Itaconate Inhibits Corticosterone-Induced Necroptosis and Neuroinflammation via Up-regulating Menin in HT22 Cells

Itaconate Inhibits Corticosterone-Induced Necroptosis and Neuroinflammation via Up-regulating Menin in HT22 Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Itaconate Inhibits Corticosterone-Induced Necroptosis and Neuroinflammation via Up-regulating Menin in HT22 Cells Jin-Yu Liang, Shan Gao, Jia-Mei Jiang, Pin Zhang, Wei Zou, Xiao-Qing Tang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2282581/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Mar, 2024 Read the published version in Journal of Physiology and Biochemistry → Version 1 posted You are reading this latest preprint version Abstract Corticosterone (CORT) damages hippocampus neurons as well as induces neuroinflammation. Tricarboxylic acid cycle metabolite itaconate has an anti-inflammatory role. Necroptosis acts as programmed cell death triggering neuroinflammation. The deficiency of Menin, a multifunctional scaffold protein, aggravates neuroinflammation. In this study, we explored whether itaconate inhibits CORT-induced neuroinflammation and necroptosis as well as the mediatory role of Menin in this protective effect of itaconate using an exposure of CORT to HT22 hippocampal neuronal cells. The viability of HT22 cells was examined by the Cell Counting Kit 8 (CCK-8). The morphology of HT22 cells was observed by transmission electron microscope (TEM). The expressions of necroptosis-related proteins (p-RIP1/ RIP1, p-RIP3/ RIP3, and p-MLKL/ MLKL) were evaluated by Western blotting. The contents of inflammatory factors were detected by an enzyme-linked immunosorbent assay kit. Our results showed that CORT increases the contents of pro-inflammatory factors (IL-1β, TNF-α) as well as decreases the contents of anti-inflammatory factors (IL4, IL10) in HT22 cells. We also found that CORT increases the expressions of necroptosis-related proteins (p-RIP1/ RIP1, p-RIP3/ RIP3, and p-MLKL/ MLKL) and decreases the cell viability in HT22 cells, indicating that CORT induces necroptosis to HT22 cells. Itaconate improves CORT-induced neuroinflammation and necroptosis. Furthermore, itaconate upregulates the expression of Menin in CORT-exposed HT22 cells. Importantly, silencing Menin abolishes the antagonistic effect of itaconate on CORT-induced necroptosis and neuroinflammation. In brief, these results indicated that itaconate protects HT22 cells against CORT-induced neuroinflammation and necroptosis via upregulating Menin. itaconate Menin corticosterone necroptosis neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Chronic stress is one of the crucial risk factors for depression [ 1 – 3 ] . Stress exposure activates the hypothalamic-pituitary-adrenal (HPA) axis [ 4 , 5 ] , thereby increasing the secretion of corticosterone (CORT) [ 5 ] . It is confirmed that sustained CORT exposure damages hippocampus neurons and induces depression-like behaviors [ 6 – 8 ] , as a result of triggering neuroinflammation [ 9 – 11 ] . But the underlying mechanism of CORT-induced neuroinflammation remains unclear. It is of great significance to further clarify the potential mechanism of CORT-caused neuroinflammation and find the corresponding therapeutic strategy for the prevention and treatment of depression. Necroptosis is a lytic cell death distinct from apoptosis [ 12 ] . Gene Ontology (GO) enrichment analysis showed that necroptosis is activated in an animal model of chronic stress-induced depression [ 13 ] . Whereas inhibiting necroptosis improves depression-like behaviors [ 14 ] . Additionally, necroptosis induces neuroinflammation [ 15 – 17 ] , while inhibition of RIP1, one of the main mediators of necroptosis, ameliorates neuroinflammation [ 18 ] . Therefore, we made a bold conjecture that CORT-induced neuroinflammation is triggered by necroptosis. Itaconate, an immunomodulator metabolite, is derived from the tricarboxylic acid (TCA) cycle intermediate cis-aconitate [ 19 – 21 ] . The level of itaconate obviously ascends after inflammatory stimulation, to restrict the progression of inflammation [ 22 ] . The endogenous metabolite itaconate has been reported to inhibit NLRP3 inflammasome [ 23 ] and the release of inflammation cytokines [ 24 ] . Importantly, itaconate attenuates neuroinflammatory response in BV2 microglia cells [ 25 ] and reduces proinflammatory factors in the brains of AD model mice [ 26 ] . Thus, the present study observed whether itaconate alleviates CORT-induced neuroinflammation. Furthermore, studies implicated necroptosis as an essential role in driving inflammation due to its pro-inflammatory features [ 27 ] . Therefore, we will explore whether itaconate prevents CORT-induced necroptosis and uncover the possible mechanism of itaconate exerting anti-inflammatory effects. What is the mechanism for itaconate to inhibit necroptosis under CORT- exposure? Menin is a scaffold protein [ 28 ] , which is highly expressed in the brain [ 29 ] . But little is known about its function in the nervous system. Recent studies found that the expression of Menin is down-regulated by chronic stress [ 30 ] and that Menin deficiency enhances neuroinflammation [ 31 ] . Upregulating Menin in astrocytes antagonizes neuroinflammation [ 32 ] . These results indicated that upregulation of Menin in the brain is a potential therapeutic candidate for the prevention of neuroinflammation. Therefore, the present work will further explore the mechanism underlying the antagonistic role of itaconate in CORT-induced necroptosis from the insight of Menin. Here, our study illustrated that itaconate protects neurons against CORT-induced neuroinflammation and necroptosis as well as up-regulates the expression of Menin in HT22 cells. Furthermore, silencing Menin reversed the improving effect of itaconate on CORT-elicited necroptosis and neuroinflammation. Therefore, our study demonstrated that itaconate prevents CORT-induced neuroinflammation and necroptosis through up-regulation of Menin. 2. Materials And Methods 2.1 Antibodies and Regents Antibodies: anti-βactin (20536-1-AP), anti-Tubulin (10094-1-AP), anti-GAPDH (60004-1-Ig), Goat anti-mouse antibody (SA0001-1), Goat anti-rabbit antibody (SA0001-1) were purchased from Proteintech company in the United States. Anti-Menin (ab31902) was purchased from Epitomics-an Abcam Company in the United States. Anti-p-RIP1 (65746) and anti-RIP1 (3493) were obtained from Cell Signaling Technology in the United States. Anti-P-RIP3 (AF7443), anti-RIP3 (AF7942), anti-P-MLKL (AF7420), and anti-MLKL (AF7412) were purchased from Affinity Biosciences in the United States. Regents: Corticosterone (C0388, TCL Chemical Industry Development Co., Ltd., Shanghai). 4-Octyl Itaconate (3133-16-2, MedChemExpress, USA), DMSO (D2650, Sigma, USA), Trypsin (T4049, Sigma, USA), CCK-8 (Dongren Chemical Technology Co., Ltd., Shanghai), MSD (Dongren Chemical Technology Co., Ltd., USA) 2.2 Cells culture HT22 cells were obtained from Shunran Biotechnology Co., Ltd (Shanghai, China) and were incubated in a DMEM medium with 10% FBS and 1% Penicillin streptomycin. And cultured in the 37 centigrade, 5% CO2 incubator. Replace DMEM medium every 1–2 days. Subculture and frozen cells when the cells are in the logarithmic growth phase. 2.3 Cells viability assay The viability of HT22 cells was determined by Cell Counting Kit-8. HT22 cells were seeded in the 96-well plates. The experiment was carried out according to the instruction. The optical density of each well was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, USA). The viability of HT22 cells was calculated by (Corrected absorbance value of experimental group/Corrected absorbance value of control group) × 100% 2.4 Transmission electron microscope observation assay The morphology of HT22 cells was observed with a transmission electron microscope (TEM). The samples were fixed with 2.5% glutaraldehyde overnight and 1% forge acid for 2–3 hours. After dehydration, it was embedded overnight at room temperature and stained with lead citrate. 2.5 RNA interference analysis MEN1-siRNA interference sequence and control sequence were designed and synthesized by GeneChem Corporation (Shanghai, China) and were constructed into GV298 plasmid. The protein samples were collected after transfection for 24 h for western blotting. 2.6 Western blot analysis The expressions of necroptosis-related proteins and Menin were detected by western blot analysis. Total protein was prepared and separated by 10% − 12% SDS-PAGE and then transferred onto PVDF membranes (IPVH00010, Merck Millipore, Birrika, MA, USA). Blocking with Tris Buffered saline Tween (TBST, 50 mM Tris − HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) containing 5% skim milk for 2 h at room temperature, following incubated with primary antibody against RIP1, p-RIP1, RIP3, p-RIP3, MLKL, p-MLKL (1:1000), Tubulin, GAPDH (1:4000) overnight at 4 centigrade. After this, the bands were washed 5 times with TBST solution, and then the second antibody was incubated. Finally, Bands were visualized using the Tanon-5600 gel imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China) and analyzed by Image-J software (NIH, Bethesda, MD, USA). 2.7 Enzyme-linked immunosorbent assay The contents of IL-1β, TNF-α, IL-4, and IL-10 were measured with the ELISA kits and were purchased from R&D systems (Minneapolis, MN, USA), including Mouse IL-1β ELISA kit (MLB00C), Mouse TNF-α ELISA kit(MTA00B), Mouse IL-4 ELISA kit (M4000B), Mouse IL-10 ELISA kit (M1000B). Detailed methods for performing the analysis are described in the instructions of manufacturers. 2.8 Statistical analysis The experimental data were analyzed by one-way ANOVA followed by the least significant difference (LSD) post hoc test with SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Sample capacity ≥ 3, the data were expressed as mean ± standard deviation (mean ± S.E.M.). P ≤ 0.05 is considered statistically significant. 3. Results 3.1 CORT induces neuroinflammation in HT22 cells To explore whether CORT induces neuroinflammation in HT22 cells, the contents of pro-inflammatory factors (IL-1β, TNF-α) and anti-inflammatory factors (IL-4, IL-10) in HT22 cells were examined by ELISA. We found that the contents of pro-inflammatory factors, IL-1β (Fig. 1 A) and TNF-α (Fig. 1 B), in HT22 cells were increased by treatment with CORT (200, 400, and 800 µM) for 24 h. By contrast, the contents of anti-inflammatory factors, IL-4 (Fig. 1 C) and IL-10 (Fig. 1 D), in HT22 cells were decreased by treatment with CORT (200, 400, and 800 µM) for 24 h. Taken together, these results indicated that CORT induces neuroinflammation in HT22 cells. 3.2 CORT induces necroptosis in HT22 cells Next, we explored whether CORT-induced neuroinflammation is involved in necroptosis in HT22 cells. We observed the morphology of HT22 cells with a Transmission electron microscope (TEM) after being treated with CORT (400 µM) for 24 h. We found that HT22 cells are characterized by cell membrane incomplete and cytoplasm swelling (Fig. 2 A). This result is in accordance with the previous result [ 33 ] . Then, we detected the effects of CORT on the expressions of necroptosis-related proteins in HT22 cells. Treatment with CORT (400, 800 µM) for 24 h up-regulated the expressions of p-RIP1/RIP1 (Fig. 2 B), p-RIP3/RIP3 (Fig. 2 C), and p-MLKL/MLKL (Fig. 2 D) in HT22 cells. We also measured the viability of cells by CCK-8 assay and found that after treatment with CORT (200, 400, and 800 µM) for 24 h, the viability of HT22 cells was decreased in a concentration-dependent (Fig. 2 E). Taken together, these above results indicated that CORT induces necroptosis in HT22 cells. 3.3 CORT down-regulates the expression of Menin in HT22 cells It has been reported that Menin deficiency aggravates neuroinflammation [ 31 ] , so we next explored the influence of CORT on the expression of Menin in HT22 cells. As shown in Fig. 3 , the expression of Menin in HT22 cells was dramatically inhibited by treatment with CORT (400, 800 µM) for 24 h, which indicated that CORT down-regulates the expression of Menin in HT22 cells. 3.4 Itaconate prevents CORT-induced neuroinflammation in HT22 cells To determine the antagonistic effect of itaconate on CORT-induced neuroinflammation in HT22 cells, HT22 cells were pretreated with OI (25, 50, and 100 µM) for 18 h and then exposed to CORT (400 µM) for 24 h. We found that itaconate decreased the contents of pro-inflammatory factors [IL-1β (Fig. 4 A) and TNF-α (Fig. 4 B)] and increased the contents of anti-inflammatory factors [IL-4 (Fig. 4 C) and IL-10 (Fig. 4 D)]. Additionally, treatment with itaconate alone had no effect on IL-1β (Fig. 4 A), TNF-α (Fig. 4 B), IL-4 (Fig. 4 C), and IL-10(Fig. 4 D). Taken together, these results indicated that itaconate prevents CORT-induced neuroinflammation in HT22 cells. 3.5 Itaconate alleviates CORT-induced necroptosis in HT22 cells We next detected the effect of itaconate on CORT-induced necroptosis. HT22 cells were pretreated with OI (25, 50, and 100 µM) for 18 h and then exposed to CORT (400 µM) for 24 h. We found that itaconate (50, 100 µM) down-regulated the expressions of necroptosis-related proteins, p-RIP1/ RIP1 (Fig. 5 A), p-RIP3/ RIP3 (Fig. 5 B), and p-MLKL/ MLKL (Fig. 5 C), in HT22 cells exposed to CORT (400 µM) for 24 h. Moreover, itaconate alone-treated had no effect on expressions of necroptosis-related proteins [p-RIP1/ RIP1 (Fig. 5 A), p-RIP3/ RIP3 (Fig. 5 B) and p-MLKL/ MLKL (Fig. 5 C)]. Additionally, Itaconate (50, 100 µM) increased the viability of HT22 cells exposed to CORT (400 µM) (Fig. 5 D). These data indicated that itaconate antagonizes CORT-induced necroptosis. 3.6 Itaconate up-regulates the expression of Menin in HT22 cells we further explored the effect of itaconate on the expression of Menin in HT22 cells. As shown in Fig. 6 , Itaconate (50, 100 µM) increased the expression of Menin in HT22 cells with or without CORT exposure, indicating that itaconate up-regulates the expression of Menin in HT22 cells. 3.7 LV- MEN1 -siRNA silences the expression of Menin in HT22 cells To further confirm the mediatory role of Menin in the protection of itaconate against CORT-induced necroptosis, we will observe whether silencing Menin abolishes the antagonistic effect of itaconate on CORT-induced cytotoxicity, necroptosis, and neuroinflammation in HT22 cells. To this end, we transiently transfected HT22 cells with three different RNA interference chains and detected the efficiency of lentiviruses transient transfection with fluorescence microscopy and western blotting. We found that three RNA interference strands were transfected successfully (Fig. 7 A). Additionally, western blotting showed that three RNA interference strands, especially MEN1 -siRNA (2), decreased the expression of Menin, while LV- Control -siRNA did not affect the expression of Menin (Fig. 7 B). Therefore, we constructed stable cell lines with MEN1 -siRNA (2) and further confirm the stable transfection efficiency of lentivirus with fluorescence microscopy and western blotting. We found that LV- Control -siRNA and LV- MEN1 -siRNA were transfected successfully (Fig. 7 C) and MEN1 -siRNA (2) decreased the expression of Menin, while Control -siRNA did not affect the expression of Menin (Fig. 7 D). 3.8 Silencing Menin reverses the antagonism effect of itaconate on CORT-induced neuroinflammation in HT22 cells We further explored whether Menin deficiency inhibits the protective effects of itaconate against CORT-induced neuroinflammation in HT22 cells and found that silencing Menin abolished itaconate-exerted decreases in pro-inflammation factors, including IL-1β (Fig. 8 A) and TNF-α (Fig. 8 B) as well as increases in anti-inflammation factors, including IL-4 (Fig. 8 C) and IL-10 (Fig. 8 D) in HT22 cells. These data indicated that Menin deficiency abolishes the protective effect of itaconate against CORT-induced neuroinflammation. 3.9 Menin deficiency reverses the antagonistic effect of itaconate on CORT-induced necroptosis in HT22 cells We next explored whether silencing Menin reverses the antagonistic effect of itaconate on CORT-induced necroptosis. We found that silencing Menin upregulated the expressions of p-RIP1/ RIP1 (Fig. 9 A), p-RIP3/ RIP3 (Fig. 9 B), and p-MLKL/ MLKL (Fig. 9 C) in HT22 cells cotreated CORT and itaconate. Furthermore, silencing Menin decreased the viability of HT22 cells cotreated expression CORT and itaconate (Fig. 9 D). These data indicated that silencing Menin reverses the antagonistic effect of itaconate on CORT-induced necroptosis. Discussion The present work is aimed to investigate whether necroptosis involves in CORT-induced neuroinflammation as well as explore the protective effect of itaconate against CORT-induced neuroinflammation and the underlying mechanisms. We here clarified that exposure of HT22 cells to CORT not only increased the contents of pro-inflammatory factors as well as decreased the contents of anti-inflammatory factors but also increased the expression of necroptosis-related proteins and decreased the viability of cells. Notably, itaconate suppressed CORT-induced neuroinflammation and necroptosis as well as up-regulated the expression of Menin in HT22 cells. Furthermore, silencing Menin not only reversed itaconate-ameliorated necroptosis but also abolished itaconate-reduced neuroinflammation. Taken together, these results revealed that necroptosis is involved in CORT-induced neuroinflammation and that itaconate inhibits CORT-induced neuroinflammation and necroptosis via up-regulating Menin. Corticosterone, a stress hormone [36] , induces depression-like behaviors via neuroinflammation [37, 38] . Numerous studies revealed that chronic CORT exposure induces inflammatory factors IL-1β and TNF-α release and neurotoxicity [6, 11, 39] . Our study also confirmed the neuroinflammatory effect of CORT, as evidenced by CORT exposure increases pro-inflammatory factors (IL-1β and TNF-α) as well as decreases anti-inflammatory factors (IL-4 and IL-10). Necroptosis, a kind of programmed necrosis mechanism different from apoptosis and necrosis [40, 41] , is tightly implicated in neuroinflammation [27, 42, 43] . Necroptosis in microglia promotes the release of pro-inflammatory cytokines [44] . In this work, we have demonstrated that the expression of necroptosis-related proteins RIP1, RIP3, and MLKL were increased as well as the viability of cells was decreased in CORT-exposed HT22 cells, indicating that CORT triggers necroptosis. It has been confirmed that deficiency of necroptosis-related proteins RIP1 or RIP3 alleviates neuroinflammation [45] . Therefore, blocking CORT-stimulated necroptosis is a novel effective method to inhibit neuroinflammation. Furthermore, the activation of necroptosis is closely related to depression [46] . Necroptosis inhibitor (necrostatin-1) ameliorates chronic unpredictable mild stress (CUMS)-induced depression-like behaviors [46] . Based on the crucial role of neuroinflammation in the development of depression. Therefore, finding a drug that blocks necroptosis and neuroinflammation stimulated by CORT is a novel therapeutic strategy for depression. The Krebs cycle-derived metabolite itaconate is synthesized from the enzyme immune-responsive gene 1 (IRG1) [23] . Endogenous itaconate deficiency increases inflammatory levels [47] . Overexpression of IRG-1 inhibits neuroinflammation in microglia [48] . Itaconate treatment reverses LPS-induced neuroinflammation in microglia [25] . However, it remains to investigate whether exogenous administration of itaconate improves neuroinflammation in CORT-exposed HT22 cells. In our data, itaconate ameliorates CORT-increased pro-inflammatory factors IL-1β and TNF-α as well as CORT-decreased anti-inflammatory factors IL-4 and IL-10 in HT22 cells, which indicated that itaconate prevents CORT-induced neuroinflammation. Necroptosis, as one of the important ways of cell death, triggers neuroinflammation [27] . Based on these findings, our present study was designed to investigate whether itaconate inhibits CORT-induced neuroinflammation by antagonizing necroptosis. Our data revealed that itaconate increases the viability of HT22 cells and decreases the expression of necroptosis-related proteins RIP1, RIP3, and MLKL in CORT-exposed HT22 cells. In summary, our results demonstrated that itaconate inhibits necroptosis to prevent CORT-induced neuroinflammation. Although the anti-inflammatory effect of itaconate has been fully confirmed, its underlying mechanism needs to be explored. Menin is a scaffold protein encoded by the Multiple endocrine neoplasia type 1 (MEN1) gene [49, 50] . It has been confirmed that targeting Menin-regulated inflammation in astrocytes ameliorates depression-like behaviors [32] . Therefore, we focus on the effect of Menin in the protective effect of itaconate against CORT-induced necroptosis and neuroinflammation. Our finding showed that itaconate up-regulates the expression level of Menin, and silencing Menin with Lentivirus transfection cancels the protective effect of itaconate against CORT-induced necroptosis and neuroinflammation in HT22 cells. KEGG analysis verified that the differentially expressed genes after MEN1 knockdown enrich inflammation responses [51] . Menin ablation induces activation of the inflammation pathway [52] . To sum up, these results support our working hypothesis that up-regulation of Menin is indispensable to the protective effect of itaconate against CORT-induced neuroinflammation, as a result of ameliorating necroptosis. In conclusion, our present work revealed that itaconate ameliorates CORT-induced neuroinflammation and necroptosis and up-regulates Menin in CORT-exposed HT22 cells. Furthermore, we further found that blocking Menin prevents the protective effect of itaconate against necroptosis and neuroinflammation elicited by CORT. These results illustrated that Menin is significant to the protective effect of itaconate against CORT-induced necroptosis and neuroinflammation. Our results highlighted itaconate as an attractive agent for the therapy strategy for CORT-induced neuroinflammation and provided a novel insight into the mechanisms underlying the antagonistic action of itaconate in CORT-induced neuroinflammation from necroptosis and Menin. Declarations DATA AVAILABILITY STATE The datasets and primary data in this study are available from the corresponding author on reasonable request. FUNDING This work was supported by [ National Natural Science Foundation of China ] (Grant numbers [ 81971267 ]) and [ Project of Hunan Provincial Department of Education ] (Grant numbers [ 21B0429 ]). CONFLICT OF INTEREST The authors have no relevant financial or non-financial interests to disclose. ETHICS APPROVAL STATEMENT This experiment does not involve animals, patients and clinical trials. AUTHOR CONTRIBUTIONS Jin-Yu Liang: Writing-original draft; Shan Gao: Conceptualization; Mei-Jia Jiang: Investigation, Formal analysis; Ping Zhang: Validation; Wei Zou: Resources; Xiao-Qing Tang: Writing - review & editing, Supervision, Data curation, Funding acquisition; Yi-Yun Tang: Writing - review & editing, Supervision, Data curation, Funding acquisition. CONSENT TO PARTICIPATE Not applicable. 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Vandenabeele, P., et al., Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol, 2010. 11 (10): p. 700-14. Dionisio, P.A., J.D. Amaral, and C.M.P. Rodrigues, Molecular mechanisms of necroptosis and relevance for neurodegenerative diseases. Int Rev Cell Mol Biol, 2020. 353 : p. 31-82. Shao, L., et al., The Contribution of Necroptosis in Neurodegenerative Diseases. Neurochem Res, 2017. 42 (8): p. 2117-2126. Huang, Z., et al., Necroptosis in microglia contributes to neuroinflammation and retinal degeneration through TLR4 activation. Cell Death Differ, 2018. 25 (1): p. 180-189. Zhang, Y., et al., Catalytically inactive RIP1 and RIP3 deficiency protect against acute ischemic stroke by inhibiting necroptosis and neuroinflammation. Cell Death Dis, 2020. 11 (7): p. 565. Yan, Z.Y., et al., Antidepressant Mechanism of Traditional Chinese Medicine Formula Xiaoyaosan in CUMS-Induced Depressed Mouse Model via RIPK1-RIPK3-MLKL Mediated Necroptosis Based on Network Pharmacology Analysis. Front Pharmacol, 2021. 12 : p. 773562. Kuo, P.C., et al., Dimethyl itaconate, an itaconate derivative, exhibits immunomodulatory effects on neuroinflammation in experimental autoimmune encephalomyelitis. J Neuroinflammation, 2020. 17 (1): p. 138. Ni, L., et al., Immune-responsive gene 1/itaconate activates nuclear factor erythroid 2-related factor 2 in microglia to protect against spinal cord injury in mice. Cell Death Dis, 2022. 13 (2): p. 140. Guru, S.C., et al., Menin, the product of the MEN1 gene, is a nuclear protein. Proc Natl Acad Sci U S A, 1998. 95 (4): p. 1630-4. Larsson, C., et al., Multiple endocrine neoplasia type 1 gene maps to chromosome 11 and is lost in insulinoma. Nature, 1988. 332 (6159): p. 85-7. Wang, S., et al., Menin regulates lipid deposition in mouse hepatocytes via interacting with transcription factor FoxO1. Mol Cell Biochem, 2022. 477 (5): p. 1555-1568. Xu, B., et al., Menin promotes hepatocellular carcinogenesis and epigenetically up-regulates Yap1 transcription. Proc Natl Acad Sci U S A, 2013. 110 (43): p. 17480-5. Supplementary Files GraphicalAbstract.png Cite Share Download PDF Status: Published Journal Publication published 01 Mar, 2024 Read the published version in Journal of Physiology and Biochemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2282581","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":154127428,"identity":"5ed5893c-5e08-4d48-8d27-85b52953bab2","order_by":0,"name":"Jin-Yu Liang","email":"","orcid":"","institution":"Institute of neuroscience, Hengyang Medical School, University of South China, 28W Changsheng Road, Hengyang 421001, Hunan, P.R. China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jin-Yu","middleName":"","lastName":"Liang","suffix":""},{"id":154127429,"identity":"41165f56-7c79-4823-88d2-001a47870478","order_by":1,"name":"Shan Gao","email":"","orcid":"","institution":"Institute of Neuroscience, Hengyang Medical School, University of South China, 28 W Road，Hengyang 421001, Hunan, P.R. China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Gao","suffix":""},{"id":154127430,"identity":"1286643b-72b8-4158-9599-9bebaaa7d3aa","order_by":2,"name":"Jia-Mei Jiang","email":"","orcid":"","institution":"Institute of Neuroscience, Hengyang Medical School, University of South China, 28 W Changsheng Road, Hengyang 421001, Hunan, P.R. China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia-Mei","middleName":"","lastName":"Jiang","suffix":""},{"id":154127431,"identity":"e055fd4b-bead-4dc4-9dd5-7ee885235eb1","order_by":3,"name":"Pin Zhang","email":"","orcid":"","institution":"Department of Neurology, Affiliated Nanhua Hospital, University of South China, No.336 S Dongfeng Road, Hengyang, 421002, Hunan Province, P.R. China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pin","middleName":"","lastName":"Zhang","suffix":""},{"id":154127432,"identity":"f4f8e5fb-3190-4f96-9681-0cd51ceff0fd","order_by":4,"name":"Wei Zou","email":"","orcid":"","institution":"Department of Neurology, Affiliated Nanhua Hospital, University of South China, No. 336 S Dongfeng Road, Hengyang, 421002, Hunan Province, P.R. China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zou","suffix":""},{"id":154127433,"identity":"9ef4371a-4465-4886-9d9b-51975ed3d1c1","order_by":5,"name":"Xiao-Qing Tang","email":"","orcid":"","institution":"Institute of Neuroscience, Hengyang Medical School, University of South China, 28 W Changsheng Road, Hengyang 421001, Hunan, P.R. China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiao-Qing","middleName":"","lastName":"Tang","suffix":""},{"id":154127434,"identity":"0d0a8e87-8cfa-4803-96a8-7615bfb94507","order_by":6,"name":"Yi-Yun Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYHAD5gPEqeNBMNkSSNbCY0CcFnv2s4df87YxJM5vP/PxxhsGOzndBkK28OSlWYO0bDiTu9lyDkOysdkBgg7LMTPmbfufuEGCd5s0D8OBxG0EtfC/AWkBOmwGzzMitUjkGD8GaWm4wcNGpJYbb8wY55xjMN5wJs3Yco4BEX5h788x/vCmjEF2fvvhhzfeVNjJEdQCBGxSvGwQlgSxUcP88ccfmBbidIyCUTAKRsEIAwBJcTz+5c+o2AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9810-1288","institution":"Institute of Neuroscience, Hengyang Medical School, University of South China, 28 W Road, Hengyang 421001, Hunan, P.R. China.","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yi-Yun","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2022-11-17 04:16:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2282581/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2282581/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13105-024-01012-3","type":"published","date":"2024-03-01T10:04:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":29659328,"identity":"640c8a11-47a5-41e5-81f3-d7ae249aecae","added_by":"auto","created_at":"2022-11-29 15:32:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":143662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CORT on inflammatory factors in HT22 cells. \u003c/strong\u003eThe contents of pro-inflammatory factors, IL-1β (A) and TNF-α (B), as well as anti-inflammation factors, IL-4 (C) and IL-10 (D), were detected by ELISA kit after treatment of HT22 cells withCORT (200, 400, and 800 μM) for 24 h. The data are expressed as means ± S.E.M. (n=3). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eversus\u003c/em\u003e control group.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/623657f996f68110471fee0d.png"},{"id":29659332,"identity":"7d343e98-8855-4358-9903-6aa2c4835a08","added_by":"auto","created_at":"2022-11-29 15:32:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1155727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CORT on the expressions of necroptosis-related proteins in HT22 cells. \u003c/strong\u003e(A) The morphology of HT22 cells was observed with a Transmission electron microscope (TEM) after being treated with CORT (400 μM) for 24 h. The black arrowhead indicates cell membrane fragmentation and nuclear disruption; the triangle indicates swollen cells in CORT-treated HT22 cells. (B-D) The expressions of necroptosis-related proteins [p-RIP1/ RIP1 (B), p-RIP3/ RIP3 (C) and p-MLKL/ MLKL (D)] were detected by western blot after treatment of HT22 cells with different concentrations of CORT (200, 400, and 800 μM) for 24 h. (E) The viability of HT22 cells was examined by the CCK-8 kit after being treated with different concentrations of CORT (200, 400, and 800 μM) for 24 h. The data are expressed as means ± S.E.M. (n=3). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eversus\u003c/em\u003econtrol group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/52a38bd00a4896d3796dfb98.png"},{"id":29659331,"identity":"42b14093-25f1-4c01-aec5-ece4372bf1a1","added_by":"auto","created_at":"2022-11-29 15:32:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CORT on the expression of Menin. \u003c/strong\u003eThe expression of Menin in the HT22 cells was detected by western blot (WB) after being treated with different concentrations of CORT (200, 400, and 800 μM) for 24 h. The data are expressed as means ± S.E.M. (n=3). **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, \u003cem\u003eversus \u003c/em\u003econtrol group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/f3c605eab545294baabd3ec7.png"},{"id":29659335,"identity":"072813c5-5ed7-460e-85ff-9240a4a5dd53","added_by":"auto","created_at":"2022-11-29 15:32:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":167689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of itaconate on neuroinflammation in the CORT-exposed HT22 cells.\u003c/strong\u003e HT22 cells were treated with CORT (400 μM) for 24 h after being pretreated with different concentrations of itaconate (25, 50, 100 μM) for 18 h. (A-D) The contents of pro-inflammation factors IL-1β (A), TNF-α (B), and anti-inflammation factors IL-4 (C) and IL-10 (D) were examined by ELISA Kit. The data are expressed as means ± S.E.M. (n=3). ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eversus \u003c/em\u003econtrol group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01,\u003csup\u003e ###\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, \u003cem\u003eversus\u003c/em\u003e CORT group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/731bd578cdebf4e1148b9879.png"},{"id":29659336,"identity":"dee792d2-f198-49cd-bbcc-fafcc23d2f1d","added_by":"auto","created_at":"2022-11-29 15:32:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":464517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of itaconate on the necroptosis in CORT-exposed HT22 cells.\u003c/strong\u003e HT22 cells were treated with CORT (400 μM) for 24 h after being pretreated with itaconate (25, 50, 100 μM) for 18 h. (A-C) The expressions of necroptosis-related proteins [p-RIP1/ RIP1 (A), p-RIP3/ RIP3 (B) and p-MLKL/ MLKL (C)] were examined by western blot. (D) The viability of cells was detected by the CCK-8 kit. The data are expressed as means ± S.E.M. (n=3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eversus\u003c/em\u003e control group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003cem\u003eversus\u003c/em\u003e CORT group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/f87550612c1f9046a3d83988.png"},{"id":29659330,"identity":"145e55e0-2f73-4253-a25e-a68d24b027f3","added_by":"auto","created_at":"2022-11-29 15:32:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":114366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of itaconate on the expression of Menin in HT22 cells. \u003c/strong\u003eHT22 cells were treated with CORT (400 μM) for 24 h after being pretreated with OI (25, 50, and 100 μM) for 18 h. Western blotting was used to detect the expression of Menin in HT22 cells. The data are expressed as means ± S.E.M. (n=3). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003eversus\u003c/em\u003e control group,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eversus\u003c/em\u003e CORT group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/296258bad3bf72d9c59c140b.png"},{"id":29659337,"identity":"ea448b75-57f4-446e-a83d-33dea7840677","added_by":"auto","created_at":"2022-11-29 15:32:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1730357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of LV-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMEN1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-siRNA on the expression of Menin in HT22 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHT22 cells were transfected with lentiviruses carrying either control siRNA or MEN1 siRNA (continuous red staining). (A) Fluorescence microscopy was used to confirm the efficiency of lentiviruses transient transfection in HT22 cells [magnification ×200]. (B) Western blotting was used to detect the expression of Menin in HT22 cells after lentivirus transfection. (C) A fluorescence microscope was used to confirm the stable transfection efficiency of lentivirus in the HT22 cell line constructed by puromycin screening [magnification ×200]. (D) Western blotting was used to detect the stable interference efficiency of the LV-\u003cem\u003eMEN1\u003c/em\u003e-siRNA-HT22 cell line. The data are expressed as means ± S.E.M. (n=3). ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001,\u003cem\u003e versus\u003c/em\u003e control-siRNA group.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/2c2dfd61acc4186e60b68bba.png"},{"id":29660040,"identity":"1589157f-baa2-44f1-9270-0ac1085ef905","added_by":"auto","created_at":"2022-11-29 15:40:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":166924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MEN1-siRNA on itaconate-alleviated neuroinflammation in CORT-exposed HT22 cells. \u003c/strong\u003eControl-siRNA HT22 cells and MEN1-siRNA HT22 cells were treated with CORT (400 μM) for 24 h after being pretreated with OI (100 Μm) for 18 h, respectively. ELISA kits were used to detect the contents of pro-inflammation factors IL-1β (A), TNF-α (B), and anti-inflammation factors IL-4 (C) and IL-10 (D). The data are expressed as means ± S.E.M. (n=3). ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eversus\u003c/em\u003e control group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 \u003cem\u003eversus\u003c/em\u003e CORT-treated group,\u003csup\u003e \u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eversus\u003c/em\u003e OI and CORT co-treatment group in control-siRNA.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/684cfd7632ae772a47f9e81a.png"},{"id":29659334,"identity":"e7460d7d-af2c-4b20-b882-4c9bc44840a6","added_by":"auto","created_at":"2022-11-29 15:32:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":466142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MEN1-siRNA on itaconate-ameliorated necroptosis in CORT-exposed HT22 cells. \u003c/strong\u003eControl-siRNA HT22 cells and MEN1-siRNA HT22 cells were treated with CORT (400 μM) for 24 h after being pretreated with 100 μM OI for 18 h, respectively. (A-C) Western blotting was used to detect the expression of necroptosis-related proteins p-RIP1/ RIP1 (A), p-RIP3/ RIP3 (B), and p-MLKL/ MLKL (C). (D) The CCK8 kit was used to detect the viability of cells. \u0026nbsp;The data are expressed as means ± S.E.M. (n=3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eversus\u003c/em\u003e control group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003eversus\u003c/em\u003e CORT-treated group,\u003csup\u003e \u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 \u003cem\u003eversus\u003c/em\u003e OI and CORT co-treatment group in control-siRNA.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/2683cb65f9388354d8d1b237.png"},{"id":55920785,"identity":"80fe3cec-62d2-42aa-a176-f63361002eda","added_by":"auto","created_at":"2024-05-06 10:04:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3586664,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/a189def2-1cdd-426f-852a-e001957e7258.pdf"},{"id":29659329,"identity":"72167e36-3de1-4240-a502-d5981134cd03","added_by":"auto","created_at":"2022-11-29 15:32:09","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":130827,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-2282581/v1/e1454908c3c8d362d1bccf20.png"}],"financialInterests":"","formattedTitle":"Itaconate Inhibits Corticosterone-Induced Necroptosis and Neuroinflammation via Up-regulating Menin in HT22 Cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChronic stress is one of the crucial risk factors for depression\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Stress exposure activates the hypothalamic-pituitary-adrenal (HPA) axis\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, thereby increasing the secretion of corticosterone (CORT)\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. It is confirmed that sustained CORT exposure damages hippocampus neurons and induces depression-like behaviors\u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, as a result of triggering neuroinflammation\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. But the underlying mechanism of CORT-induced neuroinflammation remains unclear. It is of great significance to further clarify the potential mechanism of CORT-caused neuroinflammation and find the corresponding therapeutic strategy for the prevention and treatment of depression.\u003c/p\u003e \u003cp\u003eNecroptosis is a lytic cell death distinct from apoptosis\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Gene Ontology (GO) enrichment analysis showed that necroptosis is activated in an animal model of chronic stress-induced depression\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Whereas inhibiting necroptosis improves depression-like behaviors\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Additionally, necroptosis induces neuroinflammation\u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, while inhibition of RIP1, one of the main mediators of necroptosis, ameliorates neuroinflammation\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Therefore, we made a bold conjecture that CORT-induced neuroinflammation is triggered by necroptosis.\u003c/p\u003e \u003cp\u003eItaconate, an immunomodulator metabolite, is derived from the tricarboxylic acid (TCA) cycle intermediate cis-aconitate\u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The level of itaconate obviously ascends after inflammatory stimulation, to restrict the progression of inflammation\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. The endogenous metabolite itaconate has been reported to inhibit NLRP3 inflammasome\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e and the release of inflammation cytokines\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Importantly, itaconate attenuates neuroinflammatory response in BV2 microglia cells\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e and reduces proinflammatory factors in the brains of AD model mice\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Thus, the present study observed whether itaconate alleviates CORT-induced neuroinflammation. Furthermore, studies implicated necroptosis as an essential role in driving inflammation due to its pro-inflammatory features\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Therefore, we will explore whether itaconate prevents CORT-induced necroptosis and uncover the possible mechanism of itaconate exerting anti-inflammatory effects.\u003c/p\u003e \u003cp\u003eWhat is the mechanism for itaconate to inhibit necroptosis under CORT- exposure? Menin is a scaffold protein\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, which is highly expressed in the brain\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. But little is known about its function in the nervous system. Recent studies found that the expression of Menin is down-regulated by chronic stress\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e and that Menin deficiency enhances neuroinflammation\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Upregulating Menin in astrocytes antagonizes neuroinflammation\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. These results indicated that upregulation of Menin in the brain is a potential therapeutic candidate for the prevention of neuroinflammation. Therefore, the present work will further explore the mechanism underlying the antagonistic role of itaconate in CORT-induced necroptosis from the insight of Menin. Here, our study illustrated that itaconate protects neurons against CORT-induced neuroinflammation and necroptosis as well as up-regulates the expression of Menin in HT22 cells. Furthermore, silencing Menin reversed the improving effect of itaconate on CORT-elicited necroptosis and neuroinflammation. Therefore, our study demonstrated that itaconate prevents CORT-induced neuroinflammation and necroptosis through up-regulation of Menin.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Antibodies and Regents\u003c/h2\u003e \u003cp\u003eAntibodies: anti-βactin (20536-1-AP), anti-Tubulin (10094-1-AP), anti-GAPDH (60004-1-Ig), Goat anti-mouse antibody (SA0001-1), Goat anti-rabbit antibody (SA0001-1) were purchased from Proteintech company in the United States. Anti-Menin (ab31902) was purchased from Epitomics-an Abcam Company in the United States. Anti-p-RIP1 (65746) and anti-RIP1 (3493) were obtained from Cell Signaling Technology in the United States. Anti-P-RIP3 (AF7443), anti-RIP3 (AF7942), anti-P-MLKL (AF7420), and anti-MLKL (AF7412) were purchased from Affinity Biosciences in the United States.\u003c/p\u003e \u003cp\u003eRegents: Corticosterone (C0388, TCL Chemical Industry Development Co., Ltd., Shanghai). 4-Octyl Itaconate (3133-16-2, MedChemExpress, USA), DMSO (D2650, Sigma, USA), Trypsin (T4049, Sigma, USA), CCK-8 (Dongren Chemical Technology Co., Ltd., Shanghai), MSD (Dongren Chemical Technology Co., Ltd., USA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cells culture\u003c/h2\u003e \u003cp\u003eHT22 cells were obtained from Shunran Biotechnology Co., Ltd (Shanghai, China) and were incubated in a DMEM medium with 10% FBS and 1% Penicillin streptomycin. And cultured in the 37 centigrade, 5% CO2 incubator. Replace DMEM medium every 1\u0026ndash;2 days. Subculture and frozen cells when the cells are in the logarithmic growth phase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cells viability assay\u003c/h2\u003e \u003cp\u003eThe viability of HT22 cells was determined by Cell Counting Kit-8. HT22 cells were seeded in the 96-well plates. The experiment was carried out according to the instruction. The optical density of each well was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, USA). The viability of HT22 cells was calculated by (Corrected absorbance value of experimental group/Corrected absorbance value of control group) \u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Transmission electron microscope observation assay\u003c/h2\u003e \u003cp\u003eThe morphology of HT22 cells was observed with a transmission electron microscope (TEM). The samples were fixed with 2.5% glutaraldehyde overnight and 1% forge acid for 2\u0026ndash;3 hours. After dehydration, it was embedded overnight at room temperature and stained with lead citrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RNA interference analysis\u003c/h2\u003e \u003cp\u003eMEN1-siRNA interference sequence and control sequence were designed and synthesized by GeneChem Corporation (Shanghai, China) and were constructed into GV298 plasmid. The protein samples were collected after transfection for 24 h for western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blot analysis\u003c/h2\u003e \u003cp\u003eThe expressions of necroptosis-related proteins and Menin were detected by western blot analysis. Total protein was prepared and separated by 10% \u0026minus;\u0026thinsp;12% SDS-PAGE and then transferred onto PVDF membranes (IPVH00010, Merck Millipore, Birrika, MA, USA). Blocking with Tris Buffered saline Tween (TBST, 50 mM Tris\u0026thinsp;\u0026minus;\u0026thinsp;HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) containing 5% skim milk for 2 h at room temperature, following incubated with primary antibody against RIP1, p-RIP1, RIP3, p-RIP3, MLKL, p-MLKL (1:1000), Tubulin, GAPDH (1:4000) overnight at 4 centigrade. After this, the bands were washed 5 times with TBST solution, and then the second antibody was incubated. Finally, Bands were visualized using the Tanon-5600 gel imaging system (Tanon Science \u0026amp; Technology Co., Ltd., Shanghai, China) and analyzed by Image-J software (NIH, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Enzyme-linked immunosorbent assay\u003c/h2\u003e \u003cp\u003eThe contents of IL-1β, TNF-α, IL-4, and IL-10 were measured with the ELISA kits and were purchased from R\u0026amp;D systems (Minneapolis, MN, USA), including Mouse IL-1β ELISA kit (MLB00C), Mouse TNF-α ELISA kit(MTA00B), Mouse IL-4 ELISA kit (M4000B), Mouse IL-10 ELISA kit (M1000B). Detailed methods for performing the analysis are described in the instructions of manufacturers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were analyzed by one-way ANOVA followed by the least significant difference (LSD) post hoc test with SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Sample capacity\u0026thinsp;\u0026ge;\u0026thinsp;3, the data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M.). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 is considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e3.1 CORT induces neuroinflammation in HT22 cells\u003c/h2\u003e\n \u003cp\u003eTo explore whether CORT induces neuroinflammation in HT22 cells, the contents of pro-inflammatory factors (IL-1\u0026beta;, TNF-\u0026alpha;) and anti-inflammatory factors (IL-4, IL-10) in HT22 cells were examined by ELISA. We found that the contents of pro-inflammatory factors, IL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) and TNF-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), in HT22 cells were increased by treatment with CORT (200, 400, and 800 \u0026micro;M) for 24 h. By contrast, the contents of anti-inflammatory factors, IL-4 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) and IL-10 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD), in HT22 cells were decreased by treatment with CORT (200, 400, and 800 \u0026micro;M) for 24 h. Taken together, these results indicated that CORT induces neuroinflammation in HT22 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003e3.2 CORT induces necroptosis in HT22 cells\u003c/h2\u003e\n \u003cp\u003eNext, we explored whether CORT-induced neuroinflammation is involved in necroptosis in HT22 cells. We observed the morphology of HT22 cells with a Transmission electron microscope (TEM) after being treated with CORT (400 \u0026micro;M) for 24 h. We found that HT22 cells are characterized by cell membrane incomplete and cytoplasm swelling (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). This result is in accordance with the previous result\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Then, we detected the effects of CORT on the expressions of necroptosis-related proteins in HT22 cells. Treatment with CORT (400, 800 \u0026micro;M) for 24 h up-regulated the expressions of p-RIP1/RIP1 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), p-RIP3/RIP3 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), and p-MLKL/MLKL (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD) in HT22 cells. We also measured the viability of cells by CCK-8 assay and found that after treatment with CORT (200, 400, and 800 \u0026micro;M) for 24 h, the viability of HT22 cells was decreased in a concentration-dependent (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Taken together, these above results indicated that CORT induces necroptosis in HT22 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.3 CORT down-regulates the expression of Menin in HT22 cells\u003c/h2\u003e\n \u003cp\u003eIt has been reported that Menin deficiency aggravates neuroinflammation\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, so we next explored the influence of CORT on the expression of Menin in HT22 cells. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the expression of Menin in HT22 cells was dramatically inhibited by treatment with CORT (400, 800 \u0026micro;M) for 24 h, which indicated that CORT down-regulates the expression of Menin in HT22 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.4 Itaconate prevents CORT-induced neuroinflammation in HT22 cells\u003c/h2\u003e\n \u003cp\u003eTo determine the antagonistic effect of itaconate on CORT-induced neuroinflammation in HT22 cells, HT22 cells were pretreated with OI (25, 50, and 100 \u0026micro;M) for 18 h and then exposed to CORT (400 \u0026micro;M) for 24 h. We found that itaconate decreased the contents of pro-inflammatory factors [IL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) and TNF-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB)] and increased the contents of anti-inflammatory factors [IL-4 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) and IL-10 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD)]. Additionally, treatment with itaconate alone had no effect on IL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), TNF-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), IL-4 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), and IL-10(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). Taken together, these results indicated that itaconate prevents CORT-induced neuroinflammation in HT22 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.5 Itaconate alleviates CORT-induced necroptosis in HT22 cells\u003c/h2\u003e\n \u003cp\u003eWe next detected the effect of itaconate on CORT-induced necroptosis. HT22 cells were pretreated with OI (25, 50, and 100 \u0026micro;M) for 18 h and then exposed to CORT (400 \u0026micro;M) for 24 h. We found that itaconate (50, 100 \u0026micro;M) down-regulated the expressions of necroptosis-related proteins, p-RIP1/ RIP1 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), p-RIP3/ RIP3 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), and p-MLKL/ MLKL (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), in HT22 cells exposed to CORT (400 \u0026micro;M) for 24 h. Moreover, itaconate alone-treated had no effect on expressions of necroptosis-related proteins [p-RIP1/ RIP1 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), p-RIP3/ RIP3 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB) and p-MLKL/ MLKL (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC)]. Additionally, Itaconate (50, 100 \u0026micro;M) increased the viability of HT22 cells exposed to CORT (400 \u0026micro;M) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). These data indicated that itaconate antagonizes CORT-induced necroptosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003e3.6 Itaconate up-regulates the expression of Menin in HT22 cells\u003c/h2\u003e\n \u003cp\u003ewe further explored the effect of itaconate on the expression of Menin in HT22 cells. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, Itaconate (50, 100 \u0026micro;M) increased the expression of Menin in HT22 cells with or without CORT exposure, indicating that itaconate up-regulates the expression of Menin in HT22 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003e3.7 LV-\u003cem\u003eMEN1\u003c/em\u003e-siRNA silences the expression of Menin in HT22 cells\u003c/h2\u003e\n \u003cp\u003eTo further confirm the mediatory role of Menin in the protection of itaconate against CORT-induced necroptosis, we will observe whether silencing Menin abolishes the antagonistic effect of itaconate on CORT-induced cytotoxicity, necroptosis, and neuroinflammation in HT22 cells. To this end, we transiently transfected HT22 cells with three different RNA interference chains and detected the efficiency of lentiviruses transient transfection with fluorescence microscopy and western blotting. We found that three RNA interference strands were transfected successfully (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Additionally, western blotting showed that three RNA interference strands, especially \u003cem\u003eMEN1\u003c/em\u003e-siRNA (2), decreased the expression of Menin, while LV-\u003cem\u003eControl\u003c/em\u003e-siRNA did not affect the expression of Menin (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). Therefore, we constructed stable cell lines with \u003cem\u003eMEN1\u003c/em\u003e-siRNA (2) and further confirm the stable transfection efficiency of lentivirus with fluorescence microscopy and western blotting. We found that LV-\u003cem\u003eControl\u003c/em\u003e-siRNA and LV-\u003cem\u003eMEN1\u003c/em\u003e-siRNA were transfected successfully (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC) and \u003cem\u003eMEN1\u003c/em\u003e-siRNA (2) decreased the expression of Menin, while \u003cem\u003eControl\u003c/em\u003e-siRNA did not affect the expression of Menin (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003e3.8 Silencing Menin reverses the antagonism effect of itaconate on CORT-induced neuroinflammation in HT22 cells\u003c/h2\u003e\n \u003cp\u003eWe further explored whether Menin deficiency inhibits the protective effects of itaconate against CORT-induced neuroinflammation in HT22 cells and found that silencing Menin abolished itaconate-exerted decreases in pro-inflammation factors, including IL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA) and TNF-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB) as well as increases in anti-inflammation factors, including IL-4 (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC) and IL-10 (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD) in HT22 cells. These data indicated that Menin deficiency abolishes the protective effect of itaconate against CORT-induced neuroinflammation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec20\"\u003e\n \u003ch2\u003e3.9 Menin deficiency reverses the antagonistic effect of itaconate on CORT-induced necroptosis in HT22 cells\u003c/h2\u003e\n \u003cp\u003eWe next explored whether silencing Menin reverses the antagonistic effect of itaconate on CORT-induced necroptosis. We found that silencing Menin upregulated the expressions of p-RIP1/ RIP1 (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA), p-RIP3/ RIP3 (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB), and p-MLKL/ MLKL (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eC) in HT22 cells cotreated CORT and itaconate. Furthermore, silencing Menin decreased the viability of HT22 cells cotreated expression CORT and itaconate (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD). These data indicated that silencing Menin reverses the antagonistic effect of itaconate on CORT-induced necroptosis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present work is aimed to investigate whether necroptosis involves in CORT-induced neuroinflammation as well as explore the protective effect of itaconate against CORT-induced neuroinflammation and the underlying mechanisms. We here clarified that exposure of HT22 cells to CORT not only increased the contents of pro-inflammatory factors as well as decreased the contents of anti-inflammatory factors but also increased the expression of necroptosis-related proteins and decreased the viability of cells. Notably, itaconate suppressed CORT-induced neuroinflammation and necroptosis as well as up-regulated the expression of Menin in HT22 cells. Furthermore, silencing Menin not only reversed itaconate-ameliorated necroptosis but also abolished itaconate-reduced neuroinflammation. Taken together, these results revealed that necroptosis is involved in CORT-induced neuroinflammation and that itaconate inhibits\u0026nbsp;CORT-induced neuroinflammation and necroptosis via up-regulating Menin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCorticosterone, a stress hormone\u003csup\u003e[36]\u003c/sup\u003e, induces depression-like behaviors via neuroinflammation\u003csup\u003e[37, 38]\u003c/sup\u003e. Numerous studies revealed that chronic CORT exposure induces inflammatory factors IL-1\u0026beta; and TNF-\u0026alpha; release and neurotoxicity\u003csup\u003e[6, 11, 39]\u003c/sup\u003e. Our study also confirmed the neuroinflammatory effect of CORT, as evidenced by CORT exposure increases pro-inflammatory factors (IL-1\u0026beta; and TNF-\u0026alpha;) as well as decreases anti-inflammatory factors (IL-4 and IL-10). Necroptosis, a kind of programmed necrosis mechanism different from apoptosis and necrosis\u003csup\u003e[40, 41]\u003c/sup\u003e, is tightly implicated in neuroinflammation\u003csup\u003e[27, 42, 43]\u003c/sup\u003e. Necroptosis in microglia promotes the release of pro-inflammatory cytokines\u003csup\u003e[44]\u003c/sup\u003e. In this work, we have demonstrated that the expression of necroptosis-related proteins RIP1, RIP3, and MLKL were increased as well as the viability of cells was decreased in CORT-exposed HT22 cells, indicating that CORT triggers necroptosis. It has been confirmed that deficiency\u0026nbsp;of necroptosis-related proteins RIP1 or RIP3 alleviates neuroinflammation\u003csup\u003e[45]\u003c/sup\u003e. Therefore, blocking CORT-stimulated necroptosis is a novel effective method to inhibit neuroinflammation. Furthermore, the activation of necroptosis is closely related to depression\u003csup\u003e[46]\u003c/sup\u003e. Necroptosis inhibitor (necrostatin-1) ameliorates chronic unpredictable mild stress (CUMS)-induced depression-like behaviors\u003csup\u003e[46]\u003c/sup\u003e. Based on the crucial role of neuroinflammation in the development of depression. Therefore, finding a drug that blocks necroptosis and neuroinflammation stimulated by CORT is a novel therapeutic strategy for depression.\u003c/p\u003e\n\u003cp\u003eThe Krebs cycle-derived metabolite itaconate is synthesized from the enzyme immune-responsive gene 1 (IRG1)\u003csup\u003e[23]\u003c/sup\u003e. Endogenous itaconate deficiency increases inflammatory levels\u003csup\u003e[47]\u003c/sup\u003e. Overexpression of IRG-1 inhibits neuroinflammation in microglia\u003csup\u003e[48]\u003c/sup\u003e. Itaconate treatment reverses LPS-induced neuroinflammation in microglia\u003csup\u003e[25]\u003c/sup\u003e. However, it remains to investigate whether exogenous administration of itaconate improves neuroinflammation in CORT-exposed HT22 cells. In our data, itaconate ameliorates CORT-increased pro-inflammatory factors IL-1\u0026beta; and TNF-\u0026alpha; as well as CORT-decreased anti-inflammatory factors IL-4 and IL-10 in HT22 cells, which indicated that itaconate prevents CORT-induced neuroinflammation. Necroptosis, as one of the important ways of cell death, triggers neuroinflammation\u003csup\u003e[27]\u003c/sup\u003e.\u0026nbsp;Based on these findings, our present study was designed to investigate whether itaconate inhibits CORT-induced neuroinflammation by antagonizing necroptosis. Our data revealed that itaconate increases the viability of HT22 cells and decreases the expression of necroptosis-related proteins RIP1, RIP3, and MLKL in CORT-exposed HT22 cells.\u0026nbsp;In summary, our results demonstrated that itaconate inhibits necroptosis to prevent CORT-induced neuroinflammation.\u003c/p\u003e\n\u003cp\u003eAlthough the anti-inflammatory effect of itaconate has been fully confirmed, its underlying mechanism needs to be explored. Menin is a scaffold protein encoded by the Multiple endocrine neoplasia type 1 (MEN1) gene\u003csup\u003e[49, 50]\u003c/sup\u003e. It has been confirmed that targeting Menin-regulated inflammation in astrocytes ameliorates depression-like behaviors\u003csup\u003e[32]\u003c/sup\u003e. Therefore, we focus on the effect of Menin in the protective effect of itaconate against CORT-induced necroptosis and neuroinflammation. Our finding showed that itaconate up-regulates the expression level of Menin, and silencing Menin with Lentivirus transfection cancels the protective effect of itaconate against CORT-induced necroptosis and neuroinflammation in HT22 cells. KEGG analysis verified that the differentially expressed genes after MEN1 knockdown enrich inflammation responses\u003csup\u003e[51]\u003c/sup\u003e. Menin ablation induces activation of the inflammation pathway\u003csup\u003e[52]\u003c/sup\u003e. To sum up, these results support our working hypothesis that up-regulation of Menin is indispensable to the protective effect of itaconate against CORT-induced neuroinflammation,\u0026nbsp;as a result of ameliorating necroptosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, our present work revealed that itaconate ameliorates CORT-induced neuroinflammation and necroptosis and up-regulates Menin in CORT-exposed HT22 cells. Furthermore, we further found that blocking Menin prevents the protective effect of itaconate against necroptosis and neuroinflammation elicited by CORT. These results illustrated that Menin is significant to the protective effect of itaconate against CORT-induced necroptosis and neuroinflammation. Our results highlighted itaconate as an attractive agent for the therapy strategy for CORT-induced neuroinflammation and provided a novel insight into the mechanisms underlying the antagonistic action of itaconate in CORT-induced neuroinflammation from necroptosis and Menin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe datasets and primary data in this study are available from the corresponding author on reasonable request.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis work was supported by [\u003c/em\u003eNational Natural Science Foundation of China\u003cem\u003e] (Grant numbers [\u003c/em\u003e81971267\u003cem\u003e]) and [\u003c/em\u003eProject of Hunan Provincial Department of Education\u003cem\u003e] (Grant numbers [\u003c/em\u003e21B0429\u003cem\u003e]).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eETHICS APPROVAL STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis experiment does not involve animals, patients and clinical trials.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJin-Yu Liang: Writing-original draft; Shan Gao: Conceptualization; Mei-Jia Jiang: Investigation, Formal analysis; Ping Zhang: Validation; Wei Zou: Resources; Xiao-Qing Tang: Writing - review \u0026amp; editing, Supervision, Data curation, Funding acquisition; Yi-Yun Tang: Writing - review \u0026amp; editing, Supervision, Data curation, Funding acquisition.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eCONSENT TO PARTICIPATE\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNot applicable.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll\u0026nbsp;authors\u0026nbsp;have\u0026nbsp;approved\u0026nbsp;the\u0026nbsp;manuscript\u0026nbsp;and\u0026nbsp;agreed\u0026nbsp;to\u0026nbsp;submission\u0026nbsp;to\u0026nbsp;the\u0026nbsp;journal.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEGEMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNot applicable.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang, H.L., et al., \u003cem\u003eAdolescent stress increases depression-like behaviors and alters the excitatory-inhibitory balance in aged mice.\u003c/em\u003e Chin Med J (Engl), 2019. \u003cstrong\u003e132\u003c/strong\u003e(14): p. 1689-1699.\u003c/li\u003e\n\u003cli\u003eSeo, J.S., et al., \u003cem\u003eCellular and molecular basis for stress-induced depression.\u003c/em\u003e Mol Psychiatry, 2017. \u003cstrong\u003e22\u003c/strong\u003e(10): p. 1440-1447.\u003c/li\u003e\n\u003cli\u003eDing, Y. and J. 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Tricarboxylic acid cycle metabolite itaconate has an anti-inflammatory role. Necroptosis acts as programmed cell death triggering neuroinflammation. The deficiency of Menin, a multifunctional scaffold protein, aggravates neuroinflammation. In this study, we explored whether itaconate inhibits CORT-induced neuroinflammation and necroptosis as well as the mediatory role of Menin in this protective effect of itaconate using an exposure of CORT to HT22 hippocampal neuronal cells. The viability of HT22 cells was examined by the Cell Counting Kit 8 (CCK-8). The morphology of HT22 cells was observed by transmission electron microscope (TEM). The expressions of necroptosis-related proteins (p-RIP1/ RIP1, p-RIP3/ RIP3, and p-MLKL/ MLKL) were evaluated by Western blotting. The contents of inflammatory factors were detected by an enzyme-linked immunosorbent assay kit. Our results showed that CORT increases the contents of pro-inflammatory factors (IL-1β, TNF-α) as well as decreases the contents of anti-inflammatory factors (IL4, IL10) in HT22 cells. We also found that CORT increases the expressions of necroptosis-related proteins (p-RIP1/ RIP1, p-RIP3/ RIP3, and p-MLKL/ MLKL) and decreases the cell viability in HT22 cells, indicating that CORT induces necroptosis to HT22 cells. Itaconate improves CORT-induced neuroinflammation and necroptosis. Furthermore, itaconate upregulates the expression of Menin in CORT-exposed HT22 cells. Importantly, silencing Menin abolishes the antagonistic effect of itaconate on CORT-induced necroptosis and neuroinflammation. In brief, these results indicated that itaconate protects HT22 cells against CORT-induced neuroinflammation and necroptosis via upregulating Menin.\u003c/p\u003e","manuscriptTitle":"Itaconate Inhibits Corticosterone-Induced Necroptosis and Neuroinflammation via Up-regulating Menin in HT22 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-29 15:32:04","doi":"10.21203/rs.3.rs-2282581/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b45ee0fc-353a-4de1-ae4a-2c210005c87d","owner":[],"postedDate":"November 29th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-06T10:04:29+00:00","versionOfRecord":{"articleIdentity":"rs-2282581","link":"https://doi.org/10.1007/s13105-024-01012-3","journal":{"identity":"journal-of-physiology-and-biochemistry","isVorOnly":false,"title":"Journal of Physiology and Biochemistry"},"publishedOn":"2024-03-01 10:04:29","publishedOnDateReadable":"March 1st, 2024"},"versionCreatedAt":"2022-11-29 15:32:04","video":"","vorDoi":"10.1007/s13105-024-01012-3","vorDoiUrl":"https://doi.org/10.1007/s13105-024-01012-3","workflowStages":[]},"version":"v1","identity":"rs-2282581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2282581","identity":"rs-2282581","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}