Uticaj sintetske i prirodne žučne kiseline na oksidativni stres i apoptozu hepatocita

Abstract

Žučne kiseline (ŽK) su strukturno raznoliki molekuli, koji pored uloge koju ostvaruju putem žuči, deluju i kao signalni molekuli i ostvaruju kako endokrina tako i parakrina dejstva. Činjenica da je do sada u terapijske svrhe primenjivana samo ursodeoksiholna kiselina (UDK), posledica je brojnih ograničenja u mogućnosti primene ostalih prirodnih ŽK, i ističe potrebu za otkrivanjem novih sintetskih ŽK i liganda. Cilj istraživanja bio je ispitivanje sintetske 12-monoketoholne kiseline (MK) i prirodne UDK u modelu holestaze i aloksanom izazvanog dijabetesa. Ispitivanja su vršena na pacovima soja Wistar. Analizirana je telesna masa, glikemija, pokazatelji jetrene funkcije (AST; ALT, γ-GT, ukupni i direktni bilirubin), a iz homogenate jetre određen je intenzitet lipidne peroksidacije i aktivnost antioksidativnih enzima (CAT, GSH-Px, GSH-R, GSH-ST). Isečci tkiva jetre su histološki obrađeni i bojeni hematoksilin-eozin metodom i histohemijskim metodama (retikulin, Mallory, Periodic Acid Schiff- Alcian Blue (PAS/AB)). Imunohistohemijski je ispitana proliferacija hepatocita (Ki-67), markeri apoptoze (p53, Bcl-2, Bcl-X, Bax) i ekspresija nuklearnog farnesoid X receptora (FXR). Rezultati istraživanja pokazuju da ispitivane ŽK pomažu očuvanje telesne mase u holestazi i dijabetesu, i značajno snižavaju glikemiju kod dijabetičnih jedinki. Parametri jetrene funkcije u holestazi i dijabetesu su regulisani primenom MK i UDK. Obe ŽK u značajnoj meri smanjuju intenzitet lipidne peroksidacije i pojačavaju enzimsku antioksidativnu odbranu hepatocita u holestazi i dijabetesu. Ekspresija markera apoptoze nije značajno promenjena izazvanjem modela holestaze i dijabetesa, kao ni primenom ispitivanih ŽK. Nasuprot tome, izazivanje holestaze i dijabetesa značajno smanjuje proliferaciju hepatocita, dok primena MK i UDK poništava ovaj efekat i značajno povećava proliferaciju hepatocita. Hiperglikemija u aloksanskom dijabetesu nije dovela do pojačane ekspresije FXR. Izazivanje holestaze kod zdravih i dijabetičnih životinja dovelo je do porasta ekspresije FXR, koja je redukovana primenom MK i UDK. Na osnovu dobijenih rezultata može se zaključiti da sintetska 12-monoketoholna kiselina pokazuje slična hipoglikemijska, hepatoprotektivna i antioksidativna dejstva kao i prirodna ursodeoksiholna kiselina.

Bile acids (BAs) are structurally diverse molecules, which have theroles in the digestive system, which are exercised through the bile. Beside those, BAs act as a signaling molecules and achieve endocrine and paracrine effects. In addition to its own metabolism, bile acids modulate the metabolism of lipids and glucose. The fact that so far only ursodeoxycholic acid (UDC) is used for therapeutic purposes, speak clearly about of numerous limitations on the application of other natural BAs, and highlights the need to develop new synthetic Bas and ligands. The aim of this study was to investigate the influence of synthetic 12-monoketocholic acid (MC) and natural bila acid UDC in the model of cholestasis and alloxan-induced diabetes. Tests were performed on male Wistar rats. We analyzed the body mass, glucose, liver function tests (AST, ALT, γ-GT, total and direct bilirubin). Using liver tissue homogenates we determined intensity of lipid peroxidation (by concentration of malondilaldehyde) and the activity of antioxidant enzymes (CAT, GSH-Px, GSH -R, GSH-ST). Liver tissue were histologically processed and stained with hematoxylin-eosin method and histochemical methods (reticulin, Mallory, Periodic Acid Schiff- Alcian Blue (PAS / AB)). Imunohistochemical examination included hepatocyte proliferation (Ki-67), markers of apoptosis (p53, Bcl-2, Bcl-X, Bax), and expression of the nuclear farnesoid X receptor (FXR). Results of the research show that MC prevented decrease in body mass during cholestasis and diabetes, and significantly reduced glycemia in diabetic animals. The liver function tests in cholestasis and diabetes are normalised by MC and UDC aplication. Both BAs significantly reduce lipid peroxidation and enhance enzymatic antioxidant defense of hepatocytes in cholestasis and diabetes. The expression of markers of apoptosis was not significantly changed in models of cholestasis and diabetes, as well as the application of the tested BAs. In contrast, in cholestasis and diabetes model, the proliferation of hepatocytes was significantly reduced, while the use of MC and UDC reversed this effect and significantly increased the proliferation of hepatocytes. Hyperglycemia in alloxan-induced diabetes did not lead to overexpression of FXR. Induction of cholestasis in healthy and diabetic animals resulted in an increase in the expression of FXR, which is reduced by using the MK and the UDC. Based on these results we can conclude that a synthetic 12-monoketocholic acid shows similar hypoglycemic, hepatoprotective and antioxidant effects as natural ursodeoxycholic acid.