Uloga inflamasoma, nekroptoze i piroptoze u kroničnoj opstrukcijskoj plućnoj bolesti

master's thesis

• Title (english): Role of inflammasome, necroptosis and pyroptosis in chronic obstructive pulmonary disease
• Author: Ivana Marinić
• Mentor: Marija Grdić Rajković (mentor)
• Committee member: Marija Grdić Rajković (predsjednik povjerenstva)
• Committee member: Dubravka Vitali Čepo (član povjerenstva)
• Committee member: Petra Turčić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2019-09-26, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Kronična opstrukcijska plućna bolest (KOPB) je bolest karakterizirana nedostatnim protokom zraka kroz pluća. U patogenezi KOPB-a bitnu ulogu imaju oksidacijski stres i upalni procesi, jer do opstrukcije dišnih putova dolazi zbog kronične upale dišnih putova i razaranja parenhima čime se gubi alveolarna povezanost i elastičnost pluća. Patogeneza KOPB-a povezana je s različitim oblicima stanične smrti te štetnim tvarima od kojih se cigaretni dim smatra najopasnijim.
Istraživanje je provedeno na 16-HBE bronhijalnim epitelnim stanicama. Ispitano je djelovanje rhHsp70 (0,3, 1 i 3 μg/mL), LPS-a (0,1 μg/mL), LTA (1 μg/mL) i CSE-a ( 2,5 i 15 %) te njihovih kombinacija. Cilj 24 satnog tretiranja bio je utvrditi dolazi li, i u kojim koncentracijama ovih agenasa, do aktivacije NLRP3 inflamasoma, nekroptoze i piroptoze. Aktivacija NLRP3 inflamasoma i upalni odgovor ispitani su određivanjem ekspresije mRNA za NLRP3, kaspazu-1 i upalni citokin IL-1β kvantitativnim PCR-om. Vijabilnost stanica ispitana je određivanjem aktivnosti LDH izlučene u medij iznad stanica, a njihov način umiranja ispitan je određivanjem ekspresije mRNA za kaspazu-1 i IL-1β, aktivacije proteina RIPK1 i MLKL te ekspresije proteina gasdermina D Western blot analizom.
Do pojačane ekspresije mRNA za NLRP3, IL-1β i kaspazu-1 dolazi nakon pojedinačnog tretiranja stanica s rhHsp70, LPS-om i LTA-om te nakon tretiranja CSE-om pojedinačno i u kombinaciji s rhHsp70. To ukazuje na aktivaciju NLRP3 inflamasoma i upalu. Katalitička aktivnost LDH povećana je samo nakon tretiranja s CSE-om i kombinacijama CSE i rhHsp70 što znači da CSE narušava integritet stanične membrane i aktivira staničnu smrt. Povećanje ekspresije mRNA za IL-1β i kaspazu-1 te povećanje ekspresije proteina gasdermina D ukazuju na aktivnost piroptoze. RIPK1 i MLKL eksprimirane su u svim stanicama no do aktivacije MLKL dolazi samo nakon tretiranja s CSE-om pojedinačno i u kombinacijama s rhHsp70 što upućuje na nekroptozu.

Abstract (english)

Chronic obstructive pulmonary disease (COPD) is a disease characterized by insufficient flow of air through the lungs. In the pathogenesis of COPD, oxidative stress and inflammatory processes have an essential role due to chronic inflammation of the airways and destruction of parenchyma which leads to lost of alveolar connectivity and lung elasticity. COPD pathogenesis is associated with various forms of cell death and harmful substances of which cigarette smoke is considered the most dangerous.
The study was conducted on 16-HBE bronchial epithelium cells. The effects of rhHsp70 (0.3, 1 and 3 μg/mL), LPS (0.1 μg/mL),LTA (1 μg/mL) and CSE ( 2,5 and 15%) and their combinations were assessed. The aim of 24-hour treatment was to determine whether, and at what concetrations of these agents, the activation of NLRP3 inflammasomes, necroptosis and pyroptosis occur. Activation of NLRP3 inflammasome and inflammatory response were assessed by determining the level of expression for NLRP3, caspase-1, and inflammatory cytokine IL-1β mRNA by quantitative PCR. Cell viability was determinated by LDH activity in the media above the cells and their mode of dying was explored by determining the expression of mRNA for caspase-1 and IL-1β, as well as activation of RIPK1 and MLKL proteins and expression of the protein gasdermin D by Western blot analysis. The increased expression of mRNA for NLRP3, IL-1 β and caspase-1 comes after individual treatment of cells with rhHsp70, LPS and LTA and after treatment with CSE individually and in combination with rhHsp70. This indicates the activation of NLRP3 inflammasome and inflammation. The catalytic activity of LDH is increased only after treatment with CSE and CSE and rhHsp70 combination, which means that CSE disrupts cell membrane integrity and activates cellular death. An increase in the expression of mRNA for IL-1 β and caspase-1 and an increase in the expression of gasdermin D protein indicate the activity of pyroptose. RIPK1 and MLKL are exported to all cells but the MLKL activation only occurs after treatment with the CSE individually and in combinations with rhHsp70, indicating necroptosis.

• Keywords: kronična opstrukcijska plućna bolest; KOPB; NLRP3 inflamasom; nekroptoza; piroptoza
• Keywords (english): chronic obstructive pulmonary disease; COPD; NLRP3 inflammasome; necroptosis; pyroptosis
• Language: croatian
• URN:NBN: urn:nbn:hr:163:750739
• 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
• Created on: 2021-04-23 08:05:44