Regulation And Mechanism Of Astrocyte CB2R On Lipid Accumulation And Neuroinflammation In POCD

With the development of global medical technology and the acceleration of the aging of the population,the number of elderly patients undergoing surgery is increasing,so the number of patients with cognitive impairment caused by surgery is also increasing.Postoperative cognitive dysfunction is a common postoperative complication,which is more common in elderly patients and manifests as transient memory loss or long-term cognitive impairment.POCD is the result of a variety of factors,such as patient age,type of surgery,and narcotic drugs.With the deepening of research,people have learned about the deeper pathogenic mechanisms,including glial cell-mediated inflammatory response,damage to mitochondrial function,and autophagy dysfunction.Among them,the inflammatory response mediated by glial cells has attracted much attention.In neurodegenerative diseases related to brain nerve damage similar to POCD,neuroinflammation caused by accumulated lipid droplets was found to accelerate the pathological process.In POCD,accumulation of lipid droplets on glial cells was also found,accompanied by an elevated inflammatory response.Therefore,in POCD,understanding the neuroinflammatory response caused by lipid droplet aggregation in glial cells is an important breakthrough in the treatment of POCD.Astrocytes are the most numerous glial cells in the brain,and their role in maintaining the homeostasis of the brain cannot be underestimated.On the one hand,astrocytes can secrete a variety of inflammatory factors such as IL-6,IL-1β,etc.And amplify the inflammatory response,resulting in neuronal cell damage.On the other hand,astrocytes can secrete trophic factors and antioxidant factors,such as IL-10,SOD to neutralize the damage caused by intracellular oxidative stress and enhance the neuroprotective effect.As an important cell in the degradation of harmful substances in the brain,the catabolism of lipid droplets in astrocytes not only reduces the damage to neuronal cells,but also provides energy for cells.In addition,studies have shown that both the A1-type inflammatory phenotype and the A2-type anti-inflammatory phenotype in astrocytes are related to POCD,increasing the number of A2-type astrocytes and reducing the number of A1-type astrocytes It is an important way to relieve nerve damage in POCD.Therefore,understanding the physiological functions introduced by astrocytes and exploring the specific mechanisms of catabolizing lipid droplets and relieving neuroinflammation are the important directions for exploring the therapeutics of POCDAs a GPCR-coupled receptor,CB2 R is an important target for the treatment of various inflammatory-related diseases.In related studies of neurodegenerative diseases,it was found that the autopsy results of AD patients indicated that CB2 R was significantly up-regulated in the brain;activation of CB2 R in animal models could resist the neurotoxicity mediated by microglia.In addition,elevated CB2 R has been found in other neurodegenerative diseases such as Parkinson’s disease and Huntington’s disease.In addition,in the study of tumors,it was also found that the prognosis,survival and tissue grade of cancer were related to the high expression of CB2 R.Therefore,CB2 R,as a target with great therapeutic potential,is also found to be related to the cognitive impairment of POCD in POCD.Therefore,understanding its mechanism of action and exploring its important pathways are important directions for the study of CB2 R therapeutics.Based on the previous research,the first part of mouse astrocytes was knocked down by adeno-associated virus CB2 R on astrocytes,and the accumulation of lipid droplets on astrocytes was detected by immunofluorescence staining and lipid In addition,the expression of lipid droplet metabolism and redox-related genes in the hippocampus was detected by Q-PCR,and the cognitive ability of mice was detected by behavioral analysis.It was found that CB2 R was knocked out on astrocytes decreased lipid droplets and aggravated the accumulation of lipid droplets and cognitive impairment in POCD.At the same time,the CB2 R agonist JWH133 or the inhibitor AM630 were injected into the lateral ventricle of mice,and the BODIPY lipid probe detection,Q-PCR detection of lipid metabolism-related genes,and cognitive behavioral detection,etc.,found that the CB2 R activation not only decreased The accumulation of lipid droplets on astrocytes and the expression of the inflammatory factor IL-1β were simultaneously alleviated in mice.On this basis,in the second part of the study,we found that the activation of CB2 R reduced the loss of hippocampal neurons and alleviated the activation of astrocytes by immunohistochemical staining,Nissl staining,and Golgi silver staining.In addition,we cultured U87 cell line and established an in vitro cell inflammation model with LPS.U87 cell lipid droplet accumulation and mitochondrial function were detected by lipid droplet fluorescent probe BODIPY,mitochondrial membrane potential fluorescent probe JC-1 and mitochondrial probe Mito Tracker.The results showed that the activation of CB2 R reduced LPS-induced lipid droplet accumulation and alleviated the damage of mitochondrial function.In the third part,we studied the mechanism of CB2 R activation affecting lipid droplet accumulation.U87 cells were transfected with TFEB mutant plasmid and transfected with si RNA-PGC-1α to observe the changes of intracellular autophagy-related phenotype,lipid droplet deposition and the expression of inflammatory factors;In addition,the specific binding sites of TFEB and PGC-1 were detected by Ch IP and luciferase reporter gene experiments as the 1100bp-1400 bp and 1509bp-1850 bp fragments of the PGC-1promoter.Therefore,the study of this topic clarifies the specific mechanism of CB2 R regulating lipid droplet deposition and metabolic degradation of astrocytes in POCD mice,which provides a new theoretical basis for the subsequent treatment of POCD.Part Ⅰ: CB2R-regulated lipid droplet accumulation and neuroinflammation in relation to POCDObjective: To investigate whether CB2 R is associated with lipid metabolism and neuroinflammation in astrocytes in POCDMethods: Astrocyte-specific CB2 R knockdown adeno-associated virus was injected into the hippocampus in an in vivo mouse model.The hippocampal brain region was stained with lipid droplet probe BODIPY,and the expression of lipid droplet metabolism-related genes was detected in the hippocampal brain region by Q-PCR.At the same time,cognitive ability was detected by behavioral testing to observe the effect of CB2 R knockdown on POCD.In addition,the CB2 R agonist JWH133 and the inhibitor AM630 were injected into the lateral ventricle of mice,and the same BODIPY staining behavior assay as above was used to observe whether CB2 R activation or inhibition affects the accumulation of lipid droplets and inflammatory response in the hippocampus of mice.Results:(1)Knockdown of CB2 R on astrocytes aggravated the accumulation of lipid droplets on astrocytes in the hippocampus of POCD mice;(2)Knockdown of CB2 R on astrocytes inhibited expressions of lipid metabolism-related genes and antioxidant-related genes in the hippocampus of POCD mice;(3)Knockdown of CB2 R on astrocytes aggravated cognitive impairment in mice;(4)Activation of CB2 R reduced lipids in the hippocampus of POCD mice(5)Activation of CB2 R reduced the accumulation of lipid droplets on hippocampal astrocytes in POCD mice;(6)Activation of CB2 R inhibited the production of IL-1β on astrocytes in the hippocampus of POCD mice(7)Activating CB2 R increased the expression of lipid metabolism and antioxidant-related genes in the hippocampus of POCD mice;(8)Activating CB2 R alleviated cognitive impairment in POCD mice.Conclusion: CB2 R and the accumulation of lipid droplets in POCD are related to cognitive ability.Knockdown of CB2 R in the hippocampus aggravates the accumulation of lipid droplets in POCD mice and impairs cognitive ability;activation of CB2 R reduces the accumulation of lipid droplets in astrocytes and inflammatory factors expression,while alleviating cognitive damage.Part Ⅱ CB2 R participates in neuroprotection in POCD mice and alleviates the damage of mitochondrial functionObjective: To investigate whether CB2 R is associated with neuroprotection and mitochondrial function in POCD mice.Methods: In the in vivo animal model,the CB2 R agonist JWH133 and the inhibitor AM630 were injected into the lateral ventricle,and the methods of immunohistochemistry,Nissl staining and Golgi staining were used to observe neuronal loss and astrocyte activation between CB2 R and POCD mice.In addition,U87 cells were cultured in vitro,and the lipid droplet accumulation and mitochondrial function in U87 cells was examined by lipid droplet probe BODIPY staining,mitochondrial membrane potential probe JC-1 staining and mitochondrial probe Mito Tracker.Results:(1)Activation of CB2 R reduced the activation of astrocytes in the hippocampus of POCD mice;(2)Activation of CB2 R inhibited the decrease in the number of neurons in the hippocampus of POCD mice;(3)Activation of CB2 R alleviated synaptic damage in the hippocampal brain region of POCD mice;(4)Activation of CB2 R on U87 cells reduced LPS-induced lipid droplet accumulation;(5)Activation of CB2 R on U87 cells inhibited the reduction of mitochondrial membrane potential caused by LPS;(6)Activation of CB2 R on U87 cells inhibited the reduction of mitochondrial number caused by LPS.Conclusion:(1)Activation of CB2 R alleviated the nerve damage in POCD mice in vivo.(2)The activation of CB2 R reduced the accumulation of lipid droplets and the impairment of mitochondrial function in U87 cells in vitro.Part Ⅲ CB2R-TFEB-PGC-1α involved autophagy regulation promotes the degradation of lipid droplets and reduces the secretion of inflammatory factorsObjective: To study the specific mechanism of CB2R-TFEB-PGC1-α pathway regulating lipid droplet catabolismMethods: Firstly,the expression of TFEB in U87 cells was interfered,and the regulation of CB2R-TFEB on autophagy was detected by combining Q-PCR and transfection of autophagy flow dual fluorescent virus;Next,by transfecting plasmids GV143-S211 A and GV143-S142 A with TFEB mutation sites,Combined with the extraction of cytoplasmic nuclear proteins and immunofluorescence to observe whether TFEB enters the nucleus and participates in the regulation of downstream autophagy;finally,combined with site analysis,Ch IP and luciferase reporter gene experiments were used to verify the specific binding site of the transcription factor TFEB and the key transcription factor of lipid metabolism PGC-1α,and interfere with PGC-1α to observe the effect of CB2 R activation on lipid droplets and inflammation in U87 cells.Results:(1)Knockdown of TFEB abolished the up-regulation of m RNA levels of autophagy-related genes in U87 cells after CB2 R activation;(2)Knockdown of TFEB abolished the effect of CB2 R activation in promoting the smooth flow of autophagy in U87 cells;(3)Knockdown of TFEB abolished the effect of CB2 R activation on promoting lipid droplet degradation in U87 cells;(4)Knockdown of TFEB abolished the inhibition of the expression of inflammatory factors IL-6 and IL-1β in U87 cells after CB2 R activation;(5)Activation of CB2 R promoted the nuclear entry of TFEB in U87 cells;(6)activation of CB2 R promoted the dephosphorylation of serine No.211 of TFEB;(7)activation of CB2 R promoted the expression of PGC-1α;(8)overexpression TFEB promoted the expression of PGC-1αin U87 cells;(9)CB2R activation promoted the binding of TFEB to the1100bp-1400 bp and 1509bp-1850 bp fragments of the PGC1-α promoter region,and enhanced the transcription of PGC-1α.(10)Knockdown of PGC-1α abolished the effect of promoting lipid droplet degradation and inhibiting inflammatory response after CB2 R activation.Conclusion:(1)CB2R activation promoted TFEB-mediated autophagy,increases the breakdown of lipid droplets,and reduced the production of inflammatory factors.(2)After activation of CB2 R,it promoted the binding of TFEB to the 1100bp-1400 bp and 1509bp-1850 bp fragments in the promoter region of PGC-1α,and enhanced the metabolism of lipid droplets.To sum up,the innovations of this paper are:(1)Elucidated the correlation of CB2 R with lipid droplet accumulation and neuroinflammation in POCD;(2)Revealed the mechanism of promoting the binding of the transcription factor TFEB to the 1100bp-1400 bp and 1509bp-1850 bp fragments of the promoter region of the lipid metabolism-related gene PGC1-α after activation of CB2 R,providing a theoretical basis for the subsequent development of drugs targeting this target.