Effects Of Propofol On Hypoxia-reoxegenation Injury In Human Vascular Endothelial Cells And Its Mechanism

Now ischemia reperfusion injury(iRI), is an important program that is critical for-clinical therapies. Since ischemic precondition (IPC) was eoncepted by Murry and the colleagues in 1986, more and more studies have been progressed and found drug. precondition (DPC) had the similar protective effect with IPC, which could activate multiple endogenous substance, such as G- protein coupled receptors, ATP sensitive potassium channels and inhibited the formation of free redicals. DPC has been widely accepted in recent years because its safty and easy to control. Up to date, most of therelated investigations were concentrated on neurocytes and myocardial cells, but vascular endothelial ceils were sensitive to disturbance of circulation and became dysfunction or even suicide in the following serious disease and acute severe cases example for. cerebral arterial thrombosis, myocardial infarction and et al. The study showed that the changes in structureand function of VEC were the genesis, development and pathophysiology basis of IRI. The disorders of VEC could step to aggravate tissue damage and destruction. Although intravenous anesthetics acted as One of the important clinical medications, they had protective effects or not involved in IRI were also contradictive, and the certain mechanisms remain undefined. The purpose of this study was to assess the effect of propofol precondition which was in clinical related concentration on hypoxia -reoxygenation injury in human vascular endothelial ceils, and also to evaluated the action on the expression of intracellular mediators such as nuclear factor kappa B (NF-κB), induced nitric oxide synthase(iNOS), protein kinase C (PKC) and so On, in order to approach the reg- ulatory mechanisms at cellular level. The conclusion would refer a new evidence for the protection of tissue and organs in perioperative period.MaterialsChemical and reagents: propofol (Zneca British), Bcl -2 polyclonal antibody (Santa, America), caspase - 3, PKC polyclonal antibody (Neomarker America), DMEM medium (Gibco, America), FITC - Annexin V (Boehringer Mannheim, German), Fura-2 and propidium iodide (Sigma, America), RTPCR kit (Promega, America).Experiment instruments: inverted phase contrast microscope (OLYMPUS, Japan), desk centrifuge (SIGMA 1-13, USA), PTC-100 PCRamplificator (usa), electrophoresis image analysator (Alphainnotech ChemiImager 5500, USA), CO₂ incubator (KENDRO/HERAEUS, German), superclean bench(Suzhou, China).Methods1 Serial subcultivation of human umbilical vein endothelial cells (HUVECs)HUVECs were isolated by 0.125%. trypsin, cultured in DMEMmedium and incubated in a 95% air/5% CO₂ incubator at 37 C. The cells were transferred as 1×10⁵/mL and the the cells Of passage 3 through 4 were used for experiment. Cells were devided intogroups randomly when grown in Subsequent confluence state, then established hypoxia and reoxygenation model. The immunocytochemical method was used for detection, and the cultured cells were identiffed as vcular endothelial cells when they showed positive stain of antigen related to humanâ…§factor.2. The establishment of hypoxia and reoxigenation modelTo induce hypoxic stress, culture, medium wasaspirated off and cells were replaced with hypoxic endothelial growth medium before introduced into an hypoxia chamber. The cells were removed from the hypoxic chamber after 30min, and were replaced with warm fresh medium to begin a period of reoxygenation. Then they were placed in a 95% air/5% CO₂ incubator at 37 C for 2h, 6h, 24h, 48h. Normoxic control cells were also replaced with fresh culture medium, then incubated.3. Experiment designVECs were separated into three groups, normal control group (C group), hypoxia and reoxygenation group (HR group) and propofol precondition group (PR group). The HR group was separated into four subgroups according to the reoxygenation time-phase, as 2h, 6h, 24h, 48h. The PR group was separated into twelve subgroups further according to the different concentration of propofol (25μmol/L, 50μmol/L, 100μmol/L). The cells of HR group were subjected to 30min hypoxia and followed by reoxygenation 2h, 6h, 24h, 48h. The cells of PR group were subjected to the same experimental condition perior to which incubated with propofol of different concentration for 30min.4. Detection index(1) Apoptotic cells assayApoptotic cells were measured by cytometry using FITC-AnnexinV and PI Staining.(2) The expression of Bcl-2 and caspase-3The expression of Bcl-2 and caspase-3 were detected by immunohistochemistry staining, and Calculated the mean values by optical density detected by MetaMorph 4.5 image analysis software.(3) The expression of PKCThe quantitation of PKC was measured by Western blotting, and was analyzed by electrophoresis image analysator.(4) The intracellular calcium concentrationMarked by fura-2, the calcium was calculated through fluorescence intensity by Meta Flour software.(5) The expression of iNOS and NF-kappaB mRNAQuantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used.5. Statistical analysis All values in the text and figures were presented as mean±SE and subjected to one -factor analysis of variance using SPSS 12.0 software. Homoscedasticity was checked by LSD method and the Dunnett T3 was adopted to check the heterogeneity of variance, Differences were considered significant at P＜0.05.Results1. The effect of propofol on human VECs induced by hypoxia and reoxygenation injuryHypoxia and reoxygenation treatment increased the numbers of apoptosis. Precondition of propofol attenuated the degree of morphous injury followed by hypoxia and reoxygenation, inhibited apoptosis significantly, and showed statistic difference compared with the HR group (P＜0.01). The cells which were pretreated with 50, 100μmol/L propofol submitted more potential effect of anti -apoptosis compared with the 25μmol/L PR subgroups (P＜0.01).2. The effect propofol on the expression of Bcl - 2 in human VECs inducedby hypoxia and reoxygenation injury.The expression Bcl- 2 were low in normal cultured cells, but was significantly downregulated after hypoxia and reoxygenation treatment and approached to the lowest after 24h reoxygenation. Propofol precondition upregulated the expression of Bcl -2 and showed significant defference compared with the HR group (P＜0.01). The subgroups which were pretreated with 50, 100μmol/L propofol submitted more potential effect Compared with the 25μmol/L PR subgroup (P＜0.01).3. The effect propofol on the expression of caspase -3 in human VECs induced by hypoxia and reoxygenation injury.The expression caspase -3 were low in normal cultured cells, but was significantly upregulated after hypoxia and reoxygenation treatment. They were approached to the peak point after 24h reoxygenation. Propofol precondition downregulated caspase -3, and showed significant defference compared with the HR group (P＜0.01). The subgroups which were pretreated with 50, 100μmol/L propofol submitted more effectively compared with the 25μmol/L PR subgroup (P＜0.01).4. The effect of propofol on the intracellular calcium homostasis in human VECs induced by hypoxia and reoxygenation injuryHypoxia and reoxygenation treatment increased the concentration of calcium significantly (P＜0.01) and propofol in different concentration attenuated the intracellular calcium overload (P＜0.01),5. The effect of propofol on the expression of PKC in human VECs induced by hypoxia and reoxygenation injuryHypoxia and reoxygenation treatment decreased the expression of PKC (P＜0.01), but propofol activated it and inhibited the downregulation of PKC induced by hypoxia and reoxygenation (P＜0.01).6. The effect of propofol on the expression of iNOS mRNA in human VECs induced by hypoxia and reoxygenation injuryThe expression of iNOS mRNA was low in the C group, and hypoxia and reoxygenation treatment increased its expression significantly. Propofol inhibited the activation of iNOS, and decreased the expression predominantly, but didnt reach the normal level.7. The effect of propofol on the expression of NF - kappaB mRNA in human VECs induced by hypoxia and reoxygenation injuryHypoxia and reoxygenation activated NF - kappaB, and increased its mRNA expression. Propofol inhibited the expression of NF- kappaB and showed a significant defference compared with the HR group (P＜0.01).DiscussionIn the following cases such as cardiopulmonary resuscitation, organ transplant, thrombolysis, coronary artery bypass and serious patients, insufficient circulation often occurs and ischemia and reperfusion injury became the most se vere question for every doctor inevitablely at that moment. Ischemie precondition was first found by Murry, defined as transient ischemic and repeffusion could decrease the myocardial injury, elevate the tolerance, decrease the infarction size and show an effective myocardial protection. In recent years, because the investigations involved in prevention and cure of IRI were more deeply done, DPC generally played a central role for its easy to application. Belong to the important compose of DPC, the effect of anesthetic precondition (APC) wouldn't be ignored. At present, investigations were concentrated on volatile anesthetics involved in neurocytes and myocardial cells and concluded that pretreatment with isoflurane, sevoflurane and some others would show a protective effect on hypoxia-reoxygenation and IRI. The mechanismswere mainly included ATP sensitive potassium channel, oxygen free radicals, adenosine and et'al, but it was still not known precisely.VECs are the first barrier between blood and tissue and spread widely. The study showed VECs played an important role in IRI. The injury of structure and function in VEC appeared earlier than tissue cells and the recovery was also early. The prognosis of VEC affected the progress of tissue injury. Propofol acted as a new type of intravenous anesthetic, which had antioxidative character, was adopted smoothly in clinical anesthesia and sedation in intensive care unit, but it was still not clear about its protection on IRI by now. Therefore, we used the cultured human umbilical venous endothelial cells in vitro and produced the model of hypoxia - reoxygenation. The effect of propofol precondition on VECs induced by hypoxia reoxygenation was observed and the mechanism was approached. The results indicated propofol could attenuate the apoptosis induced by hypoxia reoxygenation in VEC by a dose related manner and affected the expression of apoptotic mediators, shown as upregulation of Bcl -2 which was an antiapoptotic protein and inhibition of caspase -3 activation. There was a time course relationship between the expression of Bcl -2 and caspase -3. At the same moment, we found propofol effectively decreased the intraceUular concentration of calcium after treated by 30min hypoxia and 6h reoxygenation, and activated PKC significantly compared with that in the HR group. That indicated propofol precondition approached to the cellular protection through relieving the calcium overload by activation of PKC pathway. These results coincided with the preveous studies that showed agonist of PKC had cardiac protection similar to IPC and antagonist of PKC could block it. All these results can document the pathway of PKC is an important link in the precondition to IRI and acts as an ef- fective mediator for maintaining the normal function of cells. Using the method of RT-PCR to measure the expressions of NF-kappaB, and iNOS mRNA. The observation showed propofol could diminish either of them significantly after hypoxia reoxygenation injury. Owing to the effect of iNOS on the numerous derivation of nitric oxide, which played a key role in mediating cell injury, the conclusion revealed the inhibition of activating NF-kappaB and decreasing the expression of iNOS were important in the antiapoptotic effect of propofol precondition.We adopted the model of hypoxia reoxygenation in human VEC, which had a better relationship to the IRI in human. Propofol in a clinical related concentration could provide a more potential reference for practice. The observations about NF-kappaB, iNOS, PKC and the calcium regulating the apoptosis induced by hypoxia reoxygenation approached the mechanism of propofol precondition at a cellular level. All these are the novel and beneficial characteristics of our study, but whether application of propofol can exert organ protection or not in clinic, the trigger and mediator of propofol are still demanded further research.Conclusion1. Hypoxia and reoxygenation injury induced apoptosis in VECs, which increased the expression of caspase-3, a apoptotic promoter, and decreased the expression of Bcl-2, a apoptotic inhibitor. The changes of the two factors showed a time-phase relationship to the hypoxia and reoxygenation injury.2. Propofol inhibited the apoptosis of VECs induced by hypoxia and reoxygenation injury by a dose-dependant manner. The protective protein, Bcl-2, upregulated and the expression of caspase-3 downregulated were the executor.3. Propofol attenuated the increasing of intracellular concentration of calcium induced by hypoxia and reoxygenation injury. PKC signaling passway participated in inhibiting the calcium overload and produced protection.4. Inhibiting NF-kappa B activated by hypoxia-reoxygenation and reducing the iNOS production played an important role in the protection of propofol.