Effects And Mechanisms Of Propofol On ACE2-Ang(1-7)-Mas Axis And μ-opioid Receptor

Propofol is a widely used sedative-hypnotics in clinic. Recent studies have shown that propofol also has the effect of lowering blood pressure and analgesia, but the mechanism has not been reported. The renin-angiotensin system (RAS) is an important humoral regulation system in human body, has an important role in the normal development of cardiovascular system and blood pressure regulation. The angiotensinogen generate angiotensin I (Ang I) by the effect of renin, and the latter may be further hydrolyzed to the the strong vasoconstriction protein AngⅡ by the angiotensin converting enzyme (ACE),at the same time ACE2can degrade AngⅡ to Ang-(1-7) which has relaxation effect. It is not clear whether propofol regulate the blood pressure by influencing the formation of ACE2-Ang-(1-7) axis.Mas receptor is a functional combination of Ang-(1-7) receptor, and it is an important component elements related to pain-producing in RAS, but the effect of propofol on Mas receptor’s expression and activity are not clear.In clinic anesthesia, propofol is easy to be control with rapid onset and other advantages in a wide range of clinical applications, but propofol also has some adverse reactions such as addiction. The μ-opioid receptor has an important role in the generation of drug tolerance and dependence. To explore the effect of propofol to μ-opioid receptors,clear the mechanism of drug dependence, is conducive to a more rational use of propofol, have important academic value and potential clinical application.Objective:To observe the influence of propofol on human pulmonary artery endothelial cells to ACE2, and to explore the influence of propofol to rat dorsal root ganglion cells Mas receptor expression and activity, and to state the mechanism of propofol on human neuroblastoma cellsμ-opioid receptor regulation.Methods:(1) Effect of propofol to ACE2of human pulmonary artery endothelial cells:original generation human pulmonary artery endothelial cells (HPAEC) for the study, cells were identified by immunofluorescence; Real-time PCR detection thee ACE2mRNA expression in HPAEC treated by propofol of different concentrations (1,10,20,40,50umol/L)and at different time points (6,12,18,24,3Oh); Western blot detection the ACE2protein expression in HPAEC treated by propofol of different concentrations (1,10,20,40,50mmol/L); detection ACE2activity on HPAEC cell membrane; Observe the influence of the expression of ACE2mRNA treated by transcriptional inhibitor Act-D in HPAEC; Observe the phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002) regulations ACE2mRNA expression in HPAEC.(2) Effects of propofol on Mas receptor expression and activity in rat dorsal root ganglion cells:rat dorsal root ganglion cells as the research object, Real-time PCR detect Mas receptor mRNA expressionin rat dorsal root ganglion cells treated by propofol of different concentrations (0.1,0.5,1.5or10mmol/L) and at different time points (0.5,1,2,4or6h); Observe Mas receptor mRNA expression in rat dorsal root ganglion cells treated by transcriptional inhibitor Act-D and propofol; Observe the Mas receptor mRNA expression in rat dorsal root ganglion cells adjusted by phosphatidylinositol3-kinase kinase inhibitor (LY294002), protein kinase C kinase inhibitor (Go6983), split activated protein kinase kinase inhibitor (PD098059) and p38mitogen-activated protein kinase inhibitor (PD169316); Western Blot assay Mas receptor protein expression treated by PD169316; Observe Mas receptor and Ang-(1-7) binding activity treated by PD169316in rat dorsal root ganglion cells.(3) Effect of propofol on μ-opioid receptor regulation mechanism in human neuroblastoma cells:human neuroblastoma cells for the study, Real-time PCR detect the μ-opioid receptor’s mRNA expression treated by propofol of different concentrations (1,5,10or20mmol/L) and at different time points (6,12or24h) in human neuroblastoma cells; Western Blot detect μ-opioid receptor protein expression treated by propofol of different concentrations (1,5,10, or20mmol/L) in human neuroblastoma cells; Observe transcriptional inhibitor Act-D regulations mu-opioid receptor mRNA in human neuroblastoma nerve cells treated by propofol; Observe the effects of propofol on the human neuroblastoma cell membrane mu-opioid receptor binding activity.Results:1. Effect of propofol on ACE2in human pulmonary artery endothelial cells(1) When propofol concentration is10-40umol/L, the process having a dose-and time-dependent manner within the time range of24h to increase ACE2mRNA levels of treated cells (P<0.05).The influence of propofol concentration of1-10mmol/L level to ACE2mRNA expression was not significantly (P>0.05).(2) Western Blot test results showed that propofol can increase the HPAEC ACE2protein expression, and a dose-dependent manner within24hours. In the cells for24h, propofol dose-dependently increase ACE2AngⅡ into Ang1-7activity in the HPAEC on the cell membrane.(3) Real-time PCR analysis showed that ACT-D can inhibit the effects of propofol on HPAEC in ACE2mRNA upregulation (P<0.05).(4) In24hours, propofol increase of Akt phosphorylation dose-dependently, and LY294002to eliminate the influence of propofol on the phosphorylation of Akt.2. Effects of propofol on Mas receptor expression and activity in rat dorsal root ganglion cells(1) propofol in concentrations ranging from0.5to10micromol/L can be a time-dependent increase Mas receptor mRNA expression in rat dorsal root ganglion cells(P<0.05). When the concentration is0.1-10umol/L,propofol had no significant effect to Mas receptor mRNA levels(P>0.05).(2) The test results showed that Mas receptor mRNA levels in rat dorsal root ganglion cells treated by the transcriptional inhibitor Act-D can be reversed significantly, Act-D can restrain propofol’s up regulation to Mas receptor (P<0.05).(3) The test results showed that the inhibitor LY294002, Go6983, PD098059had no significant effect to Mas receptor mRNA expression in rat dorsal root ganglion cells(P>0.05), and PD169316can reverse Mas receptor mRNA and protein expression up regulated by propofol in rat dorsal root ganglion cell (P<0.05).(4) propofol promotes the binding of Ang-(1-7) and Mas receptor on the cell membrane dose-dependently in4hours, and this effect can also be reversal by PD169316(P<0.05).3. Effect of propofol on μ-opioid receptor regulation mechanism in human neuroblastoma nerve cells(1)When the concentration is1-10umol/L and the treatment time is in12h, propofol increase μ-opioid receptor mRNA and protein expression in human neuroblastoma cells significantly (P<0.05).(2) propofol can enhance human neuroblastoma cell membrane of nerve cells on the μ-opioid receptor binding activity.(3) The test results show that, in medium containing actinomycin D, propofol had no significant effect to μ-opioid receptor mRNA levels in human neuroblastoma cells (P>0.05).Conclusions:1. Propofol increases the expression and activity of ACE2and mas receptor ACE2-Ang (1-7)-mas axis through the PI3K/Akt and p38MAPK signaling pathways and redresses the balance between ACE-Ang Ⅱ-AT1axis, and thus plays relaxing blood vessels and analgesic effects.2. Propofol can increase human neuroblastoma tumor cells μ-opioid receptor expression and activity may be associated with propofol addiction.