# Rhein Inhibits NLRP3 Activation and Alleviates Microglial Pyroptosis After Intracerebral Hemorrhage in Rats

## TL;DR

Rhein, a natural compound, reduces brain inflammation and injury after intracerebral hemorrhage by inhibiting microglial pyroptosis and NLRP3 activation in rats.

## Contribution

This study reveals rhein's novel protective effects against ICH by targeting NLRP3-mediated microglial pyroptosis.

## Key findings

- Rhein inhibits NLRP3 activation and reduces microglial pyroptosis in a rat model of ICH.
- Rhein promotes M2 microglial polarization and reduces pro-inflammatory cytokine expression.
- NLRP3 knockdown enhances rhein's protective effects, suggesting a key role in ICH pathology.

## Abstract

Intracerebral hemorrhage (ICH) induces severe neuroinflammation and microglial pyroptosis, exacerbating secondary brain injury. Rhein, a natural anthraquinone compound, possesses anti‐inflammatory and neuroprotective properties. However, its effects on microglial pyroptosis and the underlying mechanisms remain unclear.

A rat microglial (RM) pyroptosis model was established using LPS + ATP induction, followed by rhein intervention and NLRP3 knockdown. Cell proliferation was assessed using CCK‐8, and apoptosis was evaluated through TUNEL staining. ELISA was used to measure the expression levels of inflammatory cytokine. Immunofluorescence staining was performed to label M1/M2 microglia. RT‐qPCR and western blot were used to analyze the expression of NLRP3, ASC, Caspase‐1, GSDMD, PCNA, Cyclin D1, and CDK2. Transmission electron microscopy (TEM) was used to observe pyroptotic bodies. Additionally, a rat ICH model was established, with rhein intervention and NLRP3 knockdown/Caspase‐1 inhibition. Behavioral assessments were conducted using the Y‐maze test and open‐field test. HE staining was performed to examine brain tissue pathology. ELISA was used to measure inflammatory cytokine levels in brain tissue. Immunofluorescence staining analyzed the distribution of M1/M2 microglia. RT‐qPCR and western blot were used to detect pyroptosis‐related proteins, and TEM was used to evaluate pyroptotic body formation.

At the cellular level, rhein significantly promoted microglial proliferation and M2 polarization while inhibiting pyroptosis, inflammatory cytokine expression, M1 polarization, and NLRP3 expression. NLRP3 knockdown further enhanced the protective effects of rhein. At the animal level, ICH model rats exhibited reduced exploratory behavior, exacerbated neuroinflammation, increased pro‐inflammatory cytokine expression, increased M1 microglia, and elevated NLRP3 expression and pyroptosis levels. Rhein intervention significantly alleviated inflammation in ICH rats by reducing the expression of NLRP3 and pyroptosis‐related proteins. NLRP3 knockdown or Caspase‐1 inhibition further enhanced rhein's protective effects.

Rhein alleviates neurological dysfunction following ICH by inhibiting NLRP3 inflammasome activation, reducing microglial pyroptosis, and mitigating neuroinflammation.

Rhein treatment inhibits NLRP3 inflammasome activation, suppresses Caspase‐1/GSDMD‐mediated pyroptosis, reduces pro‐inflammatory cytokine release, promotes M2 polarization, and improves behavioral outcomes of ICH. These findings suggest that rhein protects against ICH‐induced brain injury through the inhibition of NLRP3‐dependent pyroptosis, highlighting its translational potential as a therapeutic candidate for hemorrhagic stroke.