The Mechanism Of EGCG Inhibiting TNF?/TNFR1 Mediated Lipocalin2 To Improve Neuroimmune Microenvironment

Objective: Alzheimer's disease(Alzheimer's disease,AD)is a chronic progressive neurodegenerative disease characterized by progressive memory loss,cognitive impairment and behavioral abnormalities.It is the most common type of dementia,accounting for about 60-80 percent.The main pathological hallmarks of AD include extracellular senile plaque(composed of Amyloid ?(A ?)deposition),intracellular neurofibrillary tangles(composed of hyperphosphorylated tau protein),extensive neuroinflammation(characterized by excessive activation of glial cells)and neuronal loss in the brain.Although the exact cause of AD is still unclear,more and more scholars realize that neuroinflammatory response plays an important role in the pathogenesis of AD.Astrocytes are the most abundant glial cells in the central nervous system.The activation and proliferation of astrocytes will promote the increase of many inflammatory factors such as TNF ? in the brain of patients with AD,resulting in neuroinflammatory response and neurodegenerative diseases.Lipocalin 2(Lcn2),also known as neutrophil gelatinase-associated lipid transport protein(NGAL),24p3,is a member of the lipocalin family of lipid transport proteins.It is a 25 k Da-secreted glycoprotein.It is up-regulated in trauma,infection,cancer and inflammation,and is considered to be an acute phase reaction protein.Reactive astrocytes can autocrine Lipocalin 2.Previous studies have shown that the expression of Lipocalin 2 is increased in serum of patients with mild cognitive impairment and AD,and in brain tissue of patients with AD.In vitro studies have shown that TNF ?can induce Lipocalin 2 secretion and neuronal apoptosis by activating its receptor TNFR1.At the same time,it can also aggravate the neurotoxicity induced by A ?,suggesting that Lipocalin 2 may be a new target and participate in the neuroinflammatory and apoptosis of AD.It plays an important role in neuroinflammatory,apoptosis and survival of AD.Therefore,it is of great significance to find a new drug which can improve the neuroinflammatory in the brain of AD,inhibit Lipocalin 2-mediated apoptosis,and effectively improve the symptoms of AD,and explore its related mechanism,which is of great significance for the further study of AD.(-)epigallocatechin gallate(EGCG)is one of the main polyphenols in green tea,which has the effects of antioxidant stress,anti-inflammation and anti-cancer.Our group has reported that EGCG can improve cognitive impairment and inhibit neuronal apoptosis in APP/PS1 transgenic mice by regulating Trk A/p75 NTR signal balance or inhibiting endoplasmic reticulum stress.However,there are no reports about the anti-AD effect of EGCG by inhibiting neuroinflammation and Lipocalin 2 expression.As we know,this is the first study to investigate that EGCG plays a neuroprotective effect in AD via inhibiting TNF?/TNFR1 mediated Lipocalin 2 expression.Methods: 1.The aging data set GSE53890 in GEO database and the AD data set GSE48350 and GSE1297,were used to analyze the expression of Lipocalin 2 in the elderly vs young and AD vs normal subjects.Using GEO2 R online analysis tool,according to different grouping conditions(age and Lipocalin 2 expression),differentially expressed genes were analyzed according to the criteria of | log2(fold change)| > 0.5 and p-value < 0.05,then taking intersection with Venn Diagram.For the common differentially expressed genes of GSE53890 datasets,org.hs.eg.db package(version 3.10.0)in R software(version 3.6.3)was used for ID conversion,clusterprofiler package was used for GO and KEGG enrichment analysis,GGplot2 package was used for visualizing the meaningful results.Screening condition was P adjust<0.05.For the common differentially expressed genes in the GSE48350 dataset,Clue Go(version 2.5.7)plug in Cytoscape(version 3.8.2)software was used for GO and KEGG enrichment analysis,and the screening condition was P <0.05.2.Thirty12-month-old APP/PS1 transgenic mice were randomly divided into model group,EGCG low-dose group(EGCG-2mg/kg group)and EGCG high-dose group(EGCG-6mg/kg group),and 10 C57 BL/6J mice of the same age were used as control group.The experimental group was gavaged 0.02% EGCG water solution at 2mg/kg or6mg/kg,once a day,for continuous 4 weeks.Model group and control group were gavaged with the same amount of double distilled water in EGCG treatment group,once a day,for 4 weeks.After administration,the Morris water maze test was used to examine behavioral changes related to learning and memory.At the end of the behavioral test,the animals were sacrificed.Hippocampal tissues of 5 mice in each group were collected for protein levels and other tests,and brain perfusion fixation of the other 5 mice was performed for immunohistochemistry and immunofluorescence staining.3.Immunofluorescence method was used to detect the expression and localization of A ? 1-42,GFAP,TNF ?,TNFR1 and Lipocalin 2 in mouse brain tissue.Immunohistochemical method was used to detect the expression of TNFR1,24p3 R and BIM in mouse brain tissue.,Western blot was used to detect the protein expression of A? 1-42,GFAP,TNF ?,TNFR1,Lipocalin2,24p3 R and BIM in mouse brain tissue.ELISA was used to detect the secretion of TNF ? in mouse brain tissue.4.The AD model of SVGp12 astrocytes treated with A ? 1-42 was constructed.Western blot,RT-PCR and ELISA were used to detect the effect of EGCG on the expression of TNF?,TNFR1 and Lipocalin 2.Immunofluorescence was used to detect the localization of TNF ?,TNFR1,Lipocalin 2 and GFAP.5.SVGp12 astrocytes were co-cultured with SH-SY5 Y neurons.Western blot was used to detect the expression of 24p3 R and BIM in co-cultured condition.CCK-8 and flow cytometry were used to evaluate the effect of EGCG on neuronal survival and apoptosis.6.Statistical Analysis Data were analyzed with SPSS 19.0 statistical software(SPSS Inc,Chicago,USA),expressed as mean ± standard deviation((?)±SD).P<0.05 indicates statistical significance.One-Way ANOVA and Independent t test were used for comparison between groups.Pearson ?2 and Fisher's test were used to analyze the correlations between Lcn2 expression and age or severity of AD.Results: 1.The expression of Lipocalin 2 in the brain of normal elderly was increased,and with the increase of age,the expression of Lipocalin 2 increased gradually;the expression of Lipocalin 2 in the brain tissue of patients with AD was significantly higher than that of healthy controls,and the more serious the disease of AD,the higher the expression of Lipocalin 2.Lipocalin 2 is mainly involved in the biological processes related to learning,memory and cognition,as well as the regulation of copper and zinc homeostasis and inflammation.2.The APP/PS1 model group showed significant learning and memory impairment compared with WT control group when there was no significant difference in the autonomous activity of each group.Compared with the APP/PS1 transgenic AD mice model group,the learning and memory function of EGCG group was significantly improved.3.EGCG can significantly inhibit the deposition of A ? plaque and its surrounding proliferation and activation of astrocytes in hippocampus of APP/PS1 transgenic AD mice.4.EGCG can significantly reduce the expression of autocrine inflammatory mediator Lipocalin 2and its receptor 24p3 R in astrocytes of APP/PS1 transgenic AD mice,and inhibit the expression of downstream apoptosis-related protein BIM,so as to play an anti-apoptotic effect.5.EGCG can reduce the expression of TNF ? and its receptor TNFR1 in the hippocampus of APP/PS1 transgenic AD mice.6.EGCG can significantly reduce the secretion of TNF ? and the expression of TNFR1 in SVGp12 cells induced by A ? 1-42,and then inhibit the secretion of Lipocalin 2,thus inhibit the apoptosis of SH-SY5 Y cells and play a neuroprotective role.Conclusions: 1.Lcn2 was highly expressed in the brain of normal elderly,and positively correlated with age.The expression of Lcn2 in brain tissue of patients with AD was up-regulated and positively correlated with the severity of the disease.2.EGCG can significantly improve the cognitive dysfunction of APP/PS1 transgenic AD mice,inhibit the A? deposition and activation of reactive astrocytes in the brain of APP/PS1 transgenic AD mice,and maybe play a neuroprotective role by reducing the secretion of Lipocalin 2 induced by TNF?/TNFR1 and inhibiting the apoptosis mediated by Lipocalin 2/24P3R/BIM.3.EGCG plays a neuroprotective role in promoting the survival and anti-apoptosis of SH-SY5 Y neuronal cells in cultured conditions,by inhibiting the expression of TNF?/TNFR1/Lipocalin2 in SVGP12 astrocytes probably.