Roles of metformin and pioglitazone in regulating neurotoxic astrocyte activation in EAE mice

Roles of metformin and pioglitazone in regulating neurotoxic astrocyte activation in EAE mice | 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 Roles of metformin and pioglitazone in regulating neurotoxic astrocyte activation in EAE mice Suping Qin, Bohui Yuan, Jing jing Guo, Xuejiao Zhang, Yi Ding, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5406730/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neuro-inflammation is a vital mediator involved in the pathology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Neurotoxic A1 reactive astrocytes associate with neuro-inflammatory reactivity in MS/EAE. Although Metformin and Pioglitazone, two antidiabetic drugs, inhibit inflammation, the regulation mechanisms underlying A1 astrocytes in MS remain unclear. Herein, we show that Metformin and/or Pioglitazone downregulate inflammatory response, the activation of A1 astrocytes and the AKT/mTOR/STAT3 signaling pathway in primary mouse astrocytes treated with IL-17. In EAE mice, Metformin and/or Pioglitazone decreased significantly inflammation and demyelination and ameliorated the pathological process of disease. Overall, these findings uncover that Metformin and/or Pioglitazone suppress the inflammatory response in A1 astrocytes and alleviate the pathogenesis of EAE mice in vitro and in vivo . Targeting the cytotoxicity of A1 astrocytes may be a promising strategy for treating demyelinating diseases. Multiple sclerosis Experimental autoimmune encephalomyelitis A1 astrocytes AKT/mTOR/STAT3 signaling Metformin Pioglitazone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Multiple sclerosis (MS) is a chronic autoimmune disease in the central nervous system (CNS) characterized by inflammatory reaction, glial activation and demyelination. It was reported that there was one case of MS every 5 minutes worldwide in 2020, and currently approximately 2.8 million people suffer from it [ 1 ]. Traditionally, MS is divided into three clinical courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS) [ 2 ]. CNS lesions of MS are mostly linked to myelin loss, inflammatory infiltration of immune cells, reactive gliosis, BBB disruption and axonal damage [ 3 ]. The underlying pathology mechanisms of MS refer to the activation of auto-reactive lymphocytes (CD4 + Th1, CD4 + Th17, CD8 + T cell and B cell). However, glial cells are now recognized to be important players contributing to MS, particularly in the chronic progression [ 4 , 5 ]. Astrocytes occupy a role at all stages of the MS pathology. In MS and EAE, a widely-used animal model of MS, reactive astrocytes release inflammatory cytokines and mediators to participate in the damage of the blood-brain barrier and to promote the infiltration of inflammatory cells and demyelination, which is the intrinsic neurotoxic activity in the course of MS pathogenesis [ 4 , 6 , 7 ]. Reactive astrocytes in MS/EAE lesions have been described as a neurotoxic ‘A1’ phenotype [ 7 , 8 ]. The current clinical therapies for MS target mostly the peripheral immune system, however, which have repeatedly failed in progressive MS [ 4 ]. There is limited research and development of drugs for astrocytes in MS. Metformin is comprehensively the first-line drug in the treatment of type 2 diabetes via activating AMP-activated protein kinase (AMPK), especially in patients with obesity. It is known that the main effect of metformin is the reduction of insulin resistance [ 9 , 10 ]. Furthermore, Metformin alleviates inflammatory reaction through suppressing AKT (protein kinase B) signal in macrophages and promotes diabetic wound healing via AKT/mTOR (the mammalian target of rapamycin) pathway [ 11 , 12 ]. However, new functions of Metformin are constantly being discovered. Several studies have shown that Metformin inhibits the differentiation of Th17 cells and immune responses and to protect oligodendrocyte damage to restore CNS functions against inactivating AMPK [ 13 , 14 ]. Meanwhile, Metformin has potential therapeutic function in scleroderma by inhibiting the production of pro-inflammatory mediators in fibroblasts through an AMPK-independent mTOR-STAT3 (the signal transducer and activator of transcription 3) pathway [ 15 , 16 ]. Metformin treatment improves cognitive function along with alleviation of microglial activation and astrocyte hypertrophy, reduction of pro-inflammatory cytokines production in the hippocampus [ 17 ]. However, the effect and mechanism of Metformin on astrocytes in MS/EAE are unclear. Recent evidence suggests that antidiabetic drug Pioglitazone, a highly potent agonist of the peroxisome proliferator activating factor-γ (PPARγ), suppresses mTOR signaling in neurons and the inflammatory response of astrocytes in spinal cord in the nerve-injury-induced neuropathic pain [ 18 ]. Pioglitazone improves functional recovery in MS [ 19 ]. However, the mechanism of Pioglitazone on astrocytes in MS/EAE is still not well-known. In the present study, we aim to further explore the function and mechanism of Metformin and Pioglitazone alone and in combination in regulating neurotoxic astrocytes (A1) activation and EAE process. We display that Metformin and Pioglitazone alone and in combination, inhibit the activation of A1 astrocytes, the production of inflammatory cytokines and the activation of AKT/mTOR/STAT3 in vitro and in vivo , alleviates the pathology in EAE mice. The effect of Metformin treatment alone was better than that of Pioglitazone treatment alone, and the effect of the combination of the two drugs was not significantly different from that of Metformin alone. MATERIAL AND METHODS Animal, Antibodies and Reagents Female 6- to 8-wk-old C57BL/6 mice were obtained from the Shanghai Experimental Animal Center, Chinese Academy of Sciences. All mice we housed in specific pathogen-free conditions. All animal protocols were approved by the Laboratory Animal Ethics Committee of Xuzhou Medical University. Antibodies and Reagents used in this study were as follows: anti-p-AKT (Ser473, 4060, Cell Signaling Technology), anti-AKT (4685, Cell Signaling Technology), anti-p-mTOR (Ser2448, 2971, Cell Signaling Technology), anti-mTOR (4517, Cell Signaling Technology), anti-p-STAT3 (Tyr705, BS4181, Bioworld Technology), anti-STAT3 (12640, Cell Signaling Technology), anti-GFAP (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-C3 (ab11862, Abcam). The secondary antibodies were all purchased from Sigma Biotechnology. Alexa Fluor® 488 donkey anti-mouse IgG (A21202) and Alexa Fluor® 594 donkey anti- Rabbit IgG (A21207) antibodies were from Life technologies. Myelin oligodendrocyte glycoprotein (MOG) amino acids 35–55 (MOG35-55 peptides, MEVGWYRSPFRVVHLYRNGK) were purchased from China Peptides Co. Ltd (Shanghai, China). Metformin and Pioglitazone were purchased from Med Chem Express (MCE). Recombinant mouse IL-17 was from R&D Systems. Cytokine and chemokine detection kits (Cytometric Bead Array) were from BD Biosciences. EAE Induction and Evaluation Procedures used for EAE induction are as described previously [ 20 ]. In brief, female C57BL/6 mice with age of 6–8 weeks were immunized by MOG 35 − 55 (250 µg,) emulsified in complete Freund’s adjuvant (Sigma Aldrich) with 5 mg/ml of Mycobacterium tuberculosis (H37Ra strain, Difco). Pertussis toxin (200 ng, Invitrogen) was intraperitoneally injected in mice on day 0 and day 2, then the clinical scores of EAE mice were examined by an experimentally blinded investigator daily. Clinical score assessed on a 0–5 scale as described previously. Primary Mouse Astrocyte Primary astrocytes of mice were separated and cultured as described previously in our publications [ 6 , 19 ]. Astrocytes were synchronized with non-serum culture media for 12 h prior to IL-17 treatment. Drug Treatment Animal: Metformin (Met)100 mg/kg/day and Pioglitazone (Pio) 15 mg/kg/day were suspended in DMSO/Saline solution respectively, which was administered alone or in combination by intraperitoneal injection in 100 µl volumes. Met or Pio treatment was started from the first day of established EAE to the end (peak period, 19th day post immunization), EAE mice without drug treatment received an equal volume solution, once daily. Astrocytes: 10 mM Met is administered for 2 h followed by stimulation with IL17 stimulation, and 10 µM Pio is administered for 1 h before IL17 stimulation in astrocytes. Real-Time PCR Assay Cells or spinal cords tissues were processed for RNA extraction using TRIzol reagent followed by cDNA synthesis and real-time PCR analysis. The transcription levels of gene were calculated with the 2 −ΔΔCT method. The primers are listed in the Supplementary Table 1. Cytometric Bead Array (CBA) Assay The supernatant of cultured astrocytes or the serum of mice was collected. The secretion levels of IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1), and interferon-inducible protein-10 (IP-10) were detected by CBA assay (BD Biosciences, USA) as described previously [ 19 ]. Western Blot Assay Western Blot Assay As described previously [ 19 , 20 ], the total protein was extracted from primary astrocytes and spinal cords tissues of mice, respectively. The expression of protein in samples was normalized by β-actin. Immunofluoresence Assay (IFA) Immunofluorescent staining of primary astrocytes was performed as described previously [ 6 ]. Histopathology Assay To evaluate inflammation and demyelination of EAE mice, 4 µm paraffin-embedded spinal cord sections were performed by hematoxylin and eosin (H&E) and luxol fast blue (LFB) staining, respectively [ 6 , 19 ]. Moreover, the ultrastructural of spinal cords were observed under electron microscope (EM). Statistical Analysis Data are given as mean ± S.E.M and analyzed by two-tailed unpaired Student's t test for two groups comparison or one-way multiple range analysis of variance (ANOVA) for multiple columns comparison. A Mann-Whitney test was used for nonparametric data (EAE scoring). P values were determined for at least three independent experiments in triplicates using GraphPad Prism version 8.5 software. P values < 0.05 were considered significantly. RESULTS IL-17 Induces the Activation of A1 Astrocytes and AKT/mTOR/STAT3 Pathway Studies on MS patients have confirmed that Th17 cells are abundant in peripheral blood, cerebrospinal fluid and lesions of MS patients, and the counts and inflammatory mediators of Th17 cells are further increased during relapses. Therefore, IL-17 as the cytokine highly secreted by effector Th17 cells, plays an important role in the process of MS [ 21 , 22 ]. We firstly detected the activation of A1 astrocytes and the production of inflammatory cytokines in mouse primary astrocytes stimulated by IL-17 in vitro . The results of Real time PCR showed that the mRNA transcription levels of the related gene of A1 astrocytes, including Ugt1a, H2D1, Serping1, H2T23, Srgn and C3, were significantly increased at 6 h by IL-17 stimulation and persisted until 12 h, compared with 0 h group (Fig. 1 a). Meanwhile, the mRNA level of inflammatory cytokines and chemokines such as IL-6, TNF-α, MCP-1 and IP-10 reached their peak at 6 h with IL-17 treatment and continued for 24 h (Fig. 1 b). It is suggested that IL-17 induced the activation of A1 reactive astrocytes and the production of pro-inflammatory cytokines. Due to the association of Akt/mTOR /STAT3 pathways with Th17 response and inflammation [ 15 , 16 , 18 ], we further explored the possible molecular mechanisms of IL-17- induced the activation of A1 astrocytes. Western blot assay was used to detect the activation of AKT/mTOR/STAT3 pathway. The results showed that the phosphorylation of AKT and mTOR reached their peak at 5 minutes after IL-17 stimulation (Fig. 1 c). Meanwhile, the level of STAT3 phosphorylation (p-STAT3) was increased at 3 h and maintained until 12 h (Fig. 1 d). Overall, the results suggested that IL-17 induced the activation of A1 astrocytes and the AKT/mTOR/STAT3 signaling pathway. Metformin and Pioglitazone Downregulate A1 Reactive Astrocytes in Vitro To determine the effect of Met and/or Pio on the activation of A1 astrocytes, primary mouse astrocytes were stimulated by IL-17, prior to pre-treated with Met and/or Pio. As shown in Fig. 2 a, the mRNA transcription levels of the related gene of A1 astrocytes, including Ugt1a, H2D1, Serping1, H2T23, Srgn and C3, were significantly decreased by Met and/or Pio, compared with IL-17 stimulation. The results of IFA showed that the protein expression of C3 was declined in astrocytes treated with Met and/or Pio prior to IL-17 stimulation (Fig. 2 b). These results suggested that Metformin and Pioglitazone downregulated the activation of A1 reactive astrocytes. Metformin and Pioglitazone Inhibit the Activation of AKT/mTOR/STAT3 Pathway and Production of Inflammatory Cytokines in Activated Astrocytes by IL-17 To explore the possible mechanism of Met and Pio in downregulaing A1 astrocytes activation, the activation of AKT/mTOR/STAT3 pathway was measured in primary mouse astrocytes stimulated by IL-17, prior to pre-treated with Met and/or Pio. The results presented that the phosphorylation level of AKT, mTOR and STAT3 was suppressed by Met and/or Pio, compared to IL-17 treatment (Fig. 3 a). And then, IFA was employed to observe the phosphorylation level of STAT3. The results displayed that the level of p-STAT3 was dramatically decreased and the nuclear translocation was significantly reduced in primary mouse astrocytes pre-treated with Metformin and/or Pioglitazone in comparison with IL-17 stimulation (Fig. 3 b). Furthermore, we asked whether Met and Pio disturbed Akt/mTOR/STAT3 pathways to regulate inflammatory reaction. So the mRNA and releasing levels of inflammatory cytokines and chemokines (IL-6, TNF-α、MCP-1 and IP-10) were detected. The results showed that the production and release of inflammatory cytokines and chemokines were significantly downregulated by Met and/or Pio in compared to IL-17 treatment (Fig. 4 a, b). The effect of Met treatment alone was better than that of Pio treatment alone, and the effect of the combination of the two drugs was not dramatically different from that of Met alone. Above results suggested that Metformin and Pioglitazone downregulated the activation of Akt/mTOR /STAT3 pathways in A1 astrocytes and the production of inflammatory cytokines. Metformin and Pioglitazone Inhibit A1 Astrocytes Activation and Alleviate Pathogenesis in EAE Mice To definite whether to affect the activation of A1 astrocytes in vivo , Metformin or Pioglitazone was injected alone or in combination into mice constituted by MOG 35 − 55 on day 0 post immunization (dpi) till 19th dpi. Firstly, the clinical score revealed that Met and/or Pio not only delayed the onset time of EAE but also alleviated pathogenesis (Fig. 5 a). Meanwhile, the production and release of IL-6, TNF-α、MCP-1 and IP-10 were dramatically reduced in Met and/or Pio group (Fig. 5 b, c). H&E and LFB staining exhibited that EAE mice had large number of inflammatory cell infiltration and more severe demyelination lesion in the spinal cords, in comparison with NC mice. In contrast, the EAE mice treated with Met and/or Pio were characterized by less inflammatory cell infiltration and demyelination lesion in the spinal cords than that of EAE mice (Fig. 5 d, e). The myelin sheath in EAE group was disintegrated under EM, whereas only a light loosening of the sheath was observed in Met and/or Pio group (Fig. 5 f). Finally, the phosphorylation level of AKT, mTOR and STAT3 was decreased in Met and/or Pio group, compared to EAE mice (Fig. 6 a). And then, the mRNA transcription levels of the related gene of A1 astrocytes, including Ugt1a, H2D1, Serping1, H2T23, Srgn and C3, were significantly decreased in Met and/or Pio group, compared with EAE group (Fig. 6 b). Thus, these data suggest that Metformin and Pioglitazone reduce A1 astrocytes activation, inflammatory response and myelin damage. DISCUSSION Reactive astrocytes play a vital role in recruiting inflammatory cells at the lesion sites, which is involved in the positive-feedback inflammatory loop to engender the procession of MS/EAE pathogenesis [ 7 , 23 ]. Persisting and excessive pro-inflammatory cytokines and neurotoxic mediators from reactive astrocytes exacerbate BBB dysfunction and massive infiltration of immune cells into the CNS [ 23 ]. Therefore, these combined effects of reactive astrocytes produce an inflammatory environment in the MS lesions so as to enlarge the process of illness. Herein, we find that Metformin and/or Pioglitazone, two kinds of antidiabetic drugs, alleviate the pathological process of EAE mice through downregulating A1 astrocytes and inflammatory response via inhibiting AKT/mTOR/STAT3 signals. Cytotoxic reactive astrocytes, termed as A1 astrocytes, participate in some CNS diseases, such as neurodegenerative and demyelinating diseases [ 8 , 24 , 25 , 26 ]. A1 astrocytes induced by activated microglia are deficient in enhancing neuronal survival, outgrowth, synaptogenesis and phagocytosis, in turn promoting the death of neurons and oligodendrocytes [ 8 ]. Inhibition of conversion of astrocytes to an A1 neurotoxic phenotype constructs neuroprotective properties in some neurodegenerative disorders and neurologic injuries [ 24 , 25 ]. Our results showed that IL-17 enhanced the mRNA transcription levels of the related markers of A1 astrocytes, such as Ugt1a, H2D1, Serping1, H2T23, Srgn and C3, which suggest that IL-17 induces activation of A1 reactivity astrocytes. AKT/mTOR/STAT3 pathway impacts on activation of A1 astrocytes [ 27 , 28 , 29 ]. Recent study indicated that downregulation of PI3K/AKT pathway altered the conversion of A1 phenotype [ 27 , 29 ]. Moreover, IL-10, a kind of anti-inflammatory factor, inhibited A1 phenotype through suppressing STAT3 pathway [ 28 ]. Here, IL-17 activated AKT/mTOR/STAT3 signal pathways, and promoted the production of inflammatory cytokines and chemokines including IL-6, TNF-α、MCP-1 and IP-10. Metformin is a popular oral glucose-lowering drugs, widely used to be therapy for patients with type 2 diabetes mellitus (T2DM) [ 30 ]. In terms of mechanism, Metformin possesses its capacity to interfere with AMPK and mTORC1 pathways, in turn acting on mitochondria and its antioxidant effects [ 31 , 32 ]. And Pioglitazone is another drug to be approved for the treatment of T2DM and can be administered in anti-inflammation [ 33 , 34 ]. Pioglitazone downregulates mTOR signaling in the inflammatory response of astrocytes [ 18 ]. Our evidence determined that Metformin and/or Pioglitazone inhibited A1 phenotype and the production of pro-inflammatory cytokines and alleviated the pathogenesis of EAE mice through downregulating AKT/mTOR/STAT3 signal pathways. In summary, our current evidence displays that two antidiabetic drugs, Metformin and pioglitazone alone and in combination decrease A1 phenotype through downregulating AKT/mTOR/STAT3 signal pathways and then repress the production of inflammatory cytokines and chemokines in astrocytes, in turn eventually lessening the development of EAE pathogenesis. Therefore, these findings emphasize that Metformin and Pioglitazone may play key roles in decreasing neuroinflammation derived from astrocytes and pathogenesis of MS. Declarations AUTHOR CONTRIBUTIONS Suping Qin, Bohui Yuan, Jinging Guo, Xiangyang Li, Hui Hua, Feng Zhou and Xiaomei Liu wrote the main manuscript text. Suping Qin, Jinging Guo, Yi Ding and Tianxin Zhang prepared figures 1-3 and Xiaotian Wang, Bohui Yuan, Feng Zhou and Xiaomei Liu prepared figures 4-6. All authors reviewed the manuscript. FUNDING This work was supported by the National Natural Science Foundation of China (81971179 to Liu), the Natural Science Foundation of Jiangsu Province (BK20231347 to Liu), Jiangsu Commission of Health (Z2019035 to Zhou), Jiangsu Provincial Department of Education (20KJA320004 to Zhou), the Priority Academic Program Development of Jiangsu Higher Education Institutions (2017 PAPD), and the Technology Innovation Foundation of Xuzhou City (KC23242 to Qin). INSTITUTIONAL REVIEW BOARD STATEMENT The Laboratory Animal Ethics Committee of Xuzhou Medical University, Jiangsu, China, approved all the experimental procedures for investigating these animals (an approval ID of 202209S049). INFORMED CONSENT STATEMENT Not applicable. DATA AVAILABILITY STATEMENT The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. ACKNOWLEDGMENTS The authors want to thank the Experimental Animal Center, Xuzhou Medical University, for their help with animal care. CONFLICTS OF INTEREST The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Charabati, M., et al. 2023. Multiple sclerosis: Neuroimmune crosstalk and therapeutic targeting. Cell 186 (7), 1309-1327. Kuhlmann, T., et al. 2023. Multiple sclerosis progression: time for a new mechanism-driven framework. The Lancet Neurology 22 (1), 78-88. Ruiz, F., et al. 2019. Resolution of inflammation during multiple sclerosis. Seminars in Immunopathology 41 (6), 711-726. Healy, L. M., et al. 2022. The role of glial cells in multiple sclerosis disease progression. Nature Reviews Neurology 18 (4), 237-248. Dolgin, E. 2016. Therapies: Progressive steps. Nature 540 (7631), S7-S9. Zhang, Q., et al. 2022. 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04:53:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5406730/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5406730/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69843132,"identity":"11b7a247-852e-4749-8e92-c6b2ced5b997","added_by":"auto","created_at":"2024-11-25 18:28:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":475904,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17 induces A1 reactivity astrocytes and activates AKT/mTOR/STAT3 signals \u003cem\u003ein vitro. \u0026nbsp;\u003c/em\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eThe mRNA transcription levels of the related markers of A1 astrocytes (Ugt1a, H2D1, Serping1, H2T23, Srgn and C3) and \u003cstrong\u003eb \u003c/strong\u003eof inflammatory cytokines and chemokines (IL-6 , TNF-α、MCP-1 and IP-10) were measured by real-time PCR assay in primary mouse astrocytes treated with IL-17 at different time point. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 versus 0 h group (n = 3 per group). Data are represented as the means ± SEM. \u003cstrong\u003ec-d\u003c/strong\u003e The levels of p-AKT, p-mTOR and p-STAT3 were detected by Western blot assay in primary mouse astrocytes treated with IL-17. Numbers under the bands were the relative intensity of the bands after calculating for loading control using β-actin. The relative value of proteins in 0 h or 0 min group was considered as “1.0”; same for all of following Western blotting assay.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5406730/v1/b18b67878fd8c6649cee717b.png"},{"id":69843133,"identity":"e481235e-7eae-4b26-9795-3b09a55fb985","added_by":"auto","created_at":"2024-11-25 18:28:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2216158,"visible":true,"origin":"","legend":"\u003cp\u003eMetformin and Pioglitazone suppress in A1 phenotype of astrocytes induced by IL-17. Primary mouse astrocytes were pretreated with Met and Pio alone and in combination, prior to stimulate with IL-17. \u003cstrong\u003ea\u003c/strong\u003e The mRNA transcription levels of the related markers of A1 astrocytes were detected by real-time PCR assay. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 versus DMEM group. \u003csup\u003e\u003cstrong\u003e★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e★★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e★★★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 versus DMSO + IL-17 group (n = 3 per group). The Data are from three independent experiments and represented as the means ± SEM. \u003cstrong\u003eb \u003c/strong\u003eImmunofluorescent staining for GFAP (green), C3 (red) and nuclear staining of DAPI (blue) in primary astrocytes treated with IL-17. Scale bars, 20 µm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5406730/v1/27c0fdde8a860bf979ba19e7.png"},{"id":69843134,"identity":"77fba8c1-fab0-4844-944d-83ee172e73b8","added_by":"auto","created_at":"2024-11-25 18:28:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1647134,"visible":true,"origin":"","legend":"\u003cp\u003eMetformin and Pioglitazone downregulate AKT/mTOR/STAT3 signals in astrocytes activated by IL-17. Primary mouse astrocytes were pretreated with Met and Pio alone and in combination, prior to stimulate by IL-17. \u003cstrong\u003ea\u003c/strong\u003eThe levels of p-AKT, p-mTOR and p-STAT3 were evaluated by Western blot assay in primary mouse astrocytes, respectively. \u003cstrong\u003eb\u003c/strong\u003e Immunofluorescent staining for GFAP (green), p-STAT3 (red) and nuclear staining of DAPI (blue) in primary astrocytes treated with IL-17. Scale bars, 20 µm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5406730/v1/0fb53007f4ff6f4769cae946.png"},{"id":69843325,"identity":"acb65066-5d95-430f-b16d-f382dedb4392","added_by":"auto","created_at":"2024-11-25 18:36:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":463290,"visible":true,"origin":"","legend":"\u003cp\u003eMetformin and Pioglitazone decrease the production of pro-inflammatory cytokines in astrocytes activated by IL-17. \u003cstrong\u003ea\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eThe mRNA transcription levels and \u003cstrong\u003eb\u003c/strong\u003e\u003cem\u003e \u003c/em\u003esecreting levels of inflammatory cytokines and chemokines (IL-6 , TNF-α、MCP-1 and IP-10) were examined by real-time PCR assay and ELISA in primary mouse astrocytes pretreated with Met and Pio alone and in combination, prior to stimulate with IL-17 for 6 h. Results were represented as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 versus DMEM. \u003csup\u003e\u003cstrong\u003e★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e★★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e★★★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 versus DMSO + IL-17 group (n = 3 per group).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5406730/v1/c0fb8ab63ba9d33aa77b14ed.png"},{"id":69843128,"identity":"f646a575-53ef-4fda-8045-cf2e6d980a15","added_by":"auto","created_at":"2024-11-25 18:28:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2605555,"visible":true,"origin":"","legend":"\u003cp\u003eMetformin and Pioglitazone alleviate EAE pathogenesis as well as inflammatory cytokine production\u003cem\u003e \u003c/em\u003ein mice. \u003cstrong\u003ea\u003c/strong\u003e The clinical scores of EAE mice injected with Met and Pio alone and in combination (n=15 mice per group). \u003cstrong\u003eb-c\u003c/strong\u003e The levels of IL-6, TNF-α, MCP-1, IP-10 from the spinal cords and peripheral blood in mice were analyzed by real-time PCR and CBA assay, respectively. \u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001 versus NC group;\u003csup\u003e\u003cstrong\u003e ★\u003c/strong\u003e\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e★★\u003c/strong\u003e\u003c/sup\u003ep \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e★★★\u003c/strong\u003e\u003c/sup\u003ep \u0026lt; 0.001 versus DMSO+EAE group (n = 3 per group). \u003cstrong\u003ed\u003c/strong\u003e H\u0026amp;E, \u003cstrong\u003ee\u003c/strong\u003e LFB staining and \u003cstrong\u003ef\u003c/strong\u003e electron microscope were employed to measure inflammatory cells infiltrations, the medullary sheath damage in spinal cords of mice (Scale bars, 20 µm).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5406730/v1/b86d61ded39fb261b4e08095.png"},{"id":69843324,"identity":"d46e8d3b-79b8-4bfc-a667-509acc62ba6b","added_by":"auto","created_at":"2024-11-25 18:36:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":532413,"visible":true,"origin":"","legend":"\u003cp\u003eMetformin and Pioglitazone inhibit AKT/mTOR/STAT3 signals and A1 astrocytes activity in EAE mice. \u003cstrong\u003ea\u003c/strong\u003e The levels of p-AKT, p-mTOR and p-STAT3 were evaluated by Western blot assay. \u003cstrong\u003eb\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eThe mRNA transcription levels of the related markers of A1 astrocytes (Ugt1a, H2D1, Serping1, H2T23, Srgn and C3 were detected by real-time PCR assay. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 versus NC group;\u003csup\u003e\u003cstrong\u003e ★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e★★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e★★★\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001versus DMSO + EAE group (n = 3 per group). Data are represented as the means ± SEM.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5406730/v1/757424d91ec3b1276b6db335.png"},{"id":74368085,"identity":"55e43312-abde-4fee-99a9-9cc4b6fb88cf","added_by":"auto","created_at":"2025-01-21 14:32:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8057466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5406730/v1/02230c5d-c990-4e71-be89-fa8b29610322.pdf"},{"id":69843131,"identity":"9f5d2b14-b8a6-44fb-983c-529fa2e8ba86","added_by":"auto","created_at":"2024-11-25 18:28:45","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":40186,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5406730/v1/03bf7f64e0dbb4e935ace0d7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Roles of metformin and pioglitazone in regulating neurotoxic astrocyte activation in EAE mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMultiple sclerosis (MS) is a chronic autoimmune disease in the central nervous system (CNS) characterized by inflammatory reaction, glial activation and demyelination. It was reported that there was one case of MS every 5 minutes worldwide in 2020, and currently approximately 2.8\u0026nbsp;million people suffer from it [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Traditionally, MS is divided into three clinical courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. CNS lesions of MS are mostly linked to myelin loss, inflammatory infiltration of immune cells, reactive gliosis, BBB disruption and axonal damage [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The underlying pathology mechanisms of MS refer to the activation of auto-reactive lymphocytes (CD4\u003csup\u003e+\u003c/sup\u003e Th1, CD4\u003csup\u003e+\u003c/sup\u003e Th17, CD8\u003csup\u003e+\u003c/sup\u003e T cell and B cell). However, glial cells are now recognized to be important players contributing to MS, particularly in the chronic progression [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAstrocytes occupy a role at all stages of the MS pathology. In MS and EAE, a widely-used animal model of MS, reactive astrocytes release inflammatory cytokines and mediators to participate in the damage of the blood-brain barrier and to promote the infiltration of inflammatory cells and demyelination, which is the intrinsic neurotoxic activity in the course of MS pathogenesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Reactive astrocytes in MS/EAE lesions have been described as a neurotoxic \u0026lsquo;A1\u0026rsquo; phenotype [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The current clinical therapies for MS target mostly the peripheral immune system, however, which have repeatedly failed in progressive MS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. There is limited research and development of drugs for astrocytes in MS.\u003c/p\u003e \u003cp\u003eMetformin is comprehensively the first-line drug in the treatment of type 2 diabetes via activating AMP-activated protein kinase (AMPK), especially in patients with obesity. It is known that the main effect of metformin is the reduction of insulin resistance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, Metformin alleviates inflammatory reaction through suppressing AKT (protein kinase B) signal in macrophages and promotes diabetic wound healing via AKT/mTOR (the mammalian target of rapamycin) pathway [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, new functions of Metformin are constantly being discovered. Several studies have shown that Metformin inhibits the differentiation of Th17 cells and immune responses and to protect oligodendrocyte damage to restore CNS functions against inactivating AMPK [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Meanwhile, Metformin has potential therapeutic function in scleroderma by inhibiting the production of pro-inflammatory mediators in fibroblasts through an AMPK-independent mTOR-STAT3 (the signal transducer and activator of transcription 3) pathway [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Metformin treatment improves cognitive function along with alleviation of microglial activation and astrocyte hypertrophy, reduction of pro-inflammatory cytokines production in the hippocampus [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the effect and mechanism of Metformin on astrocytes in MS/EAE are unclear.\u003c/p\u003e \u003cp\u003eRecent evidence suggests that antidiabetic drug Pioglitazone, a highly potent agonist of the peroxisome proliferator activating factor-γ (PPARγ), suppresses mTOR signaling in neurons and the inflammatory response of astrocytes in spinal cord in the nerve-injury-induced neuropathic pain [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Pioglitazone improves functional recovery in MS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the mechanism of Pioglitazone on astrocytes in MS/EAE is still not well-known.\u003c/p\u003e \u003cp\u003eIn the present study, we aim to further explore the function and mechanism of Metformin and Pioglitazone alone and in combination in regulating neurotoxic astrocytes (A1) activation and EAE process. We display that Metformin and Pioglitazone alone and in combination, inhibit the activation of A1 astrocytes, the production of inflammatory cytokines and the activation of AKT/mTOR/STAT3 in \u003cem\u003evitro\u003c/em\u003e and in \u003cem\u003evivo\u003c/em\u003e, alleviates the pathology in EAE mice. The effect of Metformin treatment alone was better than that of Pioglitazone treatment alone, and the effect of the combination of the two drugs was not significantly different from that of Metformin alone.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal, Antibodies and Reagents\u003c/h2\u003e \u003cp\u003eFemale 6- to 8-wk-old C57BL/6 mice were obtained from the Shanghai Experimental Animal Center, Chinese Academy of Sciences. All mice we housed in specific pathogen-free conditions. All animal protocols were approved by the Laboratory Animal Ethics Committee of Xuzhou Medical University.\u003c/p\u003e \u003cp\u003eAntibodies and Reagents used in this study were as follows: anti-p-AKT (Ser473, 4060, Cell Signaling Technology), anti-AKT (4685, Cell Signaling Technology), anti-p-mTOR (Ser2448, 2971, Cell Signaling Technology), anti-mTOR (4517, Cell Signaling Technology), anti-p-STAT3 (Tyr705, BS4181, Bioworld Technology), anti-STAT3 (12640, Cell Signaling Technology), anti-GFAP (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-C3 (ab11862, Abcam). The secondary antibodies were all purchased from Sigma Biotechnology. Alexa Fluor\u0026reg; 488 donkey anti-mouse IgG (A21202) and Alexa Fluor\u0026reg; 594 donkey anti- Rabbit IgG (A21207) antibodies were from Life technologies. Myelin oligodendrocyte glycoprotein (MOG) amino acids 35\u0026ndash;55 (MOG35-55 peptides, MEVGWYRSPFRVVHLYRNGK) were purchased from China Peptides Co. Ltd (Shanghai, China). Metformin and Pioglitazone were purchased from Med Chem Express (MCE). Recombinant mouse IL-17 was from R\u0026amp;D Systems. Cytokine and chemokine detection kits (Cytometric Bead Array) were from BD Biosciences.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEAE Induction and Evaluation\u003c/h3\u003e\n\u003cp\u003eProcedures used for EAE induction are as described previously [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In brief, female C57BL/6 mice with age of 6\u0026ndash;8 weeks were immunized by MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e (250 \u0026micro;g,) emulsified in complete Freund\u0026rsquo;s adjuvant (Sigma Aldrich) with 5 mg/ml of Mycobacterium tuberculosis (H37Ra strain, Difco). Pertussis toxin (200 ng, Invitrogen) was intraperitoneally injected in mice on day 0 and day 2, then the clinical scores of EAE mice were examined by an experimentally blinded investigator daily. Clinical score assessed on a 0\u0026ndash;5 scale as described previously.\u003c/p\u003e\n\u003ch3\u003ePrimary Mouse Astrocyte\u003c/h3\u003e\n\u003cp\u003ePrimary astrocytes of mice were separated and cultured as described previously in our publications [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Astrocytes were synchronized with non-serum culture media for 12 h prior to IL-17 treatment.\u003c/p\u003e\n\u003ch3\u003eDrug Treatment\u003c/h3\u003e\n\u003cp\u003eAnimal: Metformin (Met)100 mg/kg/day and Pioglitazone (Pio) 15 mg/kg/day were suspended in DMSO/Saline solution respectively, which was administered alone or in combination by intraperitoneal injection in 100 \u0026micro;l volumes. Met or Pio treatment was started from the first day of established EAE to the end (peak period, 19th day post immunization), EAE mice without drug treatment received an equal volume solution, once daily.\u003c/p\u003e \u003cp\u003eAstrocytes: 10 mM Met is administered for 2 h followed by stimulation with IL17 stimulation, and 10 \u0026micro;M Pio is administered for 1 h before IL17 stimulation in astrocytes.\u003c/p\u003e\n\u003ch3\u003eReal-Time PCR Assay\u003c/h3\u003e\n\u003cp\u003eCells or spinal cords tissues were processed for RNA extraction using TRIzol reagent followed by cDNA synthesis and real-time PCR analysis. The transcription levels of gene were calculated with the 2 \u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The primers are listed in the Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCytometric Bead Array (CBA) Assay\u003c/h2\u003e \u003cp\u003eThe supernatant of cultured astrocytes or the serum of mice was collected. The secretion levels of IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1), and interferon-inducible protein-10 (IP-10) were detected by CBA assay (BD Biosciences, USA) as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot Assay\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern Blot Assay\u003c/div\u003e \u003cp\u003eAs described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the total protein was extracted from primary astrocytes and spinal cords tissues of mice, respectively. The expression of protein in samples was normalized by β-actin.\u003c/p\u003e\n\u003ch3\u003eImmunofluoresence Assay (IFA)\u003c/h3\u003e\n\u003cp\u003eImmunofluorescent staining of primary astrocytes was performed as described previously [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology Assay\u003c/h2\u003e \u003cp\u003eTo evaluate inflammation and demyelination of EAE mice, 4 \u0026micro;m paraffin-embedded spinal cord sections were performed by hematoxylin and eosin (H\u0026amp;E) and luxol fast blue (LFB) staining, respectively [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, the ultrastructural of spinal cords were observed under electron microscope (EM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M and analyzed by two-tailed unpaired Student's t test for two groups comparison or one-way multiple range analysis of variance (ANOVA) for multiple columns comparison. A Mann-Whitney test was used for nonparametric data (EAE scoring). P values were determined for at least three independent experiments in triplicates using GraphPad Prism version 8.5 software. \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significantly.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIL-17 Induces the Activation of A1 Astrocytes and AKT/mTOR/STAT3 Pathway\u003c/h2\u003e \u003cp\u003eStudies on MS patients have confirmed that Th17 cells are abundant in peripheral blood, cerebrospinal fluid and lesions of MS patients, and the counts and inflammatory mediators of Th17 cells are further increased during relapses. Therefore, IL-17 as the cytokine highly secreted by effector Th17 cells, plays an important role in the process of MS [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We firstly detected the activation of A1 astrocytes and the production of inflammatory cytokines in mouse primary astrocytes stimulated by IL-17 \u003cem\u003ein vitro\u003c/em\u003e. The results of Real time PCR showed that the mRNA transcription levels of the related gene of A1 astrocytes, including Ugt1a, H2D1, Serping1, H2T23, Srgn and C3, were significantly increased at 6 h by IL-17 stimulation and persisted until 12 h, compared with 0 h group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Meanwhile, the mRNA level of inflammatory cytokines and chemokines such as IL-6, TNF-α, MCP-1 and IP-10 reached their peak at 6 h with IL-17 treatment and continued for 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). It is suggested that IL-17 induced the activation of A1 reactive astrocytes and the production of pro-inflammatory cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to the association of Akt/mTOR /STAT3 pathways with Th17 response and inflammation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], we further explored the possible molecular mechanisms of IL-17- induced the activation of A1 astrocytes. Western blot assay was used to detect the activation of AKT/mTOR/STAT3 pathway. The results showed that the phosphorylation of AKT and mTOR reached their peak at 5 minutes after IL-17 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Meanwhile, the level of STAT3 phosphorylation (p-STAT3) was increased at 3 h and maintained until 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Overall, the results suggested that IL-17 induced the activation of A1 astrocytes and the AKT/mTOR/STAT3 signaling pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetformin and Pioglitazone Downregulate A1 Reactive Astrocytes\u003c/b\u003e \u003cb\u003ein Vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the effect of Met and/or Pio on the activation of A1 astrocytes, primary mouse astrocytes were stimulated by IL-17, prior to pre-treated with Met and/or Pio. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, the mRNA transcription levels of the related gene of A1 astrocytes, including Ugt1a, H2D1, Serping1, H2T23, Srgn and C3, were significantly decreased by Met and/or Pio, compared with IL-17 stimulation. The results of IFA showed that the protein expression of C3 was declined in astrocytes treated with Met and/or Pio prior to IL-17 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These results suggested that Metformin and Pioglitazone downregulated the activation of A1 reactive astrocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMetformin and Pioglitazone Inhibit the Activation of AKT/mTOR/STAT3 Pathway and Production of Inflammatory Cytokines in Activated Astrocytes by IL-17\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the possible mechanism of Met and Pio in downregulaing A1 astrocytes activation, the activation of AKT/mTOR/STAT3 pathway was measured in primary mouse astrocytes stimulated by IL-17, prior to pre-treated with Met and/or Pio. The results presented that the phosphorylation level of AKT, mTOR and STAT3 was suppressed by Met and/or Pio, compared to IL-17 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). And then, IFA was employed to observe the phosphorylation level of STAT3. The results displayed that the level of p-STAT3 was dramatically decreased and the nuclear translocation was significantly reduced in primary mouse astrocytes pre-treated with Metformin and/or Pioglitazone in comparison with IL-17 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we asked whether Met and Pio disturbed Akt/mTOR/STAT3 pathways to regulate inflammatory reaction. So the mRNA and releasing levels of inflammatory cytokines and chemokines (IL-6, TNF-α、MCP-1 and IP-10) were detected. The results showed that the production and release of inflammatory cytokines and chemokines were significantly downregulated by Met and/or Pio in compared to IL-17 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). The effect of Met treatment alone was better than that of Pio treatment alone, and the effect of the combination of the two drugs was not dramatically different from that of Met alone. Above results suggested that Metformin and Pioglitazone downregulated the activation of Akt/mTOR /STAT3 pathways in A1 astrocytes and the production of inflammatory cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMetformin and Pioglitazone Inhibit A1 Astrocytes Activation and Alleviate Pathogenesis in EAE Mice\u003c/h2\u003e \u003cp\u003eTo definite whether to affect the activation of A1 astrocytes \u003cem\u003ein vivo\u003c/em\u003e, Metformin or Pioglitazone was injected alone or in combination into mice constituted by MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e on day 0 post immunization (dpi) till 19th dpi. Firstly, the clinical score revealed that Met and/or Pio not only delayed the onset time of EAE but also alleviated pathogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Meanwhile, the production and release of IL-6, TNF-α、MCP-1 and IP-10 were dramatically reduced in Met and/or Pio group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). H\u0026amp;E and LFB staining exhibited that EAE mice had large number of inflammatory cell infiltration and more severe demyelination lesion in the spinal cords, in comparison with NC mice. In contrast, the EAE mice treated with Met and/or Pio were characterized by less inflammatory cell infiltration and demyelination lesion in the spinal cords than that of EAE mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e). The myelin sheath in EAE group was disintegrated under EM, whereas only a light loosening of the sheath was observed in Met and/or Pio group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Finally, the phosphorylation level of AKT, mTOR and STAT3 was decreased in Met and/or Pio group, compared to EAE mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). And then, the mRNA transcription levels of the related gene of A1 astrocytes, including Ugt1a, H2D1, Serping1, H2T23, Srgn and C3, were significantly decreased in Met and/or Pio group, compared with EAE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Thus, these data suggest that Metformin and Pioglitazone reduce A1 astrocytes activation, inflammatory response and myelin damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eReactive astrocytes play a vital role in recruiting inflammatory cells at the lesion sites, which is involved in the positive-feedback inflammatory loop to engender the procession of MS/EAE pathogenesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Persisting and excessive pro-inflammatory cytokines and neurotoxic mediators from reactive astrocytes exacerbate BBB dysfunction and massive infiltration of immune cells into the CNS [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, these combined effects of reactive astrocytes produce an inflammatory environment in the MS lesions so as to enlarge the process of illness. Herein, we find that Metformin and/or Pioglitazone, two kinds of antidiabetic drugs, alleviate the pathological process of EAE mice through downregulating A1 astrocytes and inflammatory response via inhibiting AKT/mTOR/STAT3 signals.\u003c/p\u003e \u003cp\u003eCytotoxic reactive astrocytes, termed as A1 astrocytes, participate in some CNS diseases, such as neurodegenerative and demyelinating diseases [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A1 astrocytes induced by activated microglia are deficient in enhancing neuronal survival, outgrowth, synaptogenesis and phagocytosis, in turn promoting the death of neurons and oligodendrocytes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Inhibition of conversion of astrocytes to an A1 neurotoxic phenotype constructs neuroprotective properties in some neurodegenerative disorders and neurologic injuries [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our results showed that IL-17 enhanced the mRNA transcription levels of the related markers of A1 astrocytes, such as Ugt1a, H2D1, Serping1, H2T23, Srgn and C3, which suggest that IL-17 induces activation of A1 reactivity astrocytes.\u003c/p\u003e \u003cp\u003eAKT/mTOR/STAT3 pathway impacts on activation of A1 astrocytes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Recent study indicated that downregulation of PI3K/AKT pathway altered the conversion of A1 phenotype [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Moreover, IL-10, a kind of anti-inflammatory factor, inhibited A1 phenotype through suppressing STAT3 pathway [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Here, IL-17 activated AKT/mTOR/STAT3 signal pathways, and promoted the production of inflammatory cytokines and chemokines including IL-6, TNF-α、MCP-1 and IP-10.\u003c/p\u003e \u003cp\u003eMetformin is a popular oral glucose-lowering drugs, widely used to be therapy for patients with type 2 diabetes mellitus (T2DM) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In terms of mechanism, Metformin possesses its capacity to interfere with AMPK and mTORC1 pathways, in turn acting on mitochondria and its antioxidant effects [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. And Pioglitazone is another drug to be approved for the treatment of T2DM and can be administered in anti-inflammation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Pioglitazone downregulates mTOR signaling in the inflammatory response of astrocytes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our evidence determined that Metformin and/or Pioglitazone inhibited A1 phenotype and the production of pro-inflammatory cytokines and alleviated the pathogenesis of EAE mice through downregulating AKT/mTOR/STAT3 signal pathways.\u003c/p\u003e \u003cp\u003eIn summary, our current evidence displays that two antidiabetic drugs, Metformin and pioglitazone alone and in combination decrease A1 phenotype through downregulating AKT/mTOR/STAT3 signal pathways and then repress the production of inflammatory cytokines and chemokines in astrocytes, in turn eventually lessening the development of EAE pathogenesis. Therefore, these findings emphasize that Metformin and Pioglitazone may play key roles in decreasing neuroinflammation derived from astrocytes and pathogenesis of MS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuping Qin, Bohui Yuan, Jinging Guo, Xiangyang Li, Hui Hua, Feng Zhou and Xiaomei Liu wrote the main manuscript text. Suping Qin, Jinging Guo, Yi Ding and Tianxin Zhang prepared figures 1-3 and Xiaotian Wang, Bohui Yuan, Feng Zhou and Xiaomei Liu prepared figures 4-6. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (81971179 to Liu), the Natural Science Foundation of Jiangsu Province (BK20231347 to Liu), Jiangsu Commission of Health (Z2019035 to Zhou), Jiangsu Provincial Department of Education (20KJA320004 to Zhou), the Priority Academic Program Development of Jiangsu Higher Education Institutions (2017 PAPD), and the Technology Innovation Foundation of Xuzhou City (KC23242 to Qin).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eINSTITUTIONAL REVIEW BOARD STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;Laboratory Animal Ethics Committee of Xuzhou Medical University, Jiangsu, China, approved all the experimental procedures for investigating these animals (an approval ID of 202209S049).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eINFORMED CONSENT STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors want to thank the Experimental Animal Center,\u0026nbsp;Xuzhou Medical\u0026nbsp;University, for their help with animal care.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCharabati, M., et al. 2023. 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Pioglitazone: The forgotten, cost-effective cardioprotective drug for type 2 diabetes. \u003cem\u003eDiabetes and Vascular Disease Research 16\u003c/em\u003e(2), 133-143.\u003c/li\u003e\n\u003cli\u003ePapanas, N., et al. 2011. Pioglitazone: a valuable component of combination therapy for type 2 diabetes mellitus. \u003cem\u003eExpert Opinion on Pharmacotherapy 12\u003c/em\u003e(10), 1457-1461.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Multiple sclerosis, Experimental autoimmune encephalomyelitis, A1 astrocytes, AKT/mTOR/STAT3 signaling, Metformin, Pioglitazone","lastPublishedDoi":"10.21203/rs.3.rs-5406730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5406730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeuro-inflammation is a vital mediator involved in the pathology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Neurotoxic A1 reactive astrocytes associate with neuro-inflammatory reactivity in MS/EAE. Although Metformin and Pioglitazone, two antidiabetic drugs, inhibit inflammation, the regulation mechanisms underlying A1 astrocytes in MS remain unclear. Herein, we show that Metformin and/or Pioglitazone downregulate inflammatory response, the activation of A1 astrocytes and the AKT/mTOR/STAT3 signaling pathway in primary mouse astrocytes treated with IL-17. In EAE mice, Metformin and/or Pioglitazone decreased significantly inflammation and demyelination and ameliorated the pathological process of disease. Overall, these findings uncover that Metformin and/or Pioglitazone suppress the inflammatory response in A1 astrocytes and alleviate the pathogenesis of EAE mice \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Targeting the cytotoxicity of A1 astrocytes may be a promising strategy for treating demyelinating diseases.\u003c/p\u003e","manuscriptTitle":"Roles of metformin and pioglitazone in regulating neurotoxic astrocyte activation in EAE mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 18:28:40","doi":"10.21203/rs.3.rs-5406730/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":"551b6e8e-35d0-4e3b-bcc6-0542ed7f0a72","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-21T14:24:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-25 18:28:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5406730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5406730","identity":"rs-5406730","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}