Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia

Summary

The present protocol describes an integrated strategy for exploring the key targets and mechanisms of Fructus Phyllanthi against hyperlipidemia based on network pharmacology prediction and metabolomics verification.

Abstract

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug against hyperlipidemia in Traditional Chinese Medicine (TCM) and Indian Medicine theories, however the potential mechanism requires further exploration. The present research aims to reveal the mechanism of FP against hyperlipidemia based on an integrated strategy combining network pharmacology prediction with metabolomics validation. A high-fat diet (HFD)-induced mice model was established by evaluating the plasma lipid levels, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Network pharmacology was applied to find out the active ingredients of FP and potential targets against hyperlipidemia. Metabolomics of plasma and liver were performed to identify differential metabolites and their corresponding pathways among the normal group, model group, and intervention group. The relationship between network pharmacology and metabolomics was further constructed to obtain a comprehensive view of the process of FP against hyperlipidemia. The obtained key target proteins were verified by molecular docking. These results reflected that FP improved the plasma lipid levels and liver injury of hyperlipidemia induced by a HFD. Gallic acid, quercetin, and beta-sitosterol in FP were demonstrated as the key active compounds. A total of 16 and six potential differential metabolites in plasma and liver, respectively, were found to be involved in the therapeutic effects of FP against hyperlipidemia by metabolomics. Further, integration analysis indicated that the intervention effects were associated with CYP1A1, AChE, and MGAM, as well as the adjustment of L-kynurenine, corticosterone, acetylcholine, and raffinose, mainly involving tryptophan metabolism pathway. Molecular docking ensured that the above ingredients acting on hyperlipidemia-related protein targets played a key role in lowering lipids. In summary, this research provided a new possibility for preventing and treating hyperlipidemia.

Introduction

Hyperlipidemia is a common metabolic disease with serious impacts on human health, and is also the primary risk factor for cardiovascular diseases¹. Recently, there has been a downward age-related trend for this disease, and younger people have become more susceptible because of long-term irregular lifestyles and unhealthy eating habits². In the clinic, various drugs have been used to treat hyperlipidemia. For example, one of the most commonly used drugs for patients with hyperlipidemia and related atherosclerotic disorders is statins. However, long-term use of statins has side effects that can't be neglected, which ....

Protocol

All procedures involving the handling of animals were conducted in accordance with the Chengdu University of Traditional Chinese Medicine Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of the Chengdu University of Traditional Chinese Medicine (Protocol number 2020-36). Male C57BL/6 mice (20 ± 2 g) were used for the present study. The mice were obtained from a commercial source (see Table of Materials).

1. Net.......

Discussion

In recent years, the incidence rate of hyperlipidemia has been increasing, mainly due to long-term unhealthy eating habits. TCM and its chemical ingredients have various pharmacological activities, which have been widely studied in recent years^37,38. FP is a kind of fruit resource, used both as medicine and food, and has an important potential for treating hyperlipidemia. However, the potential therapeutic mechanism of FP against hyperlipidemia needs further stud.......

Materials

Name	Company	Catalog Number	Comments
101-3B Oven	Luyue Instrument and Equipment Factory	\
80312/80302 Glass Slide	Jiangsu Sitai Experimental Equipment Co., LTD	\
80340-1630 Cover Slip	Jiangsu Sitai Experimental Equipment Co., LTD	\
AccucoreTM C18 (3 mm × 100 mm, 2. 6 μm)	Thermo Fisher Scientific	\
Acetonitrile	Fisher Chemical	A998	Version 1.5.6
ACQUITY UPLC HSS T3 Column (2.1 mm × 100 mm, 1.8 μm)	Thermo Fisher Scientific	\
Aethanol	Fisher Chemical	A995	Version 3.0
Ammonia Solution	Chengdu Cologne Chemicals Co., LTD	1336-21-6	Version 3.9.1
AutoDockTools	Scripps Institution of Oceanography	\
BS-240VT Full-automatic Animal Biochemical Detection System	Shenzhen Mindray Bio-Medical Electronics Co., Ltd.	\
Compound Discoverer	Thermo Fisher Scientific	\
Cytoscape	Cytoscape Consortium	\
DM500 Optical Microscope	Leica	\
DV215CD Electronic Balance	Ohaus Corporation ., Ltd	T15A63
Ethyl Alcohol	Chengdu Cologne Chemicals Co., LTD	64-17-5
Formic Acid	Fisher Chemical	A118
HDL-C Assay Kit	Nanjing Jiancheng Bioengineering Institute	A112-1-1
Hematoxylin Staining Solution	Biosharp	BL700B
High Fat Diet	ENSIWEIER	202211091031
Hitachi CT15E/CT15RE Centrifuge	Hitachi., Ltd.	\
Homogenizer	Oulaibo Technology Co., Ltd	\
Hydrochloric Acid	Chengdu Cologne Chemicals Co., LTD	7647-01-0
Image-forming System	LIOO	\
JB-L5 Freezer	Wuhan Junjie Electronics Co., Ltd	\
JB-L5 Tissue Embedder	Wuhan Junjie Electronics Co., Ltd	\
JK-5/6 Microtome	Wuhan Junjie Electronics Co., Ltd	\
JT-12S Hydroextractor	Wuhan Junjie Electronics Co., Ltd	\
KQ3200E Ultrasonic Cleaner	Kun Shan Ultrasonic Instruments Co., Ltd	\
LDL-C Assay Kit	Nanjing Jiancheng Bioengineering Institute	A113-1-1
Male C57BL/6 Mice	SBF Biotechnology Co., Ltd.	\	Version 2.3.2
Neutral Balsam	Shanghai Yiyang Instrument Co., Ltd	10021190865934
Pure Water	Guangzhou Watson's Food & Beverage Co., Ltd	GB19298
PyMOL	DeLano Scientific LLC	\	Version 14.1
RE-3000 Rotary Evaporator	Yarong Biochemical Instrument Factory ., Ltd	\
RM2016 Pathological Microtome	Shanghai Leica Instruments Co., Ltd	\	Version 26.0
SIMCA-P	Umetrics AB	\
Simvastatin	Merck Sharp & Dohme., Ltd	14202220051
SPSS	International Business Machines Corporation	\
TC Assay Kit	Nanjing Jiancheng Bioengineering Institute	A111-1-1
TG Assay Kit	Nanjing Jiancheng Bioengineering Institute	A110-1-1
UPLC-Q-Exactive Quadrupole Electrostatic Field Orbital Hydrazine High Resolution Mass Spectrometry	Thermo Fisher Scientific	\
Vortex Vibrator	Beijing PowerStar Technology Co., Ltd.	LC-Vortex-P1
Xylene	Chengdu Cologne Chemicals Co., LTD	1330-20-7