Presentation number: MG 51

MOLECULAR MECHANISMS OF THE RENOPROTECTIVE EFFECT OF EMPAGLIFLOZIN ON LLC-PK1 CELLULAR MODEL OF PROXIMAL TUBULAR CELLS

Vjera Ninčević1,2, Tea Omanović Kolarić1,2, Milorad Zjalić3, Marina Čović4, Tomislav Kizivat5,6, Lucija Kuna1, Robert Smolić1, Aleksandar Včev7, Martina Smolić1,2, Ines Bilić Ćurčić2,8

1Department of Pharmacology and Biochemistry, Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 2Department of Pharmacology, Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 3Department of Molecular Medicine and Biotechnology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, 4Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 5Clinical Institute of Nuclear Medicine and Radiation Protection, University Hospital Osijek, Osijek, Croatia, 6Department for Nuclear Medicine and Oncology, Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, 7Department of Pathophysiology, Physiology and Immunology, Faculty of Dental Medicine and Health Osijek, J.J. Strossmayer University of Osijek, 8Department of Diabetes, Endocrinology and Metabolism Disorders, University Hospital Osijek, Osijek, Croatia

Diabetic nephropathy (DN) is a chronic complication of diabetes mellitus, both type I and type II, which can lead to end-stage renal kidney failure. This research aimed to assess the effects of empagliflozin (SGLT2i) on cell viability and F-actin distribution in the LLC-PK1 model of DN. To mimic DN, cells were exposed to high glucose (HG30 mM) followed by 0,5 mM H2O2 and a combination of glucose and H2O2 for 24 hours. The cells were treated with different combinations of glucose and empagliflozin (100 and 500 nM) and combinations of glucose, H2O2, and empagliflozin. MTT colorimetric assay and Erythrosin B color exclusion test were used to determine cell viability. F-actin cytoskeleton was visualized with Phalloidin stain and subsequently quantified. MTT results revealed a significant reduction in cell viability when treated with the HG30/H2O2 combination (p<0.001). Cell viability was considerably increased with the addition of empagliflozin 100 and 500 nM to cells treated with HG30 and HG30/H2O2 compared to cells treated with HG30/H2O2 only (p<0.001). Furthermore, empagliflozin in the concentration of 100 nM decreased the total amount of F-actin in HG30 treated cells (p<0.001), while a higher dose of 500 nM had no effect. Cellular viability shows that empagliflozin has protective effects on renal injury, whereas the effect on F-actin structure was dose-dependent.

Key words: diabetic nephropathy, LLC-PK1 cell culture, empagliflozin


Presentation number: MG 52

MOLECULAR MECHANISMS OF THE RENOPROTECTIVE EFFECT OF LIRAGLUTIDE ON LLC-PK1 CELLULAR MODEL OF PROXIMAL TUBULAR CELLS

Vjera Ninčević1,2, Tea Omanović Kolarić1,2, Hrvoje Roguljić2,3, Klara Ormanac4, Dea Sabo4, Ana Ninčević5, Robert Smolić1, Aleksandar Včev6, Martina Smolić1,2, Ines Bilić Ćurčić2,7

1Department of Pharmacology and Biochemistry, Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 2Department of Pharmacology, Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 3Department of Internal Medicine, University Hospital Osijek, Osijek, Croatia, 4Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 5School of Medicine, University of Zagreb, Zagreb, Croatia, 6Department of Pathophysiology, Physiology and Immunology, Faculty of Dental Medicine and Health Osijek, J.J. Strossmayer University of Osijek, Osijek, Croatia, 7Department of Diabetes, Endocrinology and Metabolism Disorders, University Hospital Osijek, Osijek, Croatia

Transforming growth factor-beta (TGF-β) has recently been associated with diabetic nephropathy (DN) development. It causes cell apoptosis induced by oxidative stress and cell proliferation and migration triggered by hyperglycemia and inflammation. Liraglutide is an antihyperglycemic agent that has a direct renoprotective effect. This study aimed to evaluate the effects of liraglutide on cell viability and TGF-β expression in the LLC-PK1 model of DN. Cell viability was determined by colorimetric MTT assay and erythrosine B color exclusion assay. The expression of mRNA TGF- β1 was measured by RT-PCR and β-actin was used as an internal control. LLC-PK1 cell culture was treated with different concentrations of glucose (1.5, 30 mM) and the combination of glucose and H2O2 (0.5 mM) for 24 hours. To study the renal effect of liraglutide, cells were treated for 24 hours with different combinations of glucose and liraglutide (10, 20 nM) and combinations of glucose, H2O2, and liraglutide. A significant decrease in MTT levels compared to control (p < 0.01; p < 0.001) was observed after treatment with a combination of HG30/ H2O2 and HG30 alone. Cell viability was improved by the addition of liraglutide (10 nM) to cells treated with HG30, while 20 nM had no effect. There was no significant difference in cell survival with the addition of HG30 and HG30/ H2O2 compared to control. The addition of liraglutide at both concentrations to cells treated with HG30 and HG30/H2O2 improved cell survival, although significance was only numerical, not reaching statistical significance. TGF-β1 expression levels were significantly increased in cells treated with HG30 (p < 0.001). Liraglutide inhibited TGF-β1 expression except in HG30/H2O2 treated cells. Our results support a protective role of liraglutide in LLC-PK1 cells, mediated by inhibition of TGF-β1, thus reducing oxidative stress damage.

Key words: diabetic nephropathy, LLC-PK1 cell culture, liraglutide


Presentation number: MG 53

THE ACTIVITY OF GARLIC EXTRACTS INHIBITS EPITHELIAL DAMAGE CAUSED BY BILE SALT IN A CELLULAR MODEL OF PEPTIC ULCER DISEASE

Lucija Kuna1,2, Milorad Zjalic3, Tomislav Kizivat4, Hrvoje Roguljic1,2, Vjera Nincevic1,2, Tea Omanovic Kolaric1,2, Ana Petrovic1,2, Aleksandar Vcev1,2, Catherine H. Wu5, Martina Smolic1,2, Robert Smolic1,2

1Department of Pharmacology and Biochemistry, Faculty of Dental Medicine and Health Osijek, J. J. Strossmayer University of Osijek, Osijek, Croatia, 2Department of Pharmacology, Faculty of Medicine Osijek, J. J. Strossmayer University of Osijek, Osijek, Croatia, 3Department of Medical Biology and Genetics, Faculty of Medicine, J. J. Strossmayer University of Osijek, Osijek, Croatia, 4Department of Nuclear Medicine and Oncology, Faculty of Medicine Osijek, J. J. Strossmayer University of Osijek, Osijek, Croatia, 5Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, CT, USA

Peptic ulcer disease (PUD) is a common digestive disorder and global problem with a lifetime risk of development ranging from 5% to 10%. Proton pump inhibitors such as lansoprazole (LPZ) are used as a first-line therapy to treat gastric ulcers worldwide. On the other hand, garlic extracts (GE) have been shown in several studies to be beneficial in the treatment of ulcers. The goal of this research was to establish a cell culture model of ulcer disease using bile salts; sodium taurocholate (NaT), to investigate the effects of pretreatment with GE and addition of LPZ on oxidative stress and F-actin distribution in a cell model of PUD. The establishment of the NaT model was determined by the MTT test. The ability of GE to protect the gastric cells against the damage induced by NaT was performed by determining glutathione (GSH) and prostaglandin E2 (PGE2) levels by ELISA, proliferation of the human gastric cell line by cell counting, expression of nuclear factor kappa B subunit 2 (NFKB2), thioredoxine 1 (TRX 1) by RT PCR, and visualization of the F-actin cytoskeleton by semi-quantification of rhodamine-phalloidin staining. Our results showed that gastric cells pretreated with LPZ (p<0.001) and increasing concentrations of GE (p<0.001), exhibited a significant reduction in cell damage after incubation with NaT. In a cell culture model of PUD, pretreatment with LPZ and various concentrations of GE increased PGE2 and GSH levels (p<0.001). Positive correlation of NFKB2 (p<0.01), and TRX 1 (p<0.001) with LPZ and GE pretreatment was confirmed. Treatment with NaT as oxidative stress on F actin structure was less pronounced, while the highest concentration of GE led to a statistically significant increase of total amount of F-actin (p<0.001). In a cell model of PUD, pretreatment with GE showed a gastroprotective effect. However, further experiments are needed to confirm protective role of GE in PUD.

Key words: peptic ulcer disease, garlic extracts, lansoprazole, sodium taurocholate


Presentation number: MG 54

ETA POLYCAPROLACTONE (ε-PCL) IMPLANTS APPEAR TO CAUSE A PARTIAL DIFFERENTIATION OF BREAST CANCER LUNG METASTASIS IN A MURINE MODEL

Benjamin Benzon1, Sandra Marijan2, Matij Pervan3, Angela Mastelić2, Vedrana Čikeš Ćulić2

1University of Split, School of Medicine, Dpt. of Anatomy, Histology and Embryology, Split, Croatia, 2University of Split, School of Medicine, Dpt. of Medical Chemistry and Biochemistry, Split, Croatia, 3University of Split, School of Medicine, Medical doctor education program

Cells in every epithelium can be roughly divided in three compartments: stem cell (SC) compartment, transient amplifying cell (TA) compartment and mature or functional cell (FC) compartment. Maturation of stem cells is characterized epithelial stromal interaction and sequential maturational movement of stem cell’s progeny through those compartments. In this work we hypothesize that providing an artificial stroma, which murine breast cancer metastatic cells can infiltrate, will induce their differentiation. BALB/c female mice were injected with 106 isogenic 4T1 breast cancer cells labeled with GFP. After 20 days primary tumors were removed, and artificial ε-PCL implants were implanted on the contralateral side. After 10 more days mice were sacrificed and implants along with lung tissue were harvested. Mice were divided in four groups: tumor removal with sham implantation surgery (n=5), tumor removal with ε-PCL implant (n=5), tumor removal with VEGF enriched ε-PCL implant (n=7) and mice without tumor with VEGF enriched ε-PCL implant (n=3). Differentiational status of GFP+ cells was assessed by Ki67 and activated caspase 3 expression, thus dividing the population in SC like cells (Ki67+ aCasp3-), TA like cells (Ki67+ aCasp3+) and FC like cells (Ki67- aCasp3+/-) on flow cytometry. Lung metastatic load was reduced by 20% in mice with simple ε-PCL implant when compared to tumor bearing group with no implant (p<0.0001). Mice with VEGF enriched implants had 90% increase in lung metastatic load in comparison to tumor bearing mice with no implants (p<0.0001). Likewise, amount of GFP+ cells doubled in simple ε-PCL implant in comparison to VEGF enriched implants (p<0.0001). Differentiation wise, simple implants in comparison to sham group reduced the SC like cells by 25% and VEGF enriched implants reduced it further by another 20 % (i.e., 45% reduction in total) (p<0.0001). On the other hand, TA like cells were increased by amounts identical to SC-like cells decrease. Effects of both type of implants on FC like cells were minute. Both types of implants cause lung metastasis differentiation by shifting cancer cells from SC to TA compartment, leaving the FC compartment unaffected. VEGF enriched ε-PCL implants appear to decrease further migration of lung metastasis.

Key words: metastasis, breast cancer, differentiation, ε-PCL implant

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Published: June 21st, 2022;

Copyright: © 2022 ISABS & IAR Publishing. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.