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 therapy, surgery, and adjuvant therapy². Medications often include analgesics, non-steroidal anti-inflammatory drugs, and chronic arthritis drugs to reduce pain and control inflammation. Physical therapy includes hot compresses, cold compresses, massage, and specific rehabilitation exercises to improve joint motor function and reduce pain. Surgical treatment can include arthroscopic surgery, artificial joint replacement, or joint bone morphological repair to restore joint structure and function. Moreover, complementary therapies such as acupuncture, physiotherapy, and traditional Chinese medicine are also widely used.

However, although these treatments provide some relief from symptoms and the condition of knee osteoarthritis, there are still limitations. Drug treatment may have adverse reactions and dependence problems; Physical therapy requires an ongoing rehabilitation process, and the results vary from individual difference; Surgical treatment is risky, the recovery process is long and needs carefully consideration; and, although adjuvant therapy has received widely attention and application, there is still limited scientific supports for its efficacy, deeply study the regulatory mechanism of adjuvant therapy needs to be studied in depth. In adjuvant therapy, traditional Chinese medicines for the treatment of knee osteoarthritis have been widely used in many areas and have proven useful. There must be some components of traditional Chinese medicine that are not yet well known, but have been proven through long-term practice and follow-up studies. Currently, many Chinese medicines have accumulated rich experience in treating knee osteoarthritis, such as Cibotium barometz³, Ligusticum wallichii Franchat⁴, Angelica pubescens⁵, and others. These herbs have analgesic and anti-inflammatory effects that can effectively relieve pain and reduce inflammation.

OST is a coumarin-like compound widely used in medicine as a natural herbal extract⁶. It has many pharmacological activities and is supported by numerous studies. Studies have shown that cnidium has many pharmacological activities, including anti-tumor, anti-inflammation, hyperglycemia, anti-platelet aggregation, and neuroprotection, and has potential therapeutic effects on many other diseases⁷. It has been reported that OST can promote apoptosis of cancer cells by regulating autophagy, which OST inhibits ovarian carcinoma cells apoptosis through LC3-mediated autophagy and GSDME-dependent pyroptosis⁸; OST induces HT‑29 cells apoptosis via endoplasmic reticulum stress and cell autophagy⁹; OSTpromotes the apoptosis of colorectal cancer via suppressing AMPK/Akt signaling, inducing cell autophagy, ROS, and ferroptosis¹⁰; OST may inhibit the growth of colorectal cancer by regulating autophagy and mitochondria-mediated signal transduction¹¹. At the same time, OST is particularly significant in relieving joint pain and improving joint function, mainly by reducing inflammatory response and alleviating joint pain. And its active ingredients can reduce the release of inflammatory substances and inhibit the degeneration of joint cartilage caused by arthritis. Compared with common western drugs, OST is safer and has low toxicity, and long-term use will not produce drug resistance. It has been reported that osthole enhances osteogenesis in osteoblasts by elevating transcription factor osterix via cAMP/CREB signaling in vitro and in vivo¹²; OST suppresses knee osteoarthritis development by enhancing autophagy activated via the AMPK/ULK1 pathway¹³; Cnidium lactone inhibits osteoclast differentiation through p38 MAPK and PI3K-Akt and c-Fos/ NFATc1 signaling pathway¹⁴; Cnidium lactone prevents bone loss in an ovariectomized rat model through the estrogen-α/BMP-2/Smad signaling pathway¹⁵; OST improves osteoporosis through increasing autophagy of mesenchymal stem cells¹⁶. However, the observation and analysis indicated that most studies stayed in the phenotype, or failed to reveal the signaling pathway regulation mechanism of drug participation at the molecular level, which was also one of the reasons that compounds extracted from traditional Chinese medicine are difficult to be widely accepted. Therefore, it is of great significance to further explore the biological regulation mechanism of Osthole at the molecular level for its patent medicine and clinical application.

miRNA is a small RNA that regulates biological function by inhibiting the expression of target genes. Many traditional Chinese medicines are known to exert their biological effects by regulating miRNA. Similarly, OST has also been reported to influence miRNA expression. First, OST plays an anti-tumor biological role through miRNA. It has been reported that OST inhibited cell proliferation and induced cell apoptosis through decreasing CPEB2 expression via upregulating miR-424 in endometrial carcinoma¹⁷; OST suppresses the proliferation and accelerates the apoptosis of human glioma cells via the up-regulation of miR-16 and downregulation of MMP-9¹⁸; OST exhibits an anti-tumor effect in retinoblastoma through inhibiting the PI3K/AKT/mTOR pathway and regulating the hsa_circ_0007534/miR-214-3p axis¹⁹. Second, OST can also affect the physiological processes of body organs through the regulation of miRNA, which OST alleviates pulmonary vascular remodeling by modulating miR-22–3p mediated lipid metabolic reprogramming²⁰; OST protects rats against myocardial ischemia/reperfusion injury via down-regulating miR-30a and promoting autophagy²¹; OST protects H9c2 cardiomyocytes against trastuzumab-induced damage by enhancing autophagy through the p38MAPK/mTOR signaling pathway²². Third, OST can play a neuroprotective role through miRNA, which OST stimulated neural stem cells differentiation into neurons in an Alzheimer's disease cell model via upregulation of miR-9 and rescued the functional impairment of hippocampal neurons in APP/PS1 transgenic mice²³; OST decreases beta amyloid levels through upregulation of miR-107 in Alzheimer’s disease ²⁴. However, it has not been reported that miRNA can be regulated by OST to treat knee osteoarthritis. The literature showed that miR-34a enhances chondrocyte apoptosis and facilitates the development of osteoarthritis by targeting DLL1 and regulating the PI3K/AKT pathway²⁵. Long non-coding RNA MEG3 regulates the progression of osteoarthritis by regulating the miR-34a/Klotho axis²⁶; Downregulation of miR-34a promotes proliferation and inhibits apoptosis of rat osteoarthritic cartilage cells by activating PI3K/Akt pathway²⁷. Thus, could KOA be treated with OST via miR-34a regulation? Therefore, it is hypothesized that OST can treat knee osteoarthritis by regulating miRNA.

In summary, OST is a natural compound with extensive pharmacological activities²⁸. Earlier, the research group confirmed that OST can improve knee osteoarthritis in rats, but the cellular signal transduction and molecular mechanisms underlying this biological effect have not been fully clarified. In this article, by further studying the miRNA, autophagy, and apoptosis signaling pathways of OST, it is intended to clarify the mechanism of OST in the treatment of knee osteoarthritis and to provide new ideas and directions for its patent medicine and clinical application.

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 experiments were performed with randomization. SD rats were grouped into four groups(n = 8 per group): Normal group (no surgery, normal feeding) (Normal group), Sham group (sham surgery: skin and muscle incision without anterior cruciate ligament transection [ACLT], followed by saline gavage), KOA/NC group (ACLT surgery+saline gavage), KOA/OST group (ACLT surgery+OST gavage). The KOA model of the rat knee was constructed using the anterior cruciate ligament transection (ACLT) method as previously described²⁹. The saline group was administered saline via gavage, and the OST group was administered OST via gavage for 4 weeks. The OST dose is 50 mg/kg body weight (Dissolved in PEG 400 and diluted with physiological saline), and the administration frequency is once daily for 4 weeks in the Rats subsection. Then, rats were euthanized for analysis (following institutionally approved protocols). Tissues were either immediately frozen in liquid nitrogen or fixed in 4% paraformaldehyde.

2. Cell culture

Human chondrocyte cell line (C28/I2) was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured according to the ATCC guidelines and used within 6 months.

Culture conditions: DMEM/F-12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, incubated at 37 °C in a 5% CO₂ humidified incubator.

Treatments: Cells were seeded at 1 × 10⁶ cells/well in 6-well plates; after 24 h of adhesion, they were treated with LPS (1 µg/mL) alone or with OST (100 µM) for 24 h.

3. Assays for cell viability

Chondrocyte cells were co-incubated with OST or LPS in different concentrations in 96-well plates. The CCK8 assay reflects cell viability by detecting absorbance (OD450 nm) using a microplate reader, with higher OD values indicating more viable cells.

OST concentration gradient: 0, 25, 50, 100, 200, 400 µM (incubated for 24 h). The CCK8 assay showed >90% cell viability at all concentrations, confirming the compound's non-toxicity.

LPS concentration gradient: 0, 0.1, 0.5, 1, 2 µg/mL (incubated for 24 h). The 1 µg/mL LPS group had ~50% cell viability (vs. the Ctrl group), so this concentration was chosen for subsequent experiments.

4. Hematoxylin and eosin (H&E) staining and Safanin O staining

Tissues were paraffin-embedded, dewaxed, rehydrated, and stained with H&E and Safranin O/Fast Green reagents according to the manufacturer's directions. Staining conditions: AO working concentration was 1 µg/mL; cells were incubated at 37 °C for 5 min in the dark.

Quantitative method: AVO positivity rate was calculated by counting orange puncta-containing cells in 5 random fields (×400 magnification) per sample using ImageJ software. Then, sections were captured under a BX43 microscope (magnification ×400).

5. Flow cytometry

Cells were harvested and resuspended in Annexin-V FITC/PI solution for apoptosis analysis. Incubation conditions: Cells were resuspended at 1 × 10⁶ cells/mL in Annexin-V FITC/PI solution (KeyGEN) and incubated at room temperature for 15 min in the dark.

Gating strategy: Forward scatter (FSC) vs. side scatter (SSC) gates were used to exclude cell debris; Annexin-V FITC⁺/PI⁻ cells were defined as early apoptotic, and Annexin-V FITC⁺/PI⁺ cells as late apoptotic. The gating plot is included in Supplementary File 1 (raw flow cytometry data). Cells were collected by a flow cytometer. Data were analyzed using Flow Jo software.

6. Transfection of plasmids and mimic

MiR-34a mimic (34a-mimic), nonspecific control-mimic (NC) were obtained from a commercial source. Cells were transfected with plasmids using LipofectamineTM 3000 according to the manufacturer’s instructions.

7. Acridine orange staining

Autophagy was usually evaluated by Acridine orange staining in cells. After cells were treated with LPS or OST at specific concentrations for 24 h, they were incubated with AO (1 µg/mL) at 37 °C for 5 min in the dark. Images were captured using a laser confocal microscope. AVO positivity rate was calculated by counting orange puncta-containing cells in 5 random fields (×400 magnification) per sample using ImageJ software. Data are reported as mean ± SD from three independent experiments.

8. Transmission electron microscope observation

Samples were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, then dehydrated, rinsed, and impregnated. After polymerization, samples were cut into 70-nm-thick sections using an ultramicrotome. The ultrastructure of cells was observed by transmission electron microscopy.

9. Immunoblotting

Mouse monoclonal antibody (Abs) against Bcl-2 (#68103), rabbit polyclonal antibody against LC3 (#14600-1-AP), IL-6 (21865-1-AP), and mouse monoclonal antibody against β-Actin (#66009-1-lg) were purchased from Proteintech. Secondary Abs conjugated to IRDye 800CW Goat (polyclonal) anti-Rabbit IgG (H+L) or IRDye 800CW Goat (polyclonal) anti-Mouse IgG (H+L) were obtained commercially. Cells were lysed in a whole-cell lysis assay, separated by electrophoresis on SDS-polyacrylamide gel electrophoresis. Proteins were detected using primary Abs and corresponding secondary Abs. Protein loading was 30 µg per lane; primary antibody dilutions: Bcl-2 (1:1000), LC3 (1:1500), β-tubulin (1:5000); secondary antibodies (IRDye 800CW) were used at 1:10,000, IL-6 (1:800). Then, the protein bands were captured by the enhanced chemiluminescence laser imaging scan system. Protein bands were quantified by ImageJ software.

10. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)

Total RNAs were isolated from cells using the Trizol reagent. cDNAs derived from total RNAs were synthesized using the cDNA regent kit and analyzed by SYBR Green methods. cDNAs derived from miRNAs were synthesized using miRcute plus miRNA first-strand cDNA kit and analyzed by miRcute plus miRNA qPCR kit. All procedures were performed according to the manufacturer’s protocol. The expression of the target transcript was calculated using the 2^-△△CT method. GADPH and U6 were used as the loading control for mRNA and miRNA, respectively. The primer sequence is listed in Table 1.

11. Luciferase reporter assays

Wild-type psiCHECK2- Bcl-2-3′UTR reporter plasmid (WT) and psiCHECK2- Bcl-2-3′UTR reporter plasmid with a mutant at the miR-34a binding site (Mut). The reporters and miR-34a mimic were co-transfected into chondrocyte cells using LipofectamineTM 3000. Cells were harvested and lysed after 24 h of transfection. Luciferase intensity was then measured using the Dual-LumiTM Luciferase Reporter Gene Assay kit according to the manufacturer’s instructions.

12. Gait analysis tests

The MGT-PR system was used to observe the running limb movements of rats in each group. Before testing, the rats were allowed to adapt to the runway environment. Gait data during free walking were recorded, with three consecutive valid walking trials captured for each rat. The analyzed parameters included step length, swing duration, and coordination between the right and left hindlimbs.

13. Statistical analysis

The experiments were performed in triplicate and independently repeated at least three times. Statistical analysis was carried out using GraphPad Prism 9 software. Data were shown as mean ± SD. Statistical differences were evaluated by one-way ANOVA (Tukey’s post hoc test) for multiple comparisons, or by two-tailed Student’s t test for two experimental group comparisons. Differences were accepted as significant at p < 0.05.

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