Hua-Shi-Bai-Du decoction inactivates NLRP3 inflammasome through inhibiting PDE4B in macrophages and ameliorates mouse acute lung injury.

BACKGROUND

Hua-Shi-Bai-Du decoction (HSBD) exerts significant effects on the prevention and treatment of COVID-19 in China. The activation of the NLRP3 inflammasome of macrophages plays a vital role in COVID-19 pathology. However, no previous studies have focused on this pathological process to explore the effect of HSBD.

PURPOSE

Our aim is to uncover the effect of HSBD on NLRP3 inflammasome activation and the underlying mechanisms.

METHODS

The NLRP3-activated J774A.1 cells primed by LPS and activated by nigericin/ATP/MSU were used to evaluate NLRP3 activation in vitro. ASC oligomerization and speck formation were assessed by western blot and immunofluorescence imaging. Intracellular K levels were determined by the colorimetric assay. Mitochondrial ROS (mtROS) level was detected by the flow cytometry and the fluorescence spectrophotometry. The intracellular cAMP level was determined by chemiluminescence method and ELISA, while phosphodiesterase (PDE) activity was measured using the fluorescent substrate MANT-cAMP. siRNA was applied to knockdown PDE4B. Two in vivo mouse models, MSU-induced peritonitis and LPS-induced acute lung injury (ALI), were used to evaluate the effects of HSBD on IL-1β and other inflammatory cytokines. Pathological changes in lung tissue were observed by histopathological examination.

RESULTS

HSBD not only decreased supernatant IL-1β, caspase-1 p20, and cleaved gasdermin D (GSDMD) in NLRP3-activated J774A.1 cells, but also reduced IL-1β in the peritoneal lavage fluid of mice with MSU-induced peritonitis, demonstrating the suppressive effect on NLRP3 inflammasome activation. The mechanism study showed that HSBD blocked ASC oligomerization and speck formation without affecting K efflux or mtROS production. Furthermore, it prevented the decrease of intracellular cAMP by inhibiting PDE4B activity. And in the PDE4B-deficient cells, its suppressive effect on IL-1β release was abolished. In LPS-induced ALI mice, oral administration of HSBD decreased several proinflammatory cytokines (IL-1β, IL-6, TNF-α, and CXCL-1) and attenuated the pathological damage to the lung.

CONCLUSION

HSBD suppresses the activation of NLRP3 inflammasome by inhibiting PDE4B activity to counteract the decrease of intracellular cAMP, thereby blocking ASC oligomerization in macrophages. Our findings may provide new insight into the clinical effets of HSBD for the treatment of COVID-19.