| Part 1 Effects of propofol on serum sleep-related inflammatory factors and cognitive function in rats after sleep deprivationOBJECTIVE:To investigate the effects of propofol-induced sleep on sleep-related inflammatory factors and cognitive function in rats after sleep deprivation(SD)by establishing a rapid eye movement sleep SD model.METHODS:A total of 100 male Sprague-Dawley rats aged 9?12 weeks,healthy and weighing 260?320 g were randomly divided into normal control group(C group),SD group,SD+intralipid group(SD-F group),SD+30 mg/kg propofol group(P30 group),SD+60 mg/kg propofol group(P60 group),20 rats in each group.A modified multi-platform aqueous phase SD model for rats was used,and persisted SD for 96 hours.Each rat in SD-F group was intraperitoneally injected with 1 mL of 10%intralipid after SD.Assessments were performed after an hour.The rats in P30 group and P60 group were given intraperitoneal injection of propofol 30 mg/kg or 60 mg/kg.Assessments and behavioral tests were performed after spontaneous recovery for an hour.Morris water maze was used to detect the cognitive function.The hypothalamus,hippocampus,adrenal gland and liver were observed by HE staining.Serum sleep related inflammatory factors(IL-?,TNF-?)and cytochrome C(Cyt-C)were detected by ELISA.The hippocampal neurons were observed by transmission electron microscopy.RESULTS:Behavioral test results of water maze:Compared with group C,the escape latency of SD group and SD-F group was prolonged and the times of spatial exploration decreased(P<0.05).Compared with SD group,the numbers of escape latency and space exploration of SD-F group were no significant difference.The escape latency of both P30 and P60 were decreased and the times of spatial exploration increased(P<0.05).HE detection showed that the hypothalamic,hippocampus,adrenal gland and liver structure in the C group were normal with arranged cells and clear nucleous.Compared with C group,the morphological features of hypothalamic cells in other groups were homologours,The cells of adrenal and liver were swelling and loosen in other groups.The pyramidal cells in hippocampal CA1 area were reduced and widened in both SD group and SD-F group.The arrangement and density of pyramidal cells in hippocampal CA1 area of P30 group and P60 group were higher than in the SD group and SD-F group.Transmission electron microscopy showed that the neurons in the hippocampal CA1 area of group C were intact,nuclear morphology and chromatin distribution were uniform.Neurons in hippocampal CA1 area of SD group and SD-F group showed nuclear pyknosis,fragmentation and heterochromatin.The phenomenon of abundance and other phenomena showed apoptotic bodies;the nuclear membrane of hippocampal neurons in P30 group and P60 group was still intact,the chromatin distribution was relatively uniform and without apoptotic bodies.Compared with C group on the expression levels of IL-1? and TNF-? in serum,they were increased in SD group,SD-F group,P30 group and P60 group(P<0.05).Compared with SD,the expression levels of IL-1?,TNF-? and Cyt-C in SD-F group were no difference,the levels of IL-1? and TNF-? of both P30 group and P60 group were increased(P<0.05),and the leves of Cyt-C were decreased(P<0.05).Compared with group C,the levels of Cyt-C of both SD group and SD-F group were increased(P<0.05),and were no difference in both P30 group and P60 group.Conclusion:Sleep deprivation might caused multiple organ damage in rats.Propofol-induced sleep might increased the levels of serum IL-1? and TNF-? in rats after sleep deprivation,decreased the level of Cyt-C,alleviated neuroapoptosis in hippocampal CA1 region,and improved cognitive function in rats after sleep deprivation.Part 2 Propofol improved sleep to attenuate mitochondrial damage in hippocampal neurons after sleep deprivation in ratsOBJECTIVE:To investigate the effects of propofol on hippocampal neuronal apoptosis in rats after sleep deprivation(SD),and to explore the possible mechanism of propofol improving hippocampal neurons.METHODS:A total of 87 male Sprague-Dawley rats aged 9?12 weeks,healthy and weighing 260?320 g were randomly divided into normal control group(group C),SD+intralipid group(SD-F group),SD+30 mg/kg propofol group(P30 group),SD+60 mg/kg propofol group(P60 group),15 rats in each group.SD group(SD group)had 27 rats.A modified multi-platform aqueous phase SD model for rats was used and maintained SD for 96 hours.Some rats in SD group were sacrificed for detecting hippocampal inflammatory factors(IL-1?and TNF-?)and cytochrome C(Cyt-C)at 48 and 72 hours after SD.Each rat in SD-F group was intraperitoneally injected with 1 mL of 10%intralipid after SD.Assessments were performed after an hour.The rats in P30 group and P60 group were given intraperitoneal injection of propofol 30 mg/kg or 60 mg/kg.Assessments were performed after spontaneous recovery.ELISA was used to detect the levels of IL-1?,TNF-?,Cyt-C and ATP in hippocampus tissue.Immunohistochemistry was used to detect the positive cells of cleaved caspase-3 in hippocampal CA1 region.Western blot was used to detect the relative expression of apoptosis-related proteins Bcl-2,Bax and cleaved caspase-3.The morphological changes of hippocampal neurons were observed by transmission electron microscopy.RESULTS:By the transmission electron microscopy,the mitochondria were uniform in size with smooth outer membrane but no vacuoles in C group.In the SD group and the SD-F group,the mitochondria were different and swelling with rough outer membrane and lots of vacuoles,and the signs of autophagy were observed.In P30 group and P60 group,the mitochondria were clearly visible,little swelling without vacuoles.Compared with C group,the relative levels of TNF-? and the ratio of positive cells with cleaved caspase-3 protein were increased in other groups(P<0.05).The relative expression levels of Cyt-C,Bax and cleaced caspase-3 were increased and the relative expression levels of IL-1?,ATP and Bcl-2 were decreased in both SD group and SD-F group(P<0.05).The relative expression levels of TNF-? were increased in both P30 group and P60 group(P<0.05).Compared with SD group,the relative levels of IL-1?,TNF-?,Cyt-C,ATP,Bcl-2,Bax,cleaved caspase-3 and ratio of positive cells with cleaved caspase-3 protein in hippocampal of SD-F group were no difference.The relative expression levels of IL-1? in hippocampus were increased and the relative expression levels of TNF-?,Cyt-C,Bax,cleaved caspase-3 and the ratio of positive cells with cleaved caspase-3 protein were decreased in both P30 group and P60 group(P<0.05).Compared with group T0,the levels of IL-1? in hippocampus of rats were decreased,the levels of TNF-? were increased after 48 h,72 h and 96 h SD(P<0.05),and levels of Cyt-C were increased after 72 h and 96 h SD(P<0.05).Compared with T48 group,the relative levels of IL-1? in hippocampus of rats were decreased after 96 h SD(P<0.05),the relative levels of TNF-? in hippocampus of rats were increased after 96 hours SD(P<0.05),the relative levels of Cyt-C in hippocampus of rats were increased after 72 h and 96 h SD(P<0.05).Conclusion:The intervention of propofol might increased the levels of IL-1? and decreased the levels expression of TNF-?,Cyt-C,Bax and cleaved caspase-3.Propofol-induced sleep might attenuated hippocampal neuronal apoptosis for sleep deprivation in ratsPart 3 Propofol improved sleep to attenuate the sleep deprivation induced hippocampal neuronal injury by pinkl/parkin autophagy signaling pathway in ratsOBJECTIVE:To investigate the role of PINK1/Parkin autophagy signaling pathway on propofol alleviating the neuronal impairment of rat hippocampal neurons after sleep deprivation(SD).METHODS:A total of 181 male Sprague-Dawley rats aged 9?12 weeks,healthy and weighing 260?320 g were selected.The effects of different dosage of propofol on autophagy protein Beclin1 were studied.Rats were divided into normal control group(group C),SD group(SD group),SD+propofol 5 mg/kg group(P5 group),SD+propofol 10 mg/kg group(P10 group),SD+propofol 15 mg/kg group(P15 group),SD+propofol 30 mg/kg group(P30 group),SD+propofol 45 mg/kg group(P45 group)and SD+propofol 60 mg/kg group(P60 group),the relative expression level of the autophagy-related protein Beclin1 was detected by Western blotting.Secondly,to detect the effects of autophagy on hippocamp1 neurons after SD,rats were divided into normal control group(C group),SD group(SD group),SD+intralipid group(SD-F group),SD+propofol 30 mg/kg group(P group),SD+rapamycin 0.5 mg/kg group(R group),SD+rapamycin 0.5 mg/kg+propofol 30 mg/kg group(PR group),SD+3-MA 0.5 mg/kg Group(3-MA group)and SD+3-MA 0.5 mg/kg+propofol 30 mg/kg group(PM group),The behavioral changes of rats were detected by water maze behavior.The morphological changes of hippocampal neurons were observed by transmission electron microscopy.The relative expression of autophagy-related proteins Beclinl and LC3?/? in hippocampus was detected by Western blotting.Finally,to detect the role of PINK 1/Parkin autophagy signaling pathway on propofol alleviating the neuronal impairment of rat hippocampal neurons after SD,rats were divided into:control group(group C),SD+intralipid group(SD-F group),SD+30 mg/kg propofol group(P30 group),SD+60 mg/kg propofol group(P60 group).A modified multi-platform aqueous phase SD model for rats was used,and maintained SD for 96 hours.The expressions of autophagy-related proteins PINK1,Parkin and LC3 gene were detected by qRT-PCR.The positive cells with the proteins of PINK1,Parkin and LC3 in hippocampal CA1 neurons were detected by immunohistochemistry.The relative levels of autophagy-related proteins of PINK1,Parkin,Beclinl,LC3?/? and p62 were detected by Western blot.RESULTS:?The effect of various dosages of propofol on the relative expression levels of autophagy related protein Beclinl:Compared with group C,the relative expression levels of Beclin1 protein in SD group,P5 group,P10 group and P15 group were increased(P<0.05).Compared with SD group,the relative expression of Beclinl protein in P30 group,P45 group and P60 group decreased(P<0.05).Compared to P5 group,P10 group and P15 group,the relative expression of Beclin1 protein in P30 group,P45 group and P60 group were reduced(P<0.05).?The results of autophagy enhancer rapamycin and autophagy inhibitor 3-MA showed that the relative expression of LC3?/? and Beclin1 protein in SD group,SD-F group and R group were higher than that in group C(P<0.05).Compared to SD group,SD-F group,R group and PR group,the relative expression of Beclin1 and LC3?/? protein in P group,PM group and 3-MA group were decreased(P<0.05).The behavioral results of MWM on the 5th day:compared with C group,the escape latency of SD group,SD-F group,PR group and R group were prolonged(P<0.05).Compared to SD group,PR group and R group,the escape latency of P group and PM group were decreased(P<0.05).Compared with R group,the escape latency of 3-MA grop was decreased(P<0.05).Compared with C group,the times of crossing platfroms of SD group,SD-F group,PR group and R group were decreased(P<0.05).Compared to SD group and R group,the times of crossing platforms of P group,PM group and 3-MA were increased(P<0.05).To observe by TEM,the mitochondrial morphology was normal in C group,P group and 3-MA group.The mitochondrial autophagy were found that mitochondrial membrane structure were surrounded by phagocytic vacuoles with wrapped and fragmented mitochondria in SD group,SD-F group,R group and PR group.?To investigate the PINK1/Parkin autophagy signaling pathway in the hippocampal neuronal injury induced by propofol in rats after sleep deprivation:To detcte by qRT-PCR,compared with group C,the relative expression levels of mRNA of PINK1,Parkin and LC3 gene in hippocampus were increased in both SD group and SD-F group(P<0.05),and were decreased in both P30 group and P60 group(P<0.05).Compared with SD group,the expression levels of mRNA of PINK1,Parkin and LC3 gene in hippocampus of both P30 group and P60 group were decreased(P<0.05).Compared with C group,the relative expression levels of PINK 1,Parkin,Beclinl,p62 and LC3?/? protein were increased in SD(P<0.05).Compared with SD group,the relative expression of PINK 1,Beclin1,p62 and LC3?/? protein in P30 group and P60 group decreased(P<0.05).Compared with C group,the ratio of positive cells with protein of PINK1,Parkin and LC3 in hippocampal CA1 region were increased in SD group,SD-F group and P30 group(P<0.05).Compared with SD group,the ratio of positive cells with protein of PINK1,Parkin and LC3 in hippocampal CA1 region were decreased in both P30 group and P60 group(P<0.05).Conclusion:SD enhanced mitophagy of hippocampus in rats and impaired cognitive function.Propofol alleviated mitophagy and improved cognitive dysfunction after SD by PINK1/Parkin signaling pathway. |
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