Hydroxysafflor Yellow A Attenuates LPS-Induced Toxicity in Dopaminergic Neurons of Zebrafish by Regulating the TLR4/NF-ĸB Signaling Pathway

Summary

This protocol aims to test whether hydroxysafflor yellow A (HSYA) attenuates LPS-induced neurotoxicity in zebrafish. Motor function was evaluated using a Visual Vision system. Dopaminergic nerve injury was examined by immunohistochemistry. Molecules involved in the TLR4/NF-κB pathway were analyzed using Western blot.

Abstract

Lipopolysaccharide (LPS)-induced inflammation plays a crucial role in triggering and perpetuating the neurodegenerative diseases, including Parkinson's disease (PD). Hydroxysafflor yellow A (HSYA) is the main component of Carthamus tinctorius L.. This study aims to observe whether HSYA could ameliorate LPS-induced toxicity in dopaminergic neurons of zebrafish. Zebrafish were exposed to LPS and then treated with HSYA. Tests of behavior were conducted to evaluate the motor function of zebrafish. Dopaminergic neuronal injury and dopamine level were examined. TLR4, NF-κB, IL-1β, and TNF-α were also measured. It demonstrated that LPS (60 µg/mL, final concentration) induced toxicity in zebrafish and caused motor dysfunction and damage to tyrosine hydroxylase (TH)-positive neurons. LPS exposure not only decreased the content of dopamine but also increased levels of IL-1β, TNF-α, and expression of TLR4 and NF-κB. HSYA ameliorated the LPS-induced motor dysfunction. In addition, HSYA attenuated the loss of TH-positive neurons, which was accompanied by an increase in dopamine content. HSYA treatment also inhibited the expression of TLR4, NF-κB, and production of IL-1β, TNF-α. In conclusion, this study demonstrates that HSYA attenuates inflammation-induced injury in dopaminergic neurons of zebrafish by regulating the TLR4/NF-κB pathway, which provides an experimental basis for the prevention of inflammatory injury in PD.

Introduction

The pathogenesis of Parkinson's disease (PD) is the deterioration of motor activity due to injury of dopaminergic neurons in the brain. The manifestations of PD include motor dysfunction, including resting tremor, bradykinesia, postural instability, and muscle tone. Inflammation is a prominent pathological hallmark of PD. Cytokine levels in cerebrospinal fluid and post-mortem nigrostriatal brain regions of individuals with PD are elevated compared with age-matched healthy controls¹. The mechanisms by which dopaminergic neuronal injury is caused by inflammation have been well established. In PD, midbrain dopaminergic neurons are sensitive to....

Protocol

The experiments were conducted in accordance with the National Institutes of Health Guidelines for the Use of Laboratory Animals (Publication 86-23, revised in 1986). And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20220623). The reagents and the equipment used in this study are listed in the Table of Materials.

1. Maintenance of zebrafish

AB strain zebrafish (Danio rerio) were kept in a recirculating water system at a water temperature of 27-28.5 °C (pH 7.0), on a 14/10 h light/dark cycle. Zebrafish were fed twice a day with shrimp. ....

Results

Effects of LPS on the motor function of zebrafish
The mean speed, total distance, and maximum acceleration of zebrafish in the LPS (60 µg/mL and 90 µg/mL) groups were reduced (P < 0.01) compared to the control group. The motor dysfunction was also manifested as frequent changes in the direction of movement. The absolute turning angle of swimming of zebrafish in the LPS 60 µg/mL and LPS 90 µg/mL groups was significantly increased (P < 0.01) (

Discussion

Medicinal plants have a long track record of anti-inflammatory and neuroprotective applications¹⁴. Our previous studies showed that HSYA had a protective effect in neurotoxic agent-induced PD animal models. Using LPS-induced toxicity in zebrafish, this study demonstrates that HSYA attenuates the inflammation-induced injury in dopaminergic neurons of zebrafish by regulating the TLR4/NF-κB signaling pathway.

Not only are sleep disorders and cognitive dysfunction clos.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCA Protein Concentration Measurement Kit	Beyotime Biotechnology	P0012S
Chemiluminescent imaging system	Beijing Sage Venture Technology Co.	ChampChemi 910
Dopamine hydrochloride	Sigma–Aldrich	PHR1090
Electronic balance	Shanghai OHAUS Instrument Co.	AR-2140
ELISA kits for IL-1β	COIBO Biotechnology	CB10018-FI
ELISA kits for TNF-α	COIBO Biotechnology	CB10148-FI
GAPDH antibody	Cell Signaling Technology	5174S
GraphPad Prism 5.0	GraphPad statistical Software, Boston, MA, USA, www.graphpad.com	Prism 5.0
High-performance liquid chromatography–electrochemical detection (HPLC–ECD)	GL Sciences, Ibaraki, Japan	ED723
High-speed freezing centrifuge	Beijing Dalong Xingchuang Experimental Instrument Co.	D3024R
HPLC–ECD	GL Sciences	ED723
HSYA	Luye Pharma Group Ltd.	LY202206
Image Quant	GE Healthcare, Tokyo, Japan	LAS 4000
Lamin B1 antibody	Cell Signaling Technology	13435S
LPS	Sigma–Aldrich	L2630
Microscope	Leica, Wetzlar	DM1000LED
Microscope	Leica, Germany	DM1000LED
Multifunctional Enzyme Labeler	Shanghai Meigu Molecular Instrument Co.	SpectraMax Paradigm
NF-κB antibody	Cell Signaling Technology	8242S
Noldus Visual Vision XT system	Beijing Noldus Biotechnology Co., Ltd.	EthoVision XT15
Nuclear and Cytoplasmic Protein Extraction Kit	Beyotime Biotechnology	P0028
PMSF	Beyotime Biotechnology	ST506
TAK-242	Merck	614316
Thermostat	Shenzhen Galaxy Instrumentation Co.	12810129
TLR4 antibody	Abcam	ab13556
Zebrafish	Northern Center for National Zebrafish Model Animals	AB strain
Zebrafish recirculating water system	Beijing Aisheng Biotechnology Co., Ltd.	ESEN-AW-S1