Mechanism and Experimental Validation of Total Flavonoids of Rhizoma Drynariae in Treating Gouty Arthritis

Summary

Total flavonoids of Rhizoma Drynariae (TFRD) alleviate gouty arthritis (GA) by suppressing inflammatory cell infiltration and reducing TNF-α, IL-6, and IL-17A levels, primarily through inhibition of AKT, MAPK, and NF-κB signaling pathways, highlighting its anti-inflammatory and immunomodulatory therapeutic potential.

Abstract

Gouty arthritis (GA) is an inflammatory joint disease caused by the deposition of monosodium urate (MSU) crystals within the joint space and surrounding tissues. In traditional Chinese medicine, Rhizoma Drynariae (Gusuibu) has long been widely used in the clinical treatment of GA, and flavonoids are considered its key bioactive constituents. This research employed network pharmacology to construct a component-target network of total flavonoids of Rhizoma Drynariae (TFRD) against GA, thereby identifying key components, core targets, and related pathways. Rat models were established by intra-articular injection of a monosodium urate crystal suspension and treated with TFRD or the positive control, colchicine, by oral gavage. After sample collection, network pharmacology-based prediction results were subsequently validated using rat serum metabolomics, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Network pharmacology analysis indicated that the anti-GA effects of TFRD are mediated through key targets, including IL6, AKT1, TNF, EGFR, JUN, and PTGS2, and are mainly associated with inflammation, immune, and apoptosis-related pathways, such as the IL-17, TNF, NF-κB, MAPK, PI3K-AKT, JAK-STAT, and T-cell receptor signaling pathways. Similarly, metabolomics also uncovered the pivotal roles of the inflammatory response. Hematoxylin and eosin (H&E) staining confirmed that TFRD reduced infiltration of inflammatory cells. ELISA assay confirmed that the TFRD group significantly inhibited the expression of inflammatory factors TNF-α, IL-6, and IL-17A in synovial tissue. Western blot analysis revealed that TFRD inhibited the GA-induced hyperphosphorylation of AKT, MAPK p38, and NF-κB p65 in rat synovial tissue. TFRD can effectively ameliorate the inflammation-triggered changes in the GA rats by directly modulating related inflammatory factors and pathways.

Introduction

Gouty arthritis (GA) is an inflammatory joint disease triggered by the deposition of monosodium urate (MSU) crystals within and around the joints, which stimulate the joint area^1,2. The pathogenesis of GA involves a complex interplay of genetic predisposition, environmental influences, and metabolic dysregulation characterized primarily by hyperuricemia resulting from either excessive uric acid production or insufficient renal excretion^3,4. This leads to the systemic accumulation of urate crystals in renal tissues and articular spaces, where they ....

Protocol

This study was reviewed and approved by the Animal Ethics Committee of China-Japan Friendship Hospital (Approval No: zryhyy21-22-08-10) and conducted following institutional guidelines for the ethical care and use of laboratory animals.

Prediction and Screening of TFRD-related and GA-associated Targets
The chemical constituents of Rhizoma Drynariae were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) using “Rhizoma Drynariae” as the search term²⁹. Screening for potentially bioactive constituents was perfor....

Results

Key components of TFRD
Potential bioactive ingredients of Rhizoma Drynariae were retrieved from the TCMSP database, yielding 71 candidates. These 71 active constituents were then subjected to a screening process based on oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18), which resulted in the identification of 18 compounds. Further refinement of this list revealed that 10 of these active compounds were flavonoids, including: (2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one, Au.......

Discussion

GA is a common form of inflammatory arthritis. Currently, clinical management mainly relies on nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and urate-lowering agents⁴². However, the combined administration of these drugs inflicts a range of adverse effects on patients and imposes a significant metabolic burden on the liver and kidneys⁴³. This is particularly detrimental for patients with pre-existing hepatic or renal impairment. Flavonoids, a class of pol.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% Paraformaldehyde	Solarbio	P1110
5X Protein Loading Buffer	Solarbio	P1040
AKT Antibody	Cell Signaling Technology	4691S
Antibody Diluent Solution	Beyotime Biotechnology	P0268
Bioconductor database		https://www.bioconductor.org/
colchicine	Shanghai Yuanye Co., Ltd
GeneCards database		https://www.genecards.org/
Hematoxylin and Eosin (H&E) Staining Kit	Solarbio	G1120
MetaboAnalyst 6.0 online platform		https://www.metaboanalyst.ca/
Mouse IL-17A ELISA Kit	4a Biotech Co Ltd	CME0041
Mouse IL-6 ELISA Kit	4a Biotech Co Ltd	CME0006
Mouse TNF-α ELISA Kit	4a Biotech Co Ltd	CME0004
NF-κB p65 Antibody	Cell Signaling Technology	8242T
Non-fat Dry Milk	Solarbio	D8340
p38 MAPK Antibody	Abclonal	A14401
Phosphatase Inhibitor Cocktail	Beyotime Biotechnology	P1081
Phospho-AKT (Ser473) Antibody	Cell Signaling Technology	4060S
Phospho-NF-κB p65 Antibody	Cell Signaling Technology	3031S
Phospho-p38 MAPK Antibody	Abclonal	AP0526
Phenylmethylsulfonyl Fluoride (PMSF)	Solarbio	R0010
Polyvinylidene Fluoride (PVDF) Membrane	Millipore	IPVH00010
PubChem databas		https://pubchem.ncbi.nlm.nih.gov/
R software		www.r project.org/
RIPA Lysis Buffer	Solarbio	R0010
RNase-free water	Ambion	AM9937
SD rats	Beijing Vital River Laboratory Animal Technology Co., Ltd.
SDS-PAGE Electrophoresis Buffer	Servicebio	G2144
STRING Database		https://string-db.org/
TBST Buffer (10X)	Solarbio	T1085
TCMSP		https://old.tcmsp-e.com
Tris-Glycine Transfer Buffer	Servicebio	G2017
TFRD	Beijing Qihuang Pharmaceutical Co., Ltd.
UniProt database		https://www.uniprot.org/
β-Actin Antibody	Zhongshan Jingqiao Biotechnology	TA-09