Mechanistic Insights into Salidroside's Mitochondrial Protection via AMPK/Sirt1/HIF-1&#945; Pathway in Hypoxic HT22 Cells

Xiaobo Wang; Yating Zhang; Ya Hou; Hong Jiang; Tingting Kuang; Rui Li; Xianli Meng

doi:10.3791/66923

Research Article

Mechanistic Insights into Salidroside's Mitochondrial Protection via AMPK/Sirt1/HIF-1α Pathway in Hypoxic HT22 Cells

DOI:

10.3791/66923

⸱

April 25th, 2025

Xiaobo Wang¹^,²^,³ , Yating Zhang¹ , Ya Hou¹ , Hong Jiang¹ , Tingting Kuang¹ , Rui Li³ , Xianli Meng¹^,²^,³

¹School of Pharmacy/School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, ²Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine, ³Research Service Office, Traditional Chinese Medicine Hospital of Meishan

Summary

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The present study identified a unique mechanism by which salidroside exerts mitochondrial protective effects on hypoxic HT22 cells, partly through the AMPK/Sirt1/HIF-1α pathway.

Abstract

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Salidroside (Sal), an active ingredient of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba has been found to exert mitochondrial protective effects by improving metabolism and enhancing the energy supply of brain cells under hypoxic conditions. However, its mechanism of action has not been fully clarified. In the present study, high-performance liquid chromatography was first employed to analyze the effects of Sal on nucleotide (ATP, ADP, and AMP) levels. The cellular thermal shift assay (CETSA), a widely used molecular interaction method for validating and quantifying drug target engagement in cells and tissues across different species, was then chosen to confirm the affinity of Sal for AMPK/Sirt1/HIF-1α pathway-related proteins. The results revealed that Sal increased ATP and ADP levels in hypoxic HT22 cells while reducing AMP levels. Moreover, Sal exhibited stable binding to AMPKα, p-AMPKα, Sirt1, and HIF-1α proteins. In conclusion, Sal may exert mitochondrial protective effects by modulating the AMPK/Sirt1/HIF-1α pathway to regulate nucleotide content. This study provides a methodological reference for nucleotide content analysis in cell samples and contributes to the identification and discovery of targets for compounds derived from traditional Chinese medicine.

Introduction

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The brain is highly sensitive to oxygen due to its high metabolic demands, limited glycolytic capacity, and dependence on oxidative phosphorylation. As a result, exposure to a low-oxygen environment at high altitudes can readily lead to hypobaric hypoxic brain injury (HHBI)¹^,². Epidemiological studies indicate that when individuals unacclimated to high altitudes ascend rapidly to high-altitude regions, the incidence of acute mountain sickness can reach up to 75%, with a fatality rate of approximately 1% for severe cases. Furthermore, in the absence of medical care, mortality rates for high-altitude cerebral or....

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Protocol

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The commercial details of the reagents and the equipment used in this study are provided in the Table of Materials.

1. Solution preparation

Prepare complete Dulbecco's modified Eagle medium (DMEM) by adding 10% fetal bovine serum and 1% penicillin-streptomycin.
Prepare a 20 mM Sal solution by dissolving 3 mg of Sal in 500 µL of phosphate-buffered saline (PBS)²¹.
Filter 500 mL of chromatographically pure acetonitrile (mobile phase A) using a 0.45 µm organic membrane.
Prepare 1000 mL of mobile phase B by adding 3.5 mL of reagent I....

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Results

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The standard curves for ATP, ADP, and AMP detected by HPLC were Y = 7006.5X - 222.99, Y = 5217.3X - 17.796, and Y = 9280.1X + 22.749, respectively (Figure 1A-C). The nucleotide contents measured in each group by HPLC were calculated using the standard curves (Figure 1D-I). It was found that CoCl₂ significantly reduced ATP and ADP levels in HT22 cells compared to the control group (

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Discussion

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Mitochondria are key organelles involved in the therapeutic prevention of HHBI²³^,²⁴^,²⁵. Previous studies by the group have confirmed that Sal regulates AMPK, Sirt1, and HIF-1α protein expression, enhancing neuronal mitochondrial function and protecting against HHBI²¹^,²⁴. However, the direct effect of Sal on nucleotides in hypoxic cells requires further investigation.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by the National Natural Science Foundation of China (82274207 and 82474185), the Science & Technology Department of Sichuan Province (2024NSFSC1845), the Science Foundation for Youths of Science & Technology Department of Sichuan Province (2023NSFSC1776), the Key Research and Development Program of Ningxia (2023BEG02012), Youth Talent Support Project of the China Association of Chinese Medicine for 2024-2026 (2024-QNRC2-B07) and the Xinglin Scholar Research Promotion Project of Chengdu University of TCM (XKTD2022013 and QJJJ2024027).

AUTHOR CONTRIBUTION:
Xiaobo Wang, Yating Zhang, Ya Ho....

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Materials

List of materials used in this article
Name	Company	Catalog Number
Acetonitrile	Aladdin	A104440
0.22 µm aqueous membrane	Jinteng	JTMF0445
0.22 µm needle type microporous filter membrane	Jinteng	JTSFM013001
0.45 µm organic membrane	Jinteng	JTMF0448
Agilent OpenLab software	Agilent	Version 2.X
Antibody-AMPKα	Cell Signaling Technology	#2532
Antibody-HIF-1α	Cell Signaling Technology	#41560
Antibody-p-AMPKα	Cell Signaling Technology	#50081
Antibody-Sirt1	Cell Signaling Technology	#2028
Antibody-β-actin	Cell Signaling Technology	#4970
BCA protein concentration assay kit	Boster Biological Technology	17E17B46
Bovine serum albumin	Cywin Innovation (Beijing) Biotechnology Co., Ltd.	SW127-02
Broad-spectrum phosphatase inhibitor (100×)	Boster Biological Technology	AR1183
Chromatographic column	Agilent	SB-C₁₈
CoCl₂	Sigma	15862
Decolorization shaker	Kylin-Bell	TS-2
Dorsomorphin	MedChemExpress (MCE)	HY-13418A
Dulbecco's modified eagle medium	Gibco	8121587
Electrophoresis buffer	NCM Biotech	20230801
Fetal bovine serum	Gibco	2166090RP
Goat Anti-rabbit IgG H&L (TRITC)	ZenBioScience Co., Ltd.	511202
GraphPad Prism software	GraphPad software, LLC	Version 9.0.0
High performance liquid chromatography	Agilent	1260 Infinity II Prime
High speed refrigerated centrifuge	Thermo Fisher Scientific	Legend Micro 17R
HRP conjugated affinipure goat anti-rabbit IgG(H+L)	Boster Biological Technology Co., Ltd.	BA1054
HT22 cells	Guangzhou Jennio Biotech Co., Ltd.	JNO-02001
Hypersensitive ECL chemiluminescence kit	NCM Biotech	P10300
Image J software	National Institutes of Health	v1.8.0
Metal heating temperature control instrument	Baiwan Electronictechnology Co., Ltd.	HG221-X3
Methanol	Aladdin	M116118
Nucleotide (ATP, ADP, and AMP) content assay kit	Beijing Solarbio Science & Technology Co., Ltd.	BC5114
PAGE gel rapid preparation kit	Biosharp	PL566B-5
Penicillin-streptomycin	NCM Biotech	C125C5
Phosphate buffered saline (1×)	Gibco	8120485
Pre-stained color protein marker (10-180 kDa)	Cywin Innovation (Beijing) Biotechnology Co., Ltd.	SW176-02
Protein loading buffer (5x)	Boster Biological Technology	AR1112
PVDF (0.45 μm)	Cywin Innovation (Beijing) Biotechnology Co., Ltd.	SW120-01
Rapid membrane transfer solution	Cywin Innovation (Beijing) Biotechnology Co., Ltd.	SW171-02
RIPA lysate	Boster Biological Technology Co., Ltd.	AR0105
Salidroside	Chengdu Herbpurify Co., Ltd.	RFS-H0400191102
TBS buffer	NCM Biotech	23HA0102
Transmembrane buffer	NCM Biotech	23CA2000
Trypsin (0.25%, 1×)	HyClone	J210045
Tween 20	Shanghai Canspec Scientific Instruments Co., Ltd.	PM12012
Ultrasonic cell disruption apparatus	Ningbo Xinyi ultrasonic equipment Co., Ltd.	JY92-IIDN
Visionworks imaging system	Analytik Jena	UVP ChemStudio
Vortex mixer	Kylin-Bell	XW-80A

References

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Marutani, E., Ichinose, F. Sulfide catabolism ameliorates hypoxic brain injury. Nat Commun. 12 (1), 3108(2021).
Chen, X., Ma, W., Li, Y. Current situation of Chinese and Western medicine research on hypoxic brain injury at high altitude. Jilin Medical J....

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Mechanistic Insights into Salidroside's Mitochondrial Protection via AMPK/Sirt1/HIF-1α Pathway in Hypoxic HT22 Cells

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Summary

Abstract

Introduction

Protocol

Results

Discussion

Disclosures

Acknowledgements

Materials

References

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