A Mendelian randomization study: Association of Cathepsin with Osteoarticular Muscle Diseases

doi:10.21203/rs.3.rs-3763757/v1

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A Mendelian randomization study: Association of Cathepsin with Osteoarticular Muscle Diseases

https://doi.org/10.21203/rs.3.rs-3763757/v1

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Objective: The cathepsin present in lysosomal cells may be closely related to bone, muscle & joint diseases (BMJD)including osteoporosis and osteoarthritis, but its specific mechanism of action still needs further research and exploration. The aim of this study is to use a two-sample Mendelian randomization (MR) comprehensive analysis to explore the causal relationship between cathepsin and the risk of BMJD.

Method: Based on the published genetic data, this study used a two-sample Mendelian randomization (MR) comprehensive analysis method to explore the causality of morbidity risk between 9 cathepsins with osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, peripheral arthritis(Crohn's disease, ulcerative colitis, and postdysentery joint disease), psoriatic joint disease, lower back pain, spinal stenosis, osteoporosis, bone necrosis, and connective tissue disease, cartilage diseases, benign tumors, myositis, synovitis and tenosynovitis (radial styloid tenosynovitis), shoulder periarthritis, neuromuscular junction diseases, and muscle atrophy. Meanwhile, in order to further reveal the possibility of reverse causality, we conducted a reverse Mendelian randomization analysis,using Cochran's Q test and MR Egger intercept test to evaluate the heterogeneity, horizontal pleiotropy,and stability of SNPs.

Result: The study found that cathepsin B may be negatively correlated with ulcerative colitis arthritis, cathepsin E may be positively correlated with osteoarthritis, cathepsin E may be negatively correlated with musculoskeletal connective tissue diseases, cathepsin G may be positively correlated with ankylosing spondylitis, and cathepsin L2 may be positively correlated with cartilage diseases, Cathepsin S may be positively correlated with rheumatoid arthritis; through reverse MR analysis, we found that osteoporosis may lead to an increase in cathepsin B. In addition, we did not find any relationship between synovitis, tenosynovitis, shoulder periarthritis, bone necrosis, neuritis, osteoporosis,Crohn's arthritis,dysentery arthritis, psoriatic arthritis, low back pain,and cathepsin.

Conclusion: Based on large-scale population studies of GWAS data, we conducted MR analysis and demonstrated a close relationship between cathepsins and BMJD from a genetic perspective, which may provide a basis for the pathogenesis of BMJD and provide new directions for future treatment strategies.

Biological sciences/Genetics

Health sciences/Diseases

Health sciences/Pathogenesis

Health sciences/Rheumatology

Health sciences/Risk factors

bone, muscle & joint diseases (BMJD) are the most common diseases in orthopedic clinical practice,with a wide range of occurrences.In addition to the discomfort symptoms caused by the disease itself,there are also many complications,including skeletal deformities,cardiovascular diseases,neurological disorders,etc.At present,the occurrence of BMJD may be related to gender,age,living environment,dietary structure,genetics,hormone changes,cytokines,and inflammatory pathways,but the pathogenesis is still not very clear.The production and function of osteoclasts,the degradation and remodeling of bone,cartilage,and muscle tissues,as well as metastasis of bone tumor cells,may be closely related to cathepsins.However,it should be pointed out that the relationship between BMJD and cathepsin is not a simple linear relationship,and the interaction may be influenced by many other factors,such as genes,environment, lifestyle, etc.Therefore, it is necessary to explore the relationship between cathepsin and BMJD.

Cathepsin, as one of the main members of the cysteine protease family,is a type of protease widely present in animal tissue cells,and is more commonly found in the lysosome.In the biological world,more than 20 types of cathepsins have been discovered,of which 11 are mainly present in the human body.These proteases play important roles under normal physiological conditions,participating in many key biochemical reactions and processes within cells.With the deepening of research,tissue proteins are increasingly being valued by everyone.Existing research indicates that cathepsin B (CTSB) is a cysteine proteolytic enzyme present in lysosomes,widely present in bacteria,viruses,and mammals,and can catalyze through cysteine (Cys) and histidine (His);the cathepsin B in bone, nerve cells, interstitial fibroblasts, macrophages and other tissues can maintain a precise balance between protein synthesis and degradation;in addition, it is associated with tumor infiltration and metastasis,one existing study suggests that CTSB may be associated with Ewing's sarcoma¹,another study shows that cathepsins B and D can promote the invasion and migration of polyploid giant cancer cells (PGCC)².Cathepsin D (CTSD) is an aspartic lysosomal endonuclease widely present in vertebrates.Its main function is to hydrolyze proteins in the acidic environment of lysosomes.It can degrade hormones,peptide precursors,peptides,structural and functional proteins.It activates transglutaminase-1 (TG-1) through hydrolysis,thereby promoting cross-linking of keratinized membrane proteins,ultimately inducing skin epidermal differentiation and regeneration, participating in cell apoptosis;in addition, studies have shown that cathepsin D is also related to congenital muscle disease and atherosclerosis,which may be related to the differentiation of osteoclasts and bone marrow macrophages³.Cathepsin K (CTSK) is significantly expressed in osteoclasts and is also activated and expressed in tissues such as skin,heart, and skeletal muscle.It is associated with osteoporosis and the activation and expression of osteoclasts.An animal experimental study⁴ have shown that CTSK can down-regulation the expression of osteoclast specific genes⁵;In addition,it also participates in the degradation of type I and II collagen,especially type I collagen.Therefore,BMJD may be related to abnormal expression of cathepsins,and further research is needed to elucidate the causal relationship between different types of cathepsins and the risk of BMJD.

With the continuous progress of genomics, a large amount of evidence shows that genetic factors play an important role in the pathogenesis of diseases. Mendelian randomization (MR) is an emerging statistical method based on genome-wide association studies (GWAS), which uses genetic variation as an instrumental variable to evaluate the causal relationship between exposure and outcomesand,and conducts epidemiological study design and data analysis based on Mendelian independent distribution law,proving the causal hypothesis of disease^6–7.Previous observational studies have found many associations between cathepsins and BMJD,supporting the hypothesis of a correlation between them. This study use two-sample MR analysis to explore the causal relationship between cathepsin and BMJD. Subsequently,we further explored the possibility of reverse causal relationships,followed by reverse Mendelian randomization analysis.

Study design

Based on two-sample MR analysis,we evaluated the causal relationship between nine cathepsin and osteoarthritic muscle disease.Mendelian randomization (MR) is a genetic epidemiological method that uses instrumental variables (IVs) to study the causal relationship between exposure and outcome, and to address confounding factors. Therefore, Figure 1 illustrates that effective instrumental variables in causal inference must satisfy the following three key assumptions: (1) genetic variation is directly related to exposure; (2) Genetic variation is not associated with any confounding factors associated with exposure and outcomes (3).Genetic variation may affect outcomes,but can only affect outcomes through exposure⁸.

Figure 2 illustrates the entire idea of our research.We first selected 9 types of cathepsins based on publicly available GWAS data,and based on GWAS metabolomics,we selected instrumental variables for each cathepsin.Then,using GWAS data from European populations on BMJD,we conducted two-sample MR analyses to estimate the causal effects of cathepsins on BMJD.To confirm the possibility of reverse causality,a reverse Mendelian randomization analysis was then conducted.

GWAS summary measurement of BMJD

In order to obtain reliable conclusions on the causal relationship between cathepsin and the risk of BMJD,we selected a large-scale genome-wide association study (GWAS) from European ancestry as the outcome measure.Our GWAS data is sourced from the European Bioinformatics Center（http://www.ebi.ac.uk/services）GWASCatalog database（https://www.ebi.ac.uk/gwas/）FinnGen database（https://www.finngen.fi/en/access_results）And the UK Biobank database（https://www.ukbiobank.ac.uk/）We use the IEUOpenGWAS database through the R software package TwoSampleMR (v0.5.6)（https://gwas.mrcieu.ac.uk/）Conduct online analysis.We have sorted out a variety of BMJD,including osteoarthritis (NA cases, NA controls), rheumatoid arthritis (1605cases, 359589controls),ankylosing spondylitis (968cases, 336191controls),Crohn's disease (116cases,217013controls),ulcerative colitis (149 cases,217013controls),and postdysenteral joint disease (149cases,217013 controls),Psoriatic arthritis (1553cases,147221controls),lower back pain (1423cases,461587controls),spinal stenosis (9169cases,164682controls),osteoporosis (3203cases,209575controls), osteonecrosis (604 cases,209575controls),connective tissue disease (115768cases,103024controls),cartilage disease (420cases,209575controls),Benign bone tumors (1190cases,217602controls),myositis (330cases,167641controls),radial styloid tenosynovitis (768cases,167641controls),shoulder periarthritis (NA cases, NA controls),neuromuscular junction diseases (1736cases,217056controls),muscle atrophy (82cases,167641controls).

Genetic instrumental variable selection

In order to obtain a reliable conclusion on the causal relationship between cathepsins and the risk of BMJD,we conducted an INTERVAL study⁹, which included 3301 European individuals,all of whom signed informed consent for the experiment,and obtained approval from the National Research Ethics Service (11/EE/0538).Summary data can be queried at https://gwas.mrcieu.ac.uk.

We selected 9 types of cathepsins,including cathepsinsB,E,F,G,H,L2,O,S,and Z,as exposure data for whole genome SNPs.Then, for each cathepsin,we conducted a rigorous screening procedure to select the qualified instrument variables for each cathepsin.Firstly,we selected SNPs (P<5e-6) that are significantly associated with specific cathepsins as candidate instrumental variables for further MR analysis.Secondly,to ensure that the instrumental variables for each exposed phenotype are independent,we used linkage disequilibrium (LD)-based clustering to remove SNPS with strong linkage disequilibrium (r2 threshold<0.001,window size=10000kb).Based on the European Thousand Genome Project as a standard reference group, the analysis process provides auxiliary information such as coordinate annotation, phylogenetic judgment, and allele frequency for the dataset. For SNPs that were not exist in GWAS data for BMJD,we used the LDlink tool to search for the most relevant proxy SNPs using the European Thousand Genome Project (r2>0.8)¹⁰. We also discarded uncorrectable SNPs with inconsistent alleles and duplicated ambiguous palindromic SNPs, which were strictly filtered and used as instrumental variables for subsequent MR analysis.To determine whether there is weak instrumental bias, we calculated the F-statistic to quantify the strength of the instrumental variable, where the F-statistic is at least greater than 10 to effectively avoid bias caused by weak instrumental variables^11-12.Use the R software package Two-Sample MR (v0.5.6)¹³ for all instrument variable selection and quality control steps.

Statistical analysis

We used R software for data analysis (version number:3.5.3 http://www.example.com）.In order to investigate the causal relationship between 9 types of cathepsins and BMJD,we mainly used the Two-Sample MR software package (version 0.5.6),MREgger,Weighted media,Inverse valuation weighted (IVW),Simplemode,and Weightedmode methods^14-18.In the case of inconsistent data,we mainly used the Inversevaluation weighted (IVW) method and took corresponding p-values to prove the correlation between exposure and outcome.We used Cochran's Q test and corresponding p-values to test for heterogeneity between instrumental variables¹⁹.If I equal>50%,use random effects IVW instead of fixed effects IVW,otherwiseuse fixed effects model.To exclude the influence of horizontal pleiotropy,we used the MR-Egger method and corresponding p-values. If p＜0.05, this means that if the intercept term is significant,there is horizontal pleiotropy;otherwise, we consider that there is no horizontal pleiotropy²⁰. In addition,we also used scatter plots and funnel plots.Scatter plots display whether there is a quantitative correlation trend between variables and observe whether there are outliers, inorder to analyze the impact of these outliers on data analysis.The forest map displays the effect sizes and confidence intervals of each study, demonstrating the possibility of heterogeneity.

In order to investigate the causal relationship between various cathepsins and different BMJD,we first selected GWAS data of 9 cathepsins (cathepsinsB,E,F,G,H,L2,O,S,andZ)as exposure factors,which were associated with osteoarthritis,rheumatoid arthritis,ankylosing spondylitis,and peripheral arthritis (Crohn's disease,ulcerative colitis,and postdysenteral joint disease),Two-Sample MR analysis was conducted on GWASdata of psoriatic arthritis,lower back pain,spinal stenosis,osteoporosis,bone necrosis,connective tissue disease,cartilage disease,benign tumors,myositis,synovitis and tenosynovitis(radial styloid tenosynovitis),shoulder periarthritis,neuromuscular junction disease,and muscle atrophy.

We have conducted a series of studies and visualized the results obtained (Table 1, Fig. 3) to reveal that our study found a positive correlation between high levels of cathepsinE and osteoarthritis (inverse variance weighted(IVW): p = 0.006810154,OR = 1.062864,95%confidence interval(CI) = 1.0169496–1.110852),and a positive correlation between high levels of cathepsin F and radial styloid tenosynovitis (inverse variance weighted(IVW): p = 0.04593187),OR = 1.2128231,95%confidence interval(CI) = 1.0034879 − 1.465827),high levels of cathepsin G are positively correlated with ankylosing spondylitis(inverse variance weighted(IVW): p = 0.03940755,OR = 1.000778,95%confidence interval(CI) = 1.000377 − 1.001519), and high levels of cathepsin L2 are positively correlated with cartilage disease (inverse variance weighted(IVW):p = 0.03305462,OR = 1.608014,95%confidence interval(CI) = 1.0389409 − 2.488793),high levels of cathepsin S are positively correlated with rheumatoid arthritis (inverse variance weighted (IVW): p = 0.003952175,OR = 1.000632,95%confidence interval(CI) = 1.00022-1.0001062);high levels of cathepsin B are negatively correlated with ulcerative colitis arthritis (inverse variance weighted(IVW): p = 0.00813986,OR = 0.5807975,95% confidence interval(CI) = 0.388367–0.8685747), while high levels of cathepsin E are negativelycorrelated with musculoskeletal connective tissue disease (inverse variance weighted (IVW):p = 0.04510052,OR = 0.9717213,95%confidence interval(CI) = 0.9448335 − 0.9993743),High levels of cathepsin E are negatively correlated with spinal stenosis (inverse variance weighted(IVW):p = 0.03176659,OR = 0.9300667, 95%confidence interval(CI) = 0.8705126 − 0.9936951),while high levels of cathepsin F are negatively correlated with muscle atrophy (inverse variance weighted (IVW):p = 0.02719006,OR = 0.5246858,95%confidence interval(CI) = 0.2960375 − 0.9299333),high levels of cathepsin H are negatively correlated with benign bone tumors (inverse variance weighted(IVW):p = 0.0025007,OR = 0.8610156, 95%confidence interval(CI) = 0.7814085 − 0.9487327), while high levels of cathepsin L2 are negatively correlated with myositis (inverse variance weighted(IVW):p = 0.004498949,OR = 0.4895089,95%confidence interval (CI) = 0.2990322 − 0.801315).

In order to explore the possibility of a reverse causal relationship between cathepsins and BMJD,researchers conducted reverse MR analysis.The results indicate that osteoporosis may lead to elevated levels of cathepsin B (inverse variance weighted(IVW):p = 0.03801203,OR = 1.099211,95%confidence interval(CI)=1.0052443–1.201961) and cathepsin H(inverse variance weighted (IVW): p = 0.007021102,OR = 1.1308304,95%confidence interval(CI) = 1.0341299–1.236573).

Muscle nerve junction diseases may lead to elevated levels of cathepsin F (inverse variance weighted (IVW):p = 0.002637921),OR = 1.095448,95%confidence interval(CI) = 1.0322522–1.162513) levels increase, ulcerative colitis joint disease may lead to a decrease in cathepsin B (inverse variance weighted(IVW): p = 0.0114182, OR = 0.9850488,95% confidence interval(CI) = 0.9736182–0.9966136) levels, synovitis and tenosynovitis may lead to a decrease in cathepsin S (inverse variance weighted (IVW):p = 0.02021788) levels, The levels of cathepsin Z (inverse variance weighted (IVW):p = 0.007957709, OR = 2.08E-06,95%confidence interval(CI) = 3.13E-11 − 1.29E-01) and cathepsin Z (inverse variance weighted (IVW):p = 0.007957709,OR = 3.08E-07,95%confidence interval(CI) = 4.78E-12 − 1.98E-02) decreased.

The pathogenesis of BMJD is a result of multiple factors, which may be closely related to protein hydrolysis,and cathepsins play a crucial role in this process. We searched for articles on cathepsins and found that there are few Mendelian reports on the causal relationship between cathepsins and skeletal muscle diseases. In order to comprehensively analyze the causal relationship between the two,we used genetic variations strongly correlated with exposure factors as instrumental variables to systematically analyze the causal relationship between nine different cathepsins and the risk of various skeletal muscle diseases.To the best of our knowledge,this is an MR analysis based on genome-wide sequencing data (GWAS) from large-scale cohort studies,aimed at establishing causal relationships between cathepsins and BMJD.

Through analysis, we found that cathepsin E is positively correlated with osteoarthritis, cathepsin F is positively correlated with radial styloid tenosynovitis,cathepsin G is positively correlated with ankylosing spondylitis, cathepsin L2 is positively correlated with cartilage disease, and cathepsin S is positively correlated with rheumatoid arthritis;Cathepsin B is negatively correlated with ulcerative colitis arthritis, cathepsin E is negatively correlated with musculoskeletal connective tissue disease, cathepsin E is negatively correlated with spinal stenosis,cathepsin F is negatively correlated with muscle atrophy,cathepsin H is negatively correlated with benign bone tumors,and cathepsin L2 is negatively correlated with myositis. Through reverse MR analysis,we found that osteoporosis may lead to elevated levels of cathepsin B and cathepsin H, muscular nerve junction disease may lead to elevated levels of cathepsin F, ulcerative colitis joint disease may lead to decreased levels of cathepsin B, synovitis and tenosynovitis may lead to decreased levels of cathepsin S and cathepsin Z.

CatheE is an intracellular aspartic protease belonging to the peptidase A1 family of proteases, mainly distributed in immune related cells and epithelial cells²¹.Osteoarthritis is a degenerative cartilage disease characterized by the degeneration, destruction, and periarticular bone hyperplasia of articular cartilage²².Fibrous synovial cells in patients with osteoarthritis regulate the expression of Th17 cell transcription factor Foxp3 by expressing IL-6, which can induce osteoclast maturation²³. Another study showed that macrophages can secrete inflammatory cytokines,differentiate into osteoclasts, and promote osteolysis²⁴.However,in an animal experiment,it was shown that in a rat model,cathepsin E showed a high level of preferential immune response in active osteoclasts, while neighboring osteoblasts lacked its immune response.This feature leads to loss of extracellular matrix, extensive cartilage loss, and fibrosis, resulting in degeneration of articular cartilage and ultimately complete loss of cartilage surface²⁵.These pieces of evidence strongly suggest that cathepsin E is involved in the extracellular degradation of bone organic matrix and the intracellular breakdown of substances ingested by osteoclasts, which is closely related to the occurrence of osteoarthritis. We did not find direct evidence to support the correlation between cathepsin E and musculoskeletal connective tissue diseases. However,we found that in a rat experiment, the application of estradiol and progesterone to ovariectomized rats significantly increased the activity of cathepsin E,which provided a protective effect on uterine fibroconnective tissue²⁶.Another study showed that in mice with cathepsin E deficiency, developmental defects in adipose tissue27 were found²⁷.This seems to support our viewpoint,but a large number of experiments are still needed to further draw conclusions.

Cathepsin G (CathG) is one of the effector factors of inflammation²⁸,which can regulate the inflammatory response by stimulating the production of cytokines and chemokines.These cytokines and chemokines can activate immune cells and transfer them to the site of pathogen or tissue damage, thereby producing an immune response²⁹.In addition,tissue protein G is also involved in antigen presentation and specific immune responses. Ankylosing spondylitis is a chronic inflammatory disease that mainly affects the sacroiliac joint,spinal axis joints,ligaments,and tendon adhesions.It is an autoimmune disease characterized by pain, stiffness, and limited mobility. The etiology of this disease is not fully understood and may be related to multiple factors such as genetics,immunity,and infection^30–31.Research has shown that NK cells are closely related to the pathogenesis of AS,and cathepsin G can enhance the cytotoxicity of NK cells while binding to them,thereby participating in the development of AS.

Cathe B is a cysteine protease that exists in the late endosomes and lysosomes of mammals. It is widely present in liver,spleen,kidney,bone,nerve cells,interstitial fibroblasts, macrophages,and other parts,and is also overexpressed in many cancer cells.As a widely distributed cathepsin in immune cells,it is associated with some immune diseases³².A study showed that³³ after feeding mice with a high-fat diet for six weeks,the activity of cathepsin B in the adipose tissue of mice was significantly enhanced,with a large amount of F4/80 + CD11b macrophages infiltrating, producing a large amount of pro-inflammatory factors,and ultimately leading to the death of adipocytes.This indicates that the activation of cathepsin B is related to the production of pro-inflammatory factors.A clinical study has shown that³⁴ cathepsin B is associated with gastrointestinal tumors and ulcerative colitis, and may be one of the biomarkers for disease diagnosis and prognosis.Another study also showed that³⁵ there is an increasing trend of CTSB mRNA expression in IBD mucosa; Ulcerative colitis, as an inflammatory bowel disease, can present with extraintestinal manifestations in 20–35% of patients, and up to 25% of patients may have prior intestinal symptoms. Among these extraintestinal manifestations, peripheral arthritis is the most common³⁶.

Cathepsin H (CatH) is a cysteine protease that exists in the cells of animal tissues, especially in the lysosomal part.It has unique aminopeptidase activity and is widely expressed in the lungs, pancreas,thymus, and skeletal muscles.Due to its specific enzyme activity,CatH has multiple physiological functions, participating in cell apoptosis, tissue remodeling, and immune response, and is closely related to tumor cells³⁷.A study on animals has shown that cathepsin H is involved in regulating mRNA expression in mouse bone morphogenetic protein 4 (BMP-4), which may be related to the occurrence of osteosarcoma³⁸.However, a study has shown the presence of cathepsins B,H,K,L,and SmRNA in human chondrosarcoma, which also suggests a possible association between cathepsins H and some bone tumors³⁹.The causal relationship between cathepsins H and the occurrence of bone tumors is still not fully determined. However, some studies have shown that the expression of cathepsin in malignant tumors is significantly higher than that in precancerous and benign lesions⁴⁰.

Cathepsin L (CTSL) is a major member of the lysosomal cysteine protease family and belongs to the papain family. Participated in the release of antigenic peptides and maturation of MHCII class molecules, as well as in the turnover of elastin fibrils, intracellular antigen presentation, T cell sorting, cell apoptosis, embryonic development, and extracellular substrate lysis⁴¹.The expression of cathepsin L is mainly related to chondrocyte hypertrophy⁴².Research on some mammals has found that CTSL is involved in the processing of various proteins and hormonal precursors, as well as the maturation process of functional proteins. Some studies have shown that cathepsins have a damaging effect on articular cartilage in osteoarthritis and rheumatoid arthritis^43–46. Cathepsin L is abundant in various types of cells in degenerative intervertebral discs. A study has shown that CTSL and CTSD are positive in the fibrous ring and cartilage of osteoarthritis and rheumatoid arthritis, providing direct evidence for the involvement of cathepsin L in human intervertebral disc cartilage degeneration⁴⁷.On the other hand, we found that the activity of active cathepsin L also showed an upward trend in the injured supraspinatus tendon⁴⁸.

CatheS, a cysteine protease belonging to the papain superfamily,is highly expressed on antigen-presenting cells.It has catalytic hydrolysis activity against various proteins including MHCII constant chains and extracellular matrix components, especially elastin and collagen. Cathepsin S is involved in a variety of diseases, including Alzheimer's disease, atherosclerosis, tumors, osteoarthritis, neuralgia and a variety of autoimmune diseases ^49–50.In neutral or slightly acidic environments, cathepsin S has the potential to degrade proteoglycans⁵¹. Expression of cathepsin S can be observed on synovial macrophages in rheumatoid arthritis (RA). During the inflammatory process, this enzyme is secreted into the cartilage matrix, which may disrupt the integrity of the cartilage aggregation protein glycan-CII network tissue, which has the function of forming functional cartilage⁵².A study confirmed that the levels of cathepsin S and cathepsin L in the serum and synovial fluid of ACPA and RF positive patients were correlated with DAS28 and CRP, but not in serum negative patients⁵³.Another human experiment based on probe detection also demonstrated that more cathepsin S was detected in the synovial fluid and serum of patients with rheumatoid arthritis. Compared to osteoarthritis, the correlation between cathepsin S and rheumatoid arthritis was more significant⁵⁴.

We found that osteoporosis may lead to elevated levels of cathepsin B.Cathepsin B is closely related to many serious diseases,such as cancer,osteoporosis,and autoimmune diseases.Some studies have shown that osteoporosis is a disease characterized by an imbalance between osteoblasts and osteoclasts,leading to decreased bone mass,destruction of bone tissue microstructure, and increased bone fragility, which may be related to overexpression of cathepsin B (CatB)⁵⁵.Some research reports suggest that the levels of pathogenic bone loss and osteoclast generation induced by osteoclast differentiation factor (RANKL) are positively correlated with increased cathepsin B and K activity⁵⁶.The decrease in the expression level of cathepsin B can inhibit bone mineralization. Administering parathyroid hormone (PTH) that promotes bone mineralization can reduce the expression of cathepsin B. However,it can promote the gene expression and protein level of tissue-specific alkaline phosphatase (TNALP), phosphoethanolamine/phosphatidylcholine phosphatase (PHOSPHO1), and exonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1),inducing mineralization processes in osteoids. Simultaneously stimulating the expression of the phosphate regulator (PHEX) and bone morphogenetic protein (SIBLING) family to maintain a balance between matrix protein accelerated synthesis and mineralization⁵⁷ may further support our conclusion.

Finally,we regret to find that some of our results cannot be explained by existing theories, such as the positive correlation between cathepsin F and radial styloid tenosynovitis,the positive correlation between cathepsin F and osteoporosis,the negative correlation between cathepsin F and muscle atrophy,the negative correlation between cathepsin E and spinal stenosis,and the negative correlation between cathepsin L2 and myositis,There is a positive correlation between osteoporosis and cathepsin H,a positive correlation between muscle nerve junction disease and tissue protein F,a negative correlation between ulcerative colitis arthritis and cathepsin B, and a negative correlation between synovitis and tenosynovitis and cathepsin S and cathepsin Z. We consider that the following situations may occur due to; Firstly, due to the relatively small impact of genetic variation on risk factors in MR design (i.e.,the generation of a small portion of variation),this may result in lower statistical power of our analysis and a risk of false negative results⁵⁸.Secondly, perhaps due to the small sample size of this study, our partial statistical significance and accuracy are insufficient⁵⁹.Thirdly, we speculate that there may be some cathepsin efficacy that needs further exploration. Finally, we found that some cathepsins, such as cathepsins S and H,may be involved in the activation of the human immune response⁶⁰.Some proteases are associated with inflammasomes or participate in certain inflammatory pathways ^61–62,which may be related to the pathogenesis of BMJD, inflammation, and fibrosis.

We searched relevant literature and found that this is the first MR study to systematically investigate the causal relationship between cathepsin and the risk of BMJD in a European population. This study contributes to a better understanding of the association between cathepsins and BMJD. With the deepening research on BMJD, the exploration of related biomarkers is also receiving increasing attention. Cathepsin has demonstrated its potential research value in this field. This study utilized advanced genetic analysis methods to explore the causal relationship between different cathepsins and various BMJD. Through the integration of Mendelian randomization analysis and reverse Mendelian randomization analysis, we strive to ensure the accuracy of the results. This series of research methods provide us with robust research conclusions, thereby strengthening the causal relationship between cathepsin and BMJD.

It is worth noting that this study is mainly based on individuals of European ancestry, therefore, further research is needed on its applicability to other ethnic groups. In addition, due to sample size limitations or uneven random grouping, experimental results may deviate. However, our study still provides some valuable findings, such as the correlation between cathepsin B and ulcerative colitis arthritis, cathepsin E and osteoarthritis, cathepsin E and skeletal connective tissue disease, cathepsin G and ankylosing spondylitis, cathepsin L2 and cartilage disease, and cathepsin S with rheumatoid arthritis. These preliminary research results not only provide us with a deeper understanding of BMJD, but may also provide new biomarkers for future disease prediction, early diagnosis, and prognosis evaluation. In addition, research on inhibitors of cathepsins related to specific BMJD may also provide new directions for future treatment strategies.

Acknowledgements

We would like to thank the European Bioinformatics Center, GWASCatalog database, FinnGen database, UK Biobank database, IEUOpenGWAS database, and INTERVAL research for sharing GWAS data. We would like to thank anonymous commentators for their constructive feedback.

Authors’ contributions

Authors Yang Wei, Han Xiuzhen,Cui Miao, and Guan Mengqi contributed to the conceptualization and design of this study. Li Zhenhua supervises the research process. Yang Wei, and Yang Peng conducted data analysis.Yang Wei wrote the manuscript. Han Xiuzhen and Yu Qingyuanrevised and polished the manuscript. All authors have revised and approved the final manuscript.

Funding

There was no financial support for the research.

Availability of data and materials

The GWAS summary statistics of cathepsin can be obtained by querying the IEUOpenGWAS database on the website (https://gwas.mrcieu.Ac. uk/) query obtained. The GWAS summary statistical data of bone, muscle & joint diseases can be obtained by querying the IEUOpenGWAS database on the website（ https://gwas.mrcieu.ac.uk/ ）

Ethics approval and consent to participate.

These analyses were conducted based on the INTERVAL study, which included a total of 3301 European individuals. All participants signed informed consent for the experiment and obtained approval from the National Research Ethics Service (11/EE/0538).

Author details

1.College of Traditional Chinese Medicine, Changchun University of Traditional Chinese Medicine, No. 1035, Boshuo Road, Changchun, 130117

2.Jiangsu Province Hospital of Traditional Chinese Medicine，No. 155 Hanzhong Road, Nanjing, 210029.

3.Basic College of Changchun University of Traditional Chinese Medicine, No. 1035 Boshuo Road, Changchun City, 130117

4.College of Integrated Traditional Chinese and Western Medicine, Changchun University of Traditional Chinese Medicine, No. 1035 Boshuo Road, Changchun City, 130117

5.School of Physics, South China Normal University, No. 378 Outer Ring West Road, Guangzhou, 510006

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A Mendelian randomization study: Association of Cathepsin with Osteoarticular Muscle Diseases

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