Protective Effect Of Activated Protein Kinase C On Donor Lung In Rats

Background: Lung transplantation (LT) has recently become one of thestandard therapies to prolong life and lung capacity for the patients with end-stage pulmonary diseases. Despite of advances in lung transplantation, there is 15%-20% post-transplantation patients suffered from severe lung injury, inevitable injury of ischemic/reperfusion, increased acute rejection and long term dysfunction of donor lung. Moreover, the patients who are waiting for transplantation are increasing, but 20%-30% of donor lungs only are available.The purpose of the lung preservation is to find a new method to preserve and increase the availability of donor lungs, to prevent the injury of ischemic/reperfusion and to decrease the short or long term complications.Ischemic preconditioning (IP) is a phenomenon by which a brief episode(s) of ischemia increases the ability of the ischemic organ or tissue to tolerate a subsequent prolonged period of ischemic injury. After two decades of extensive research, it has been demonstrated that IP not only exist in different mammalians, but can also protect many organs and tissues. The effect of this phenomenon is affirmative, and served as an endogenous protective mechanism.Nevertheless, this method of organ-protection has not been widely used in the clinic, especially in the surgical field, because of its own shortfalls. To circumvent this, some researchers have used a brand-new organ-protective method - pharmacological preconditioning, on the base of ischemic preconditioning.It has been demonstrated that transient ischemic can release the endogenous triggers, these triggers bind to the G protein coupled receptors on the myocyte surface, activate phospholipase c (PLC), which hydrolyzes phosphatidylinositol 4,5-diphosphate and phosphatidylcholine to produce 1,2-diacylglycerol and inositol-l,4,5-triphosphate. Increased levels of diacylglycerol then activate protein kinase c (PKC) and the translocation of PKC from cytosol to the cell membrane. Activated PKC open the ATP-sensitive potassium channel (KAtp channel) and phosphorylates other effector proteins. This is the early phase of ischemic preconditioning (first window of protection). PKC can also translocate into nucleus and activate transcriptional regulator nuclear factor-? B (NF-?B), and increases the transcription of protective proteins, some of which have been identified so far. For example, superoxide dismutase, which is an important antioxidant enzyme, and heat shock proteins, are upregulated and are important in delayed preconditioning. This is the late phase of ischemic preconditioning (second window of protection.). Most researchers agree that translocation of the PKC plays an important role in triggering initial intracellular signaling cascade and ultimately increased transcription of cardioprotective genes. In some animal models inhibition of PKC alone during the index ischaemia aborts preconditioning, suggesting that PKC is a common step of the ischemic preconditioning but not a trigger.The purpose of this project is to directly test this hypothesis in the lung transplantation using protein kinase c activator. The activator will directly stimulate PKC translocation, trigger intracellular signaling cascade, and ultimately increases transcription of protective proteins. We will determine whether this will protect the donor lung or not.Phorbol myristate acetate (PMA) is an analogue of DAG and is a specific agonist of PKC. The molecular weight of PMA is 616. It has been demonstrated that PKC is the mainreceptor and target of PMA;In contrast to the short-lived DAG, PMA is stable and has the same active group just as DAG.The experimental design is to develop an animal model for the left lung transplantation on rats, perfusing PMA via the pulmonary artery before retrieving the donor lung, preserve the lung in modified EC solution, and then observe the effect of PMA.Part onePurpose: To improve the former cuff techniques and establish a new simplified effective rat lung orthotopic transplantation model.Methods: Preparation of the donor lung: after perfusing the donor lung, wecut the pulmonary artery, pulmonary vein and bronchus in the proximal end, and then pick out the most proper cuff tubes and stent tube. The vessels were drawn through the cuff tubes and everted over the cuff, then tied by 5-0 sutures. Half length of the stent tube was inserted into the donor bronchus and tied by 5-0 sutures.Operation of the recipient: Cut the distal part of the left pulmonary artery ^ vein and bronchus. Anastomose the vessels using traditional cuff technique and anastomose bronchus using stent technique.Results: The transplanted lungs have good and satisfied results. They meetthe criteria of a standard successful transplantation.Conclusion: This model (two-cuff-and-one-stent technique) is much easier and more economical to use on studying lung preservation/transplantation.Part twoPurpose: Based on the ischemic preconditioning mechanism, in which activation of PKC is the common step of the ischemic preconditioning, we perfuse thedonor lung with PMA, the exogenous activator of PKC, and assess the lung preservative effects by analyzing blood gas, SOD, HSP70, apoptotic index, the expression of Bcl-2, and the morphological change of alveolar, and to observe whether PMA can mimic the protective effects of ischemic preconditioning.Methods: (1) SD rats were divided into four groups:Group C (control group, n=10 pairs): Modified EC solution was perfused into donor's pulmonary artery directly after anesthesia, orotracheal intubation and thoracotomy, and then the donor lung was retrieved and the two-cuff-and-one-stent technique was used. After stored in cold modified EC solution for 12 hrs, the donor lung was transplanted into recipient. 30 min after the transplantation was finished;blood was drawn from left pulmonary vein for blood gas analysis and the specimen was collected for later analysis. Group P (preconditioning group, n=10 pairs): The protocol was similar to Group C, except left lung was ischemic preconditioned before perfusing modified EC solution from pulmonary artery. Group B (blockade group, n=10 pairs): The protocol was just like Group P,except polymyxin B was perfused after ischemic preconditioning. Group A (PMA group, n=10): The protocol was just like Group C, except perfusing PMA before perfusing modified EC solution from pulmonary artery.(2)Indicators tested: 1) Blood gas analysis: Blood was drawn from left pulmonary vein after transplantation and records the PO2. 2) Transmission electron microscopy: Morphological and ultrastructural changes of lung tissue were observed. 3) Level of SOD, HSP70 mRNA: To measure the expression of SOD and HSP70 mRNA in the lung tissue. 4) Apoptotic index and the expression of Bcl-2: Apoptotic index was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of Bcl-2 in lung tissue was measured by quantitative immunohistochemistry.(3)Statistical analysis: All data were represented as Mean ± Standard Deviation. Statistical differences between groups were evaluated by one-way ANOVA. A value of p<0.05 was considered significant. All the statistical analysis was carried on using SPSS 11.5 program.Results: (1) PO2 of left pulmonary vein: PO2 in Group P and A weresignificantly higher than that of Group C (P<0.05). In contrast, Group P was not significantly different compared to Group A (P>0.05), but Group B was significantly lower (P<0.05). (2) SOD activity: Compared to Group C, Group P and A were significantly higher (p<0.05). Compared to Group A, all other groups have significantly difference (P<0.05). (3) The expression of HSP70 mRNA: Compared to Group C, Group P and Group A were significantly higher (P<0.05). Compared to Group A, Group B> C were significantly lower (p<0.05). (4) Apoptotic index: Compared to Group C, Group A and C were significantly difference (P<0.05). Compared to Group A, Group C and B had significantly difference (P<0.05). (5) The expression of Bcl-2: Compared to Group C, Group P and A were significantly difference (P<0.05). Compared to Group A, Group C and B had significantly difference (P<0.05). (6) H & E stain: the lung tissues in Group P and A showed a little edema and swelling (both in interstitial and alveolar space), and a little congestion in interstitial space. These changes became more prominent in Group C and B. (7)Transmission electron microscopy: In Group C and B, the cells of alveolar were smaller, and the nuclear chromatin was condensed, appeared as "semi-lunar" shape. There were less organelles were found, including mitochondria and lamellar body. Fewer changes were found in Group P and A.Conclusions:1. Ischemic preconditioning has lung protective effect.2. Activating PKC by perfusing PMA can mimic the protective effect of ischemic preconditioning. In the aspect of SOD activity, pharmacological preconditioning is even better than ischemic preconditioning.3. PKC is the mediator of the ischemic preconditioning. The ischemicpreconditioning can be blocked if PKC is inactivated;even the preconditioning has been triggered.4. Once the pharmacological preconditioning started, preconditioning can continue to have effect on this process even though the prime trigger disappeared.5. The activity of SOD in Group A are higher than that of Group P. It was demonstrated that SOD might be the direct effector of PKC.6. The underlying mechanism of the pharmacological preconditioning might be that the triggers activate PKC, up-regulate SOD, HSP and Bcl-2, ameliorate the injury of ischemic/reperfusion and inhibit apoptosis, which will protect the construction and the function of the lung.