Based On AMPK/PGC-1? Pathway To Explore The Role Of Methylene Blue In The Regulation Of Brain Energy Metabolism In Sepsis

Objective: The purpose of this study was to observe mitochondrial damage in Sepsis-associated Encephalopathy(SAE),investigate the regulation effect of Methylene Blue(MB)on SAE brain energy metabolism through AMPK/PGC-1? pathway.The main research includes: 1)preparing a cell experimental model of sepsis-related encephalopathy,observing mitochondrial damage,adenosine triphosphate(ATP)levels,apoptosis;2)exploring the role of MB in AMPK/ PGC-1? activation and energy regulation in the SAE cell model;3)establishing a sepsis model of Wistar rats and exploring the activation and energy regulation of AMPK/PGC-1? pathway of SAE Wistar rats by methylene blue in SAE animal models.Methods: 1)to isolate and purify astrocytes from the cerebral cortex of Wistar newborn rats(within 3 days)by using primary cell culture technology.After purity identification,different concentrations of Lipopolysaccharide(LPS)and 20 ng/mL Interferon-?(IFN-?)combined incubation,according to the incubation time(6h,12 h,24h,48h)and the concentration of LPS(50 ng/mL,100 ng/mL,200 ng/mL),to simulate the duration and severity of the disease.The in vitro models of SAE were confirmed by comparing cell viability,TNF-?,IL-6,NO,ROS,and change in the shape of the astrocytes.To observe the mitochondrial damage of astrocytes in SAE cell model by observing the apoptosis,mitochondrial network morphology and mitochondrial membrane potential.To observe mitochondrial biogenesis and dynamics by Western blot,including peroxisome proliferator-activated receptor gamma coactivator-1?(PGC-1?),mitochondrial transcription factor A(TFAM),nuclear respiratory factor-1(NRF1),optic atrophy l gene protein(OPA1),dynamin-related protein(DRP1)and to measure the ATP;2)based on the first part of the study,the primary cultured astrocytes were incubated with 100 ng/mL of LPS combined with 20 ng/mL of IFN-? for 6h and 48 h,respectively,simulating the early and late stages of in vitro model of SAE.Both two SAE in vitro model were administered with MB(0.01 nmol/mL)and MB(0.01 nmol/mL)+ Compound C(10 nmol/mL).To explore the molecular mechanisms of MB activation of AMPK/PGC-1? pathway in the early and late stages of SAE cell model,and to observe the mitochondrial-related indicators at the cell research level,including PGC-1?,TFAM,NRF1,and ATP,apoptosis;3)An adult male model of Wistar rats weighing 180-200 g was used to perform cecal ligation and puncture(CLP)to prepare an experimental model of sepsis.24 hours after CLP,extensive and long-lasting ? waves according to the EEG and neurobehavioral scores were confirmed as SAE models.Methylene blue(0.15mg/100g)was injected intraperitoneally to the animals in the SAE group randomly.After 6hours,the animals in each group were treated,serum was collected,and rat cerebral cortex protein was extracted.Western blot experiments were used to observe changes in mitochondrial biogenesis-related protein levels,in addition to that,apoptosis indicators,and ATP in the cerebral cortex of SAE rats were observed.Results: 1)Primary cultured astrocytes were incubated with LPS and IFN-? to prepare SAE in vitro experimental models.By comparison,the viability of SAE cells decreased,IL-6,TNF-?,ROS,and NO increased,and the astrocyte cell body of the SAE model became larger and the protrusions became thicker,confirming that the model was successfully prepared;2)To observe the in vitro model of SAE,it was found that the levels of mitochondrial biogenesis genes(PGC-1?,TFAM,NRF1),mitochondrial fusion and division-related proteins(OPA1 and DRP1)of astrocytes which were incubated in LPS and IFN-? for 6hours,the expression gradually increased with the increase of LPS concentration.After 12 hours of combined incubation,the above indicators showed an increase and then a decrease with the increase of LPS concentration.At 24 and 48 hours of combined incubation,the above indicators decreased followed LPS concentration.The intracellular ATP content showed a relatively close change trend;3)In the in vitro SAE cell experimental model,compared with the control group,MB increased p-AMPK expression at 6h and 48 h groups,enhanced cell mitochondrial biogenesis,increased intracellular ATP content,and also the apoptosis.In the 48 h group,no decrease in apoptosis was observed;4)In the in vivo SAE experimental model,it was found that MB increased the expression of p-AMPK in the cerebral cortex of SAE rats 24 hours after CLP,and enhanced mitochondrial biogenesis expression,increase intracellular ATP content,and improve apoptosis.Conclusion: mitochondrial biogenesis and ATP levels in primary cultured astrocytes were enhanced early and weakened in SAE in in vitro experimental models.Studies at the cellular and tissue levels have shown that MB can be enhanced by activating the AMPK/PGC-1? pathway mitochondrial biogenesis occurs to improve the energy level and apoptosis of SAE cells.