Puerarin ameliorates acute lung injury by modulating NLRP3 inflammasome-induced pyroptosis

Abstract

We commenced to analyze putative anti-pyroptosis effects of puerarin (PU) as mediated by the PP2A-HDAC1-NLRP3 pathway in acute lung injury (ALI). ALI animal and cell models were constructed, followed by treatment of PU. Then, the effect of HDAC1, PP2A, and NLRP3 on cell inflammation and pyroptosis was explored. The interaction between HDAC1 and PP2A as well as between PP2A and NLRP3 was analyzed. Our findings suggested that PU downregulated HDAC1 expression to alleviate symptoms of ALI. HDAC1 overexpression promoted inflammation induced by LPS, which reversed the inhibitory effect of PU on ALI. HDAC1 overexpression also decreased PP2A expression, suggesting that PP2A was involved in the effects of HDAC1 on LPS-induced inflammation. PP2A exerted inhibitory effects on NLRP3. Meanwhile, PU hindered the progression of ALI by silencing HDAC1 or overexpressing PP2A both in vivo and in vitro. Taken together, PU restrained pyroptosis of cells induced by NLRP3 inflammasome to abate ALI.

Fig. 1. HDAC1 is expressed at a low level in lung tissues of PU-treated ALI mice.

A Pathological changes of lung tissues in animal model, detected by HE staining (×200). B Total number of cells and neutrophils in BALF of animal model. C TNF-α, IL-1β, INF-γ and IL-6 levels in the BALF of animal model detected by ELISA. D HDAC1 expression in lung tissues of animal model detected by western blot analysis. *p < 0.05. n = 10.

Fig. 2. PU exerts inhibitory effect on LPS-induced inflammation by HDAC1.

A HDAC1 protein expression in RAW 264.7 cells. B The level of inflammatory factors (TNF-α, IL-1β, INF-γ and IL-6) in RAW 264.7 cell supernatant assayed by ELISA. C The expression of HDAC1 in RAW 264.7 cells after transduction assayed by western blot analysis. D The level of lung inflammatory factors (TNF-α, IL-1β, INF-γ, and IL-6) in the transduced RAW 264.7 cell supernatant. *p < 0.05.

Fig. 3. HDAC1 promotes LPS-induced cell inflammation through PP2A.

A PP2A protein expression in lung tissues of animal models assayed by western blot analysis (n = 10). B The expression of HDAC1 and PP2A in RAW 264.7 cell models, checked by western blot analysis. C The interaction between HDAC1 and IKZF1 in 293 T cells, detected by Co-IP. D The interaction between HDAC1 and IKZF1 in RAW 264.7 cells detected by Co-IP. E The enrichment of HDAC1 in the PP2A HDAC1 bound to the promoters region, verified by ChIP. F The effect of HDAC1 on the expression of HDAC1 and PP2A in RAW 264.7 cells assessed by western blot analysis. G The effect of PP2A overexpression in RAW 264.7 cells checked by RT-qPCR. H The levels of lung inflammatory factors (TNF-α, IL-1β, INF-γ and IL-6) in RAW 264.7 cell supernatant after transduction evaluated by ELISA. *p < 0.05.

Fig. 4. The inhibitory effect of overexpressed PP2A on LPS-induced cellular inflammation and pyroptosis.

A The expression of NLRP3 inflammasome-related indicators in lung tissues of animal models (n = 10), detected by western blot analysis. B The expression of NLRP3 inflammasome-related indicators in cell models, detected by western blot analysis. C The expression of PP2A and NLRP3 inflammasome-related indicators after overexpression of PP2A, detected by western blot analysis. D The levels of lung inflammatory factors (TNF-α, IL-1β, INF-γ and IL-6) after overexpression of PP2A, detected by ELISA. E The formation of pyrosomes, detected by immunofluorescence (× 400). *p < 0.05.

Fig. 5. Overexpressed HDAC1 or silencing PP2A suppresses the protective effect of PU on ALI in mice.

A The expression of HDAC1 in lung tissues of ALI mice after PU, oe-HDAC1 or sh-PP2A treatment, assayed by RT-qPCR. B The expression of PP2A in lung tissues of ALI mice after PU, oe-HDAC1 or sh-PP2A treatment, assayed by RT-qPCR. C The pathological changes in animal models and lung tissues after transfection (× 200), assessed by HE staining. D The total number of cells and the number of neutrophils in BALF in animal models after transfection. E The expression of PP2A and NLRP3 inflammasome-related indicators in lung tissues of animal models after transfection, assayed by Western blot analysis. F The levels of inflammatory factors (TNF-α, IL-1β, INF-γ and IL-6) in BALF of animal models after transfection, assayed by ELISA. * p < 0.05. n = 10.

Fig. 6. Schematic map of role of Puerarin in ALI.

Puerarin ameliorated ALI by modulating NLRP3 inflammasome-induced pyroptosis.