Osteoarthritis (OA), the most prevalent joint illness, is a degenerative condition marked by joint inflammation and the loss of cartilage (1). OA mainly impacts load-bearing joints, such as the knee and hip, causing chronic pain and dysfunction in the affected joints, and thus seriously affecting the patient’s quality of life (2). Although the pathogenetic mechanisms of OA are diverse, previous studies have shown that inflammatory responses and joint immune disorders are significantly involved in the formation and development of osteoarthritis and affect the clinical presentation and prognosis of patients to varying degrees (3). In recent years, as the research on OA has gradually intensified, the relationship between OA and other diseases has gradually attracted the attention of researchers (4).

Corona Virus Disease 2019 (COVID-19) refers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing pneumonia, first discovered in 2019 (5). The COVID-19 pandemic poses serious challenges to the global economy and development (6). As research on COVID-19 progresses, increasing evidence suggests that COVID-19 is closely associated with immune dysregulation and abnormal inflammatory responses (7, 8). For example, Schimke et al. found that patients with severe COVID-19 were characterized by excessive neutrophil activation and that the degree of activation correlated with the severity of COVID-19 patients (9). Therefore, the relationship between COVID-19 and inflammatory disease and immune-related diseases has been extensively studied, for example, Hu et al. uncovered the relationship between COVID-19 and RA and identified genes that are significantly different and play an important role in both diseases (10). However, the shared molecular pathways and therapeutic strategies between OA and COVID-19 are rarely mentioned.

In a predictive model examining the survival of COVID-19 patients, we found OA to be a predictor associated with this model, which drew our attention (11). Interestingly, Paxlovid, a drug widely used to treat COVID-19, has been shown to accelerate cartilage degeneration in patients, thereby triggering or exacerbating osteoarthritis (12). In addition, Lauwers et al. found in their study that both COVID-19 and OA patients had dysfunction of the renin-angiotensin system and inflammatory response (13). Based on these findings, the potential association between COVID-19 and OA and its molecular mechanisms urgently needs to be further explored to find effective therapeutic plans or drugs to deal with OA caused or exacerbated by SARS-CoV-2 infection. For this purpose, in this study, we used RNA-sequencing (RNA-seq) data from OA and COVID-19 patients for bioinformatics, machine learning algorithms, and single-cell analysis to explore shared molecular pathways and therapeutic strategies between OA and COVID-19 for the first time. Finally, we identified key genes that contribute to the development of both OA and COVID-19 and then used network pharmacology to filter out potentially effective therapeutic agents for the OA patients infected with SARS-CoV-2.

Since the onset of the COVID-19 outbreak, the cumulative number of confirmed cases worldwide has exceeded 700 million, of which over 6 million have died (https://covid19.who.int/). For the treatment of COVID-19 patients, several brand-new antiviral drugs have been created. However, these antiviral drugs can cause damage to the skeletal muscle system, such as cartilage degeneration (17). In addition, studies have shown that immune dysregulation is prevalent in patients with severe COVID-19 (18). Interestingly, cartilage degeneration and disturbances in the immune microenvironment are the main features of OA, which is the most common joint disease. Although researchers have noted a common clinical presentation and some potential associations between COVID-19 and OA patients, further studies on the shared molecular pathways and therapeutic strategies between these two diseases are needed (19).

In a predictive model examining the survival of COVID-19 patients, we found OA to be a predictor associated with this model, which drew our attention (11). Therefore, to investigate the potential link between COVID-19 and OA, we researched all the literature related to COVID-19 and OA and found that most studies focused on changes in treatment, diagnosis, and recovery status of OA patients during COVID-19 isolation (20, 21). However, our attention was drawn to the study by Veronesi et al. who hypothesized that COVID-19 infection causes a strong inflammatory response that leads to damage to the skeletal muscle system of patients (22). Unfortunately, to this date, no studies have reported a potential connection between OA and COVID-19 in terms of shared molecular changes and their mechanisms and therapeutic strategies. Therefore, in this paper, we explored for the first time the potential links and molecular mechanisms between OA and COVID-19 using bioinformatics, machine learning algorithms, and single-cell analysis. Firstly, the DEGs between OA and COVID-19 patients and normal subjects were identified separately. Then, we used WGCNA to identify genes in the modules most associated with OA and COVID-19 and crossed them with DEGs to find 26 common genes involved in OA and COVID-19 occurrence. Functional and pathway enrichment analysis is an important research method in bioinformatics, which helps to investigate the molecular mechanisms and regulatory roles of genes. Therefore, we performed functional and pathway enrichment analyses of 26 common genes, including GO enrichment analyses, KEGG pathway analyses, Reactome pathway analyses, and WikiPathway pathway analyses. The results indicate that the common genes of OA and COVID-19, in terms of GO, are mainly enriched in biological functions related to immunity, such as macrophage differentiation, T cell receptor complex, and MHC class II protein binding, and in KEGG pathway enrichment analysis are significantly enriched in pathways such as Primary immunodeficiency. These results are consistent with our finding that immune dysregulation is involved in both OA and COVID-19 patients. In addition, Mitochondrial Uncoupling in Reactome pathway analysis results in the involvement of OA onset and progression, as well as in the oxidative stress regulation and status of inflammation in COVID-19 patients (23, 24). Similarly, in WikiPathway pathway analysis results, SARS-CoV-2 altering angiogenesis via NRP1 and Endochondral Ossification are involved in the progression of COVID-19 and OA, respectively. Furthermore, a study showed that curcumin, which has anti-inflammatory and antioxidant effects, can be used to treat a variety of diseases, including COVID-19 and OA, which also suggests that inflammation and oxidation may be the shared pathogenic mechanisms in COVID-19 and OA (25). More importantly, in one study, Lauwers et al. found there were abnormal inflammatory responses and pain in COVID-19 patients, which is also characteristic of OA, due to endothelial dysfunction and adipose tissue dysfunction mediated by ACE2. Therefore, they hypothesized that dysfunction of the renin-angiotensin system and inflammatory response are common pathogenic molecular pathways in patients with COVID-19 and OA (13). Based on the above results, we are more certain that there are similar pathological processes and molecular changes between OA and COVID-19, which are mainly related to excessive inflammatory response. To further explore the molecular changes that play the most critical role in the formation and development of two diseases, we used the machine learning algorithm, LASSO logistic regression analysis, to screen for four key genes, DDIT3, MAFF, PNRC1, and STX11, out of 26 common genes. Differential gene expression is important for revealing the occurrence of disease at the genetic level, so we performed differential expression analysis of key genes in the train set GSE114007 and GSE147507 of OA and COVID-19, and verified the results in the validation sets GSE55235 for OA and GSE171110 for COVID-19. In a further step, we plotted ROC curves to analyze the predictive power of key genes for OA and COVID-19 in the train and validation sets. However, we found that the differential expression of STX11 was not statistically significant in GSE55235 and GSE147507. We, therefore, decided to abandon STX11 and carried out further research on DDIT3, MAFF, and PNRC1. Moreover, we noticed that DDIT3, MAFF, and PNRC1 were better predictors for OA, with AUC values greater than 0.85 in both the training and validation sets of OA, and may serve as new targets and perspectives for the diagnosis and treatment of OA. Therefore, we compared them with the diagnostic markers of OA identified in other published studies. In the study of Xia et al, ferroptosis-related genes such as CDKN1A and EGR1 were found to be potential diagnostic markers for OA, with the highest diagnostic value being JUN, which had AUC values as high as 0.989 and 0.96 in the ROC curves of the experimental and validation groups, but its diagnostic value was still lower than that of DDIT3 and MAFF, which we found to be the highest diagnostic markers for OA, with AUC values as high as 1.000, 0.997, 0.960, and 1.000 in the experimental and validation groups, respectively (26). In the study of Hu et al., we found the same result that DDIT3, MAFF, and PNRC1 had better diagnostic values (27). Although the diagnosis of COVID-19 is currently based on antigen testing and SARS-CoV-2 nucleic acid testing, the three key genes identified in our study are still important for the diagnosis of patients who suffer from both COVID-19 and OA. DNA damage-inducible transcription 3 (DDIT3), which increases apoptosis and is associated with adipogenesis and erythropoiesis, is significantly overexpressed in nasal epithelial cells with a high SARS-CoV-2 viral load and upregulates the TNF- a pathway (28). However, the pathogenic mechanism of DDIT3 in OA has not been elucidated, which may provide a new direction for future research on OA. MAFF, a key transcription factor associated with inflammation and lipid metabolism, has been shown to be significantly downregulated and play a critical role in OA (29). In addition, high MAFF expression can upregulate ACE2 and thus increase the inflammatory response in COVID-19 patients, resulting in poor prognosis (30). In contrast, the role of PNRC1, a marker with high diagnostic value for both OA and COVID-19, has not been investigated in OA and COVID-19, which provides a new perspective to study the potential connection and common pathogenesis between OA and COVID-19.

As we found in the functional analysis that immune disorders may be a shared pathological process and molecular changes between OA and COVID-19, we sought to explore the relationship between the expression of 3 key genes and pathological alterations in the immune microenvironment of OA and COVID-19 patients. To begin with, using the HPA database to explore the expression of three key genes in normal human blood immune cells, we discovered that DDIT3 was highly expressed in neutrophils, MAFF was highly expressed in basophils, NK cells, and neutrophils, but PNRC1 was not specifically expressed in immune cells. In addition, we investigated the correlation between the expression of key genes and the level of 22 immune cell infiltration in OA and COVID-19 patients with the CIBERSORT algorithm, which is shown in detail in the results. However, the CIBERSORT algorithm, which is mainly based on mRNA-seq data to analyze the immune cell composition of samples, applies to the analysis of immune cell composition in all diseases and normal subjects, which determines that its calculation results in different diseases will have a certain bias. Therefore we repeated this calculation 1000 times in the hope of minimizing bias. In addition, since it mainly calculates the immune cell composition of the sample and cannot express the gene expression of different immune cells in a given disease, we further performed single-cell analysis to investigate the expression patterns of DDIT3, MAFF, and PNRC1 in the immune cells of COVID-19 patients. Subsequently, to explore the specific role of the three key genes in the pathogenesis of COVID-19 development, we performed the single-cell analysis of scRNA-seq data from COVID-19 patients using the DISCO database. Comparing the results of the analysis with those of the HPA database, we found that the expression of DDIT3, MAFF, and PNRC1 in immune cells was the same as normal, indicating that the expression pattern of key genes in immune cells did not change during the progression of COVID-19. Increased neutrophils and decreased lymphocytes in the blood have been shown in many studies to be characteristic of severe COVID-19 patients and, more importantly, SARS-CoV-2 activates and induces the release of large numbers of neutrophil extracellular traps (NETs) from aggregated neutrophils, which can directly lead to lung epithelial cell damage and immune thrombosis, resulting in poor prognosis and increased risk of mortality (31, 32). In addition, Schimke et al. found that patients with severe COVID-19 were characterized by excessive neutrophil activation and that the degree of activation correlated with the severity of COVID-19 patients (9). Interestingly, the results of our study indicate that DDIT3, MAFF, and PNRC1 are all highly expressed in neutrophils, which strongly supports the importance of DDIT3, MAFF, and PNRC1 in the pathogenesis of COVID-19, and provides a new target for the treatment of severe COVID-19 patients with immune thrombosis caused by the substantial release of NETs. On the contrary, the role of macrophages and their transformation in the development of OA has been extensively studied, while the role of neutrophils has often been neglected. However, an increasing number of studies have shown that neutrophils are also involved in OA development and are required for OA progression (33, 34). Therefore, the key genes identified in this paper may provide new insights into the role of neutrophils in the pathogenesis of OA and may further contribute to the understanding of the pathogenesis of OA.

The regulatory networks of common genes - TF and common genes - miRNAs were built to identify the major variants common to OA and COVID-19 at the transcriptional level and to gain more insight into the regulation of common genes by transcription factors and post-transcriptional modification mechanisms of common genes by miRNAs. The networks showed that GATA2, FOXC1, CREB1, YY1, and TFAP2A were identified as the TFs most associated with common gene regulation, and hsa-mir-26b-5p, hsa-mir-26a-5p, hsa-mir-335-5p, hsa-mir-17-5p, and hsa-mir-98-5p were found to be the miRNAs with the highest correlation to common genes. Similarly, Huang et al. in a study identified YY1, CREB1, and GATA2 to be critical TFs involved in the regulation of COVID-19 patients at the genetic level in their pathogenesis (35). In addition, He et al. found that high expression of miR-204-5p downregulated FOXC1 expression in OA patients thereby suppressing the inflammatory response of synovial fibroblasts of OA (36). Paniri et al. discovered that hsa-mir-26b-5p might affect the susceptibility of COVID-19 patients to antiviral drugs (37). In addition, Rasheed et al. found that hsa-mir-26a-5p might be a new drug target for the treatment of OA due to its ability to modulate the NF-κB pathway in OA patients (38). In a study on key miRNAs for SARS-CoV-2 infection, hsa-mir-17-5p and hsa-mir-98-5p were found to play a key role in COVID-19 (39). The mechanism of action of these TFs and miRNAs in OA patients infected with SARS-CoV-2 has to be further studied, although several studies indicate they are important for the development and treatment of OA or COVID-19.

Since many antiviral drugs, such as Paxlovid, have been shown to accelerate cartilage degeneration in patients, thereby triggering or exacerbating OA, we tried to find suitable drugs for OA patients infected with SARS-CoV-2. Finally, we identified niclosamide, ciclopirox, and ticlopidine as potential therapeutic agents for the treatment of OA patients infected with SARS-CoV-2 by performing drug prediction on the shared genes of OA and COVID-19, and molecular docking and the free energy of binding estimation of predicted drugs with DDIT3, MAFF, and PNRC1 using Autodock software. Niclosamide, a widely used anthelmintic, has been shown in numerous studies to be a potential therapeutic agent for COVID-19. In a cellular experiment, Jeon et al. found that niclosamide had extremely high anti-SARS-CoV-2 efficacy (40). Brunaugh et al. similarly demonstrated the therapeutic efficacy of niclosamide on COVID-19 in a mouse model of COVID-19 (41). In addition, clinical trials conducted by Backer et al. and Cairns et al. have demonstrated the safety and efficacy of niclosamide for the treatment of SARS-CoV-2 patients (42, 43). Significantly, the treatment of patients with severe COVID-19 is still dominated by hormonal therapy such as dexamethasone, however, heavy hormone therapy can trigger secondary bacterial infections and immune dysregulation in patients, which is the main cause of death in COVID-19 patients. Whereas in studies for the treatment of COVID-19, niclosamide has shown not only the effects of inhibition on SARS-CoV-2 replication but also extremely strong bacteriostatic and immunomodulatory properties, which can greatly reduce the risk of death in patients with heavy COVID-19 (44–46). Although niclosamide has not been reported for the treatment of OA, we can speculate that niclosamide has an important value for the treatment of OA patients infected with SARS-CoV-2 based on its unique modulatory effect on the over-activation of the immune system, which is one of the mechanisms for the development of OA. Severe COVID-19 patients have a large number of lung microthrombi due to disruption of the immune system and severe destruction of alveolar endothelial cells, which can cause hypoxemia resulting from alveolar ventilation dysfunction and increase the risk of death in patients (47, 48). Moreover, immune system disorders can lead to a hypercoagulable state and arteriovenous thrombosis, which is responsible for the high rate of thrombotic complications in COVID-19 patients (49). Therefore, prophylactic use of anticoagulants in severe COVID-19 patients may improve patient survival (50). Interestingly, ticlopidine, a platelet inhibitor, not only can be used as a prophylactic anticoagulant in patients with severe COVID-19, but was also shown to counteract the intense endoplasmic reticulum stress caused by the SARS-CoV-2 virus infection, which leads to severe COVID-19, in a study by Tesei et al. and therefore has therapeutic value in patients with COVID-19 (51). Although the use of ticlopidine for OA treatment has not been reported, our study found that the free energy of binding between ticlopidine and the three shared key genes of OA and COVID-19 was extremely low, implying a stronger binding power, which may provide a new perspective for thromboprophylaxis and molecular therapy in OA patients infected with SARS-CoV-2. In terms of ciclopirox, as a broad-spectrum antifungal agent, studies for the use in the treatment of both OA and COVID-19 have not been reported. However, as we discussed before, severe COVID-19 patients with heavy hormone therapy can cause bacterial and fungal infections, and OA patients whose pain cannot be relieved by common medications are also commonly treated clinically with joint cavity injections of hormonal drugs to relieve patient pain, which can cause fungal septic arthritis (52). Therefore, future studies of Ciclopirox for the treatment of fungal infections secondary to COVID-19 or OA are interesting.

Notably, the present study focused only on the immunologically relevant role of key genes in the development of OA and COVID-19, but its role in other dimensions of OA and COVID-19 pathogenesis, as well as in SARS-CoV-2-infected OA patients, awaits further investigation.

In this study, we identified 26 common genes between OA and COVID-19 by WGCNA and screened three key genes DDIT3, MAFF, and PNRC1 using machine learning algorithms. The ROC curves showed high diagnostic power of the 3 key genes for both diseases In addition, using the single cell analysis we uncovered that key genes are involved in the pathogenesis of OA and COVID-19 through high expression in neutrophils. Finally, we identified niclosamide, ciclopirox, and ticlopidine as potential therapeutic agents for the treatment of OA patients infected with SARS-CoV-2 by drug prediction and simulated docking patterns to calculate the free energy of binding between key genes and drugs.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

YZ and ZD designed the study and performed the computational analysis. YG, TX, and YF collected the study data and wrote the manuscript draft. GL revised the draft and offered financial support for the project. All authors contributed to the article and approved the submitted version.