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 cytokines such as tumor necrosis factor (TNF)-ɑ². Lipopolysaccharide (LPS) is a component of bacteria. LPS is identified as a ligand of Toll-like receptor 4 (TLR-4). Once binding to TLR-4, LPS leads to an inflammatory response and shows toxicity to tissue. TLR-4 is located on microglia in the central nervous system. LPS activates TLR-4 and therefore produces TNF-α and IL-1β. Central or peripheral exposure to LPS will induce toxicity to the central nervous system. There is evidence demonstrating that LPS-induced toxicity plays a role in triggering and perpetuating the neurodegenerative diseases, including PD^3,4,5.

Recently, zebrafish have been commonly used as a model for studying nervous system diseases. Zebrafish and humans have 80% genetic homology. And zebrafish have almost the same number of chromosomes as humans. Furthermore, the main structure of the brain of the zebrafish and its signalling pathways are similar to those of mammals. Zebrafish can also produce large numbers of transparent embryos, develop rapidly, and are prone to genetic manipulation⁶. These advantages make zebrafish an excellent model for investigating the pathogenesis of PD and developing high-throughput drug screening in PD.

Hydroxysafflor yellow A (HSYA) is a main ingredient of Carthamus tinctorius L. Previous studies have shown that HSYA has anti-inflammatory properties. In primary mesencephalic cultures, HSYA at 20 µM to 640 µM did not show any toxicity. And HSYA concentration ranging from 40 µM to 640 µM reduced LPS-induced dopaminergic neuronal damage⁷. C57BL/6J mice were exposed to 6-hydroxydopamine to induce a model of PD. HSYA not only attenuates impaired neurological function but also protects dopaminergic neurons in the brain. Furthermore, HSYA reduced the expression of cyclooxygenase-2, inducible nitric oxide synthase, and NF-κB in the substantia nigra⁸.

To date, there are no reports demonstrating the property of HSYA to reduce the toxicity of LPS in vivo. It showed that HSYA attenuated the LPS-induced toxicity in vitro⁷. However, cell-based evaluation does not represent all steps of the complex process of neurotoxicity. Zebrafish prove particularly valuable for unravelling the mechanism of toxicity. In recent years, it has also been shown that HSYA is able to inhibit NF-κB inflammatory pathway activation by decreasing the expression of protein kinase B, thereby ameliorating the inflammatory response, and that HSYA also inhibits the activator protein 1-associated inflammatory signalling pathway, which affects cell proliferation and tumour necrosis factor α-induced inflammatory injury^9,10. This study aims to investigate whether HYSA can ameliorate LPS-induced toxicity in dopaminergic neurons of zebrafish.

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. After natural mating, fertilized embryos were collected in Petri dishes containing E3 medium with deionized water, KCl, NaCl, MgSO₄·7H₂O, and CaCl₂.

2. LPS-induced toxicity in zebrafish

Zebrafish embryos at 24 h post-fertilization (hpf) were harvested and assigned to control and LPS groups, with 60 embryos in each group. The LPS powder was weighed and added to a certain amount of PBS (pH = 7.4) solution for dissolution, prepared as a 2 mg/mL LPS stock solution, placed in -20 °C for storage and standby. During the experiment, the LPS reserve solution was diluted to 30 µg/mL, 60 µg/mL, and 90 µg/mL of LPS solution. LPS solution at a final concentration of 30 µg/mL, 60 µg/mL, or 90 µg/mL was added to Petri dishes containing 24 hpf zebrafish embryos; the volume of solution for each experimental well was 3 mL. In the control group, zebrafish embryos were cultured in E3 medium without LPS. Petri dishes were placed in a constant temperature incubation chamber at 28 °C. Exposure to LPS was repeated following the same procedure mentioned above for five consecutive days.

3. Effect of TLR4 inhibitor on LPS-induced toxicity in zebrafish

Zebrafish embryos at 24 hpf were assigned to control, LPS, and TAK-242 groups. A number of studies have been conducted to validate the regulatory effect of TAK-242 at a final concentration of 1 µM on the regulation of the TLR4 signalling pathway, among which some studies have shown that TAK-242, by inhibiting LPS-induced cell proliferation and the release of inflammatory factors, provides a new way of solving the problems related to the reduction of glaucoma in the postoperative period¹¹. In the TAK-242 group, TAK-242 (1 µM, final concentration) was added to a Petri dish containing 24 hpf zebrafish embryos; the volume of solution for each experimental well was 3 mL. After 30 min, LPS (60 µg/mL, final concentration) was added to induce toxicity, and LPS concentrations did not cause zebrafish mortality. Embryos in the control group were maintained in E3 medium only. Petri dishes were placed in a constant temperature incubation chamber at 28 °C. The above procedure was repeated for five consecutive days.

4. Effect of HSYA on LPS-induced toxicity in zebrafish

Zebrafish embryos at 24 hpf were assigned to control, LPS, and HSYA groups. HSYA at a final concentration of 20 µM, 40 µM, or 80 µM was added to Petri dishes containing 24 hpf zebrafish embryos; the volume of solution for each experimental well was 3 mL. After 30 min, LPS (60 µg/mL, final concentration) was added. Embryos in the control group were maintained in E3 medium only. Petri dishes were placed in a constant temperature incubation chamber at 28 °C. The above procedure was repeated for five consecutive days.

5. Behavioral evaluation

Zebrafish were transferred to the middle 60 wells of a 96-well plate, with one zebrafish larva per well. E3 culture medium (250 µL) was added to each well to allow free swimming. Locomotor activity was recorded using a vision system. Pure water was added to the dark box (30 cm × 40 cm × 60 cm), which was set to a heating state with the temperature adjusted to 28 °C.

Once the dark box temperature reached 28 °C, the 96-well plate containing zebrafish was placed inside, and the Visual Vision system was activated to record locomotor activity. A new folder was created to store the data. Homemade Video File was selected as the video source to enter the experimental settings interface. In the Experiment Settings section, the number of observation zones was set to 60, allowing simultaneous observation of 60 zebrafish. In Arena Settings, the image of the 96-well plate captured by the lens appeared on screen, along with positioning circles. The modules in the Arrange Arenas interface were adjusted so that the lens capture area aligned with each well. In Arena Hardware Mapping, the dark box light was set to Light on.

In Trial Control Settings, a new program was created with a 10 min preparation period and a 10 min detection period, which was then applied to all parallel experiments. Detection Settings was used to confirm that the lens capture function was operating normally. In Trial List, data from 60 zebrafish in each group were displayed. The number of trials in the toolbar was used to generate a data table containing all experimental groups, ensuring data completeness.

All settings were confirmed, and the dark box temperature was maintained at 28 °C. After verifying that each well contained one zebrafish with vital signs, the experiment was initiated. During the experiment, the behavioral instrument's lens recorded the movement trajectory, duration, and related parameters. After 20 min of the first recording, the system automatically ended and prepared for the next trial. The dark box was opened, and the 96-well plate was replaced with a new one containing zebrafish for the subsequent experiment.

At the end of the experiment, Results was selected from the Analysis module. Experimental parameters such as average speed, distance, maximum acceleration, and absolute steering angle were extracted from the results module and exported. The data were then compiled for analysis of behavioral differences among zebrafish groups¹².

6. Measurement of pro-inflammatory cytokines

Zebrafish were collected. Specimens were weighed and PBS (0.01 M, pH = 7.4) was added at a gravimetric ratio of 1:9 and homogenised (5000 r/min, 30 s) at 4 °C. Homogenates were centrifuged at 4 °C, 5000 x g, 20 min, and supernatants were harvested. IL-1β and TNF-α were assayed with fish ELISA kits according to the instructions provided by the manufacturer.

7. Immunohistochemistry

Zebrafish were fixed with 5 mL 4% (v/v) paraformaldehyde. Embedded in paraffin and cut 5 µm-thick sections. Sections were incubated with citrate buffer (200 µL) in the microwave for 10 min. They were incubated in 0.3% hydrogen peroxide and methanol solution for 30 min to block endogenous peroxidases. Washed with PBS 3 times, 5 min each time. Non-immune serum was incubated for 30 min at 37 ºC. Primary anti-TH antibody (1:100) was incubated for 30 min at 37 ºC. Washed 3 times with PBS. Incubated with secondary antibody linked with horseradish peroxidase (1:200) at 37 ºC for 30 min. Then, 3,3´-diaminodbenzidine was added. Sections were imaged using a brightfield microscope.

8. Neurotransmitters

HPLC-ECD was employed to assay the dopamine level. Dopamine hydrochloride was dissolved in 0.1 M perchloric acid (HClO₄) to obtain a concentration gradient solution at 3.90625 ng/mL, 7.8125 ng/mL, 15.625 ng/mL, 31.25 ng/mL, 62.5 ng/mL, and 125 ng/mL. A series of dopamine standard solutions was analyzed by HPLC-ECD. Linear regression was performed using dopamine concentration and dopamine peak area. A standard curve was plotted. Zebrafish tissues were homogenized with 0.1 M HClO₄¹³. Homogenates were centrifuged at 4 °C, 20,000 x g, 15 min. Next, samples were separated on an ODS2 column (E2118047; 4.6 × 250 mm, 5 µm). Buffer was prepared: 25 mmol/L sodium acetate, 25 mmol/L citric acid, 0.01 mmol/L ethylenediaminetetraacetic acid disodium salt, 1 mmol/L sodium 1-octanesulfonate, and acetic acid, pH adjusted to 3.35. The mobile phase was acetonitrile: buffer (1:9, v/v). After vacuum filtration with a 0.22 µm filter membrane, the sample was degassed by ultrasonic degassing for 10 min. The temperature of the column oven was 30 °C. The flow rate was 1 mL/min. The sample was injected at a volume of 20 µL. The electrochemical detector was operated at a voltage of 1000 mV with a 10 µA range. The amount of dopamine in zebrafish tissues was calculated based on the regression equation.

9. Western blot

Proteins were extracted from zebrafish tissue, and the concentration was assayed (final concentration: 10 mg/mL). Proteins (50 µg/5 µL) were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The transfer membrane was conducted at 110 V for 1 h. Membranes were blocked with 5% milk for 1 h. Incubated at 4 °C with primary antibodies: rabbit anti-TLR4 (1:1000), rabbit anti-NF-κB (1:2000), rabbit anti-lamin B1 (1:2000), or rabbit anti-GAPDH (1:1000). After washing with TBST (3 times for 5 min each), the membranes were incubated with secondary antibody for 1 h. Bands were visualized using an ECL Chemiluminescent Substrate Kit and quantified using a gel imaging system. Lamin B1 and GAPDH were used as loading controls.

10. Statistical analysis

All data were expressed as mean ± SD and analyzed with statistical software. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey's test. A P-value of <0.05 was statistically significant.

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