Uticaj bioaktivnih molekula laktobacila na procese autofagije i inflamacije u in vitro i in vivo sistemima

Abstract

Izolacija i karakterizacija bakterijskih biomolekula koji interaguju sa receptorima ćelija domaćina predstavlja kljuĉ za razumevanje mehanizama probiotiĉkog dejstva laktobacila. Savremena istraţivanja probiotiĉkih bakterija usmerena su ka identifikaciji biomolekula koji mogu modulisati razliĉite signalne puteve u humanim ćelijama. Svi molekuli koji su poreklom iz probiotika i odgovorni su za njihov pozitivan efekat na zdravlje domaćina nazvani su postbiotici. Zbog slabijeg imunostimulišućeg potencijala, njihova primena predstavlja bezbednu alternativu primeni ţivih bakterija. Ovo istraţivanje je imalo za cilj da testira mogućnost primene postbiotika u ublaţavanju simptoma bola i neţeljenih efekata koji nastaju kao posledica primene analgetika. Taĉnije, definisana su dva specifiĉna cilja istraţivanja: (i) ispitati uticaj bioaktivnih molekula odabranih sojeva laktobacila na proces autofagije u hepatocitima in vitro, kao i protektivan efekat ovih molekula kod toksiĉnosti izazvane paracetamolom i (ii) ispitati potencijalni imunomodulatorni efekat egzopolisaharida (EPS-CG11) izolovanog iz soja Lactobacillus paraplantarum BGCG11, u in vivo eksperimentalnim modelima inflamacije. Eksperimenti u kulturi hepatocita ukljuĉili su praćenje stepena oštećenja HepG2 ćelija izloţenih toksiĉnoj koncentraciji paracetamola i procesa autofagije, sa ciljem identifikacije potencijalnog mehanizma delovanja postbiotika. Metodološki, vijabilnost HepG2 ćelija analizirana je MTT i LDH esejima. Autofagija je praćena Western blot analizom odreĊivanjem ekspresije p62/SQSTM1 proteina i akumulacijom liposolubilne forme LC3 proteina. Dodatno, autofagija je analizirana praćenjem ekspresije BECN1, Atg5, p62/SQSTM1 i PINK1 gena i autofagnog fluksa. Za analizu imunomodulatornog efekta EPS-CG11 korišćena su dva in vivo modela inflamacije izazvane karageninom: model inflamatornog bola i model peritonitisa kod pacova Wistar soja. U eksperimentu inflamatornog bola praćeni su: vremenski tok razvoja hiperalgezije i edema šapica nakon profilaktiĉke sistemske primene EPS-CG11, ekspresija medijatora inflamacije (IL-1β, TNF-α, IL-6 i iNOS), infiltracija neutrofila (ekspresija MPO enzima) i aktivacija/infiltracija monocita (ekspresija CD14 markera)...

Isolation and characterization of bacterial biomolecules involved in the interaction with the receptors of the host cells represent the key factor for understanding the mechanisms of probiotic action of lactobacilli. Novel studies regarding probiotic bacteria have been focused on the identification of biomolecules which can modulate different signaling pathways in human cells. All molecules that originate from probiotics which are responsible for its positive effects on the host’s health are called postbiotics. Their application represents the safe alternative to the use of live bacteria and its immunostimulating potential. This research aimed to test the possibility of using postbiotics in alleviation of pain symptoms and analgesics side effects. More precisely, two main objectives of this research were: (i) to examine the influence of bioactive molecules of selected strains of lactobacilli on the autophagy process in the hepatocytes, in vitro, as well as protective effect of these molecules in paracetamol-induced toxicity and (ii) to examine the potential immunomodulatory effect of exopolysaccharide (EPS-CG11) isolated from Lactobacillus paraplantarum BGCG11 strain, in in vivo experimental models of inflammation. Experiments in the hepatocytes culture included monitoring the degree of damage of HepG2 cells exposed to the toxic paracetamol concentration and the autophagy process, with the aim of identification of potential mechanism of postbiotic action. Methodologically, the cell viability was monitored by MTT and LDH assays. Autophagy was monitored by Western blot analysis, in order to determine the expression of the p62/SQSTM1 protein and the accumulation of the liposoluble form of the LC3 protein. Further, the autophagy was analyzed by monitoring the expression of BECN1, Atg5, p62/SQSTM1 and PINK1 genes and the autophagy flux. For the analysis of the immunomodulatory effect of EPS-CG11, two in vivo models of carrageenan-induced inflammation were used: an inflammatory pain model and a peritonitis model in the Wistar rats...