Emodin relieves the inflammation and pyroptosis of lipopolysaccharide-treated 1321N1 cells by regulating methyltransferase-like 3 -mediated NLR family pyrin domain containing 3 expression

Abstract

Sepsis brain injury (SBI) is a major cause of death in critically ill patients. The present study aimed to investigate the role of emodin in SBI development. Human astrocyte 1321N1 cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) to establish an SBI model in vitro. Flow cytometry was performed to measure the cell pyroptosis. The protein expression levels of syndecan-1 (SDC-1), NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and the N-terminal fragment of gasdermin D (GSDMD-N) were measured using Western blotting. Interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α levels in cells were measured using enzyme-linked immunosorbent assay kits. The N⁶-methyladenosine (m⁶A) modification was analyzed using the methylated RNA immunoprecipitation assay. NLRP3 activator, nigericin, was used to overexpress NLRP3. LPS treatment significantly enhanced the pyroptosis in 1321N1 cells, increased the levels of TNF-α, IL-1β, and IL-6, and decreased the levels of IL-10. The protein expression levels of NLRP3, SDC-1, GSDMD-N, and Caspase-1 were also increased. Emodin treatment decreased the levels of TNF-α, IL-1β, IL-6, NLRP3, SDC-1, GSDMD-N, and Caspase-1, while increasing the levels of IL-10 in LPS-treated 1321N1 cells. Nigericin reversed the effects of emodin. Furthermore, emodin upregulated m⁶A levels in NLRP3 by increasing the expression of methyltransferase-like 3 (METTL3). Meanwhile, knockdown of METTL3 reversed the effects of emodin on the mRNA expression and stability of NLRP3. Therefore, emodin inhibits the inflammation and pyroptosis of LPS-treated 1321N1 cells by inactivating METTL3-mediated NLRP3 expression.

Keywords: NLRP3; Sepsis; emodin; inflammation; pyroptosis.

Graphical abstract

Figure 1.

Lipopolysaccharides (LPSs) promoted the pyroptosis of 1321N1 cells. (a) Dead 1321N1 cells treated with LPS (1, 10, and 100 ng/mL) were analyzed using propidium iodide (PI) staining. **P < 0.01. (b) Protein expression levels of NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), pro-caspase-1, caspase-1-p20, gasdermin D (GSDMD), and N-terminal fragment of gasdermin D (GSDMD-N) in LPS (100 ng/mL)-treated 1321N1 cells were measured using Western blotting. **P < 0.01.

Figure 2.

Emodin inhibited the pyroptosis of LPS-treated 1321N1 cells. (a) After 20 μM emodin and 100 ng/ml LPS treatment, the dead 1321N1 cells were analyzed using PI staining. **P < 0.01, ***P < 0.001. (b) After 20 μM emodin and 100 ng/ml LPS treatment, the protein levels of NLRP3, caspase-1-p20, and GSDMD-N in 1321N1 cells were determined using Western blotting. *P < 0.05, **P < 0.01.

Figure 3.

Emodin relieved the inflammation in LPS-treated 1321N1 cells. (a-d) After 20 μM emodin treatment, interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α levels in LPS (100 ng/mL)-treated 1321N1 cells were analyzed. *P < 0.05, **P < 0.01. (e-f) After 20 μM emodin treatment, SDC-1 protein expression levels in LPS (100 ng/mL)-treated 1321N1 cells were determined using Western blotting. *P < 0.05, **P < 0.01.

Figure 4.

Nigericin reversed the effects of emodin on the pyroptosis of LPS-treated 1321N1 cells. (a) After 20 μM emodin and 20 μM nigericin treatment, dead LPS (100 ng/mL)-treated 1321N1 cells were analyzed using PI staining. **P < 0.01, ***P < 0.001. (b) After 20 μM emodin and 20 μM nigericin treatment, the protein levels of NLRP3, caspase-1-p20, and GSDMD-N in LPS (100 ng/mL)-treated 1321N1 cells were determined using Western blotting. *P < 0.05, **P < 0.01. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Nigericin reversed the effects of emodin on the cytokines levels and SDC-1 protein levels in LPS-treated 1321N1 cells. (a-d) After 20 μM emodin and 20 μM nigericin treatment, IL-1β, TNF-α, IL-6, and IL-10 levels in LPS (100 ng/mL)-treated 1321N1 cells were analyzed. *P < 0.05, **P < 0.01, ***P < 0.001. (e-f) After 20 μM emodin and 20 μM nigericin treatment, SDC-1 protein expression levels in LPS (100 ng/mL)-treated 1321N1 cells were determined by Western blotting. **P < 0.01, ***P < 0.001.

Figure 6.

Emodin downregulated NLRP3 expression by increasing the N⁶-methyladenosine (m⁶A) RNA methylation regulator, methyltransferase-like 3 (METTL3), in LPS-treated 1321N1 cells. (a) After treatment with 20 μM emodin, m⁶A levels in LPS (100 ng/mL)-treated 1321N1 cells were detected. ***P < 0.001. (b) After 20 μM emodin treatment, mRNA expression levels of the fat mass and obesity-associated protein (FTO), METTL3, and METTL14 in LPS (100 ng/mL)-treated 1321N1 cells were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). **P < 0.01. (c) After METTL3 and sh-METTL3 transfection, NLRP3 m⁶A methylation levels were determined. **P < 0.01. (d) After 20 μM emodin and sh-METTL3 treatment, NLRP3 m⁶A methylation levels in LPS (100 ng/mL)-treated 1321N1 cells were measured. **P < 0.01, ***P < 0.001. (e) After 20 μM emodin and sh-METTL3 treatment, the mRNA stability of NLRP3 in LPS (100 ng/mL)-treated 1321N1 cells was analyzed using RT-qPCR. *P < 0.05, **P < 0.01. (f) After 20 μM emodin and sh-METTL3 treatment, mRNA expression levels of NLRP3 in LPS (100 ng/mL)-treated 1321N1 cells were measured using RT-qPCR. **P < 0.01.