Abstract | Ciljevi: Transformirajući faktor rasta beta (TGF-β) uključen je u razvoj dijabetičke nefropatije (DN) koja uzrokuje staničnu apoptozu uzrokovanu oksidativnim stresom, proliferacijom i migracijom stanica izazvane hiperglikemijom i upalom. GLP 1 RA i SGLT2i dvije su klase antihiperglikemijskih lijekova za koje se pokazalo kako imaju izravne renoprotektivne učinke, iako molekularni mehanizmi za oba lijeka još uvijek nisu jasni. Cilj ovog istraživanja bio je procijeniti učinke Liraglutida (GLP 1 RA) i Empagliflozina (SGLT2i) na vijabilnost stanica, GSH razine, ekspresiju i koncentraciju TGF-β, razine proteina uključenih u signalni put inzulina i oštećenje stanica te distribucija F aktina u LLC-PK1 modelu dijabetičke nefropatije. Tijek istraživanja: Kako bi se oponašala dijabetička nefropatija, stanice su bile izložene visokoj glukozi (HG30 mM) uz dodavanje 0,5 mM H2O2 i kombinaciji glukoze i H2O2 tijekom 24 sata. U prvom dijelu pokusa stanice su tretirane različitim kombinacijama glukoze i Liraglutida te kombinacijama glukoze, H2O2 i Liraglutida. U drugom dijelu stanice su tretirane različitim kombinacijama glukoze i Empagliflozina te kombinacijama glukoze, H2O2 i Empagliflozina. Materijali i metode: Vijabilnost stanica određena je MTT kolorimetrijskim testom i bojom Eritrozin B. Glutation (tGSH), ekspresija ECM i TGF-β1 koncentracija mjereni su pomoću spektrofotometrijskog/mikroploče čitača i ELISA kompleta. RT-PCR mjerio je ekspresiju mRNA TGF-β1, a β aktin korišten je kao interna kontrola. Western blot korišten je za otkrivanje razine proteina Akt, pAkt, GSK3β, pGSK3β, p53, p-p53, pSTAT3, SMAD7 i PPAR-. F-aktinski citoskelet vizualiziran je s faloidin bojom i naknadno kvantificiran. Rezultati: Vijabilnost stanica i GSH razine smanjene su u DN modelu stanične kulture. Liraglutid i Empagliflozin općenito su poboljšali vitalnost stanica i razine GSH. Ekspresija razina TGF-β1 bila je značajno povećana u stanicama tretiranim HG30. Liraglutid je smanjio ekspresiju TGF-β1 osim u stanicama tretiranim HG30/H2O2. Zabilježeno je značajno smanjenje SMAD 7 i pGSK3β u DN modelu u usporedbi s kontrolom. U stanicama tretiranim HG30/H2O2/Empa500 uočeno je značajno povećanje razina p53, pp53, PPAR-γ, pSTAT3, pGSK3BETA, GSK3BETA, SMAD 7 i p AKT, osim za AKT. Uočeno je značajno povećanje razine TGF-β1 u ozlijeđenim stanicama, a liječenje Liraglutidom i Empagliflozinom dovelo je do smanjenja razine TGF-β1. Liraglutid je smanjio nakupljanje ECM-a izazvano visokom glukozom i oksidativnim stresom, ali Empagliflozin nije imao značajan učinak. Dodavanje Liraglutida svim tretiranim skupinama stanica dovelo je do značajnog smanjenja, dok je Empagliflozin djelomično doveo do smanjenja distribucije F-aktina. Zaključak: Ozljeda bubrežnih tubula u LLC-PK DN modelu omogućena je kroz TGF beta koji pretežno potiče oštećenje stanica oksidativnim stresom. Inhibicija TGF beta uglavnom je odgovorna za bubrežne pozitivne učinke Liraglutida i Empagliflozina. |
Abstract (english) | Objectives: Transforming growth factor-beta (TGF- β) has been recently implicated in the development of diabetic nephropathy (DN) causing cell apoptosis induced by oxidative stress, cell proliferation, and migration triggered by hyperglycemia and inflammation. GLP 1 RA and SGLT2i are two antyhyperglycemic drug classes that have direct renoprotective effects, although molecular mechanisms for both drugs are still not clear. This research aimed to assess the effects of Liraglutide (GLP 1 RA) and Empagliflozin (SGLT2i) on cell viability, GSH levels, TGF-β expression and concentration, protein levels involved in insulin signaling pathway and cell damage, and F actin distribution in the LLC-PK1 model of DN. Study design: In order to mimic DN, cells were exposed to high glucose (HG30 mM) followed by 0,5 mM H2O2 and a combination of glucose and H2O2 during 24 hours. In the first set of the experiment, the cells were treated with different combinations of glucose and Liraglutide and combinations of glucose, H2O2, and Liraglutide. In the second set, cells were treated with different combinations of glucose and Empagliflozin and combinations of glucose, H2O2 and Empagliflozin. Materials and methods: Cell viability was determined by MTT colorimetric test and Erythrosin B color exclusion test. Glutathione (tGSH), ECM expression, and TGF-β1 concentration were measured using spectrophotometric/microplate reader assay and ELISA kit, respectively. Expression of mRNA TGF- β1 was measured by RT-PCR and β actin was used as an internal control. Western blotting was used to detect the protein level of Akt, pAkt, GSK3β, pGSK3β, p53, p-p53, pSTAT3, SMAD7, and PPAR-. F-actin cytoskelet was visualized with Phalloidin stain and subsequently quantified. Results: Cell viability and GSH levels were decreased in the DN model of cell culture. Liraglutide and Empagliflozin generally improved cell viability and GSH levels. Expression of TGF- β1 levels were significantly increased in HG30 treated cells. Liraglutide reduced expression of TGF-β1 except in HG30/H2O2 treated cells. A significant reduction of SMAD 7 and pGSK3β in the DN model was noted compared to the control. In HG30/ H2O2/Empa500 treated cells significant increase in p53, pp53, PPAR-γ, pSTAT3, pGSK3BETA, GSK3BETA, SMAD 7, and p AKT levels was observed except for AKT. A significant increase of TGF-β1 levels in injured cells was observed and Liraglutid and Empagliflozin treatment led to a decrease of TGF- β1 levels. Liraglutide reduced ECM accumulation induced by high glucose and oxidative stress, but Empagliflozin did not have any significant effect. The addition of Liraglutide to all treated cell groups led to a significant decrease, while Empagliflozin partially led to a decrease in the distribution of F-actin. Conclusion: Renal tubular injury in the LLC-PK model of DN is facilitated through TGF beta predominantly stimulating oxidative stress cell damage. Inhibition of TGF beta is mainly responsible for renal benefits of Liraglutide and Empagliflozin. |