Loganin Alleviates Gout Inflammation by Suppressing NLRP3 Inflammasome Activation and Mitochondrial Damage

Abstract

Gout is a type of inflammatory arthritis caused by the deposition of monosodium uric acid (MSU) crystals in tissues. The etiology of gout is directly linked to the NLRP3 inflammasome, since MSU crystals are NLRP3 inflammasome activators. Therefore, we decided to search for a small-molecule inhibitor of the NLRP3 inflammasome for the prevention of gout inflammation. We found that loganin suppressed MSU crystals-induced caspase-1 (p20) and interleukin (IL)-1β production and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks formation in mouse primary macrophages, showing its ability to inhibit the NLRP3 inflammasome. In an air pouch inflammation model, oral administration of loganin to mice prevented MSU crystals-induced production of mature IL-1β and IL-18 in air pouch exudates, resulting in decreased neutrophil recruitment. Furthermore, oral administration of loganin suppressed MSU crystals-induced gout inflammation in a mouse foot gout model, which was accompanied by the inhibition of the NLRP3 inflammasome. Loganin blocked de novo synthesis of mitochondrial DNA in air pouches and foot tissues injected with MSU crystals. Consistently, loganin prevented MSU crystals-induced mitochondrial damage in macrophages, as it increased mitochondrial membrane potential and decreased the amount of mitochondrial reactive oxygen species. These data demonstrate that loganin suppresses NLRP3 inflammasome activation by inhibiting mitochondrial stress. These results suggest a novel pharmacological strategy to prevent gout inflammation by blocking NLRP3 inflammasome activation and mitochondrial dysfunction.

Keywords: cytokine; inflammation; innate immunity; mitochondria; pharmacological inhibitor.

Figure 1

Loganin inhibits monosodium urate (MSU) crystals-induced activation of the NLRP3 inflammasome in primary macrophages. (A) Chemical structure of loganin. (B–D) Bone marrow-derived mouse primary macrophages (BMDMs) were primed with lipopolysaccharide (LPS) (100 ng/mL) for 4 h. The cells were treated with loganin for 1 h and then stimulated with MSU crystals (500 μg/mL) for (B) 6 h or (C–E) 4.5 h. (B) Cell culture supernatants and cell lysates were analyzed by immunoblotting for pro-caspase-1, caspase-1(p20), pro-interleukin (IL)-1β, and mature IL-1β. (C) Cell culture supernatants were analyzed for secreted IL-1β by ELISA. (D) BMDMs were fixed, permeabilized, and stained for apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC, green), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). The arrows indicate ASC specks. Scale bars = 10 µm. (E) The bar graph represents the number of ASC specks per 100 μm × 100 μm, which was obtained from different fields of view (n = 3). (F) Cell viability of BMDMs was determined by the MTT assay after the cells were treated with loganin at the indicated concentrations for 7 h. The values in the bar graphs represent the means ± SEM (n = 3); # significantly different from vehicle alone, p < 0.05; * significantly different from MSU alone, p < 0.05; ND, not detected.

Figure 2

Oral administration of loganin inhibits MSU crystals-induced NLRP3 inflammasome activation and inflammation in a mouse air pouch model. Air pouches were formed on the mouse back by injecting air twice. Loganin (5 or 30 mg/kg) or colchicine (1 mg/kg) was orally administered. After 1 h, MSU crystals (3 mg/mL in PBS/mouse) or PBS alone were injected into the air pouches. After 6 h, the pouch exudates were harvested and the supernatants were analyzed. (A) Experimental scheme. (B) ELISA for IL-1β. (C) Immunoblotting for IL-1β. (D) ELISA for IL-18. (E) Myeloperoxidase (MPO) activity, which reflects neutrophil recruitment, was assessed in the air pouch exudates. The values in the bar graphs represent the means ± SEM (n = 5 mice); # significantly different from vehicle alone, p < 0.05; * significantly different from MSU alone, p < 0.05.

Figure 3

Oral administration of loganin alleviates MSU crystals-induced inflammatory symptoms and NLRP3 inflammasome activation in a mouse acute gout model. Mice were orally administered loganin (5 or 30 mg/kg) or colchicine (1 mg/kg). After 1 h, MSU crystals (2 mg/0.1 mL of PBS/mouse) or PBS alone were subcutaneously injected into the hind footpad of each mouse. After 24 h, the foot tissues were collected for analysis. (A) Time course of foot thickness changes. (B) MPO activity in the foot tissues was analyzed for neutrophil infiltration. (C) Immunoblotting of foot tissues for pro-caspase-1, caspase-1 (p20), and IL-1β expression. (D,E) ELISA for IL-1β and IL-18 expression in foot tissues. The values in (A,B,D,E) represent the means ± SEM (n = 5 mice); # significantly different from vehicle alone, p < 0.05; * significantly different from MSU alone, p < 0.05.

Figure 4

Oral administration of loganin suppresses mitochondrial DNA synthesis in gout tissues. The samples were obtained from the mice described in Figure 2 and Figure 3. The levels of mitochondrial DNA such as D-loop and non-nuclear mitochondrial DNA (NUMT) in (A,B) air pouch tissues and (C,D) foot tissues were quantified by qPCR analysis. The levels of nuclear DNA were measured to normalize mitochondrial DNA levels. The values represent the means ± SEM (n = 3); # significantly different from vehicle alone, p < 0.05; * significantly different from MSU alone, p < 0.05.

Figure 5

Loganin prevents mitochondrial dysfunction induced by MSU crystals in primary mouse macrophages. After LPS priming, BMDMs were treated with loganin for 1 h. Then, the cells were further stimulated with MSU crystals (500 μg/mL) for (A) 6 h or (B–E) 2 h. (A) The levels of mitochondrial DNAs such as D-loop and non-NUMT were determined by qPCR analysis. (B,C) For the measurement of mitochondrial membrane potential, BMDMs were stained with JC-1 (2 μM). (B) Red/green fluorescence ratios were determined with a fluorescence plate reader or (C) fluorescent images were obtained by confocal microscopy (magnification ×400). DAPI was used for nuclei staining. (D,E) For the measurement of mitochondrial reactive oxygen species, BMDMs were loaded with MitoSOX-Red (5 μM). (D) Fluorescence was determined by a fluorescence plate reader, or (E) fluorescent images were obtained by confocal microscopy (magnification ×400). The values in (A,B,D) represent the means ± SEM (n= 3); # significantly different from vehicle alone, p < 0.05; * significantly different from MSU alone, p < 0.05.