| Abstract | Dim cigareta smatra se jednim od glavnih čimbenika u razvoju kronične opstrukcijske plućne bolesti (KOPB). Upalnom odgovoru
pridonosi izvanstanični protein toplinskog šoka 70 (Hsp70) aktivacijom receptora 2 i 4 sličnih Tollu (TLR2 i TLR4), što dovodi do
kaskadne aktivacije protein-kinaza aktiviranih mitogenom (MAPK), poput protein-kinaza reguliranih izvanstaničnim signalima
(ERK) i p38. Suprotno tome, unutarstanični Hsp70 potiskuje upalni odgovor. Cilj ovog rada bio je ispitati utjecaj izvanstaničnog
Hsp70, ekstrakta dima cigareta (CSE), lipopolisaharida (LPS) i lipoteikoične kiseline (LTA) na ekspresiju receptora TLR2 i TLR4 te unutarstaničnog Hsp70, kao i na aktivaciju MAPK u 16HBE i NHBE stanicama humanog bronhijalnog epitela. Citotoksičnost je
ispitana MTS testom nakon 24 h tretiranja stanica. Aktivacija i ekspresija MAPK (ERK i p38) te ekspresija unutarstaničnog Hsp70 određena je Western blot tehnikom nakon 30 minuta, 2 i 8 sati tretiranja, dok je ekspresija receptora TLR2 određena kvantitativnom lančanom reakcijom polimeraze u stvarnom vremenu (qPCR) nakon 24 sata tretiranja. Rezultati pokazuju da CSE u nižim koncentracijama povećava vijabilnost 16HBE stanica, ali da je u višim koncentracijama smanjuje. Suprotno tome 15 % CSE povećao je vijabilnost NHBE stanica. Nadalje, u 16HBE stanicama ERK pokazuje aktivaciju nakon 30 minuta kod gotovo svih tretiranja, a nakon 8 sati se aktivira uz izlaganje nižoj koncentraciji CSE-a, LPS-u i LTA-u. CSE je u svim ispitivanim vremenima i koncentracijama doveo do povećane aktivacije p38, do čega je dovelo i 30-minutno izlaganje stanica LPS-u i LTA-u. Do smanjenja unutarstaničnog Hsp70 doveli su LPS i LTA, dok je CSE uglavnom povećavao njegovu ekspresiju. Na aktivaciju MAPK i ekspresiju Hsp70 u NHBE stanicama utjecao je samo 15 % CSE. U 16HBE stanicama LPS i LTA ne utječu značajno na mRNA ekspresiju receptora TLR2, dok je rhHsp70 smanjuje. S druge strane, rhHsp70 pokazuje sinergistički učinak s LPS-om i LTA-om na povećanje ekspresije mRNA za receptor TLR2 u odnosu na stanice tretirane samim LPS-om ili LTA-om. CSE povećava ekspresiju mRNA za receptor TLR2 u individualnim i u kombiniranim tretiranjima. Kombinirano tretiranje 15 % CSE-om i rhHsp70 pokazuje značajno smanjenje ekspresije mRNA za receptor TLR2. Ovo ukazuje na antagonistički učinak rhHsp70 i viših koncentracija CSE-a. U ovome istraživanju potvrđeno je citotoksično djelovanje viših koncentracija CSE-a, dok bi niže koncentracije CSE-a mogle uzrokovati poremećenu proliferaciju stanica plućnog epitela. Isto tako, rezultati pokazuju da rhHsp70, LPS, LTA i CSE mogu aktivirati MAPK ovisno o vrsti stanica, koncentraciji te vremenu tretiranja. Nadalje, primijećeno je međudjelovanje rhHsp70 te LPS-a, LTA-a i CSEa u modulaciji ekspresije mRNA za receptor TLR2. NHBE stanice pokazale su manju osjetljivost na primijenjena tretiranja u odnosu na 16HBE stanice. |
| Abstract (english) | Cigarette smoke is considered one of the main risk factors in the development of chronic obstructive pulmonary disease (COPD). Extracellular heat shock protein 70 (Hsp70) stimulates inflammatory responses through activation of Toll like receptors 2 and 4 (TLR2 and TLR4), followed by induction of mitogen-activated protein kinases (MAPKs) pathways, including extracellular signal-regulated kinases (ERK) and p38. Contrary to this, intracellular Hsp70 has an anti-inflammatory role inside the cell. The aim of this study was to assess the effect of extracellular Hsp70, cigarette smoke extract (CSE), lipopolysaccharide (LPS), lipoteichoic acid (LTA) and their combinations on the expression of receptors TLR2 and TLR4 as well as intracellular Hsp70, and the activation of MAPKs in 16HBE and NHBE human bronchial epithelial cells. Cytotoxicity was determined by MTS viability assay after 24 h treatment. The activation
of MAPKs (ERK and p38) and the expression of Hsp70 were determined by Western blot analysis after 30 min, 2 and 8 hours treatments. The expression of TLR2 was determined by quantitative polymerase chain reaction (qPCR) after 24 h treatment. The results indicate that lower doses of CSE increase the viability of 16HBE cells, but higher concentrations have a significant cytotoxic effect. 15% CSE increased the viability of NHBE cells. Furthermore, ERK was activated in 16HBE cells by almost all treatments after 30 minutes of exposure as well as by lower doses of CSE, LPS and LTA after 8 h of treatment. p38 was activated by LPS and LTA after 30 minutes as well as by CSE after all time periods examined. Hsp70 showed lower expression after being exposed to LPS and LTA, but CSE mostly increased its expression. In NHBE cells, 15% CSE was the only treatment that had any effect on the activation of MAPKs and expression of Hsp70. Neither LPS nor LTA had a significant effect on mRNA expression of receptor TLR2, while rhHsp70 caused its down-regulation. On the other hand, with rhHsp70 and LTA or LPS combined treatments a significant synergistic effect inducing an increase in mRNA expression of TLR2 was detected compared to untreated cells as well as to cells treated with LPS or LTA alone. CSE
caused an increase in TLR2 mRNA expression after all treatments, but there was a suppressive antagonistic effect on TLR2 mRNA expression observed after combined treatment with 15% CSE and rhHsp70 when compared to untreated cells as well as cells treated with 15% CSE alone. Our results confirm the cytotoxic effects of higher concentrations of CSE, while lower concentrations of CSE could cause alterations in lung epithelial cells proliferation. In addition, results show that rhHsp70, LPS, LTA and CSE can activate MAPKs, and this was dependent on cell type, concentration, and duration of the treatment. Furthermore, interactions of rhHsp70, LPS, LTA and CSE can modulate mRNA expression of TLR2. NHBE cells are less susceptible to applied treatments compared to 16HBE cells. |
