Jingna Wu1, Xiaoting Chen2, Nan Pan2, Bei Chen2, Jinhua Zhang3, Zhiyu Liu4. 1. Xiamen Key Laboratory of Marine Medicinal Natural Products Resources, Xiamen Medical College, Xiamen 361023, PR China; Fujian Universities and Colleges Engineering Research Center of Marine Biopharmaceutical Resources, Xiamen Medical College, Xiamen 361023, PR China. Electronic address: wjn@xmmc.edu.cn. 2. Fisheries Research Institute of Fujian, Xiamen 361013, PR China. 3. Xiamen Key Laboratory of Marine Medicinal Natural Products Resources, Xiamen Medical College, Xiamen 361023, PR China; Fujian Universities and Colleges Engineering Research Center of Marine Biopharmaceutical Resources, Xiamen Medical College, Xiamen 361023, PR China. 4. Fisheries Research Institute of Fujian, Xiamen 361013, PR China. Electronic address: 13906008638@163.com.
Abstract
AIMS: We investigated the anti-inflammatory activity of 3β-hydroxycholest-5-en-7-one from Hippocampus trimaculatus leach and provided a theoretical basis for identifying its therapeutic targets. MAIN METHODS: Small-RNA libraries were constructed for untreated control RAW 264.7 cells and cells treated with lipopolysaccharide (LPS; 1.0 μg/mL) or 10 μM 3β-hydroxycholest-5-en-7-one +1.0 μg/mL LPS. We constructed and tested a miR-98-5p-interfering lentivirus to evaluate the role of miR-98-5p in the 3β-hydroxycholest-5-en-7-one-dependent regulation of inflammatory responses in LPS-induced macrophage and murine inflammation models. The small-RNA libraries were analyzed using high-throughput sequencing. KEY FINDINGS: Among the differentially expressed microRNAs, miR-98-5p showed the most significant difference. Bioinformatics tools were used to identify the potential regulatory targets of miR-98-5p, which were tested using dual-luciferase reporter assays. Our results demonstrated that 3β-hydroxycholest-5-en-7-one exerted an anti-inflammatory effect via miR-98-5p, which negatively regulated the expression of its target gene TNFAIP3. The results indicate that miR-98-5p interference and 3β-hydroxycholest-5-en-7-one treatment significantly upregulated the low TNFAIP3 expression induced by LPS stimulation, thereby inhibiting TRAF6, RIP, NF-κB, IL-1β, and TNF-α secretion. SIGNIFICANCE: 3β-Hydroxycholest-5-en-7-one alleviates inflammation by downregulating miR-98-5p and upregulating TNFAIP3, thereby blocking NF-κB pathway activation. These results reveal the specific anti-inflammatory mechanism of 3β-hydroxycholest-5-en-7-one, providing a foundation for developing new drugs and identifying drug targets.
AIMS: We investigated the anti-inflammatory activity of 3β-hydroxycholest-5-en-7-one from Hippocampus trimaculatus leach and provided a theoretical basis for identifying its therapeutic targets. MAIN METHODS: Small-RNA libraries were constructed for untreated control RAW 264.7 cells and cells treated with lipopolysaccharide (LPS; 1.0 μg/mL) or 10 μM 3β-hydroxycholest-5-en-7-one +1.0 μg/mL LPS. We constructed and tested a miR-98-5p-interfering lentivirus to evaluate the role of miR-98-5p in the 3β-hydroxycholest-5-en-7-one-dependent regulation of inflammatory responses in LPS-induced macrophage and murineinflammation models. The small-RNA libraries were analyzed using high-throughput sequencing. KEY FINDINGS: Among the differentially expressed microRNAs, miR-98-5p showed the most significant difference. Bioinformatics tools were used to identify the potential regulatory targets of miR-98-5p, which were tested using dual-luciferase reporter assays. Our results demonstrated that 3β-hydroxycholest-5-en-7-one exerted an anti-inflammatory effect via miR-98-5p, which negatively regulated the expression of its target gene TNFAIP3. The results indicate that miR-98-5p interference and 3β-hydroxycholest-5-en-7-one treatment significantly upregulated the low TNFAIP3 expression induced by LPS stimulation, thereby inhibiting TRAF6, RIP, NF-κB, IL-1β, and TNF-α secretion. SIGNIFICANCE: 3β-Hydroxycholest-5-en-7-one alleviates inflammation by downregulating miR-98-5p and upregulating TNFAIP3, thereby blocking NF-κB pathway activation. These results reveal the specific anti-inflammatory mechanism of 3β-hydroxycholest-5-en-7-one, providing a foundation for developing new drugs and identifying drug targets.