Influence of bioactive molecules of lactobacilli on autophagy and inflamation processes in in vitro and in vivo systems

Izolacija i karakterizacija bakterijskih biomolekula koji interaguju sa receptorimaćelija domaćina predstavlja kljuĉ za razumevanje mehanizama probiotiĉkog dejstvalaktobacila. Savremena istraţivanja probiotiĉkih bakterija usmerena su ka identifikacijibiomolekula koji mogu modulisati razliĉite signalne puteve u humanim ćelijama. Svimolekuli koji su poreklom iz probiotika i odgovorni su za njihov pozitivan efekat nazdravlje 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 uublaţavanju simptoma bola i neţeljenih efekata koji nastaju kao posledica primeneanalgetika. Taĉnije, definisana su dva specifiĉna cilja istraţivanja: (i) ispitati uticajbioaktivnih molekula odabranih sojeva laktobacila na proces autofagije u hepatocitima invitro, kao i protektivan efekat ovih molekula kod toksiĉnosti izazvane paracetamolom i (ii)ispitati potencijalni imunomodulatorni efekat egzopolisaharida (EPS-CG11) izolovanog izsoja Lactobacillus paraplantarum BGCG11, u in vivo eksperimentalnim modelimainflamacije. Eksperimenti u kulturi hepatocita ukljuĉili su praćenje stepena oštećenjaHepG2 ćelija izloţenih toksiĉnoj koncentraciji paracetamola i procesa autofagije, sa ciljemidentifikacije potencijalnog mehanizma delovanja postbiotika. Metodološki, vijabilnostHepG2 ćelija analizirana je MTT i LDH esejima. Autofagija je praćena Western blotanalizom odreĊivanjem ekspresije p62/SQSTM1 proteina i akumulacijom liposolubilneforme LC3 proteina. Dodatno, autofagija je analizirana praćenjem ekspresije BECN1, Atg5,p62/SQSTM1 i PINK1 gena i autofagnog fluksa. Za analizu imunomodulatornog efektaEPS-CG11 korišćena su dva in vivo modela inflamacije izazvane karageninom: modelinflamatornog bola i model peritonitisa kod pacova Wistar soja. U eksperimentuinflamatornog bola praćeni su: vremenski tok razvoja hiperalgezije i edema šapica nakonprofilaktiĉke sistemske primene EPS-CG11, ekspresija medijatora inflamacije (IL-1β,TNF-α, IL-6 i iNOS), infiltracija neutrofila (ekspresija MPO enzima) i aktivacija/infiltracijamonocita (ekspresija CD14 markera)...Isolation and characterization of bacterial biomolecules involved in the interactionwith the receptors of the host cells represent the key factor for understanding themechanisms of probiotic action of lactobacilli. Novel studies regarding probiotic bacteriahave been focused on the identification of biomolecules which can modulate differentsignaling pathways in human cells. All molecules that originate from probiotics which areresponsible for its positive effects on the host’s health are called postbiotics. Theirapplication represents the safe alternative to the use of live bacteria and itsimmunostimulating potential.This research aimed to test the possibility of using postbiotics in alleviation of painsymptoms and analgesics side effects. More precisely, two main objectives of this researchwere: (i) to examine the influence of bioactive molecules of selected strains of lactobacillion the autophagy process in the hepatocytes, in vitro, as well as protective effect of thesemolecules in paracetamol-induced toxicity and (ii) to examine the potentialimmunomodulatory effect of exopolysaccharide (EPS-CG11) isolated from Lactobacillusparaplantarum BGCG11 strain, in in vivo experimental models of inflammation.Experiments in the hepatocytes culture included monitoring the degree of damage ofHepG2 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. Autophagywas monitored by Western blot analysis, in order to determine the expression of thep62/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 theimmunomodulatory effect of EPS-CG11, two in vivo models of carrageenan-inducedinflammation were used: an inflammatory pain model and a peritonitis model in the Wistarrats..