Farmakološko uravnavanje izražanja in delovanja interlevkina-6 v kulturi človeških skeletnomišičnih celic

Zupanc, Maja (avtor), Pirkmajer, Sergej (mentor), Miš, Katarina (komentor)

Uvod: Skeletna mišica je z izločanjem miokinov pomemben endokrini organ in mišična vlakna so eno izmed najbolj presnovno aktivnih tkiv. Interlevkin-6 (IL-6) je pleiotropen protein, ki ga izloča širok spekter celic in tkiv. Vnetje in presnovni učinki so odvisni od raznolikega časovno ali tkivno specifičnega delovanja IL-6. Vnetni učinki kronično povišanega IL-6 poslabšujejo prognozo bolezenskih stanj, medtem ko akutno izločen IL-6 pri fizioloških prilagoditvah v splošnem deluje pozitivno na zdravje in izboljšuje presnovo. Zaviralci delovanja IL-6 so pomembni pri zdravljenju vnetnih revmatičnih bolezni. Namen: Razumevanje uravnalnih mehanizmov IL-6 nam širi možnosti optimizacije terapevtskih pristopov in razvoja zdravil naslednjih generacij. Zaradi pomanjkanja natančnih definicij dejavnikov, ki privedejo do dvojne vloge IL-6, nas zanima uravnavanje njegove sinteze, izločanja in delovanja. Raziskovali smo mehanizme izražanja IL-6, receptorske signalizacije za IL-6 ter učinke terapevtske blokade delovanja IL-6 v skeletnih mišicah. Glavni namen je bil preveriti učinke zaviralcev IL-6 na njegovo izražanje in delovanje v kulturi skeletnomišičnih celic. Hipoteze: 1.) TLR4 in ERK1/2 sta pomembna za bazalno izražanje IL-6 v kulturi človeških skeletnomišičnih celic. 2.) Zaviralci IL-6 ne vplivajo na izražanje in izločanje IL-6 v kulturi človeških skeletnomišičnih celic. 3.) Zaviralci IL-6 in inzulin zmanjšajo bazalno fosforilacijo STAT3 v človeških skeletnomišičnih celicah. 4.) Učinek zaviralcev IL-6 na bazalno in z inzulinom spodbujeno aktivnost signalnih poti Akt in ERK1/2 je odvisen od koncentracije in trajanja izpostavitve IL-6. 5.) Zaviralci IL-6 zmanjšajo aktivnost z AMP aktivirane protein kinaze v človeških skeletnomišičnih celicah. Metode: Poskuse smo izvedli na primarni kulturi človeških mišičnih cevčic. Za določanje izražanja IL-6 mRNA v mišičnih celicah smo uporabili metodo kvantitativne verižne reakcije s polimerazo v realnem času. Izločen IL-6 smo določili z encimskoimunskim testom. Signalne odzive celic smo spremljali z metodo prenos western, ki smo jo uporabili za analizo fosforilacije ključnih signalnih proteinov; STAT3. AKT, ERK1/2, AS160, GSK-3α/β, PAS in ACC. Rezultati: Izmerili smo bazalno izločanje IL-6. LPS aktivira TLR4, kar spodbudi izražanje IL-6 v skeletnomičičnih celicah. Dejavniki, ki zavirajo to pot, zavirajo izražanje IL-6 ob dodatku LPS. Noben uporabljen zaviralec ni spodbudil ali zavrl izražanja IL-6. Tocilizumab in zaviralec tofacitinib nista spodbudila ali zavrla fosforilacije s presnovo povezanih signalnih proteinov. Pri kronični izpostavitvi skeletnomišičnih celic z IL-6 smo opazili zmanjšanje inzulinskega odziva. Tofacitinib je učinkovito zaviral tudi bazalno fosforilacijo STAT3, zaviralni učinek tocilizumaba pa smo izmerili samo ob akutni izpostavitvi IL-6. Pri zaviralcih IL-6 nismo izmerili učinka na delovanje inzulina. Zaključki: Potrdili smo endogeno izločanje IL-6 v bazalnih razmerah. Izločili smo vpletenost mehanizmov spontane aktivnosti TLR4 in delovanja ERK1/2, kot pozitivne povratne zanke IL-6 za ta pojav. Sklepamo, da IL-6 sam ne vpliva na lastno izražanje/izločanje. Naši rezultati ne kažejo bistvenega vpliva zaviranja delovanja IL-6, ali kronične izpostavljenosti IL-6, na signalne poti STAT3, Akt, ERK1/2 in AMPK v kulturi človeških skeletnomišičnih celicah. Kronična izpostavljenost IL-6 v mišici bi lahko vplivala na delovanje inzulina.

• Jezik: Slovenski jezik
• Ključne besede: Interlevkin-6, kronično vnetje, energijska homeostaza, skeletne mišice, terapevtski pristopi
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-143689
• Datum objave v RUL: 08.01.2023

ZUPANC, Maja, 2023, Farmakološko uravnavanje izražanja in delovanja interlevkina-6 v kulturi človeških skeletnomišičnih celic [na spletu]. Magistrsko delo. [Dostopano 3 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=143689

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Pharmacological regulation of interleukin-6 expression and action in cultured human skeletal muscle cells

Izvleček

Background: Skeletal muscle is highly active metabolic tissue, and with its myokine secretion an important endocrine organ. Interleukin-6 (IL-6) is a pleiotropic cytokine that is secreted from a wide spectrum of cells and tissues. Inflammatory and metabolic responses depend mostly on different physiological environment, time, and tissue specific actions of IL-6. Chronically elevated IL-6 has proinflammatory effects and is linked to negative prognosis of disease but its acute elevation under physiological conditions exerts beneficial effects on metabolism and health in general. IL-6 inhibitors are important in treating chronic inflammatory diseases. Aim: Understanding the regulatory mechanisms of IL-6 broadens our possibilities in therapeutic optimisation and development of next generation drugs. Factors that determine the IL-6 double role are not yet well defined, therefore we are interested aim to gain the understanding of the regulation of IL-6 synthesis, secretion, and its actions. We study the mechanisms of IL-6 expression, receptor signalling and effects of therapeutic targeting of IL-6 in skeletal muscle. The main aim was to examine the effects of IL-6 inhibitors on IL-6 expression and action in skeletal muscle cell culture. Hypotheses: 1.) TLR4 and ERK1/2 are important for basal IL-6 expression in human skeletal muscle cell culture.2.) IL-6 inhibitors do not affect IL-6 expression and secretion in human skeletal cell culture. 3.) IL-6 inhibitors and insulin reduce basal STAT3 phosphorylation in human skeletal muscle cells. 4.) The effect of IL-6 inhibitors on basal and insulin-stimulated Akt and ERK1/2 signalling pathway activity is dependent on the concentration and duration of IL-6 exposure. 5.) IL-6 inhibitors reduce AMP-activated protein kinase activity in human skeletal muscle cells. Methods: Experiments were taken on primary human skeletal muscle cell culture. IL-6 mRNA expression in muscle cells was determined with quantitative real time PCR. IL-6 secretion was measured with the enzyme linked immunoabsorbent assay. Cellular signalling responses were assessed with western blotting by measuring the phosphorylation of key signalling proteins; STAT3. AKT, ERK1/2, AS160, GSK-3α/β, PAS in ACC. Results: Constitutive IL-6 secretion from cultured skeletal muscle cells was measured. LPS treatment activated the receptor TLR4, which stimulated IL-6 expression in muscle cells. IL-6 expression was inhibited by TLR4 signalling inhibitors. Neither stimulation nor complete inhibition of IL-6 expression was noticed by any of the used inhibitors. Phosphorylation of metabolic signalling proteins after treatment with tocilizumab and tofacitinib was not significantly changed. Decreased insulin response was noticed after the chronic treatment of skeletal muscle cells with IL-6. Constitutive phosphorylation of STAT3 was effectively inhibited by tofacitinib. Inhibition of STAT3 phosphorylation by tocilizumab was measured only at the acute IL-6 treatment. Effect on insulin actions by IL-6 inhibitors was not detected. Conclusions: Endogenous secretion of IL-6 under basal conditions was confirmed. The mechanisms of spontaneous TLR4 activation and ERK1/2 activity as an IL-6 positive feedback loop were excluded for this state. We conclude that IL-6 itself does not influence its own expression/secretion. Our results show no significant effect of IL-6 inhibition, or chronic IL-6 exposure, on STAT3, Akt, ERK1/2 and AMPK signalling pathways in cultured human skeletal muscle cells. Chronic exposure to IL-6 in muscle could affect insulin action.

• Ključne besede: Interleukin-6, chronic inflammation, energy homeostasis, skeletal muscle, therapeutic approaches