| BackgroundReperfusion of ischemic tissues or cells is the most common pathologic process in operative surgery, leading to widespread microvascular dysfunction and tissue/cell injury which in turn may affect prognosis and may directly endanger the patients' life. Nutrients carried in the blood are released to tissues via the permeable endothelium of blood vessels, and blood vessel damage is the basic pathologic process in ischemia/reperfusion (I/R) injury. Endothelial cells are important barriers between the blood vessels and tissues. Studies have shown that the vascular endothelium is a crucial site that is affected by I/R injury, and the apoptosis of endothelial cellsis prior to that of tissue cells. Damage of vascular endothelial cells leads to the infiltration of intravascular inflammatory mediators and inflammatory cells directly into the tissue, and as a consequence aggravates tissue damage. Therefore, it is of great clinical significance to study the molecular mechanism of I/R injury.Liver damage is induced by a variety of pathological ways like liver ischemia/reperfusion and liver fibrosis. Liver ischemia/reperfusion injury is a phenomenon in which cellular damage is induced by hypoxia following the return of blood flow and restoration of oxygen delivery after transplantation surgery, tissue resections, hemorrhagic shock and so forth. Therefore, it is a crucial consideration in the management of hepatobiliary surgery, especially liver resection and liver transplantation surgery, which remains a major cause of graft dysfunction or nonfunction and results in a high mortality. Liver I/R injury usually occurs during traumatic shock or hepatic surgery. It affects the liver function and also significantly increases the risk to the circulatory system and respiratory system. All these issues make liver ischemia/reperfusion a highly important clinical problem to be studied and solved.Propofol is a common intravenous anesthetic, and it is widely used because of the minimal side effects, a quick onset, fast metabolic rate in vivo and controllable anesthetic state. Evidences have shown that propofol is involved in the protection of many organs during surgery, including brain, lung, spinal cord, cardiovascular, kidney and etc. Although many progresses have been gained in the molecular mechanisms of propofol's protective effects on tissue I/R injury, there are still many aspects to be investigated. For example, investigation of genes involved in this process is of great significance for drug development and clinical application in ischemia-reperfusion injury.MicroRNAs (miRNAs) are an evolutionarily conserved family of short noncoding RNAs at the length of 18-22 nt, which regulate genes via binding to the 3'-untranslated region (UTR) of their target mRNAs. Studies provide an overview of the role of miRNAs in changing cell activities induced by propofol treatment. For example, propofol can effectively induce apoptosis of epithelial ovarian cancer (EOC) cells by upregulating miR-let-7i. In addition, propofol may have antitumor potential in osteosarcoma, which is partly due to the downregulation of MMP-13 expression by miR-143. However, miRNAs that are associated with the protective effect of propofol on I/R injury remain largely unknown. As a member of the miR-17/92 cluster, miR-17 was initially found abnormally amplified in diffuse cell lymphomas. Subsequent studies reveal that miR-17 acts as an oncogene which is related to the development of many tumors. On the other hand, miR-17 is highly expressed in embryonic cells and has an important role in the development of many tissues including heart, lung and brain.Propofol is widely used in clinical anesthesia, and it has been reported to have a protective effect in ischemia/reperfusion injury in the heart, brain and lower limbs. Similarly, in liver transplantation, propofol is extensively used because its metabolism is not greatly affected by liver failure. Volatile anesthetic agents have been shown to preserve hepatic blood flow and cell function after ischemia of the liver. Sevoflurane, a new inhalation anesthetic, has recently been developed and is now frequently used. Sevoflurane is known to act as a bronchodilator by directly relaxing the bronchial smooth muscle through a reduction in Ca2+concentration and perturbation of calcium homeostasis, similarly to other inhalation anesthetics.In this study we intend to investigate the molecular mechanism of miR-17 in the protective effect of propofol on vascular endothelial cell I/R injury and to provide experimental evidence for clinical application of propofol as a protect drugfor suppressing I/R injury. Consequently, the aim of this study was to explore whether propofol and sevoflurane have protective effects in hepatic ischemia reperfusion in a rat model, and subsequently the mechanisms through which they play their roles to provide basic evidence for their clinical use.Part?Propofol Inhibits MicroRNA-17 Expression to Activate STAT3 Signaling Pathway in HUVECS during Hypoxia-reoxygenationObjectiveThis study is to investigate the role of miR-17 in propofol induced protective effects in vascular endothelial cells.Methods1. Hypoxia-reoxygenation (H/R) model was established in human umbilical vein endothelial cells (HUVECs) to mimic the in vivo situation of ischemia-reperfusion; For hypoxia, the culture media was replaced by synthetic ischaemia solution as described before and the HUVECs were then placed in hypoxic conditions with5% CO2 and 95% N2 at 37? for 24 h. After hypoxia, the medium was washed off, and the HUVECs were returned to reperfusion solution as described before and cultured with 5% CO2 and 95% O2 for 6 h. At the same time, prepared propofol was added to the medium at the concentration of 150 ?mol/L;2. Cell viability was detected by CCK8 to confirm the role of propofol induced protective effects in vascular endothelial cells;3. Annexin V-FITC staining and flow cytometry analysis was performed to examined the uppression of miR-17 inhibits HUVECs cell apoptosis;4. SDS-PAGE and Western blot assay was performed to analyze Protein abundance of Bcl-2, Bax and GAPDH, in oder to analyze effect of miR-17 inhibitor on cell apoptosis;5. Real-time PCR was used to analyze the expression level of miR-17 in HUVECs after propofol treatment.6. Western blot assay was further performed to analyze expression of STAT3 protein;7. Luciferase reporter gene assay was performed to determine whether the observed reduction of STAT3 expression is directly driven by miR-17.Results1. CCK-8 assay showed that after exposure to H/R, propofol treatment significantly increased cell viability. Transfection with miR-17 inhibitor resulted in upregulation of cell viability after exposure to H/R. These results suggested that administration of propofol could protect HUVECs against H/R, and this protec-tion may dependent on the regulation miR-17 expression;2. To confirm the inhibitory effect of miR-17 inhibitor on cell apoptosis, HUVECs were transfected with miR-17 inhibitor or scramble, stained with Annexin V-FITC and propidium iodide, and then analyzed by flow cytometry after exposure to H/R. There was an obvious reduction in the number of apoptotic cells when propofol was administrated compared with the untreated H/R group. A similar reduction in the number of apoptotic cells was observed when cells were transfected with miR-17 inhibitor compared with that transfected with scramble;3. We then examined the apoptosis-related proteins by Western blot. After H/R exposure, apoptotic protein Bax was significantly downregulated while anti-apoptotic protein Bcl-2 was upregulated upon propofol treatment. In addition, the expression of these proteins was in the same tendency upon miR-17 inhibitor transfection after hypoxia treatment;4. Using Real-time PCR analysis, we found that miR-17 expression was not obviously changed in normal HUVECs and HUVECs subjected to H/R. However, its expression was significantly reduced after propofol treatment in both normal cells and cells stimulated with H/R. These results indicated that miR-17 may play a role in the protection of invascular endothelial cell injury induced by H/R;5. STAT3 activation results in dysregulation of cell cycle control and abnormal expression of apoptosis genes, leading to cell apoptosis. Therefore, miR-17 may inhibit endothelial cell apoptosis through targeting STAT3. To determine whether miR-17 could directly target STAT3 in HUVECs, we performed Western blot and found that expression of STAT3 protein is significantly downregulated upon miR-17 mimic transfection;6. The 3'-UTR of STAT3 was cloned into the pMIR-REPOR Luciferase control vector. In addition, we generated a mutated reporter construct in which the miR-17 seed match sequence in STAT3 3'-UTR was mutated. Co-transfection of the STAT3 3'-UTR construct and miR-17 yielded a significantly reduced relative luciferase activity.In contrast, the mutated STAT3 3'-UTR construct was not affected by overexpression of miR-17 compared with NC. These data demonstrated a direct interaction between STAT3 mRNA and miR-17.ConclusionPropofol ameliorates invascular endothelium damage after ischemia-reperfusion, and one possible mechanism is the activation of STAT3 signal pathway mediated by downregulation of miR-17. Further studies are still needed to reveal the specific downstream molecular mechanisms.Part ?The Effects of Two Anesthetics, Propofol and Sevoflurane, on Liver Ischemia/Reperfusion InjuryObjectiveThis study aims to investigate and compare the effects of propofol and sevoflurane on liver ischemia/reperfusion and the precise molecular mechanism.Methods1. Male Sprague-Dawley rats weighing 250-280 g were obtained from the Vital River Company. Rats were randomized into four groups:the sham group, I/R group, propofol treatment group (infused with 1% propofol at 500 ?g·kg-1·min-1), and sevoflurane treatment group (infused with 3%(2 L/min) sevoflurane);2. Liver tissues were collected and fixed in 10% formalin for more than 24 hours, embedded and sectioned at 4 ?m thickness. Subsequently, the sections were stained with hematoxylin-eosin. Inflammation and tissue damage were evaluated in histopathological analysis by light microscopy.3. To assess liver injury, we used several markers, including AST (aspartate aminotransferase), ALT (alanine aminotransferase), and LDH (lactate dehydrogenase), released into the serum;4. Enzyme-linked immunosorbent assay (ELISA) analysis was performed to detect the concentrations and levels of the inflammatory mediators (TNF-a, IL-1, IL-10 and IL-6) in the serum;5. The mRNA expression levels of TNF-?, IL-1. IL-6 and IL-10 in liver tissues were determined by real-time PCR in each group;6. SDS-PAGE and Western blot assay was performed to analyze the effects of propofol and sevoflurane on p-p65 and I?B?, expression;7. The levels of oxidative stress biomarkers MDA and NO and the antioxidant marker SOD in liver tissue were evaluated using specific detection kits for rats according to the manufacturer's protocols;8. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed to detect apoptosis in the liver tissues according to the manufacturer's instructions. Quantitative analysis is presented as a percentage of TUNEL-positive hepatocyte nuclei per total nuclei in each experimental group;9. Western blot assay was further performed to analyze the effects of propofol and sevoflurane on Bcl-2 family, AKT and p38 MAPK signaling pathways;10. Immunohistochemical staining for p-Akt and p-p38 was performed on liver sec-tions from rats to analyze the effects of propofol and sevoflurane treatment influenced the expression of p-Akt and p-p38 proteins in hepatic I/R injured rats.Results1. Histological evaluation of the damage in the liver was performed by hematoxylin-eosin staining. Pathological changes in the IR group, indicated by Suzuki's score, revealed the severe damage induced by IR; similarly, propofol and sevoflurane depressed the damage indicated by Suzuki's score;2. AST, ALT, and LDH released into the serum. These enzyme levels in the I/R group were increased significantly, while both propofol and sevoflurane decreased the leakage of all three enzymes;3. Inflammatory cytokines, such as TNF-a, IL-1, IL-6 and IL-10, play key roles in the pathophysiology of hepatic I/R injury. To determine whether propofol and sevoflurane reduce the levels of these cytokines, we used ELISA and QRT-PCR to measure the level and the mRNA expression in the liver tissue. Propofol and sevoflurane both reduced the release and mRNA expression of TNF-a, IL-1, and IL-6 and increased IL-10;4. Propofol showed significantly lower IL-1 and higher TNF-? release compared with sevoflurane. Subsequently, the expression of NF?B was detected by western blot to investigate whether the alteration of inflammatory cytokine release is regulated by NF?B. As expected, the elevated phosphorylation of P65 induced by I/R is suppressed by propofol and sevoflurane treatment. In contrast, the expression of I?B? was upregulated by propofol and sevoflurane. Propofol exhibited a stronger inhibitory effect on NF-?B expression;5. The MDA, SOD and NO levels were assayed to evaluate the effect of propofol and sevoflurane on oxidative stress. The levels of MDA and NO were significantly increased during ischemia/reperfusion and recovered by propofol and sevoflurane treatment. The level of SOD was increased in the propofol and sevoflurane treatment groups compared with the I/R group;6. The TUNEL assay was used to detect apoptosis in the ischemic liver. The apoptotic rate in the I/R group was significantly increased, and propofol and sevoflurane both attenuated the apoptosis;7. Propofol and sevoflurane inhibited the downregulation of antiapoptotic proteins such as bcl-2 and bcl-xl while decreasing the upregulation of proapoptotic proteins such as bak and bax in ischemic liver injur, ultimately downregulating the expression of cytochrome c and caspase protein (cleaved-caspase 9, cleaved-caspase 3), which induce apoptosis in liver tissue. In addition, we found that sevoflurane had a better effect in regulating the expression of the proteins mentioned above, except for caspase 3;8. We investigated the total expression and phosphorylation of AKT, BAD and p-38, which play crucial roles in ischemia/reperfusion-induced apoptosis. Propofol enhanced the phosphorylation of AKT while inhibited the phosphorylation of BAD. Sevoflurane significantly reduced the phosphorylation of p-38 induced in liver ischemia reperfusion, while no changes were found in the total expression or phosphorylation of AKT and BAD;9. Immunohistochemical detection of p-AKT and p-p38 in the liver tissue was consistent with previous findings. The phosphorylation of AKT was elevated in propofol treatment, while the phosphorylation of p38 was inhibited in sevoflurane treatment.ConclusionPropofol and sevoflurane both protect the liver from ischemia/reperfusion injury through modulation of several vital pathophysiological processes such as altered cytokine release; oxidative stress with changed ROS generation; and apoptosis. These processes are interrelated and throw light on the precise mechanism of protection.. Further research in this field is warranted which will provide the necessary information for the clinical use of propofol and sevoflurane. |
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