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[The Effect of Interference of NLRP3 with shRNA on AGEs-induced Inflammatory Response in Myocardial Cell]
He-Lei Jia1, Chang-Qing Lu1, Juan Wang1
1Department of Emergency, Henan Traditional Chinese Medicine Hospital (the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine), Zhengzhou 450002, China.
Objective:
This study aims to explore the effect of silence of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome on advanced glycation end products (AGEs)-induced myocardial injury.
Methods:
The myocardial injury model was indued by AGEs. NLRP3 was silenced by shRNA. H9c2 cells were divided into four groups: H9c2 (control group); AGEs group; AGEs+sh-Ctrl group; AGEs+sh-NLRP3 group. The latter two groups of cells will first shRNA control (sh-Ctrl) and shRNA-NLRP3 (sh-NLRP3) plasmids were transfected into H9c2 cells, the last 3 cells were then treated for 24 h with 100 mg/L AGEs, establishment of H9c2 damage model, control cells were treated with solvent for 24 h; Apoptosis was measured by Hoechst33258 staining. The levels of interleukin (IL)-6, IL-18 and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The protein levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-3, Caspase-9, nuclear factor κB (NF-κB) P65 and p-P65 were tested by Western blot. The nuclear NF-κB P65 levels were detected by immunofluorescent staining.
Results:
The expressions of NLRP3, ASC, Caspase-3 and Caspase-9 in AGEs group and AGEs+sh-Ctrl group was higher than control group ( P<0.05). Compared with AGEs group, the expressions of NLRP3, ASC, Caspase-3 and Caspase-9 in AGEs+sh-NLRP3 group was decreased ( P<0.05). Compared with control group, the apoptosis and the levels of IL-6, IL-18 and IL-1β in AGEs group and AGEs+sh-Ctrl group were elevated ( P<0.05). The apoptosis and the levels of IL-6, IL-18 and IL-1β in AGEs+sh-NLRP3 group were lower than those of AGEs group ( P<0.05). The phosphorylation of NF-κB P65 and nuclear NF-κB P65 in AGEs group and AGEs+sh-Ctrl group were higher than control group ( P<0.05). Compared with AGEs group, the phosphorylation of NF-κB P65 and nuclear NF-κB P65 in AGEs+sh-NLRP3 group were reduced ( P<0.05). Conlusion Silencing of NLRP3 inflammasome alleviates AGEs-induced apoptosis and inflammatory response in myocardial cell via inhibiting NF-κB P65 activation.
Insights
Silencing the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome reduces advanced glycation end product (AGE)-induced myocardial apoptosis and inflammation. This protective effect is mediated by inhibiting nuclear factor-kappa B (NF-κB) activation.
Area of Science:
- Cardiovascular Biology
- Immunology
- Molecular Medicine
Background:
- Advanced glycation end products (AGEs) contribute to myocardial injury.
- The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome plays a role in inflammatory responses.
- Understanding the role of NLRP3 inflammasome in AGEs-induced cardiac damage is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the impact of silencing the NLRP3 inflammasome on AGEs-induced myocardial injury.
- To elucidate the underlying molecular mechanisms, particularly the involvement of NF-κB signaling.
Main Methods:
- H9c2 cells were treated with AGEs to induce myocardial injury.
- NLRP3 was silenced using short hairpin RNA (shRNA).
- Apoptosis, inflammatory cytokine levels (IL-6, IL-18, IL-1β), and key protein expressions (NLRP3, ASC, Caspase-3, Caspase-9, NF-κB P65, p-P65) were assessed.
Main Results:
- Silencing NLRP3 significantly decreased AGEs-induced expression of NLRP3, ASC, Caspase-3, and Caspase-9.
- Apoptosis and levels of IL-6, IL-18, and IL-1β were reduced in cells with silenced NLRP3.
- Phosphorylation and nuclear translocation of NF-κB P65 were inhibited by NLRP3 silencing.
Conclusions:
- NLRP3 inflammasome activation is involved in AGEs-induced myocardial apoptosis and inflammation.
- Silencing NLRP3 alleviates myocardial injury by inhibiting the NF-κB signaling pathway.
- Targeting NLRP3 may offer a therapeutic approach for AGEs-related cardiovascular diseases.
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