# Sulfated Pelvetia siliquosa Polysaccharides Attenuate Pyroptosis via NF-κB Pathway Inhibition Against Calcium Oxalate Stone Formation

## TL;DR

This study shows that sulfated polysaccharides from Pelvetia siliquosa reduce kidney stone formation by inhibiting cell death and inflammation.

## Contribution

The novel finding is that sulfated Pelvetia siliquosa polysaccharides inhibit pyroptosis and NF-κB pathways to prevent calcium oxalate stone formation.

## Key findings

- Sulfated polysaccharides reduced pyroptosis in HK-2 cells by up to 6.35%.
- PSP3 showed higher protection efficiency than PSP0 due to increased sulfation.
- Polysaccharides inhibited NLRP3/ASC/caspase-1/IL-1β pathways and reduced crystal adhesion.

## Abstract

Objective: The formation of calcium oxalate (CaOx) kidney stones is accompanied by the pyroptosis of renal epithelial cells. The risk of kidney stone formation can possibly be reduced through pyroptosis inhibition. Methods: Pyroptosis of HK-2 cells induced by 3 µm CaOx monohydrate (COM-3 µm) was inhibited by Pelvetia siliquosa polysaccharides before and after sulfation (PSP0 and PSP3, with −OSO3− contents of 1.04% and 36.12%, respectively). The inhibitory efficiency and mechanism of PSP0 and PSP3 were evaluated via caspase-1/PI double staining and Western blot detection of pathway proteins in pyroptosis cells. The potential anti-stone effect of polysaccharides was evaluated through measurement of the extent of crystal adhesion on the cell surface. Results: The proportion of pyroptosis cells induced by COM-3 µm reached 17.87%. After protection by PSP0 and PSP3, the percentage of pyroptosis cells was reduced to 12.7% and 6.35%. The levels of NLRP3, ASC, gasdermin D, IL-1β, and IL-18 related to pyroptosis were downregulated. In addition, the activation of the NF-κB pathway was considerably inhibited. During inhibition of pyroptosis, reactive oxygen species and lactate dehydrogenase levels were decreased, the integrity of zonula occludens-1 protein was restored, and the expressions of CaOx-specific adhesion proteins (ANXA3 and CD44) were substantially decreased. As a result, the adhesion of COM crystals on the cell surface was reduced. PSP3 exhibited a higher protection energy efficiency than PSP0. Conclusions: PSP0 and PSP3 inhibited the pyroptosis of HK-2 cells through the NLRP3/ASC/caspase-1/IL-1β pathway, which caused the inhibition of cell inflammation and injury, reduced the expressions of adhesion proteins, and reduced the risk of CaOx crystal adhesion and stone formation. The biological activity of PSP0 and PSP3 after sulfation modification increased.