Journal
AQUACULTURE
Volume 555, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.aquaculture.2022.738220
Keywords
NLRP3; Inflammation; Glucose metabolism; Gene cloning; Megalobrama amblycephala
Categories
Funding
- National Natural Science Foundation of China [32002361]
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This study cloned and characterized the NLRP3 gene from Megalobrama amblycephala and investigated its function in high-glucose-induced inflammation. The results showed that NLRP3 played a role in the regulation of HG-induced inflammation, and high-leucine levels could aggravate this inflammation via NLRP3 activation in hepatocytes of M. amblycephala.
The nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) has emerged as a key regulator of glucose homeostasis in mammals, while relevant information are still poorly interpreted in aquatic animals. Herein, we cloned and characterized NLRP3 gene from Megalobrama amblycephala, and analyzed its function in high glucose (HG)-induced inflammation. Full-length cDNA of NLRP3 measured 4380 bp with 3570 bp open reading frame encoding 1189 amino acids, compared with Pimephales promelas, it displayed 90.3% homology. Structural analysis revealed that M. amblycephala NLRP3 processed characteristic domains of pyrin, FISNA, NACHT, CARD and LRRs. Spatial expression analysis displayed NLRP3 was ubiquitously expressed in eight organs/tissues (gill, liver, spleen, intestine, head kidney, white muscle, heart and brain) with largest magnitude in head kidney. After a 13-week nutritional experiment, carbohydrate-enriched (45%) diets significantly upregulated NLRP3 mRNA levels in the liver and white muscle than that of the control (30%) diets. Glucose loading caused a significantly increased NLRP3 mRNA levels in the white muscle and liver, with maximum values got at 1 and 2 h, respectively. Subsequently, the mRNA levels rapidly returned to the basal level at 12 h. As for the in-vitro experiments, both ATP and high-glucose treatments remarkably increased the expression of NLRP3 and pro-inflammatory cytokines (IL18 and IL6) in hepatocytes. Additionally, high-leucine (30 mM) levels remarkably increased NLRP3 protein content and transcriptions of NLRP3, NF-kappa B, IL18 and IL6 than those of the HG and HG + low leucine (10 mM) treatment groups, while the opposite was true for IL8 and IL10. Overall, our findings indicated that M. amblycephala NLRP3 is highly conserved compared with other vertebrates. NLRP3 participated in the control of HG-induced inflammation, and high-leucine levels could aggravate HG-induced inflammation in hepatocytes of M. amblycephala via NLRP3 activation.
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