +/- 0.5). NF-kappaB activity was the highest in D group, and that in A group was 331 +/- 113, which was decreased significantly as compared with B, C and D groups (455 +/- 63, 478 +/- 74, 645 +/- 162, all P < 0.05). The mRNA expression of TNF-alpha and IL-10 and their concentrations in lung homogenates in A group were lower than those in B, C and D groups. In A group, the concentrations of MPO and MDA in lung homogenates were significantly lower than those in B, C and D groups. CONCLUSION Lung protective ventilation strategy can inhibit lung inflammation and may improve lung injury in ARDS, but low tidal volume with high PEEP may increase lung inflammation.
Positive End-Expiratory Pressure
Diffuse alveolar damage
Malondialdehyde
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