INSL3 suppresses LPS-induced inflammation in N9 microglia cells

Yıl 2024, Cilt: 49 Sayı: 2, 489 – 496, 30.06.2024

Dilek Şaker , Gülfidan Coşkun , Sait Polat

https://doi.org/10.17826/cumj.1455491

Öz

Amaç: İnsülin Benzeri Peptid 3 (INSL3) reseptörünü de içeren G-protein kaplı reseptör (GPCR) ailesi, inflamasyonda Nükleer Faktör kappa B (NF-κB) aracılı yolakta yer almaktadır. Bu bakımdan INSL3’ün NF-κB yolu üzerinden inflamasyonda rol oynadığı düşünülebilir. Bu çalışmada INSL3’ün, lipopolisakkarit (LPS) ile indüklenen N9 mikroglia hücre hattında inflamasyon ve hücre canlılığı üzerindeki etkisini araştırdık.
Gereç ve Yöntem: N9 mikroglial hücreleri, 2 saat boyunca INSL3 ile ön işleme tabi tutuldu, ardından 6 saat boyunca LPS ile işleme tabi tutuldu. Hücre canlılığı WST-8 analizi ile belirlendi. İnterlökin-1β (IL-1β), Tümör nekroz faktörü (TNF)-α ve NF-κB düzeyleri de immünhistokimyasal yöntemlerle değerlendirildi.
Bulgular: LPS grubundaki hücrelerde morfolojide dejeneratif değişiklikler ve hücre canlılığında azalma görüldü. INSL3+LPS (1.21±0.06) grubunda hücrelerin genel görünümü ve canlılığı LPS (0.61±0.05) grubuna göre kontrol grubuna (1.92±0.04) daha benzerdi. INSL3’ün LPS kaynaklı IL-1β, TNF-α ve NF-κB seviyelerindeki artışı önlediği ve hücre ölümünü azalttığı belirlendi.
Sonuç: INSL3’ün inflamasyonu baskılayarak hücresel iyileşmeyi desteklediği ve inflamasyonu azaltan terapötik bir ajan olarak değerlendirilebileceği sonucuna varıldı.

Anahtar Kelimeler

Inflamasyon, insülin benzeri peptid 3, lipopolisakkarit, mikroglia

Kaynakça

• Medina-Rodriguez EM, Lowell JA, Worthen RJ, Syed SA, Beurel E. Involvement of innate and adaptive immune systems alterations in the pathophysiology and treatment of depression. Front Neurosci. 2018;12:547.
• Bachiller S, Jimenez-Ferrer I, Paulus A, Yang Y, Swanberg M, Deierborg T et al. Microglia in neurological diseases: a road map to brain-disease dependent-inflammatory response. Front Cell Neurosci. 2018;12:488.
• Casano AM, Peri F. Microglia: multitasking specialists of the brain. Dev Cell. 2015;32:469–77.
• Eren E, Tufekci KU, Isci KB, Tastan B, Genc K, Genc S. Sulforaphane inhibits lipopolysaccharide-induced inflammation, cytotoxicity, oxidative stress, and miR-155 expression and switches to mox phenotype through activating extracellular signal-regulated kinase 1/2-nuclear factor erythroid 2-related factor 2/antioxidant response element pathway in murine microglial cells. Front Immunol. 2018;9:36.
• Park J, Min JS, Kim B, Chae UB, Yun JW, Choi MS et al. Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-kappaB pathways. Neurosci Lett. 2015;584:191–6.
• Steardo L Jr, Bronzuoli MR, Iacomino A, Esposito G, Steardo L, Scuderi C. Does neuroinflammation turn on the flame in Alzheimer’s disease? Focus on astrocytes. Front Neurosci. 2015;9:259.
• von Bernhardi R, Eugenin-von Bernhardi L, Eugenin J. Microglial cell dysregulation in brain aging and neurodegeneration. Front Aging Neurosci. 2015;7:124.
• Otani T, Mizokami A, Hayashi Y, Gao J, Mori Y, Nakamura S, Takeuchi H, Hirata, M. Signaling pathway for adiponectin expression in adipocytes by osteocalcin. Cell Signal. 2015;27:532–44.
• Oury F, Ferron M, Huizhen W, Confavreux C, Xu L, Lacombe J et al. Osteocalcin regulates murine and human fertility through a pancreas-bone-testis axis. J Clin Invest. 2013;123:2421–33.
• Oury F, Sumara G, Sumara O, Ferron M, Chang H, Smith CE et al. Endocrine regulation of male fertility by the skeleton. Cell. 2011;144:796–809.
• Rochira V. Late-onset Hypogonadism: Bone health. Andrology. 2020;8:1539–50.
• Samuel CS, Royce SG, Hewitson TD, Denton KM, Cooney TE, Bennett RG. Anti-fibrotic actions of relaxin. Br J Pharmacol. 2017;174:962–76.
• Santora K, Rasa C, Visco D, Steinetz BG, Bagnell CA. Antiarthritic effects of relaxin, in combination with estrogen, in rat adjuvant-induced arthritis. J Pharmacol Exp Ther. 2007;322:887–93.
• Santos CR, Benjamin ACA, Chies AB, Domeniconi R F, Zochio GP. Spadella MA. Adjuvant-induced arthritis affects testes and ventral prostate of wistar rats. Andrology. 2020;8:473–85.
• Righi M, Mori L, De Libero G, Sironi M, Biondi A, Mantovani A, Donini SD, Ricciardi-Castagnoli P. Monokine production by microglial cell clones. Eur J Immunol. 1989;19:1443–8.
• Şaker D, Sencar L, Yılmaz DM, Polat S. Relationships between microRNA-20a and microRNA-125b expression and apoptosis and inflammation in experimental spinal cord injury. Neurol Res. 2019;41:991-1000.
• Rosadini CV, Kagan JC. Early innate immune responses to bacterial LPS. Curr Opin Immunol. 2016;44:14–9.
• Leifer CA, Medvedev AE. Molecular mechanisms of regulation of toll like receptor signaling. J Leukoc Biol. 2016;100:927–41.
• Brown GC, Neher JJ. Inflammatory neurodegeneration and mechanisms ofmicroglial killing of neurons. Mol Neurobiol. 2010;4:242–7.
• Kim J, Lee HJ, Park JH, Cha BY, Hoe HS. Nilotinib modulates LPS-induced cognitive impairment and neuroinflammatory responses by regulating P38/STAT3 signaling. J Neuroinflammation. 2022;19:187.
• Menassa DA, Gomez-Nicola D. Microglial dynamics during human brain development. Front Immunol. 2018;9:1014.
• Wu X, Yu T, Xu H, Sun X, Kou D, Li L. Morphological and functional changes of microglia cultured under different oxygen concentrations and the analysis of related mechanisms. Exp Ther Med. 2018;15:2015–9.
• Zhou X, Zhao R, Lv M, Xu X, Liu W, Li X et al. ACSL4 promotes microglia-mediated neuroinflammation by regulating lipid metabolism and VGLL4 expression. Brain Behav Immun. 2023;109:331-43.
• Zhou S, Guo X, Chen S, Xu Z, Duan W, Zeng B. Apelin-13 regulates LPS-induced N9 microglia polarization involving STAT3 signaling pathway. Neuropeptides. 2019;76:101938.
• Li P, Zhao G, Chen F, Ding Y, Wang T, Liu S et al. Rh-relaxin-2 attenuates degranulation of mast cells by inhibiting NF-κB through PI3K-AKT/TNFAIP3 pathway in an experimental germinal matrix hemorrhage rat model. J Neuroinflammation. 2020;17:250.
• Truong SHT, Bonnici B, Rupasinghe S, Kemp-Harper BK, Samuel CS, Broughton BRS. Post-stroke administration of H2 relaxin reduces functional deficits, neuronal apoptosis and immune cell infiltration into the mouse brain. Pharmacol Res. 2023;187:106611.
• Kohsaka T, Yoneda Y, Yoshida T, Minagawa I, Pitia AM, Iwasawa A, Ikegaya N. Relaxin exerts a protective effect during ischemia-reperfusion in the rat model. Andrology. 2022;10:179-189.
• Aragón-Herrera A, Couselo-Seijas M, Feijóo-Bandín S, Anido-Varela L, Moraña-Fernández S, Tarazón E et al. Relaxin-2 plasma levels in atrial fibrillation are linked to inflammation and oxidative stress markers. Sci Rep. 2022;12:22287.
• Aragón-Herrera A, Feijóo-Bandín S, Anido-Varela L, Moraña-Fernández S, Roselló-Lletí E, Portolés M et al. Relaxin-2 as a potential biomarker in cardiovascular diseases. J Pers Med. 2022;12:1021.
• Henn A, Lund S, Hedtjarn M, Schrattenholz A, Porzgen P, Leist M. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX. 2009;26:83–94.
• Stansley B, Post J, Hensley K. A comparative review of cell culture systems for the study of microglial biology in Alzheimer’s disease. J Neuroinflammation. 2012;9:115.

INSL3 suppresses LPS-induced inflammation in N9 microglia cells

Yıl 2024, Cilt: 49 Sayı: 2, 489 – 496, 30.06.2024

Dilek Şaker , Gülfidan Coşkun , Sait Polat

https://doi.org/10.17826/cumj.1455491

Öz

Purpose: The G-protein coated receptor (GPCR) family, including the Insulin-Like Peptide 3 (INSL3) receptor, is involved in the Nuclear Factor kappa B (NF-κB)-mediated pathway in inflammation. In this regard, it can be thought that INSL3 plays a role in inflammation via the NF-κB pathway. In this study, we investigated the effect of INSL3 on inflammation and cell viability in the lipopolysaccharide (LPS)-induced N9 microglia cell line.
Materials and Methods: N9 microglial cells were pretreated with INSL3 for 2 hours, and then treated with LPS for 6 hours. Cell viability was identified by WST-8 assay. Immunostaining was performed to evaluate the levels of Interleukin-1β (IL-1β), Tumor necrosis factor (TNF)-α, and NF-κB.
Results: The cells in the LPS group showed degenerative changes in morphology and decreased cell viability. In the INSL3+LPS group (1.21±0.06), the general appearance and viability of the cells were more similar to the control group (1.92±0.04) compared to the LPS group (0.61±0.05). It was determined that INSL3 prevented the LPS-induced increase in IL-1β, TNF-α, and NF-κB levels and decreased cell death.
Conclusion: INSL3 suppresses inflammation and thus promotes cellular healing and can be considered a therapeutic agent that reduces inflammation.

Anahtar Kelimeler

inflammation, insulin-like peptide 3, lipopolysaccharide, microglia

Kaynakça

• Medina-Rodriguez EM, Lowell JA, Worthen RJ, Syed SA, Beurel E. Involvement of innate and adaptive immune systems alterations in the pathophysiology and treatment of depression. Front Neurosci. 2018;12:547.
• Bachiller S, Jimenez-Ferrer I, Paulus A, Yang Y, Swanberg M, Deierborg T et al. Microglia in neurological diseases: a road map to brain-disease dependent-inflammatory response. Front Cell Neurosci. 2018;12:488.
• Casano AM, Peri F. Microglia: multitasking specialists of the brain. Dev Cell. 2015;32:469–77.
• Eren E, Tufekci KU, Isci KB, Tastan B, Genc K, Genc S. Sulforaphane inhibits lipopolysaccharide-induced inflammation, cytotoxicity, oxidative stress, and miR-155 expression and switches to mox phenotype through activating extracellular signal-regulated kinase 1/2-nuclear factor erythroid 2-related factor 2/antioxidant response element pathway in murine microglial cells. Front Immunol. 2018;9:36.
• Park J, Min JS, Kim B, Chae UB, Yun JW, Choi MS et al. Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-kappaB pathways. Neurosci Lett. 2015;584:191–6.
• Steardo L Jr, Bronzuoli MR, Iacomino A, Esposito G, Steardo L, Scuderi C. Does neuroinflammation turn on the flame in Alzheimer’s disease? Focus on astrocytes. Front Neurosci. 2015;9:259.
• von Bernhardi R, Eugenin-von Bernhardi L, Eugenin J. Microglial cell dysregulation in brain aging and neurodegeneration. Front Aging Neurosci. 2015;7:124.
• Otani T, Mizokami A, Hayashi Y, Gao J, Mori Y, Nakamura S, Takeuchi H, Hirata, M. Signaling pathway for adiponectin expression in adipocytes by osteocalcin. Cell Signal. 2015;27:532–44.
• Oury F, Ferron M, Huizhen W, Confavreux C, Xu L, Lacombe J et al. Osteocalcin regulates murine and human fertility through a pancreas-bone-testis axis. J Clin Invest. 2013;123:2421–33.
• Oury F, Sumara G, Sumara O, Ferron M, Chang H, Smith CE et al. Endocrine regulation of male fertility by the skeleton. Cell. 2011;144:796–809.
• Rochira V. Late-onset Hypogonadism: Bone health. Andrology. 2020;8:1539–50.
• Samuel CS, Royce SG, Hewitson TD, Denton KM, Cooney TE, Bennett RG. Anti-fibrotic actions of relaxin. Br J Pharmacol. 2017;174:962–76.
• Santora K, Rasa C, Visco D, Steinetz BG, Bagnell CA. Antiarthritic effects of relaxin, in combination with estrogen, in rat adjuvant-induced arthritis. J Pharmacol Exp Ther. 2007;322:887–93.
• Santos CR, Benjamin ACA, Chies AB, Domeniconi R F, Zochio GP. Spadella MA. Adjuvant-induced arthritis affects testes and ventral prostate of wistar rats. Andrology. 2020;8:473–85.
• Righi M, Mori L, De Libero G, Sironi M, Biondi A, Mantovani A, Donini SD, Ricciardi-Castagnoli P. Monokine production by microglial cell clones. Eur J Immunol. 1989;19:1443–8.
• Şaker D, Sencar L, Yılmaz DM, Polat S. Relationships between microRNA-20a and microRNA-125b expression and apoptosis and inflammation in experimental spinal cord injury. Neurol Res. 2019;41:991-1000.
• Rosadini CV, Kagan JC. Early innate immune responses to bacterial LPS. Curr Opin Immunol. 2016;44:14–9.
• Leifer CA, Medvedev AE. Molecular mechanisms of regulation of toll like receptor signaling. J Leukoc Biol. 2016;100:927–41.
• Brown GC, Neher JJ. Inflammatory neurodegeneration and mechanisms ofmicroglial killing of neurons. Mol Neurobiol. 2010;4:242–7.
• Kim J, Lee HJ, Park JH, Cha BY, Hoe HS. Nilotinib modulates LPS-induced cognitive impairment and neuroinflammatory responses by regulating P38/STAT3 signaling. J Neuroinflammation. 2022;19:187.
• Menassa DA, Gomez-Nicola D. Microglial dynamics during human brain development. Front Immunol. 2018;9:1014.
• Wu X, Yu T, Xu H, Sun X, Kou D, Li L. Morphological and functional changes of microglia cultured under different oxygen concentrations and the analysis of related mechanisms. Exp Ther Med. 2018;15:2015–9.
• Zhou X, Zhao R, Lv M, Xu X, Liu W, Li X et al. ACSL4 promotes microglia-mediated neuroinflammation by regulating lipid metabolism and VGLL4 expression. Brain Behav Immun. 2023;109:331-43.
• Zhou S, Guo X, Chen S, Xu Z, Duan W, Zeng B. Apelin-13 regulates LPS-induced N9 microglia polarization involving STAT3 signaling pathway. Neuropeptides. 2019;76:101938.
• Li P, Zhao G, Chen F, Ding Y, Wang T, Liu S et al. Rh-relaxin-2 attenuates degranulation of mast cells by inhibiting NF-κB through PI3K-AKT/TNFAIP3 pathway in an experimental germinal matrix hemorrhage rat model. J Neuroinflammation. 2020;17:250.
• Truong SHT, Bonnici B, Rupasinghe S, Kemp-Harper BK, Samuel CS, Broughton BRS. Post-stroke administration of H2 relaxin reduces functional deficits, neuronal apoptosis and immune cell infiltration into the mouse brain. Pharmacol Res. 2023;187:106611.
• Kohsaka T, Yoneda Y, Yoshida T, Minagawa I, Pitia AM, Iwasawa A, Ikegaya N. Relaxin exerts a protective effect during ischemia-reperfusion in the rat model. Andrology. 2022;10:179-189.
• Aragón-Herrera A, Couselo-Seijas M, Feijóo-Bandín S, Anido-Varela L, Moraña-Fernández S, Tarazón E et al. Relaxin-2 plasma levels in atrial fibrillation are linked to inflammation and oxidative stress markers. Sci Rep. 2022;12:22287.
• Aragón-Herrera A, Feijóo-Bandín S, Anido-Varela L, Moraña-Fernández S, Roselló-Lletí E, Portolés M et al. Relaxin-2 as a potential biomarker in cardiovascular diseases. J Pers Med. 2022;12:1021.
• Henn A, Lund S, Hedtjarn M, Schrattenholz A, Porzgen P, Leist M. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX. 2009;26:83–94.
• Stansley B, Post J, Hensley K. A comparative review of cell culture systems for the study of microglial biology in Alzheimer’s disease. J Neuroinflammation. 2012;9:115.

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