Učinak izvanstaničnog Hsp70, ekstrakta dima cigareta, lipopolisaharida i lipoteikoične kiseline na aktivnosti kaspaza-3/-7, -8 i -9 u 16HBE bronhijalnim epitelnim stanicama

master's thesis

• Title (english): Effects of extracellular Hsp70, cigarette smoke extract, lipopolysaccharide and lipoteicoic acid on activity of caspases-3/-7,-8 and -9 in 16HBE bronchial epithelial cells
• Author: Veronika Jarni
• Mentor: Anita Somborac Bačura (mentor)
• Committee member: Anita Somborac Bačura (predsjednik povjerenstva)
• Committee member: Roberta Petlevski (član povjerenstva)
• Committee member: Maja Ortner Hadžiabdić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2020-07-20, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Kronična opstrukcijska plućna bolest (KOPB) je kronična upalna bolest koja uzrokuje opstrukciju protoka zraka u plućima. Jedan od glavnih uzročnika KOBP-a je dim cigarete. Molekularni mehanizmi u pozadini klasična su kombinacija urođene i stečene imunosti, a važnu ulogu imaju i proupalni medijatori i oksidacijski stres. Takav upalni odgovor dovodi do smrti stanice. Razlikujemo mnogo načina smrti stanice, ali u ovom kontekstu su najrelevantnije apoptoza, nekroza i nekroptoza, Važnu ulogu u usmjeravanju stanice u određeni oblik stanične smrti imaju proteini toplinskog šoka. LPS (lipopolisaharid) i LTA (lipoteikoična kiselina) kao gradivne komponente bakterija utječu kao molekularni obrasci povezani s patogenima (PAMPs) te također usmjeravaju stanicu u staničnu smrt preko djelovanja na TLR receptore. Cilj ovog ispitivanja bio je korištenjem izvanstaničnog Hsp70, ekstrakta dima cigareta (CSE), LPS-a i LTA te njihovih kombinacija na bronhijalnim epitelnim stanicama 16HBE utvrditi dovode li do smrti stanica mjerenjem aktivnosti pojedinih kaspaza. Ispitivanje aktivnosti kaspaza-3/7, -8 i -9 provodilo se pomoću komercijalnih kitova koji sadrže specifične luminogene supstrate za navedene kaspaze. Nastala luminescencija proporcionalna je aktivnosti kaspaza prisutnih u uzorku. Praćena je aktivnost kaspaza nakon dvosatnog i osmerosatnog trermana. Dobiveni rezultati ukazuju na to da nakon tretmana 16HBE stanica s izvanstaničnim Hsp70, CSE-om, LPS-om, LTA-om ili njihovim kombinacijama tijekom 2 ili 8 sati većinom ne dolazi do značajnijeg porasta broja stanica u apoptozi u odnosu na netretirane stanice. Pronađeno je jedino da nakon 2 sata inkubacije kombinacija 15 % CSE-a i 1 μg/mL Hsp70 značajno više aktivira kaspaze-3/7 u 16HBE stanicama u odnosu na sami 15 % CSE. Nasuprot tome, u uzorcima 16HBE stanica tretiranih samo s 15 % CSE-om tijekom 2 sata uočena je značajno smanjena aktivnosti kaspaza -8 i -9 u odnosu na netretirane stanice. Snižena aktivnost kaspaze-8 može upućivati na to da stanice izložene visokoj koncentraciji dima cigareta umiru nekim alternativnim putem (primjerice, inaktivacijom kaspaze-8 može doći do razvoja nekroptoze). Iz rezultata ovog rada može se zaključiti da izlaganje bronhijalnih epitelnih stanica 16HBE izvanstaničnom Hsp70, CSE-u, LPS-u, LTA-u ili njihovim kombinacijama uglavnom ne dovodi do aktivacije kaspaza-3/7, -8 i -9 te razvoja apoptoze. Rezultate rada bilo dobro nadopuniti određivanjem biljega drugih načina umiranja stanica (primjerice, nekroze ili nekroptoze), što bi omogućilo bolju usporedbu s rezultatima već objavljenih radova te primjerenije zaključke vezano uz moguće uzroke gubitka plućnih struktura kod oboljelih od KOPB-a, a možda čak i potencijalne nove mete lijekova.

Abstract (english)

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease that causes obstruction of air flow in the lungs. One of the main causes of COPD is cigarette smoke. Molecular mechanisms in the background are a classic combination of innate immunity and adaptive immunity. Pro-inflammatory cytokines and oxidative stress also play an important role. Such response can lead to cell death. Different types of cell death can be differentiated, but in this case the most relevant ones are apoptosis, necrosis and necroptosis. Heath shock proteins play an important role in modifying cell death. LPS (lipopolysaccharides) and LTA (lipoteicoic acid), as structural components of bacteria, behave as PAMPs (pathogen associated molecular patterns) and may modulate cell death via TLRs (Toll-like receptors). The aim of this study was to establish if the treatment of 16HBE cells with extracellular Hsp70, CSE (cigarette smoke extract), LPS, LTA or their combinations leads to cell death, that was quantified by the measurement of caspases activities. The measurement of the activities of caspases -3/-7 , -8 and -9 was done with the use of commercial kits that contain specific luminogenic substrats for these caspases. The measured luminescence is proportional to the activity of caspases. The activity was measured after a two hour treatment and after an eight hour treatment. The results imply that after the treatment of 16HBE cells with eHsp70, CSE, LPS, LTA and their combinations after 2 hours or 8 hours there were no significant increases of cells in apoptosis when compared to untreated cells. The only result that was statistically significantly different was that after a two hour treatment, a combination of 15% CSE and 1 μg/mL Hsp70 significantly increased an activity of caspases-3/7 in 16HBE cells when compared to the treatment with 15% CSE only. However, in the samples of 16HBE cells treated with 15% CSE during 2 hours, there was a significant drop in the activity of caspase-8 and caspase-9 when compared to untreated cells. The drop in the activity of caspaze-8 can implicate that exposure to high concentrations of CSE lead to an alternative type of cell death (inactivation of caspase-8 may lead to necroptosis). From the results of this study, we can conclude that exposure of bronchial epithelial cells 16HBE to extracellular Hsp70, CSE, LPS, LTA or their combinations mostly does not lead to activation of caspases-3/7, -8 or -9 and apoptosis in general. The results of this work could be completed by measuring the markers of other types of cell death (such as necrosis or necroptosis). That would make even better comparison to results of the already conducted experiments. Better comparisons are crucial for making new, more appropriate conclusions about what might be the cause of lung structure loss in COPD patients, and possibly even a discovery of new drug targets.

• Keywords: kronična opstrukcijska plućna bolest; KOPB; stanična smrt; kaspaze; cigaretni dim; Hsp70; LPS; LTA
• Keywords (english): chronic obstructive pulmonary disease; COPD; cell death; caspases; cigarette smoke extract; Hsp70; LPS; LTA
• Language: croatian
• URN:NBN: urn:nbn:hr:163:976505
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Repository: Repository of Faculty of Pharmacy and Biochemistry University of Zagreb
• Created on: 2021-04-28 09:48:21