Separation and Differential Characterization of Gut Microbial Extracellular Vesicles in Salt-Sensitive Rats under High-Salt Diet Conditions

Summary

The protocol describes the isolation of gut microbial EVs from salt-sensitive rats fed HSD using density gradient centrifugation. EVs were characterized by nanoparticle tracking, TEM, LPS/BCA assays, and 16S rRNA sequencing to analyze the size, morphology, composition, and microbiota origin.

Abstract

High salt intake is a major risk factor for hypertension, and its underlying mechanism may be closely linked to extracellular vesicles (EVs) secreted by gut microbiota. These EVs, produced by gut microbiota, carry various bioactive components that may play a crucial role in the development of hypertension induced by a high-salt diet (HSD). To investigate this mechanism, we developed an efficient extraction method based on density gradient centrifugation to isolate EVs from the gut microbiota of salt-sensitive rats fed an HSD. Through particle size analysis, transmission electron microscopy (TEM), and lipopolysaccharide (LPS) detection, we identified the gradient distribution of gut microbiota EVs and achieved precise extraction. Furthermore, 16S rRNA gene sequencing was employed to analyze the origin and compositional differences of EVs between the normal and HSD groups, revealing the impact of high salt intake on the genetic characteristics of gut microbiota EVs. This study provides valuable tools and scientific insights into the gut microbiota mechanisms underlying salt-induced hypertension and offers new perspectives for the prevention and treatment of related diseases.

Introduction

The gut microbiota, also known as gut microbiota microflora or gut microecology, is a complex of tens of thousands of microorganisms located in the biological gastrointestinal tract and plays a crucial role in the maintenance of human health¹. In recent years, with further research, it has been found that the gut microbiota can produce extracellular vesicles (EVs)². EVs are small vesicles released by cells, which carry various molecules in the cell, such as proteins, nucleic acids, and lipids^3,4. They can interact with other microbes⁵, int....

Protocol

This animal experimental study complies with the relevant ethical guidelines and international standards. Studies involving animals were approved by the Laboratory Animal Welfare and Ethics Committee of Chengdu University of Chinese Medicine (institution: Chengdu University of Chinese Medicine; protocol number: 2018-21).

1. Animal preparation and diet regime

1. Acclimatize 6-week-old male Dahl salt-sensitive rats, body weight 180-200 g, by rearing at 50% ± 10% humidity; 12 h / 12 h light cycle; 20.0 ± 2.0 °C for 1 week under specific pathogen-free conditions.
2. Subsequently, divide the rats e....

Results

EVs concentrations were determined in different fractions (Figure 2A). The experimental results showed that the concentration of the EVs exhibited a typical normal distribution pattern in a series of density gradient solutions (Figure 2B). Specifically, in fraction 9, the concentration of EVs reached its highest point (3.85 x 10⁹), suggesting that the main distribution fraction of EVs may be 9³⁶.

For.......

Discussion

In this study, we focused on the gut microbiota EVs in salt-sensitive rats on an HSD and achieved a series of key achievements. First, an efficient extraction method based on density gradient centrifugation was successfully constructed to isolate EVs from salt-sensitive rat gut microbiota on HSD, and most non-EV components were isolated through a meticulous, standardized animal experimental manipulation and sample processing process. The EVs extraction method of density gradient centrifugation is highly efficient and rep.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Essential Supplies
Centrifugal Filter (10nkDa 2 mL)	Millipore	UFC903096
Centrifuge Tude(50 mL)	BKMAN	20220404
Centrifuge Tudes	BECKMAN COULTER	Z30815SCA
Vacuum Filtration System	Biosharp	24902581
Reagents
Chromogenic LAL Endotoxin Assay Kit	Beyotime	022124240705
Coomassie Blue Fast Staining Solution	Beyotime	Z972241010
EDTA	Damas-beta	P3117308
Enhanced BCA Protein Assay Kit	Beyotime	A006241112
Ethanol	KESH
HCl
OptiPrep (60% wt/vol, iodixanol)	Serumwerk	00124
PBS	Labshark	130114005
phosphotungstic acid	RUIXIN
Sucrose	Damas-beta	P1917057
Tris (VWR)	Damas-beta	P3061764
Trypan blue staining solution (0.4%)	Beyotime	BD07242904
Equipment
Absorbance Microplate Reader	SpectraMax	ABP01690
Biomicroscope	Motic	BA210Digital
Desk centrifuge	Cence	CHT210R
Desktop high-speed micro centrifuge	DLAB	D3024
Fixed Angle Aluminum Rotor + 500 mL Centrifugal Cup	Cence
High precision electronic balance	SKR	BN-200
Laminar flow cabinet	Nantong Hunan Scientific Instrument Co., Ltd.	SW-CJ-2FDS
SW 32.1 Ti Swing bucket turn+ SW 32.1 Ti Rotor bucket	BECKMAN COULTER
Transmission electron microscope	JEOL	JEM-1400FLASH
Tube rotator
Ultracentrifuge	BECKMAN COULTER	Optima XE-100
Ultra-pure water system	ULPHW	UPR-II-15TNZ
Water-Cieculation Multifunction Vacuum Pump	Qiang Qiang	SHZ-D(III)