Osthole Improves Rat Arthritis Through the miR-34a/Bcl-2 Axis

Summary

Osthole (OST) is a natural compound with extensive pharmacological activities. This study suggests that OST alleviates the effects of Knee Osteoarthritis (KOA) via the miR-34a/Bcl-2 axis, as demonstrated in in vivo and in vitro studies in rats, and provides new ideas and directions for its patent medicine and clinical applications.

Abstract

Knee osteoarthritis (KOA), also referred to as hypertrophic or degenerative arthritis, is a prevalent joint disorder. Current clinical management strategies include pharmacological therapy, physical intervention, surgical procedures, and adjunctive treatments. While these approaches can alleviate KOA symptoms to some extent, they often come with limitations, such as side effects, limited efficacy, or invasiveness. Osthole (OST), a natural coumarin derivative, has been widely studied for its medicinal properties, particularly in alleviating joint pain and improving articular function. In this study, CCK8 assay, transmission electron microscopy (TEM), H&E staining, and serum biochemical analysis were employed to demonstrate that OST exerts protective effects on chondrocytes both in vitro and in vivo, with no significant toxicity observed. Subsequent investigations using TEM, acridine orange staining, flow cytometry, and western blotting revealed that OST promotes autophagy and suppresses apoptosis in chondrocytes. Further mechanistic studies through qRT-PCR and luciferase reporter assays indicated that OST downregulates miR-34a, thereby upregulating Bcl-2 expression and inhibiting apoptosis via the miR-34a/Bcl-2 pathway. In summary, these findings indicate that OST protects chondrocytes in both cellular and animal models, attenuating LPS-induced stress and reducing inflammation and apoptosis in KOA rats. The downregulation of miR-34a and subsequent increase in Bcl-2 expression contribute to its anti-apoptotic effect. These results provide a theoretical foundation for the development of OST-based patented drugs and their potential clinical translation in KOA therapy.

Introduction

Knee Osteoarthritis (KOA) is a common joint disease also known as hypertrophic, hypertrophic, or senile knee arthritis. It is caused by the degeneration of the articular cartilage and changes in the structure around the joint. According to epidemiological studies, knee osteoarthritis is a common disease, especially in elderly people¹. Its incidence is closely related to age, weight, genetics, joint injury, and so on. Physiological functions can also be affected, including limited joint motion, reduced muscle strength, and abnormal gait. At present, in the treatment of knee osteoarthritis, the commonly used methods include drug therapy, physical....

Protocol

All studies were approved by the Institutional Animal Care and Treatment Committee of Xuzhou Medical University. The reagents and the equipment used are listed in the Table of Materials.

1. Animal model

Adult male and female Sprague-Dawley (SD) rats weighing 250–300 g were purchased from the Experimental Animal Center of Xuzhou Medical University. Rats were bred in a special pathogen-free room. The animal experimental procedures, including treatment, care, and endpoint choice, followed the “Animal Research: Reporting In Vivo Experiments” guidelines. Animal experim....

Results

OST-protected chondrocyte cells both in vitro and in vivo
As a natural product, the safety profile of OST requires thorough investigation. This study first assessed its cytotoxicity on chondrocytes in vitro using the CCK8 assay. The results indicated that OST exhibited no toxicity toward chondrocytes even at a concentration of 400 µM (Figure 1A). To examine the anti‑inflammatory and anti‑apoptotic effects of OST on chondrocytes, cel.......

Discussion

Above all, OST protected chondrocytes from LPS-induced stress in vitro and alleviated inflammation and apoptosis in KOA rats in vivo. These protective effects were associated with the downregulation of miR-34a and a consequent increase in Bcl-2 expression, consistent with the proposed role of the miR-34a/Bcl-2 axis in mediating OST's action.

In this study, OST was found to promote chondrocyte cells' autophagy and decrease apoptosis both in vitro and in vi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1% osmium tetroxide	Ruixin Technology
2.5% glutaraldehyde	Zhongjingkeyi Technology
Acridine orange	Sigma	Amresco 0360
Annexin-V FITC/PI solution	KeyGEN	KGA107
Bcl-2	Proteintech	#68103
Cell Counting Kit-8	VICMED	39.VC5001L
chemiluminescence	Odyssey	CLX
confocal microscope	Leica	STELLARIS 5
DMEM/F-12	HyClone	12.SH30023-01
Dual-LumiTM Luciferase Reporter Gene Assay kit	Beyotime	RG088
FastKing cDNA regent kit	TIANGEN	KR116-03
Flow cytometer	BD	FACS Canto II
Human chondrocyte cell line	American Type Culture Collection	YTLK280144
LC3	Proteintech	#14600-1-AP
LipofectamineTM 3000	Invitrogen	L3000015
Lipopolysaccharides	Sigma	29.L2880
Lysis assay	KeyGEN	KGP704-100
microplate reader	BioTek	Synergy 2
miRcute plus miRNA first-strand cDNA kit	TIANGEN	KR211-02‌
miRcute plus miRNA qPCR kit	TIANGEN	FP411-02
Osthole	Medchemexpress	O815127-25g
SDS-polyacrylamide gel electrophoresis	NCM Biotech	P2012
Serum	Clark Bioscience	FB15015
SYBR Green	TIANGEN	FP217-01‌
transmission electron microscopy	FEI	Tecnai G2
ultra-microtome	Lecia	UC7
Upright fluorescence microscope	Olympus	BX43
β-Actin	Proteintech	#66009-1-lg