# A modified hot phenol-based protocol for high-purity Escherichia coli lipopolysaccharide extraction: Biochemical validation, stem cell cytotoxicity, and dose dependent multi-organ inflammation in a rat model

## TL;DR

This paper introduces a new method to extract pure LPS from E. coli, which causes inflammation in rat organs and harms stem cells.

## Contribution

The study introduces a modified hot phenol protocol with enzymatic treatment for high-purity LPS extraction and reports its cytotoxic effects on stem cells.

## Key findings

- The modified hot phenol method with enzymatic treatment yields high-purity LPS.
- Extracted LPS shows cytotoxic effects on mesenchymal and embryonic neural stem cells.
- LPS injection in rats causes dose-dependent multi-organ inflammation and tissue damage.

## Abstract

•A modified hot-phenol protocol with enzymatic treatment for high-purity Escherichia coli LPS extraction.•First report linking this extraction method to cytotoxicity in both mesenchymal and embryonic neural stem cells.•Validation of extracted LPS in a rat model shows dose-dependent multi-organ inflammation.•Provides a fully validated, reliable LPS product for inflammation research from bench to bedside.

A modified hot-phenol protocol with enzymatic treatment for high-purity Escherichia coli LPS extraction.

First report linking this extraction method to cytotoxicity in both mesenchymal and embryonic neural stem cells.

Validation of extracted LPS in a rat model shows dose-dependent multi-organ inflammation.

Provides a fully validated, reliable LPS product for inflammation research from bench to bedside.

Gram-negative bacteria contain lipopolysaccharides (LPS) in their outer membrane, which induce strong inflammatory responses. Traditional LPS extraction methods often leave residual protein and nucleic acid contamination. These impurities interfere with downstream analyses and reduce reproducibility in biological studies. This study provides a modified hot phenol method combined with enzymatic treatments using proteinase K, DNase, RNase to extract pure LPS from Escherichia coli. Purity was confirmed by gel electrophoresis using Coomassie blue and silver nitrate staining. The biological activity of isolated LPS was tested on mesenchymal and embryonic neural stem cells, demonstrating decreased viability. In Wistar rats, LPS injection elevated serum IL-6 but not TNFα or IL-1β. Histological examinations indicated liver, kidney, brain, and colon tissue damage post-injection. Our results show that the modified hot phenol method efficiently produces high-purity, biologically active LPS suitable for both in vitro and in vivo inflammation studies, supporting research into inflammatory processes and associated diseases.