S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Medicine (의학과) Theses (Ph.D. / Sc.D._의학과)
Pretreatment with low-dose fimasartan, one of angiotensin receptor blockers, inhibits NLRP3 inflammasome activation in a rat intracerebral hemorrhage model
백서 뇌출혈 모델에서 저농도 피마사탄 전처치 후 NLRP3 인플라마좀 활성화 억제를 통한 뇌손상 보호 효과에 대한 연구
- 의과대학 의학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 의학과, 2017. 2. 윤병우.
- Background and Purpose: The NLRP3 inflammasome, which is composed of NLRP3 (NOD-Like Receptor family Pyrin domain containing 3), the adaptor molecule apoptosis-associated speck-like protein containing a CARD (ASC) domain and procaspase-1, plays an important role in the immune pathophysiology of secondary damage induced by intracerebral hemorrhage (ICH). This study aims to investigate whether pretreatment with low-dose fimasartan, an angiotension II receptor blocker (ARB), has anti-neuroinflammatory effect in normotensive rats following ICH by inhibiting the activation of the NLRP3 inflammasome.
Materials and Methods: Sprague-Dawley rats were divided into the four groups as follows: sham, vehicle, low-dose (0.5 mg/kg) and regular dose (1.0 and 3.0 mg/kg) fimasartan. These rats were treated for 30 days before the induction of collagenase-induced ICH and for 2 days after surgery. Brain water content, hematoma volume, neurological functional recovery and blood pressure (BP) were evaluated according to the different doses of fimasartan. The expression of protein, mRNA, pro-inflammatory cytokines and double immunofluorescent staining of the NLRP3 inflammasome components were measured on 1 day post-ICH in the rats administrated with the low-dose fimasartan.
In vitro experiments, we stimulated astrocytes with hemolysate to induce hemorrhagic inflammation. Astrocytes were pretreated with fimasartan at different concentrations and then incubated with hemolysate. Inflammatory cell signaling pathways were assessed by western blotting. Pro-inflammatory mediators were evaluated by real time RT-PCR and ELISA.
Results: ICH rat model resulted in a large hematoma and edema accompanied by the up-regulation of NLRP3/ASC/Caspase-1 and the NF-κB signaling pathway, which subsequently contribute to the release of pro-inflammatory cytokines within one day. Long-term pretreatment with either dose of fimasartan attenuated the ICH-induced edema and improved neurological functional outcomes. BP was regulated by fimasartan in a dose-dependent manner, but with no change in the low-dose. Furthermore, the activation of the NLRP3 inflammasome components and the NF-κB pathway were markedly reduced by low-dose fimasartan. The double immunofluorescent staining of brain cells showed a progressive decrease in co-localization of NLRP3/ASC/Caspase-1 with Iba1 (microglia marker) and GFAP (astrocyte marker) positive cells.
In vitro, the stimulation by hemolysate generated a robust activation of inflammatory response in astrocytes. Pretreatment with fimasartan significantly decreased hemolysate-induced NFκB-related inflammatory pathways induced by hemolysate. This reduction of inflammatory upstream pathways decreased the expression of end-products, COX-2 and interleukin-1.
Conclusions: Our study suggests that the direct anti-inflammatory effect of fimasartan reduces acute ICH-induced damage. Specially, low-dose fimasartan improves the neurological recovery and reduce the edema by inhibiting the NLRP3 inflammasome without affecting BP. Pretreatment with fimasartan also inhibits hemolysate-induced inflammatory in astrocytes. Of translational significance, fimasartan could potentially target the inflammasome to attenuate secondary injury after ICH.