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Objective:To explore the construction and mechanism of Mindin gene specific macrophage knockout mice in acute lung injury induced by lung ischemia-reperfusion injury(IRI).Methods:Mindin gene knockout mice were constructed by CRE-LOP system, Mice were divided into four groups of C57/B6 wild-type mice sham operation(n=10), C57/B6 mice operation(n=10), Mindin-/-macrophage-specific knockout mice operation(n=10)and C57/B6 mice operation + Mindin recombinant protein intervention(n=10). And lung ischemia-reperfusion injury model was established by clamping pulmonary portal.The effects of Mindin gene knockout and recombinant protein intervention on acute lung injury were observed in vivo and in vitro.t-test and ANOVA test were employed for data processing.Results:Mindin gene macrophage specific knockout mice was successfully constructed.Surgery(Mindin-/-)group significantly reduced pulmonary edema, release of inflammatory factors(IL1β: 2.73±0.19 vs. 5.81±0.61; IL-18: 6.52±0.63 vs. 11.03±0.34; TNF-α 2.18±0.14 vs. 4.76±0.20; HMGB1: 4.57±0.33 vs. 8.76±0.87), expression of NLRP3(2.07±0.27 vs. 4.91±0.22)and secretion of GSDMD(2.78±0.37 vs. 5.78±0.29)as compared with surgery group in vivo.In surgery(WT)+ Mindin group, the expression of lung IRI, inflammatory factors and cell pyroptosis were opposite, And the results were consistent in vitro and in vivo.As compared with surgery group, the above parameters were up-regulated in surgery(WT)+ Mindin protein group.And inter-group differences were statistically significant(all P<0.05). In vitro, the expressions of NLRP3(1.00±0.36, 0.41±0.06, 4.13±0.23), GSDMD(1.00±0.17, 0.34±0.16, 6.32±0.46)and integrin β4(1.00±0.11, 0.28±0.07, 3.53±0.17)were detected in different groups including hypoxia-recovery oxygen(HR), HR+ Mindin siRNA and HR+ Mindin protein groups in macrophage cell line(J774A); As compared with HR group, the above parameters were up-regulated in HR+ Mindin protein group and down-regulated in HR+ Mindin siRNA group.And the differences were statistically significant( P<0.05). The expressions of NLRP3(1.00±0.07, 1.13±0.11, 0.51±0.14)and GSDMD(1.00±0.09, 0.87±0.16, 0.37±0.12)were detected in Mindin, Mindin protein+ vehicle and Mindin protein+ integrin β4 knockout groups.The above parameters were down-regulated in Mindin protein+ integrin β4 knockout group as compared with Mindin protein and Mindin protein + vehicle groups.And the inter-group differences were statistically significant(all P<0.05). Conclusions:During pulmonary IRI, Mindin knockdown can alleviate pulmonary IRI.Mindin gene may promote the expression of inflammatory factors, NLRP3 and GSDMD protein by activating integrin β4 and aggravate cell pyroptosis to promote the development of pulmonary IRI.
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Objective:To observe the effect of annexin A1 peptidomimetic Ac2-26 on lung injury in rats with cardiopulmonary bypass, and analysis its anti-inflammatory mechanism.Methods:Eighteen male SD rats were divided into 3 groups(n=6) according to the random number table: sham operation group(S group), ischemia reperfusion group(IR group), Ac2-26 group(A group). The sham operation group received femoral arteriovenous puncture and catheterization, only the left lung was opened and the cardiopulmonary bypass tube was not connected. The other two groups were established with cardiopulmonary bypass left lung ischemia-reperfusion injury model. The IR group was given the same volume 10 minutes before cardiopulmonary bypass with the normal saline solution. Group A was injected with Ac2-26(1 mg/kg) through the tail vein 10 minutes before cardiopulmonary bypass. The arterial blood of the three groups of rats was taken for blood gas analysis before CPB(T1), immediately after opening the left lung hilum(T2), and at the end of the experiment(T3) to calculate OI and RI. At T1 and T3, the femoral vein blood was taken to measure the number of PMN. At T3, pulmonary artery blood was taken to measure the number of PMN and VCAM-1 content, left lung tissue was taken to measure NE and MPO content by ELISA, lung tissue neutrophil apoptosis was measured by Tunel method, and lung tissue ICAM-1 and AnxA1 protein expression was measured by Western-Blot.Results:At T3, compared with group S, the number of PMN in peripheral blood and pulmonary artery blood and the content of pulmonary artery VCAM-1 in IR group were significantly higher, the content of MPO and NE in lung tissue increased, the lung tissue pathological damage score increased, OI decreased and RI increased, all P<0.05. Compared with IR group, the lung tissue pathological damage score of rats in group A was significantly reduced, the number of PMN in peripheral blood and pulmonary artery blood and the content of VCAM-1 in pulmonary artery blood were significantly reduced, the expression of AnxA1 and ICAM-1 in the lung tissue of rats decreased; the content of MPO and NE in lung tissue decreased, and the apoptosis rate of PMN increased, all P<0.05. Conclusion:Ac2-26 annexin A1 peptidomimetics can inhibit the aggregation of neutrophils in the lung, weaken the adhesion of neutrophils to the pulmonary vascular endothelium, promote the apoptosis of neutrophils in the lungs, and reduce the content of NE and MPO. It has a protective effect on lung injury after cardiopulmonary bypass in rats.
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Objective:To explore the effects and mechanism of apigenin on lung ischemia-reperfusion (I/R) injury in mice.Methods:A total of 40 male C57/B6 mice were randomized into 4 groups of sham, I/R, low-dose apigenin and high-dose apigenin (n=10 each). Lung I/R injury model was established by clipping left hilum for 1h and reperfusion for 2 h. Low/high-dose apigenin group received a gavage of apigenin (10/50 mg·kg -1·d -1) for 7 days before lung I/R.After 2-hour reperfusion, lung tissue was collected and lung injury status evaluated and scored by hematoxylin-eosin (H&E) stain; mRNA expression levels of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), high mobility group box 1 (HMGB1), prostaglandin-endoperoxide synthase 2 (PTGS2) and glutathione peroxidase 4 (GPX4) were detected by reverse transcription-polymerase chain reaction (RT-PCR); Western blot was utilized for detecting the protein expressions of Bax, Bcl-2 and HIF-1α.Finally, after morinostat activated Hif-1α, the effect of apigenin (50 μmol/L) on hypoxia-reoxygenation (H/R)-induced ferroptosis was further observed in A549 lung epithelial cells. Results:As compared with sham group, lung injury score spiked markedly in I/R group (7.05±0.6 vs.1.25±0.42), pulmonary edema worsened obviously (8.65±1.12 vs.4.17±0.54), the percentage of Tunel positive cells rose significantly (58.22±4.92 vs.8.23±1.22) and mRNA expression levels of IL-1β, TNF-α and HMGB1 increased (7.82±0.16 vs.1.00±0.14, 4.24±0.12 vs.1.00±0.19, 6.24±0.19 vs.1.00±0.11) ( P<0.05); Compared with I/R group, lung injury score declined markedly in low/high-dose apigenin group (4.88±0.31/2.11±0.29 vs.7.05±0.66)( P<0.05), pulmonary edema lessened markedly (6.42±1.03/4.88±1.62 vs.8.65±1.12)( P<0.05) and the percentage of Tunel positive cells (41.46±6.73/16.02±5.31 vs.58.22±4.92) and the mRNA expression levels of IL-1β, TNF-α and HMGB1 became obviously suppressed (5.88±0.13/3.21±0.19 vs.7.82±0.16, 3.56±0.11/2.12±0.09 vs.4.24±0.12, 4.12±0.14/3.12±0.09 vs.6.24±0.19)( P<0.05); protein expressions of HIF-1α and PTGS2 dropped sharply in low/high-dose apigenin group (2.00±0.10/0.93±0.11 vs.2.99±0.06, 4.12±0.14/2.51±0.18 vs.6.11±0.12) while GPX4 protein rose obviously (0.55±0.02/0.83±0.02 vs.0.38±0.04)( P<0.05). In vitro experiments further showed that apigenin could significantly suppress the H/R-induced protein expression of PTGS2 in A549 lung epithelial cells (1.11±0.0 vs.4.55±0.12)( P<0.05) while up-regulating the protein expression of GPX4 (0.93±0.11 vs.0.32±0.04)( P<0.05). The inhibition of PTGS2 protein (4.01±0.12 vs.1.11±0.05) and the up-regulation of GPX4 were significantly blocked (0.52±0.05 vs.0.93±0.11)( P<0.05). Conclusions:Through an inhibition of HIF-1α/ferroptosis axis, apigenin can alleviate lung injury, apoptosis and inflammatory response associated with lung I/R in mice.
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Objective:To investigate the role and regulatory mechanism of triggering receptor expressed on myeloid cell 2 (TREM2) in mice lung ischemia/reperfusion injury (LIRI).Methods:Thirty-six healthy male C57BL/6 mice were divided into six groups according to the random number method ( n = 6): normal control group, and LIRI 2, 6, 12, 24, 48 hours group. Mice LIRI models were established by clamping the left hilum. The wet/dry weight ratio (W/D) of left lung tissue was measured. Lung injury was observed and evaluated by hematoxylin-eosin (HE) staining and electron microscopy. The levels of interleukins (IL-1β, IL-18) in lung tissue were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of TREM2 and caspase-1 were determined by polymerase chain reaction (PCR). The protein expressions of TREM2, caspase-1, Gasdermin-D (GSDMD) were determined by Western blotting. Results:At 2 hours after LIRI, lung injury began to appear, the lung ultrastructure changed, and the lung injury score increased; at 6 hours, the degree of lung injury was the most serious; after 12 hours, the lung injury gradually reduced and the lung injury score gradually decreased. Compared with the normal control group, lung W/D ratio and lung injury score of LIRI 2, 6, 12, 24, 48 hours groups were significantly higher, the differences were statistically significant (lung W/D ratio: 7.06±0.52, 8.34±0.17, 6.42±0.35, 5.34±0.25, 5.59±0.45 vs. 4.69±0.23; lung injury score: 5.50±0.54, 9.75±0.89, 5.88±0.84, 3.63±0.74, 4.13±0.64 vs. 1.13±0.35, all P < 0.05). Compared with the normal control group, the levels of IL-1β and IL-18 in lung tissue were significantly increased at 2 hours after LIRI, reached a peak at 6 hours [IL-1β (ng/L): 502.76±12.25 vs. 56.50±8.07, IL-18 (ng/L): 414.02±10.75 vs. 81.63±5.29, both P < 0.05], then decreased gradually, and were still significantly higher than the normal control group at 48 hours. The PCR and Western blotting showed that the expression of TREM2 was significantly lower than that in the normal control group at 2 hours after LIRI, and reached a valley at 6 hours [TREM2 mRNA (2 -ΔΔCt): 0.47±0.05 vs. 1.02±0.05, TREM2/GAPDH: 0.23±0.13 vs. 0.48±0.17, both P < 0.05], then gradually increased, and reached the peak at 24 hours [TREM2 mRNA (2 -ΔΔCt): 3.98±0.15 vs. 1.02±0.05, TREM2/GAPDH: 0.71±0.17 vs. 0.48±0.17, both P < 0.05]. The trend of expression of caspase-1 and GSDMD were opposite to that of TREM2, which increased at first and then decreased, and reached a peak at 6 hours after reperfusion [caspase-1 mRNA (2 -ΔΔCt): 2.20±0.13 vs. 1.01±0.02, caspase-1/GAPDH: 0.64±0.02 vs. 0.20±0.06, GSDMD/GAPDH: 1.23±0.01 vs. 0.87±0.02, all P < 0.05]. Conclusions:TREM2 might be involved in LIRI in mice. The mechanism may be related to the effect of TREM2 on caspase-1-mediated pyroptosis.
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Objective To observe the effects of Yiqi Huoxue Tongluo Jiedu fang (YHTJF) on pneumocyte apoptosis after lung ischemia/reperfusion (I/R) injury (LIRI) in mice and to investigate whether c-Jun N-terminal protein kinase (JNK) is involved in the mechanism of apoptosis.Methods Seventy C57BL/6J male mice were randomly divided into seven groups:normal control group (C group),carboxyl methyl cellulose-Na+normal control group (CMC-Na+C group),CMC-Na+sham group (CMC-Na+S group),CMC-Na+I/R group (CMC-Na+I/R group) and CMC-Na+YHTJF-low,-middle,-high dose groups (CMC-Na+YL,CMC-Na+YM,CMC-Na+YH groups).C group did not undergo any processing;in CMC-Na+S group,only was chest opened without clipping the lung hilum;in the rest of the four groups,they all underwent opening of the chest and clipping the lung hilum for 30 minutes,then the clipping of artery was relieved and left lung reperfusion was carried out for 3 hours.After operation,the mice were sacrificed,the lung tissues were harvested.Under light and electron microscopes,the lung morphological and ultra-structural changes were observed,and the changes of index of quantitative evaluation for alveolar damage (IQA) were determined.The terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) was applied to evaluate the apoptosis index (AI) of the lung tissues.The protein and mRNA expressions of JNK and glucose regulating protein 78 (GRP78) in lung tissues were detected by Western Blot and reverse transcription-polymerase chain reaction (RT-PCR);the correlations between lung AI and the expressions of mRNA and protein of JNK and GRP78,IQA were analyzed.Results Compared with CMC-Na+S group,IQA,AI and mRNA and the protein expressions of JNK and GRP78 in CMC-Na+I/R group were obviously higher [IQA:(74.00 ± 7.31)％ vs.(7.00 ± 1.23)％,AI:(64.40 ± 11.97)％ vs.(5.60 ± 1.14)％,JNK mRNA (gray value):1.143 ± 0.284 vs.0.152 ± 0.128,GRP78 mRNA (gray value):0.897 ± 0.129 vs.0.284 ± 0.044,JNK protein (A value):0.428 ± 0.074 vs.0.073 ± 0.052,GRP78 protein (A value):1.075 ± 0.145 vs.0.589 ± 0.060].Compared with CMC-Na+I/R group,the IQA,AI,protein and mRNA expressions of JNK and GRP78 in CMC-Na+YL,CMC-Na+YM,CMC-Na+YH groups were all lower,and the degree of reduction in group CMC-Na+YM was the most remarkable,greater than that in CMC-Na+YL or CMC-Na+YH group [IQA:(26.20 ± 3.35)％ vs.(34.00±5.34)％,(41.20±9.18)％,AI:(29.40±3.05)％ vs.(48.20±3.83)％,(39.20±6.14)％,JNK mRNA (gray value):0.681 ± 0.130 vs.0.804 ± 0.153,0.938 ± 0.11,GRP78 mRNA (gray value):0.450 ± 0.105 vs.0.747 ± 0.231,0.566 ± 0.115,JNK protein (A value):0.188 ± 0.049 vs.0.261 ± 0.065,0.209 ± 0.063,all P ＜ 0.01],compared with the CMC-Na+I/R group,the expression of GRP78 protein was obviously higher in CMC-Na+YH,CMC-Na+YL,CMC-Na+YM groups and the most remarkably high was in CMC-Na+YH group (A value:1.429 ±0.226 vs.1.130±0.169,1.128 ±0.177,all P ＜ 0.01).The apoptosis of each group was mainly in the pulmonary vascular endothelial cells and alveolar epithelial cells,and brown particles were positive cells under light microscope.Under transmission electron microscope:nuclear pyknosis and margination under the nuclear membrane,cytoplasm condensed,lamellar bodies decreased and emptying increased,cell membrane microvilli decreased or disappeared,mitochondria swelling,inflammatory cells increased in alveolar septum and adhering onto the capillary walls could be seen in CMC-Na+I/R group.Compared with CMC-Na+I/R group,the lung tissue ultrastructural damage alleviated,ultrastructure of alveoli clearly seen,nuclear chromatin relatively uniform,cytoplasm increased,type Ⅱ alveolar epithelial cell surface microvilli relatively plenty,lamellar corpuscle number increased,mitochondria swelling ameliorated in CMC-Na+YH,CMC-Na+YL,CMC-Na+YM groups and the most remarkable one was CMC-Na+YM group.AI was significantly positive correlated with the mRNA and protein expressions of JNK,GRP78 and IQA (r =0.907,0.928,0.880,0.712,0.911,all P ＜ 0.01).Conclusions YHTJF may effectively alleviate the cell apoptosis in mice LIRI,and its mechanism may be related to the inhibition of JNK pathway.
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Objective To observe the effects of large-dose ambroxol hydrochloride on lung ischemia-reperfusion injury (LIRI)and discuss the protection of ambroxol hydrochloride on Toll-like receptor 4 (TLR4)/nuclear transcription factor-kappa B (NF-κB ) after lung ischemia-reperfusion injury in rats.Methods We randomly assigned 60 healthy SD rats into four groups (n=15 for each):control group,ambroxol hydrochloride group (0.75 g/L),ischemia-reperfusion group (I/R),and I/R+ambroxol hydrochloride group.The ambroxol hydrochloride group and I/R+ambroxol hydrochloride group were injected large dose of ambroxol hydrochloride by intravenous injection.The control group and the I/R group received normal saline.The effects of ambroxol hydrochloride on lung ischemia-reperfusion (LIR)-induced pathological changes and inflammatory cytokines release level were examined.DNA ends situ labeling assay (TUNEL)was used to detect the apoptosis of cells.NF-κBp6 5 was detected by immunohistochemistry.In addition,the TLR4 signaling pathway activation in lung tissues was detected by Western blot analysis.Results Compared with those in the control group,some hemorrhage and inflammation changes of lung tissues were observed;the W/D ratio,inflammatory cytokines,apoptosis of cells,NF-κBp6 5 and TLR4 signaling pathway protein expression in I/R group was obviously increased.Compared with I/R group,some mild hemorrhage and inflammation changes of lung tissues were observed;W/D ratio,inflammatory cytokines,apoptosis of cells, NF-κBp6 5 activity, and TLR4 signaling pathway expression were all decreased significantly in I/R+ambroxol hydrochloride group.Conclusion Large dose of ambroxol hydrochloride can protect rats with lung ischemia-reperfusion injury by downregulating TLR4 signaling pathway.
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Objective To explore the expression change of Toll Like Receptor 4 (TLR4) in lung ischemia-reperfusion injury in rabbits and the influence of clemastine fumarate on it. Methods Fifty rabbits were divided into five groups randomly (n=10): Sham (N+S), reperfusion for 2 hours (N+I1/R2), reperfusion for 4 hours (N+I1/R4), clemastine fumarate + reperfusion for 2 hours (F+I1/R2) and clemastine fumarate+reperfusion for 4 hours (F+I1/R4). The ischemia time in each group was 1 hour and normal saline was given respectively in groups of N+S , N+I1/R2 and N+I1/R4. Western blotting , RT-PCR and immunofluorescence were used to detect the expression of TLR4 in lung tissue , and the changes of ultrastructure in ischemia-reperfusion lung tissue were observed by electron microscope. Result The expression of TLR4 was elevated obviously in ischemia-reperfusion lung tissue (P<0.05), and there was positive correlation between the increased TLR4 level and reperfusion time (P<0.05), the swelled and thick-ridge mitochondria were observed in type II alveolar epithelial cells after LIRI (P<0.05); but clemastine fumarate inhibited the expression of TLR4 in lung tissue significantly caused by LIRI (P<0.05). And the mitochondria injury was reduced in the groups of clemastine fumarate. Conclusion TLR4 expression is elevated in lung tissue after LIRI; clemastine fumarate inhibits the expression of TLR4 caused by LIRI and protects the lung tissue from LIRI in rabbits.
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In order to provide us new clues to induce some endogenous protective molecular mechanisms, the changes in gene expression profile induced by ischemia-reperfusion in pulmonary tissues of rats were investigated and the dynamic mechanism of pulmonary ischemia-reperfusion in- jury was elucidated. Thirty male Wistar rats were randomly divided into 6 groups: 5 ische-mia-reperfusion (I/R) groups (I/R 0-h, I/R 1-h, I/R 3-h, I/R 6-h, I/R 24-h) and control group (n=5 ineach). An in situ ischemia-reperfusion lung injury rat model was established by occluded hilus of lung. The RatRef-12 Expression Beadchip (22 226 gene probes per array) was used to analyze the pattern of gene expression in all groups. The results showed that 648, 340, 711, 1279 and 641 genes were differentially expressed in I/R 0-, 1-, 3-, 6- and 24-h groups respectively. The differentially ex- pressed genes were classified as following 7 functional categories: cytokine, adhesion molecule, growth factor and apoptosis-related factor, oxidation and antioxidation molecule, metabolic enzyme, ion channel and aquaporin, signal transduction molecule. It was suggested that gene chip technology was an effective and quick method for screening differentially expressed genes. Many differentially expressed genes with different functions interacted each other to result in pulmonary ische- mia-reperfusion injury.
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Objective To investigate the change of caspase-3 in rabbits after lung ischemia-reperfusion injury(LIRI) and the effect of puerarin(葛根素).Methods Thirty healthy rabbits used for unilateral lung ischemia-reperfusion model were randomly divided into 3 groups(each n=10): control group(C group),lung ischemia-reperfusion group(I/R group) and puerarin group.The activity of serum superoxide dismutase(SOD),the contents of serum malondialdehyde(MDA) and nitric oxide(NO),the wet to dry weight(W/D) ratio of lung tissue and the index of quantitative assessment of histological lung injury(IQA) were measured respectively in different groups;the pneumocyte apoptosis index(AI) was achieved by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL);caspase-3 protein and mRNA expression were studied by using in situ hybridization(ISH) and immunocytochemistry(IHC) techniques in the groups mentioned above.Results The activity of SOD and content of NO were significantly lower in I/R group than those in C group(both P
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BACKGROUND: Many investigations were done about pulmonary protection in lung transplantation which is the most effective treatment of end-stage pulmonary disease. The objective of this study is to verify the effect of prostacyclin on the ischemia-reperfusion injury in terms of the change of arterial blood gas (ABGA), pulmonary vascular resistance (PVR) and pulmonary water content. METHODS: In twelve mongrel dogs weighing approximately 20 kg, double-lumen endotracheal tube was intubated and Swan-Ganz catheter was inserted. To obtain control data for water content of left lung right postcaval lobe was resected. Left hilum was snared with umbilical tape after collapse of left lung and tightened to clamp. It was maintained for 90 min. Thereafter, ventilation and perfusion of left lung were restored. To control group (n=6), prostacyclin was not given. To prostacyclin group (n=6), prostacyclin was intravenously administered at 250 ng/kg/min for 20 min, just before ischemia and just after reperfusion. We measured hemodynamic variables and analyzed arterial blood gas before and after ischemia and then at every 1 hour interval. At 4 hours after reperfusion, left lung was resected, and water content was measured with wet-dry method. RESULTS: There was no significant difference between control group and prostacyclin group in ABGA, PVR and water content of lung. However, three subjects of prostacyclin group showed higher PaO2 after reperfusion than that of others. CONCLUSION: This study shows that protective effect of prostacyclin is not uniform in severely injured canine ischemia-reperfusion model. We conclude that prostacyclin does not have pulmonary protective effect in severe ischemia-reperfusion injury.