The Causal Effects of Plasma Metabolites on Achilles Tendon Injury: A Mendelian Randomization Study

Summary

Here, we present a combined Mendelian randomization and experimental validation protocol to identify causal metabolites in Achilles tendon injury, identifying 19 plasma metabolites with causal links to Achilles tendon injury, and demonstrating the therapeutic application of 5,6-dihydrothymine and hydroxyasparagine in promoting tissue repair and reducing inflammation.

Abstract

To investigate the causal effects of plasma metabolites on the progression and recovery of Achilles tendon injury (ATI), Mendelian randomization (MR) was applied using a two-sample approach with genetic data from two large datasets. ATI data were sourced from the IEU-OpenGWAS project (ebi-a-GCST90018895), while plasma metabolite data included 1,400 metabolites. Significant instrumental variables (IVs) for metabolites were identified, and five MR methods-IVW, MR-Egger, weighted median, simple mode, and weighted mode-were applied to assess causal relationships. Reverse MR analysis was conducted to explore the impact of ATI on metabolite levels. Tissue repair and the expression of IL-6, TNF-α, VEGFA, and TGF-β1 in mouse Achilles tendon tissue were assessed on day 7 post-treatment with 5,6-dihydrothymine (Dih) and hydroxyasparagine (Hyd) using H&E staining and Western blot. MR analysis identified 19 metabolites with significant causal associations to ATI. Of these, 12 metabolites, including Dih (IVW: OR = 1.197, 95% CI: 1.074-1.334; P = 0.001) and Pregnanediol-3-glucuronide (IVW: OR = 1.098, 95% CI: 1.023-1.177; P = 0.001), were positively associated with ATI progression. In contrast, 7 metabolites, such as Hyd (IVW: OR = 0.789, 95% CI: 0.707-0.880; P < 0.001), were linked to ATI recovery. Reverse MR analysis did not indicate significant causal effects of ATI on metabolite levels, suggesting a unidirectional relationship. X-ray and H&E analyses revealed that Dih and Hyd treatment significantly enhanced Achilles tendon healing in a dose-dependent manner. Further, Dih and Hyd treatment notably reduced IL-6 and TNF-α expression (P < 0.05) while increasing VEGFA and TGF-β levels (P < 0.05), indicating a reduction in inflammation and promotion of tissue repair. This study identified potential causal relationships between specific plasma metabolites and ATI outcomes. These findings may inform future therapeutic strategies and highlight the utility of MR in exploring metabolite-disease associations.

Introduction

Achilles tendon injury (ATI) is a prevalent and debilitating musculoskeletal condition, particularly among athletes and physically active individuals^1,2. The Achilles tendon, the largest and strongest tendon in the human body, plays a pivotal role in locomotion, transmitting forces from the calf muscles to the heel during activities such as walking, running, and jumping^3,4,5,6. Its limited blood supply and the high mechanical stress it endures make it especially vulnerable to overuse....

Protocol

Ethics approval and consent to participate

All animal experimental designs were strictly conducted in accordance with the Ningxia Province Laboratory Animal Management Measures and were approved by the Ethics Committee of People's Hospital of Ningxia Hui Autonomous Region, Ningxia Medical University.

Study Design

This study investigated the causal relationships between 1,400 plasma metabolites and ATI employing a two-sample MR methodology. To ensure the validity of IVs in causal inference, three critical assumptions must be satisfied: (1) the genetic v....

Results

Causal relationship between 1,400 plasma metabolites and ATIs

A comprehensive MR analysis of 1,400 plasma metabolites was performed to assess their causal effects on ATI. The primary analysis utilized the IVW approach, with strict controls for LD and potential confounders, to identify metabolites that significantly impact ATI progression and recovery. Figure 1 displays the causal associations between 144 plasma metabolites and ATI, where an associ.......

Discussion

This study conducted a comprehensive MR analysis to explore the causal relationships between 1,400 plasma metabolites and ATI. Nineteen plasma metabolites were identified as significantly associated with ATI progression and healing, with Dih and Hyd emerging as key factors influencing ATI outcomes. Subsequent in vivo experiments confirmed the beneficial roles of these metabolites in promoting tendon healing. Treatment with Dih and Hyd resulted in significant, dose-dependent downregulation of the pro-inflammatory.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5,6-Dihydrothymine	Reagent	696-04-8	Reagent
BCA Protein Assay Kit	Reagent	P0012	Reagent
ECL Chemiluminescence Substrate Kit	Reagent	P0018S	Reagent
GAPDH Antibody	Antibody	10494-1-AP	Antibody
Hematoxylin and Eosin Staining Kit	Reagent	G1120	Reagent
Hydroxyasparagine	Reagent	H1126	Reagent
IL-6 Antibody	Antibody	26404-1-AP	Antibody
ImageJ	software	version 1.8	software
Nitrocellulose Membrane	Consumable	HATF08130	Consumable
Paraformaldehyde	Reagent	P0099-3L	Reagent
RStudio	software	version 4.5.2	software
SDS-PAGE Gel Preparation Kit	Reagent	P0012AC	Reagent
Sodium Pentobarbital	Anesthetic	200-323-9	Anesthetic
TGF-β1 Antibody	Antibody	21898-1-AP	Antibody
TNF-α Antibody	Antibody	17590-1-AP	Antibody
VEGFA Antibody	Antibody	ab46154	Antibody
X-ray system	Instrument	MX-20	Instrument