Effect Of Mitochondrial ATP-sensitive Potassium Channels Opening On Ischemia Reperfusion Injury After Rat Lung Transplantation

Part I Establishing the rat model of orthotopic left allograft lungtransplantation assisted with microscope by a single man Objectives: We established the rat model of orthotopic single left allograft lung transplantation using cuff technique assisted with the microscope by a single man, in order to do some research in lung protection and ischemia reperfusion injury(IRI). Methods: Thirty-two male SD rats were randomized into 16 pairs for donors and recipients, allocated into 2 groups(2 hours group, 24 hours group), 8 pairs in each group(n=8), average body weight was 257.5g±23.7g (from 230g to 300g). 16 orthotopic left single lung transplantations were undergone using cuff technique. The functions of the transplanted lung were tested by analysis of the blood gas from the recipient's right cervical artery and allograft left pulmonary vein. Results: Twelve rats were successfully alive more than 24 hours after transplantation. PaO₂ from right cervical artery and left pulmonary artery showed significant difference in 2 hours group after reperfusion of the transplanted lung(222.14±29.43mmHg vs 146.71±35.35mmHg, P=0.001), the same result was seen in 24 hours group after surgery(217.20±32.59mmHg vs 109.80±22.29mmHg, P<0.001). PaCO₂ from right cervical artery and left pulmonary artery showed no significant difference in 2 hours group, 24 hours group after reperfusion of the transplanted lung(P>0.05).Conclusions: It is easy to establish the orthotopic single left allograft lung transplantation model on the rat by using cuff technique by one operator. The model of IRI is stable, replicable and suitable for experiment of lung preservation and IRI. Part II Mitochondrial ATP-sensitive potassium channels opening protects ischemia reperfusion injury after rat lung transplantation Objectives: To investigate the mechanism of the protection of mitochondrial ATP-sensitive potassium channels(mitoKATP) opening on IRI after rat lung transplantation and offer the experimental and theoretical evidence for the choice of IRI treatment in clinical practice.Methods: The animal model was built as the same way of Part I. 120 male rats(SD) were randomized into 60 pairs, and divided to 5 groups as following: Sham group(group S): the rats were undergone thoracotomies without transplantation. The operation time was 90 min; control group with NS(group C): undergone lung transplantation, the recipient received 0.5ml NS by intraperitoneal injection preoperatively; control group with DMSO(group DMSO): undergone lung transplantation, the recipient received 0.5ml DMCO by intraperitoneal injection preoperatively; experimental group with diazoxide(group DA): the recipient received diazoxide 5mg/kg, added NS to 0.5ml by intraperitoneal injection preoperatively; 5-hydroxidecaprate with diazoxide group(group HD-DA): the recipient received 5-hydroxydecanoate 45 min preoperatively(5mg/kg), after 15min the rats received DA, the transplantation began 30 minutes after this injection. We selected two time point(2 and 24 hours after reperfusion of the allograft) to study the lung injury in each group(n=6). The following index were selected to reveal the function and pathophysiological changes of the allografts: blood gas analysis from the right cervical artery and the left pulmonary vein, the ratio of wet/dry lung tissue weight(W/D), the activity of malondialdehyde(MDA), total antioxidative capabilities(T-AOC) both from the serum and the lung tissue as well as the activity of myeloperoxidase (MPO) of the lung tissue. Cytokines such as interleukin-6(IL-6), tumor necrosis fator-alpha(TNF-a) from the lung and serum were quantified by ELISA. The pathological changes of the allograft were examined by microscopy and electro-microscope and apoptosis of alveolar cells were detected by TUNEL. Results:1. When compared group DA with group C, PaO₂ from the left pulmonary vein at the point of postoperative 2 hours showed significant difference(226.0±30.4 vs 152.8±41.9mmHg, P=0.009). The same result can be seen at the point of postoperative 24 hours in these two groups(206.2±36.4 vs 106.5±25.5, P<0.001). Difference of PaO₂ from the cervical artery can only be detected at the point of postoperative 24 hours in these two groups(277.7±37.0 vs 210.2±37.3, P=0.008). When compared group HD-DA(adding the blocker of DA) with group DA , statistic differences of PaO₂ from the pulmonary vein can be detected at the point of postoperative 2 hours(154.8±46.1 vs226.0±30.4, P=0.011), as well as at the point of postoperative 24 hours(111.5±23.2 vs 206.2±36.4, P<0.001).2. When it came to compare the W/D ration in each group, we found no significantdifference in group DA and group C (5.44±0.32 vs 5.69±0.25, P=0.092)at the point of postoperative 2 hours, but statistic differences were found at the point of postoperative 24 hours(5.86±0.20 vs 6.72 ± 0.34, P<0.001)in the tow groups. We found no significant difference of W/D in group HD-DA and group DA (5.72±0.20 vs 5.44±0.32, P=0.062)at the point of postoperative 2 hours, and statistic differences of W/D can be detected at the point of postoperative 24 hours in these two groups(6.65±0.26 vs 5.86±0.20, P<0.001).3. Although no statistic differences could be seen in pathological morphology evaluation in any group, morphological changes of lung injury such as congestion, edema, leukocyte infiltration and exudation were less severe in group DA than those in group C, group HD-DA at any time points. The observation was confirmed by the electronmicroscope, which found less injury of mitochondria in group DA.4. Significant decrease of MDA either in the serum or in the lung tissue of group DA was observed at the time point of postoperative 2 hours, compared with those in group C(2.46±0.46 vs 8.41±0.91nmol/ml, 0.77±0.07 vs 2.72±0.37nmol/mgprot, P<0.001). The same results could also be found at the time point of postoperative 24 hours (2.57±0.45 vs 5.80±1.07, P<0.001, 0.92±0.12 vs 1.27±0.20, P=0.001). When giving the blocker of DA, significant decrease of MDA either in the serum or in the lung tissue of group HD-DA was observed at the time point of postoperative 2 hours, compared with those in group DA(8.72±0.67 vs 2.46±0.46, 2.98±0.41 vs 0.77±0.07, P<0.001). The same results can also be found at the time point of postoperative 24 hours (6.21±0.83 vs 2.57±0.45, P<0.001,1.26±0.21 vs 0.92±0.12, P=0.001).Compared with those in group C, significant decrease of MPO levels in the lung tissue of group DA were observed at the time point of postoperative 2 hours(3.45± 0.37 vs 5.52±0.71U/g wet film, P<0.001) and 24 hours(3.50±0.35 vs 7.92±0.48, P<0.001).In the group adding DA blocker(group HD-DA), MPO levels were higher than those in group DA, either at the time point of postoperative 2 hours(5.46±0.56 vs 3.45 ± 0.37, P<0.001) or 24 hours(7.81±0.41 vs 3.50±0.35, P<0.001).Significant increase of T-AOC either in the serum or in the lung tissue of group DA was observed at the time point of postoperative 2 hours, compared with those in group C(20.98±2.32 vs 9.49±1.57 U/ml, 2.36±0.32 vs 1.4510.25 U/mgprot, P<0.001).The same results can also be found at the time point of postoperative 24 hours(l8.43±1.51 vs 8.10±1.89, 2.19±0.21 vs 1.32±0.18, P<0.001). Comparing group HD-DA with group DA, significantly decrease of T-AOC can be observed in groupHD-DA at both time point. (9.48±1.57 vs 20.98±2.32, 1.32±0.20 vs 2.36±0.32, P<0.001)(7.60±1.39 vs 18.43±1.51, 1.13±0.19 vs 2.19±0.21, P<0.001).5. Serum cytokine showed no difference in each group. But cytokines in the lung tissue were found difference with statistic value. Compared with group C, both TNF-a and IL-6 of group DA were decreased at time point postoperative 2 hours (TNF-a: 4.03±0.51 vs 12.68±0.94ng/mgprot, P<0.001, IL-6: 7.09±0.66 vs 19.92±2.66 ng/mgprot, P<0.001)and 24 hours (TNF-a: 4.14±0.57 vs 6.18±1.38, P=0.001; IL-6: 6.64±0.63 vs 11.21±1.20, P<0.001).When compared with group DA (adding the blocer of DA), the same result was observed in both TNF-a and IL-6 of group HD-DA at 2 hours (TNF-a: 11.89±0.80 vs 4.03±0.51, P<0.001, IL-6:20.41±2.46 vs 7.09±0.66, P<0.001) and 24 hours (TNF-a: 6.02±0.54 vs 4.14±0.57, P=0.002, IL-6: 11.20±0.87 vs 6.6410.63, P<0.001).6. Apoptosis Index(AI) was observed much lower in group DA than that in group C(2.40±0.61 vs 13.20±0.93, P<0.001)at the time point of postoperative 2 hours, but no significant difference at the time point of postoperative 24 hours(2.27±0.62 vs 3.27±0.45, P=0.065). The same thing happened if group HD-DA compared with group DA. At the first time point, AI was higher in group HD-DA than in group DA(12.67±1.07 vs 2.40±0.61, P<0.001), but no difference at the second time point (2.83±0.61 vs 2.27±0.62, P =0.131).Conclusions:1. DA, which opens the mitoKATP can increase the oxygenation of the allograft after transplantation.2. Capillary permeability of the lung could not be significantly improved by opening of mitoKATP, but melioration in morphological changes of lung injury can be observed.3. Lipid peroxidation and PMN aggregation could be inhibited by opening of mitoKATP, coming with the potential of anti-oxidation in the allograft.4. Paracrine and autocrine of cytokines play great role in IRI of the lung. The opening of mitoKATP can suppress this procedure and down regulation of cytokine cascade reaction and then prevention of IRI.5. Observed by our experiment, cell apoptosis is an early result of IRI. The opening of mitoKATP could decrease the apoptosis of alveolar cells at the time point of postoperative 2 hours.Part III The role of NF-κB , TNF-a mRNA and IL-6 mRNA in IRI after rat lung transplantation and their correlation with mitoKATP Objectives: Based on the research in part II, further research works were investigated to find the role of nuclear factor-kappa B(NF-kB), TNF-a mRNA and IL-6 mRNA in IRI after rat lung transplantation and their correlation with mitoKATP. Methods: On the animal model in Part II, fluorescent quantitation of TNF-a mRNA, IL-6 mRNA in the lung was tested by Real Time PCR, and activity of NF-κB was detected by ELISA. Results:1. Significant decrease of TNF-a mRNA in the lung tissue of group DA was observed at the time point of postoperative 2 hours, compared with those in group C(17.00±1.50 E-5 vs 30.82±1.37 E-5, P<0.05), similar results were observed at the time point of postoperative 24 hours(4.34±0.79 E-5 vs 8.13±0.95 E-5, P<0.05). Significant increase of TNF-a mRNA in the lung tissue of group HD-DA was observed at the time point of postoperative 2 hours(28.75±1.96 E-5 vs 11.89±0.80, P<0.05), compared with those in group DA, similar results were observed at the time point of postoperative 24 hours(8.30±0.74 E-5 vs 6.02 ± 0.54 P<0.05).2. Significant decrease of IL-6 mRNA in the lung tissue of group DA was observed at the time point of postoperative 2 hours, compared with those in group C(0.052±0.007 vs 0.138±0.015, P<0.05), but no significant difference was observed at the time point of postoperative 24 hours(0.036±0.003 vs 0.041±0.005, P>0.05). Significant increase of IL-6 mRNA in the lung tissue of group HD-DA was observed at the time point of postoperative 2 hours(0.151±0.009 vs 0.052±0.007, P<0.05), compared with those in group DA, no significant difference was observed at the time point of postoperative 24 hours(0.042±0.005 vs 0.036±0.003, P>0.05).3.Significant increase of NF-κB activity in the lung tissue was observed at any time point of postoperative. Significant decrease of NF-κB in the lung tissue of group DA was observed at the time point of postoperative 2 hours, compared with those in group C(0.49±0.08 vs 1.84±0.20, P<0.05), similar results were observed at the time point of postoperative 24 hours(0.51 ±0.09 vs 1.08±0.17, P<0.05). Significant increaseof NF-κB in the lung tissue of group HD-DA was observed at the time point of postoperative 2 hours(1.93±0.13 vs 0.49±0.08, P<0.05), compared with those in group DA, similar results were observed at the time point of postoperative 24 hours(1.11±0.18 vs 0.51 ±0.09, P<0.05). Conclutions:1. The activity of NF-κB increased after reperfusion at any time point, and it could be inhibited by opening of mitoKATP, and confirmed by the phenomenon of such change can be blocked by 5-HD.2. At any time point after reperfusion, the increase of TNF-a mRNA is correlated with the increase of TNF-a protein and activate of NF-κB. After opening of mitoKATP, down regulation of TNF-a mRNA transcription was detected, but decrease of TNF-a was not happened concomitantly. It suggests that opening of mitoKATP might inhibit priming of NF-κB and transcription of TNF-a mRNA, but there maybe have other fators in transcription in TNF-a.3. The level of IL-6 mRNA, its correlation with NF-κB and mitoKATP at the time point of postoperative 2 hours were similar as TNF-a mRNA, but different at the time point of postoperative 24 hours.