Salvia miltiorrhiza-derived exosome-like nanoparticles improve diabetic cardiomyopathy by inhibiting NLRP3 inflammasome-mediated macrophage pyroptosis via targeting the NEDD4/SGK1 axis

Abstract

Aim: Exosome-like nanoparticles mediate intercellular communication and regulate gene expression. In this study, we isolated and purified exosome-like nanoparticles from Salvia miltiorrhiza (SM-ELNs), a traditional Chinese medicinal herb, and investigated their therapeutic effects on diabetic cardiomyopathy (DCM).

Materials & methods: To investigate the effect of SM-ELNs on DCM, we established a mouse model via HFD/STZ treatment. Cardiac function was assessed by echocardiography. Cardiac hypertrophy was assessed by measuring the heart weight/body weight ratio and HE staining, while myocardial fibrosis was evaluated using Masson's trichrome staining. The role of SM-ELNs on NLRP3 inflammasome inhibition and macrophage pyroptosis were evaluated both in vivo and in vitro. The interaction between NEDD4 and SGK1 was analyzed by Co-IP and ubiquitination assays.

Results: SM-ELNs treatment alleviated cardiac function and histopathological changes in DCM mice. Moreover, SM-ELNs suppressed NLRP3 inflammasome activation and subsequent macrophage pyroptosis in both in vivo and in vitro models. Mechanistically, NEDD4 facilitated the ubiquitination and degradation of SGK1 in macrophages. Both NEDD4 depletion and SGK1 addition could counteract the SM-ELNs-induced suppression of NLRP3 inflammasome-triggered macrophage pyroptosis in LPS/ATP-treated RAW264.7 cells.

Conclusion: Our study provides the first evidence that SM-ELNs inhibit NLRP3 inflammasome-mediated macrophage pyroptosis in DCM by modulating the NEDD4/SGK1 axis.

Keywords: NEDD4; NLRP3; SGK1; SM-ELNs; inflammasome; macrophage; pyroptosis.

Figure 1.

SM-ELNs ameliorates cardiac dysfunction in DCM mice (a) effect of SM-ELNs on the EF (left ventricular ejection fraction), FS (fractional shortening), LVPW.S (left ventricular posterior wall thickness at end systole), and LVPW.D (left ventricular posterior wall thickness at end diastolic) of DCM mice hearts. (b and c) HE staining and Masson staining of DCM mice heart tissues. The experiment was divided into the following groups: sham, DCM, DCM+SM-ELNs (10 mg/kg, 200 µL). (d) The mRNA level of NLRP3, IL-1β, IL-18, and GSDMD was determined by RT-qPCR. (e) The protein level of NLRP3, pro-IL-1β, IL-1β, pro-IL-18, IL-18, full-GSDMD, and GSDMD-N was measured by western blot assay. (f) Immunofluorescence staining of heart tissues for ASC or GSDMD (red) and macrophage marker F4/80 (green). The quantification analysis of F4/80 and ASC or GSDMD-positive cells in heart tissues. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

SM-ELNs inhibited NLRP3 inflammasome-induced macrophage pyroptosis in vitro (a) cytotoxicity was assessed by CCK-8 assay in RAW264.7 cells treated with SM-ELNs (0, 25, 50, 100, and 200 μg/mL). (b) RAW264.7 cells were treated with LPS (200 ng/mL) in the presence or absence of pretreatment of SM-ELNs (0, 25, 50, and 100 μg/mL) followed by CCK-8 assay of cell viability. The experiment was divided into the following groups: control, LPS/ATP, and LPS/ATP+SM-ELNs (100 μg/mL). (c) NLRP3, IL-1β, IL-18, and GSDMD mRNA levels were detected by RT-qPCR. (d) NLRP3, pro-IL-1β, IL-1β, pro-IL-18, IL-18, full-GSDMD, and GSDMD-N protein levels were determined by western blot assay. (E) Cell pyroptosis was assessed by flow cytometry assay. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

SM-ELNs restrained macrophage pyroptosis via regulating NEDD4 (a and b) NEDD4 mRNA and protein levels were measured by RT-qPCR and western blot assays in RAW264.7 cells treated with SM-ELNs (100 μg/mL). (c and d) RT-qPCR and western blot assays were used to measure NEDD4 mRNA and protein levels in RAW264.7 cells treated with LPS (200 ng/mL) in the presence or absence of pretreatment of SM-ELNs (100 μg/mL). (e and f) NEDD4 mRNA and protein levels were detected by RT-qPCR and western blot assays in RAW264.7 cells transfected with sh-NC or sh-NEDD4. The experiment was divided into the following groups: control, LPS/ATP, LPS/ATP+SM-ELNs, and LPS/ATP+SM-ELNs+sh-NEDD4. (g) NEDD4, NLRP3, IL-1β, IL-18, and GSDMD mRNA levels were detected by RT-qPCR. (H) NEDD4, NLRP3, pro-IL-1β, IL-1β, pro-IL-18, IL-18, full-GSDMD, and GSDMD-N protein levels were determined by western blot assay. (I) Cell pyroptosis was assessed by flow cytometry assay. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

NEDD4 facilitated the ubiquitination and degradation of SGK1 in macrophage (a) the BioGRID database was performed to predict the protein interaction network of NEDD4. (b) Ubibrowser database was utilized to predict SGK1-associated ubiquitinated E3 ligases. (c-f) NEDD4 and SGK1 mRNA and protein levels were detected by RT-qPCR and western blot assays in RAW264.7 cells transfected with oe-NC or oe-NEDD4. (g) Co-IP was used to verify the binding of NEDD4 and SGK1 in macrophages. (h) The protein stability of SGK1 was examined by western blot in the presence of CHX. (i) The effects of NEDD4 overexpression and protease inhibitor MG132 on the ubiquitination of SGK1 in RAW264.7 cells by western blot assay. (j) SGK1 protein ubiquitination was detected in macrophages transfected with oe-NC or oe-NEDD4. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.

SM-ELNs inhibited macrophage pyroptosis via regulating NEDD4/SGK1 axis (a and b) SGK1 mRNA and protein levels were detected by RT-qPCR and western blot assays in RAW264.7 cells transfected with oe-NC or oe-SGK1. The experiment was divided into the following groups: control, LPS/ATP, LPS/ATP+SM-ELNs, and LPS/ATP+SM-ELNs+oe-SGK1. (c) SGK1, NLRP3, IL-1β, IL-18, and GSDMD mRNA levels were detected by RT-qPCR. (d) SGK1, NLRP3, pro-IL-1β, IL-1β, pro-IL-18, IL-18, full-GSDMD, and GSDMD-N protein levels were determined by western blot assay. (e) Cell pyroptosis was assessed by flow cytometry assay. *p < 0.05, **p < 0.01, ***p < 0.001.