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PHYSIOPATHOLOGICAL EFFECTS OF PHOENIXIN

Year 2023, Volume: 4 Issue: Ek Sayı, 258 - 265, 16.10.2023

Abstract

Phoenixin (PNX) is a newly discovered endogenous neuropeptide produced from small integral
membrane protein 20 (Smim20). The most common isoforms are PNX-14 and PNX-20 amino acid
peptides. PNX, a ligand of G protein-coupled receptor 173 (GPR173), has been isolated mainly
from brain regions such as the hypothalamus and pituitary gland, and from many peripheral tissues.
PNX plays an important role in the regulation of reproductive function by increasing the level of
gonadotropin-releasing hormone and stimulating the release of luteinizing hormone from the
pituitary cells. However, PNX not only regulates the reproductive system, but also exerts anxiolytic,
anti-inflammatory and cell-protective effects. In addition, it has many functions in the regulation of
behavior, food intake, sensory perception, memory, body fluid balance, cardiovascular functions
and energy metabolism. This review will help us to understand the molecular structure of PNX, its
physiological effects and functions on systems, and its role in the pathogenesis and treatment
processes of various diseases.

References

  • 1- Yosten GL, Lyu RM, Hsueh AJ, et al. A novel reproductive peptide, phoenixin. J Neuroendocrinol 2013;25:206-15.
  • 2. Lyu RM, Huang XF, Zhang Y, et al. Phoenixin: a novel peptide in rodent sensory ganglia. Neuroscience 2013;10:622-31.
  • 3. Rocca C, Scavello F, Granieri MC, et al. Phoenixin-14: detection and novel physiological implications in cardiac modulation and cardioprotection. Cell Mol Life Sci. 2018;75:743-756.
  • 4. Prinz P, Scharner S, Friedrich T, et al. Central and peripheral expression sites of phoenixin-14 immunoreactivity in rats. Biochem Biophys Res Commun. 2017;4:195-201.
  • 5. Billert M, Kołodziejski PA, Strowski MZ, Nowak KW, Skrzypski M. Phoenixin-14 stimulates proliferation and insulin secretion in insulin producing INS-1E cells. Biochim Biophys Acta Mol Cell Res. 2019;1866:118533.
  • 6. Billert M, Wojciechowicz T, Jasaszwili M et al. Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism. Biochim Biophys Acta Mol Cell Biol Lipids. 2018;1863:1449-1457.
  • 7. Kalamon N, Błaszczyk K, Szlaga A, et al. Levels of the neuropeptide phoenixin-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue in rat model of polycystic ovary syndrome. Biochem Biophys Res Commun. 2020;6:628-635.
  • 8. Nguyen XP, Nakamura T, Osuka S, et al. Effect of the neuropeptide phoenixin and its receptor GPR173 during folliculogenesis. Reproduction. 2019;158:25-34.
  • 9. Cowan A, Lyu RM, Chen YH, Dun SL, Chang JK, Dun NJ. Phoenixin: A candidate pruritogen in the mouse. Neuroscience. 2015;3:541-8.
  • 10. Stein LM, Haddock CJ, Samson WK, Kolar GR, Yosten GL. The phoenixins: From discovery of the hormone to identification of the receptor and potential physiologic actions. Peptides. 2018;106:45-48.
  • 11. McIlwraith EK, Belsham DD. Phoenixin: Uncovering its receptor, signaling and functions. Acta Pharmacol Sin. 2018;39:774-778.
  • 12. Stein LM, Tullock CW, Mathews SK, et al. Hypothalamic action of phoenixin to control reproductive hormone secretion in females: Importance of the orphan G protein-coupled receptor Gpr173. Am J Physiol Integr Comp Physiol. 2016;311:R489-R496.
  • 13. Treen AK, Luo V, Belsham DD. Phoenixin Activates Immortalized GnRH and Kisspeptin Neurons Through the Novel Receptor GPR173. Mol Endocrinol 2016;30:872-888.
  • 14. Matsumoto M, Saito T, Takasaki J, et al. An evolutionarily conserved G-protein coupled receptor family, SREB, expressed in the central nervous system. Biochem Biophys Res Commun 2000;272:576-582.
  • 15. Ma H, Su D, Wang Q, et al. Phoenixin 14 inhibits ischemia/reperfusion-induced cytotoxicity in microglia. Arch Biochem Biophys 2020; 689:108411.
  • 16. Haddock CJ, Almeida-Pereira G, Stein LM, Yosten GLC, Samson WK. A novel regulator of thirst behavior: phoenixin. Am J Physiol Regul Integr Comp Physiol 2020;318:R1027-R1035.
  • 17. Guvenc G, Altinbas B, Kasikci E, et al. Contingent role of phoenixin and nesfatin-1 on secretions of the male reproductive hormones. Andrologia 2019;51:e13410.
  • 18. Ullah K, Ur Rahman T, Wu DD, et al. Phoenixin-14 concentrations are increased in association with luteinizing hormone and nesfatin-1 concentrations in women with polycystic ovary syndrome. Clin Chim Acta 2017;471:243-247.
  • 19. Schalla M, Prinz P, Friedrich T, et al. Phoenixin-14 injected intracerebroventricularly but not intraperitoneally stimulates food intake in rats. Peptides 2017;96:53-60.
  • 20. Friedrich T, Schalla MA, Scharner S, et al. Intracerebroventricular injection of phoenixin alters feeding behavior and activates nesfatin-1 immunoreactive neurons in rats. Brain Res 2019;15:188-195.
  • 21. Rajeswari JJ, Blanco AM, Unniappan S. Phoenixin-20 suppresses food intake, modulates glucoregulatory enzymes, and enhances glycolysis in zebrafish. Am J Physiol Regul Integr Comp Physiol 2020;318:R917-R928.
  • 22. Wang M, Deng SP, Chen HP, et al. Phoenixin participated in regulation of food intake and growth in spotted scat, Scatophagus argus. Comp Biochem Physiol B Biochem Mol Biol 2018;226:36-44.
  • 23. Brown CH. Magnocellular Neurons and Posterior Pituitary Function. Compr. Physiol. 2016,6:1701-1741.
  • 24. Pałasz A, Tyszkiewicz-Nwafor M, Suszka-Świtek A, et al. Longitudinal study on novel neuropeptides phoenixin, spexin and kisspeptin in adolescent inpatients with anorexia nervosa - association with psychiatric symptoms. Nutr Neurosci 2021;24:896-906.
  • 25. Gasparini S, Stein LM, Loewen SP, et al. Novel regulator of vasopressin secretion: Phoenixin. Am J Physiol Regul Integr Comp Physiol 2018,314:R623-R628.
  • 26. Zhu R, Tian P, Zhang H, Wang G, Chen W. Gut microbiome-brain interactions in anorexia nervosa: Potential mechanisms and regulatory strategies. Neuropharmacology 2023;15:109315.
  • 27. Jiang JH, He Z, Peng YL, et al. Phoenixin-14 enhances memory and mitigates memory impairment induced by Aβ1-42 and scopolamine in mice. Brain Res 2015;10:298-308.
  • 28. Yuruyen M, Gultekin G, Batun GC, et al. Does plasma phoenixin level associate with cognition? Comparison between subjective memory complaint, mild cognitive impairment, and mild Alzheimer's disease. Int Psychogeriatr 2017;29:1-8.
  • 29. Ter Horst GJ, de Boer P, Luiten PG, van Willigen JD. Ascending projections from the solitary tract nucleus to the hypothalamus. A Phaseolus vulgaris lectin tracing study in the rat. Neuroscience 1989;31:785-797.
  • 30. Friedrich T, Schalla MA, Lommel R, et al. Restraint stress increases the expression of phoenixin immunoreactivity in rat brain nuclei. Brain Res 2020;1743:146904.
  • 31. Schalla MA, Goebel-Stengel M, Friedrich T, et al. Restraint stress affects circulating NUCB2/nesfatin-1 and phoenixin levels in male rats. Psychoneuroendocrinology 2020;122:104906.
  • 32. Lawson EA, Donoho D, Miller KK, et al. Hypercortisolemia is associated with severity of bone loss and depression in hypothalamic amenorrhea and anorexia nervosa. J Clin Endocrinol Metab 2009;94:4710-4716.
  • 33. Umathe SN, Bhutada PS, Jain NS, Shukla NR, Mundhada YR, Dixit PV. Gonadotropin-releasing hormone agonist blocks anxiogenic-like and depressant-like effect of corticotrophin-releasing hormone in mice. Neuropeptides 2008;42:399-410.
  • 34. Hofmann T, Weibert E, Ahnis A, et al. Phoenixin is negatively associated with anxiety in obese men. Peptides 2017;88:32-36.
  • 35. Wang J, Zheng B, Yang S, Tang X, Wang J, Wei D. The protective effects of phoenixin-14 against lipopolysaccharide-induced inflammation and inflammasome activation in astrocytes. Inflamm Res 2020;69:779-787.
  • 36. Zeng X, Li Y, Ma S, Tang Y, Li H. Phoenixin-20 Ameliorates Lipopolysaccharide-Induced Activation of Microglial NLRP3 Inflammasome. Neurotox Res 2020;38:785-792.
  • 37. Mai N, Prifti V, Kim M, Halterman MW. Characterization of neutrophil-neuronal co-cultures to investigate mechanisms of post-ischemic immune-mediated neurotoxicity. J Neurosci Methods 2020;15:108782.
  • 38. Singh V, Roth S, Veltkamp R, Liesz A. HMGB1 as a Key Mediator of Immune Mechanisms in Ischemic Stroke. Antioxid. Redox Signal 2016;24:635-651.
  • 39. Sun G, Ren Q, Bai L, Zhang L. Phoenixin-20 suppresses lipopolysaccharide-induced inflammation in dental pulp cells. Chem Biol Interact 2020;25:108971.
  • 40. Wei X, Lin H, Zhang B, et al. Phoenixin-20 Prevents ox-LDL-Induced Attachment of Monocytes to Human Aortic Endothelial Cells (HAECs): A Protective Implication in Atherosclerosis. ACS Chem Neurosci 2021;17:990-997.
  • 41. Zandeh-Rahimi Y, Panahi N, Hesaraki S, Shirazi-Beheshtiha SH. Protective Effects of Phoenixin-14 Peptide in the Indomethacin-Induced Duodenal Ulcer: An Experimental Study. Int J Pept Res Ther 2022;28(1):43.
  • 42. Yang F, Huang P, Shi L, Liu F, Tang A, Xu S. Phoenixin 14 inhibits high-fat diet-induced non-alcoholic fatty liver disease in experimental mice. Drug Design, Develop Therapy 2020;14: 3865.
  • 43. Akdu S, Can U, Polat E. Investigation of serum phoenixin levels in patients with hypertension. Rev Assoc Med Bras (1992. 2022;24;68(6):814-819.
  • 44. Yao B, Lv J, Du L, Zhang H, Xu Z. Phoenixin-14 protects cardiac damages in a streptozotocin-induced diabetes mice model through SIRT3. Arch Physiol Biochem 2021;7:1-9.

PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ

Year 2023, Volume: 4 Issue: Ek Sayı, 258 - 265, 16.10.2023

Abstract

Phoenixin (PNX), küçük integral membran protein 20’den (Smim20) üretilen yeni keşfedilmiş
endojen bir nöropeptittir. En yaygın izoformları PNX-14 ve PNX-20 aminoasit peptitleridir. G
proteinine bağlı reseptör 173’ün (GPR173) bir ligandı olan PNX, temel olarak hipotalamus ve
hipofiz bezi gibi beyin bölgesinden ve birçok periferal dokudan izole edilmiştir. PNX, gonadotropin
salgılayan hormon (GnRH) seviyesini artırarak hipofiz hücrelerinden lüteinize edici hormon (LH)
salınımını uyararak üreme işlevinin düzenlenmesinde önemli bir rol oynar. Bununla birlikte, PNX
sadece üreme sistemini düzenlemekle kalmaz, aynı zamanda anksiyolitik, anti-inflamatuar ve hücre
koruyucu etkiler de gösterir. Ayrıca davranış, gıda alımı, duyusal algı, hafıza, vücut sıvısı dengesi,
kardiyovasküler fonksiyonlar ve enerji metabolizmasının düzenlenmesinde birçok işlevi
bulunmaktadır. Bu derleme, PNX’in moleküler yapısı, sistemler üzerindeki fizyolojik etki ve
işlevleri ile çeşitli hastalıkların patogenezinde ve tedavi süreçlerindeki rolünü anlamada bize
yardımcı olacaktır.

References

  • 1- Yosten GL, Lyu RM, Hsueh AJ, et al. A novel reproductive peptide, phoenixin. J Neuroendocrinol 2013;25:206-15.
  • 2. Lyu RM, Huang XF, Zhang Y, et al. Phoenixin: a novel peptide in rodent sensory ganglia. Neuroscience 2013;10:622-31.
  • 3. Rocca C, Scavello F, Granieri MC, et al. Phoenixin-14: detection and novel physiological implications in cardiac modulation and cardioprotection. Cell Mol Life Sci. 2018;75:743-756.
  • 4. Prinz P, Scharner S, Friedrich T, et al. Central and peripheral expression sites of phoenixin-14 immunoreactivity in rats. Biochem Biophys Res Commun. 2017;4:195-201.
  • 5. Billert M, Kołodziejski PA, Strowski MZ, Nowak KW, Skrzypski M. Phoenixin-14 stimulates proliferation and insulin secretion in insulin producing INS-1E cells. Biochim Biophys Acta Mol Cell Res. 2019;1866:118533.
  • 6. Billert M, Wojciechowicz T, Jasaszwili M et al. Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism. Biochim Biophys Acta Mol Cell Biol Lipids. 2018;1863:1449-1457.
  • 7. Kalamon N, Błaszczyk K, Szlaga A, et al. Levels of the neuropeptide phoenixin-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue in rat model of polycystic ovary syndrome. Biochem Biophys Res Commun. 2020;6:628-635.
  • 8. Nguyen XP, Nakamura T, Osuka S, et al. Effect of the neuropeptide phoenixin and its receptor GPR173 during folliculogenesis. Reproduction. 2019;158:25-34.
  • 9. Cowan A, Lyu RM, Chen YH, Dun SL, Chang JK, Dun NJ. Phoenixin: A candidate pruritogen in the mouse. Neuroscience. 2015;3:541-8.
  • 10. Stein LM, Haddock CJ, Samson WK, Kolar GR, Yosten GL. The phoenixins: From discovery of the hormone to identification of the receptor and potential physiologic actions. Peptides. 2018;106:45-48.
  • 11. McIlwraith EK, Belsham DD. Phoenixin: Uncovering its receptor, signaling and functions. Acta Pharmacol Sin. 2018;39:774-778.
  • 12. Stein LM, Tullock CW, Mathews SK, et al. Hypothalamic action of phoenixin to control reproductive hormone secretion in females: Importance of the orphan G protein-coupled receptor Gpr173. Am J Physiol Integr Comp Physiol. 2016;311:R489-R496.
  • 13. Treen AK, Luo V, Belsham DD. Phoenixin Activates Immortalized GnRH and Kisspeptin Neurons Through the Novel Receptor GPR173. Mol Endocrinol 2016;30:872-888.
  • 14. Matsumoto M, Saito T, Takasaki J, et al. An evolutionarily conserved G-protein coupled receptor family, SREB, expressed in the central nervous system. Biochem Biophys Res Commun 2000;272:576-582.
  • 15. Ma H, Su D, Wang Q, et al. Phoenixin 14 inhibits ischemia/reperfusion-induced cytotoxicity in microglia. Arch Biochem Biophys 2020; 689:108411.
  • 16. Haddock CJ, Almeida-Pereira G, Stein LM, Yosten GLC, Samson WK. A novel regulator of thirst behavior: phoenixin. Am J Physiol Regul Integr Comp Physiol 2020;318:R1027-R1035.
  • 17. Guvenc G, Altinbas B, Kasikci E, et al. Contingent role of phoenixin and nesfatin-1 on secretions of the male reproductive hormones. Andrologia 2019;51:e13410.
  • 18. Ullah K, Ur Rahman T, Wu DD, et al. Phoenixin-14 concentrations are increased in association with luteinizing hormone and nesfatin-1 concentrations in women with polycystic ovary syndrome. Clin Chim Acta 2017;471:243-247.
  • 19. Schalla M, Prinz P, Friedrich T, et al. Phoenixin-14 injected intracerebroventricularly but not intraperitoneally stimulates food intake in rats. Peptides 2017;96:53-60.
  • 20. Friedrich T, Schalla MA, Scharner S, et al. Intracerebroventricular injection of phoenixin alters feeding behavior and activates nesfatin-1 immunoreactive neurons in rats. Brain Res 2019;15:188-195.
  • 21. Rajeswari JJ, Blanco AM, Unniappan S. Phoenixin-20 suppresses food intake, modulates glucoregulatory enzymes, and enhances glycolysis in zebrafish. Am J Physiol Regul Integr Comp Physiol 2020;318:R917-R928.
  • 22. Wang M, Deng SP, Chen HP, et al. Phoenixin participated in regulation of food intake and growth in spotted scat, Scatophagus argus. Comp Biochem Physiol B Biochem Mol Biol 2018;226:36-44.
  • 23. Brown CH. Magnocellular Neurons and Posterior Pituitary Function. Compr. Physiol. 2016,6:1701-1741.
  • 24. Pałasz A, Tyszkiewicz-Nwafor M, Suszka-Świtek A, et al. Longitudinal study on novel neuropeptides phoenixin, spexin and kisspeptin in adolescent inpatients with anorexia nervosa - association with psychiatric symptoms. Nutr Neurosci 2021;24:896-906.
  • 25. Gasparini S, Stein LM, Loewen SP, et al. Novel regulator of vasopressin secretion: Phoenixin. Am J Physiol Regul Integr Comp Physiol 2018,314:R623-R628.
  • 26. Zhu R, Tian P, Zhang H, Wang G, Chen W. Gut microbiome-brain interactions in anorexia nervosa: Potential mechanisms and regulatory strategies. Neuropharmacology 2023;15:109315.
  • 27. Jiang JH, He Z, Peng YL, et al. Phoenixin-14 enhances memory and mitigates memory impairment induced by Aβ1-42 and scopolamine in mice. Brain Res 2015;10:298-308.
  • 28. Yuruyen M, Gultekin G, Batun GC, et al. Does plasma phoenixin level associate with cognition? Comparison between subjective memory complaint, mild cognitive impairment, and mild Alzheimer's disease. Int Psychogeriatr 2017;29:1-8.
  • 29. Ter Horst GJ, de Boer P, Luiten PG, van Willigen JD. Ascending projections from the solitary tract nucleus to the hypothalamus. A Phaseolus vulgaris lectin tracing study in the rat. Neuroscience 1989;31:785-797.
  • 30. Friedrich T, Schalla MA, Lommel R, et al. Restraint stress increases the expression of phoenixin immunoreactivity in rat brain nuclei. Brain Res 2020;1743:146904.
  • 31. Schalla MA, Goebel-Stengel M, Friedrich T, et al. Restraint stress affects circulating NUCB2/nesfatin-1 and phoenixin levels in male rats. Psychoneuroendocrinology 2020;122:104906.
  • 32. Lawson EA, Donoho D, Miller KK, et al. Hypercortisolemia is associated with severity of bone loss and depression in hypothalamic amenorrhea and anorexia nervosa. J Clin Endocrinol Metab 2009;94:4710-4716.
  • 33. Umathe SN, Bhutada PS, Jain NS, Shukla NR, Mundhada YR, Dixit PV. Gonadotropin-releasing hormone agonist blocks anxiogenic-like and depressant-like effect of corticotrophin-releasing hormone in mice. Neuropeptides 2008;42:399-410.
  • 34. Hofmann T, Weibert E, Ahnis A, et al. Phoenixin is negatively associated with anxiety in obese men. Peptides 2017;88:32-36.
  • 35. Wang J, Zheng B, Yang S, Tang X, Wang J, Wei D. The protective effects of phoenixin-14 against lipopolysaccharide-induced inflammation and inflammasome activation in astrocytes. Inflamm Res 2020;69:779-787.
  • 36. Zeng X, Li Y, Ma S, Tang Y, Li H. Phoenixin-20 Ameliorates Lipopolysaccharide-Induced Activation of Microglial NLRP3 Inflammasome. Neurotox Res 2020;38:785-792.
  • 37. Mai N, Prifti V, Kim M, Halterman MW. Characterization of neutrophil-neuronal co-cultures to investigate mechanisms of post-ischemic immune-mediated neurotoxicity. J Neurosci Methods 2020;15:108782.
  • 38. Singh V, Roth S, Veltkamp R, Liesz A. HMGB1 as a Key Mediator of Immune Mechanisms in Ischemic Stroke. Antioxid. Redox Signal 2016;24:635-651.
  • 39. Sun G, Ren Q, Bai L, Zhang L. Phoenixin-20 suppresses lipopolysaccharide-induced inflammation in dental pulp cells. Chem Biol Interact 2020;25:108971.
  • 40. Wei X, Lin H, Zhang B, et al. Phoenixin-20 Prevents ox-LDL-Induced Attachment of Monocytes to Human Aortic Endothelial Cells (HAECs): A Protective Implication in Atherosclerosis. ACS Chem Neurosci 2021;17:990-997.
  • 41. Zandeh-Rahimi Y, Panahi N, Hesaraki S, Shirazi-Beheshtiha SH. Protective Effects of Phoenixin-14 Peptide in the Indomethacin-Induced Duodenal Ulcer: An Experimental Study. Int J Pept Res Ther 2022;28(1):43.
  • 42. Yang F, Huang P, Shi L, Liu F, Tang A, Xu S. Phoenixin 14 inhibits high-fat diet-induced non-alcoholic fatty liver disease in experimental mice. Drug Design, Develop Therapy 2020;14: 3865.
  • 43. Akdu S, Can U, Polat E. Investigation of serum phoenixin levels in patients with hypertension. Rev Assoc Med Bras (1992. 2022;24;68(6):814-819.
  • 44. Yao B, Lv J, Du L, Zhang H, Xu Z. Phoenixin-14 protects cardiac damages in a streptozotocin-induced diabetes mice model through SIRT3. Arch Physiol Biochem 2021;7:1-9.
There are 44 citations in total.

Details

Primary Language Turkish
Subjects Clinical Sciences
Journal Section Review
Authors

Muhammed Taşar 0000-0003-1894-028X

Raziye Akcılar 0000-0003-4720-1945

Early Pub Date October 16, 2023
Publication Date October 16, 2023
Published in Issue Year 2023 Volume: 4 Issue: Ek Sayı

Cite

APA Taşar, M., & Akcılar, R. (2023). PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ. Eskisehir Medical Journal, 4(Ek Sayı), 258-265.
AMA Taşar M, Akcılar R. PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ. Eskisehir Med J. October 2023;4(Ek Sayı):258-265.
Chicago Taşar, Muhammed, and Raziye Akcılar. “PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ”. Eskisehir Medical Journal 4, no. Ek Sayı (October 2023): 258-65.
EndNote Taşar M, Akcılar R (October 1, 2023) PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ. Eskisehir Medical Journal 4 Ek Sayı 258–265.
IEEE M. Taşar and R. Akcılar, “PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ”, Eskisehir Med J, vol. 4, no. Ek Sayı, pp. 258–265, 2023.
ISNAD Taşar, Muhammed - Akcılar, Raziye. “PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ”. Eskisehir Medical Journal 4/Ek Sayı (October 2023), 258-265.
JAMA Taşar M, Akcılar R. PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ. Eskisehir Med J. 2023;4:258–265.
MLA Taşar, Muhammed and Raziye Akcılar. “PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ”. Eskisehir Medical Journal, vol. 4, no. Ek Sayı, 2023, pp. 258-65.
Vancouver Taşar M, Akcılar R. PHOENİXİN’İN FİZYOPATOLOJİK ETKİLERİ. Eskisehir Med J. 2023;4(Ek Sayı):258-65.