1. Introduction

Melilotus officinalis (L.) Pall. (M. (L.) Pall.) is a traditional Chinese medicine that is widely distributed and has broad prospects for development and utilization [1]. M. (L.) Pall. has antiedema, antioxidant, and hepatoprotective properties [1–3]. In ancient China, M. (L.) Pall. was used to treat a variety of chronic diseases [4]. M. (L.) Pall. is often used to reduce postoperative edema and promote early recovery after clinical orthopedic and sports medicine procedures. However, the function of M. (L.) Pall. in chronic skeletal muscle injury is unproven.

Skeletal muscle injuries are one of the most common sports injuries, accounting for approximately 40% of sports-related injuries in older people [5]. Muscles can be damaged by external forces, biological factors, and chemical factors [6, 7]. Excessive chronic injuries will lead to scar formation and fat infiltration [8, 9]. Therefore, understanding the factors influencing muscle repair can help promote skeletal muscle repair [10, 11]. Recent topical studies have explored the spatial-positional interactions of skeletal muscle regeneration and their underlying mechanisms to find new ways to improve the repair potential of skeletal muscles [12, 13]. Recent studies have found that the positional information driving limb muscle patterns are contained in the fibroblasts of the connective tissues [14]. Our previous studies have annotated and functionally analyzed these cells [15]. These cells have extensive intercellular interactions via the ligand-receptor pathway.

Liu et al. isolated 29 compounds from M. (L.) Pall. alcoholic extracts. They have good antioxidant activity and play an important role in anti-inflammatory and antioxidant functions [16]. Recent studies have also found that M. (L.) Pall. promotes wound repair [17]. This study provides a new way to explore these effects of M. (L.) Pall. in skeletal muscles by targeting the ligand-receptor pathway, which is important for drug function [18, 19]. Studying the effects of M. (L.) Pall. in the skeletal muscle fibroblasts allows us to explore its potential mechanisms in muscle repair patterns.

In this study, network pharmacology and molecular docking approaches have been used to predict the possible regulatory targets of M. (L.) Pall. in muscle repair patterns, to reveal the potential molecular mechanisms of this compound in regulating muscle repair patterns and provide new ideas for the treatment of skeletal muscle injury.

2. Materials and Methods

3. Results

3.1. Screening for Potentially Active Compounds in M. (L.) Pall.

Figure 1 depicts the research flow of this study. The active ingredients of M. (L.) Pall. are based on previous research results [16]. Six active ingredients were screened according to the following conditions: OB ≥ 30% and DL ≥ 0.18 (Table 1).

Table 1. Six active ingredients of M. (L.) Pall. were screened.

Molecule name	Molecule ID	OD	Drug-likeness
Scoparone	MOL001999	74.75	0.09
Ferulic acid	MOL000360	39.56	0.06
Soyasapogenol E	MOL003651	37.64	0.75
Beta-sitosterol	MOL000358	36.91	0.75
Luteolin	MOL000006	36.16	0.25
Salicylic acid	MOL001801	32.13	0.03

3.2. Potential M. (L.) Pall. Targets of on the MTJ Cells

Five of the six active ingredients had 87 corresponding targets (after excluding duplicates). The “herbal-active-component-disease target gene” regulatory network was constructed (Figure 2(a)). The red oval in the diagram represents the gene corresponding to the target protein. The green triangle represents the active ingredient. MTJ cells from the GSE168153 dataset were used for ligand-receptor analysis. All 246 potential ligand-receptor key genes were extracted. Cross-talk analysis revealed that M. (L.) Pall. might regulate the proteins of five genes (Figure 2(b)). Correlation analysis revealed that the expressions of CCL2, VEGFA, MET, MMP2, and EGFR were positively correlated with each other (Figure 2(c)).

3.3. Potential Effects of M. (L.) Pall. on MTJ Cells

As previously described, the data and cellular annotations for ligand-receptor maker analysis were obtained from GSE168153 database and our previous study, respectively. The receptor-ligand marker analysis of the MTJ cells was performed using the CellphoneDB algorithm (v2.1.2) (Figure 3(a)). The intersection analyses between the target enrichment pathways of M. (L.) Pall. and ligand receptor-related pathways of MTJs are shown in Figure 3(b). These target genes were found to be enriched in 28 KEGG pathways (EGFR tyrosine kinase inhibitor resistance, cancer-related pathway, epithelial cell signaling in Helicobacter pylori infection, rheumatoid arthritis, endocrine resistance; ErbB, MAPK, PI3K−Akt, Rap1, HIF−1, relaxin, estrogen, GnRH, and AGE−RAGE signaling pathways in diabetic complications; and so on). This suggests that M. (L.) Pall. may target some intercellular signaling pathways, such as neuropeptide-related, tumor-related, and stress direction-related pathways (Figures 2(b) and 2(c)). These genes were also enriched in metabolic functions (MF), including transmembrane receptor protein tyrosine kinase, receptor-ligand, and growth factor activities. Moreover, these genes were found to be enriched in cellular functions (CC), including coated vesicle, clathrin-coated vesicle membrane, and coated vesicle membrane. In biological processes (BP), these genes are enriched in the positive regulation of protein kinase B signaling, peptidyl-tyrosine modification, and peptidyl-tyrosine phosphorylation (Figures 4(a) and 4(b)).

3.4. Regulatory Relationship and Molecular Docking Analysis of Active Ingredient-Target Proteins of M. (L.) Pall.

Pharmacological database analysis suggests that scoparone, the active ingredient of M. (L.) Pall. targets CCL2 and luteolin targets VEGFA, MMP2, MET, and EGFR (Figure 5(a)). CCL2, VEGFA, MMP2, MET, and EGFR were molecularly docked to the two key pharmacodynamic components (scoparone, luteolin) of M. (L.) Pall. The lowest docking binding energy is shown in Figure 5(b). These molecular dockings are visualized in Figures 6(a)–6(e). The hydrogen bonds are indicated using dashed lines, and the distances between the hydrogen bonds and the compounds are also marked. The scoparone target binding CCL2 protein has a free energy of -6.5 kJ/mol. The free energies of luteolin target binding energy for VEGFA, MMP2, MET, and EGFR proteins are -8.9, -10.8, -7.7, and -6.9 kJ/mol, respectively. The minimum binding energies of these two key components to CCL2, VEGFA, MMP2, MET, and EGFR were less than -5.0 kJ/mol suggesting that M. (L.) Pall. exerts its effects mainly by targeting these components through scoparone and luteolin.

3.5. Molecular Dynamics Simulations of MMP2 and Luteolin

In the above molecular docking analysis, MMP2 and luteolin had the minimum binding free energy. The free energy of luteolin target binding energy for MMP2 is -10.8 kJ/mol. To further assess the binding efficacy of MMP2 and luteolin, MD simulations were performed (Figure 7(a)). Due to the interaction between MMP2 and luteolin, the root mean square displacement (RMSD) was found to increase at first and then stabilise (Figure 7(b)). The radius of gyration (Rg) of the MMP2-lutelin complex was also found to stabilise with the time passing (Figure 7(c)). In addition, the number of hydrogen bonds formed by MMP2 with luteolin remained in a relatively stable range (Figure 7(d)). The overall free energy in the system was also found to be stable (Figure 7(e)). Finally, MD simulations revealed that luteolin can stably target binding to MMP2.

4. Discussion

To the best of our knowledge, this study is the first to assess the impact of M. (L.) Pall. on the musculotendinous junction. According to the TCMSP database, M. (L.) Pall. has five active ingredients (OB ≥ 30% and DL ≥ 0.18) potentially acting on 87 targets. Therefore, the “herb-active ingredient-disease target gene” network was constructed. We found that M. (L.) Pall. influences the interaction of fibroblasts in muscle-tendon junctions and affects muscle repair patterns through the modulation of five ligand receptor-related proteins (CCL2, VEGFA, MMP2, MET, and EGFR) using the active ingredients luteolin and scoparone. And MD simulations revealed that luteolin can stably target binding to MMP2.

Scoparone is a naturally occurring coumarin found in green plants. It is purified from a lipid-lowering herb that reduces the proliferation of human peripheral blood mononuclear cells, scavenges reactive oxygen species, inhibits tyrosine kinase, and enhances the production of prostaglandins [36]. Recent studies have confirmed that scoparone has various biological activities such as antifibrosis, antioxidant, and fat differentiation inhibition [37–39]. Scoparone was also found to inhibit high-glucose-induced activation of the ERK1/2 signaling pathway in thylakoid cells and played an active role in inhibiting the accumulation of extracellular matrix in the high-glucose microenvironment [40]. In this study, scoparone was found to target CCL2. CCL2 is a ligand for CCR2. Inhibition of CCR2 after injury promotes skeletal muscle regeneration and function recovery [41, 42]. Our study suggests that scoparone may inhibit the function of CCR2 by binding to CCL2, and this may be a potential mechanism for M. (L.) Pall. to promote skeletal muscle regeneration.

Recent studies have found that luteolin protects skeletal muscles from attrition caused by inflammation and downregulates the expression of proteins associated with muscle catabolism [43]. Luteolin increased muscle strength in fatigued subjects and improved skeletal muscle contraction during ischemia and reperfusion [44]. In this study, luteolin targeted VEGFA, MMP2, MET, and EGFR. Luteolin inhibits VEGFA and affects microvascular networks formed during neovascularization in mice [45]. In tumors, luteolin inhibits MMP2 and MET [46–48]. Luteolin also inhibits downstream signaling molecules activated by EGFR, especially the Akt and MAPK signaling pathways [49].

Luteolin was found to target MMP2 in MD simulations. Elevated levels of MMP2 expression are associated with insulin resistance due to extracellular matrix (ECM) remodelling in skeletal muscle [50]. This suggests that luteolin targeting of MMP2 may have the potential to improve insulin resistance due to ECM. MMP2 has been found to be widespread in skeletal muscle, and therefore, the study of its function is important for exploring the repair of skeletal muscle after injury [51]. For example, both exogenous hydrogen sulphide and electroacupuncture treatments can improve skeletal muscle injury and reduce skeletal muscle fibrosis by downregulating MMP2 and related pathways [5, 52]. In the present study, luteolin was found to target MMP2, which may contribute to skeletal muscle injury repair.

This study provides new data supporting the treatment of sports injuries using M. (L.) Pall. and provides a theoretical basis for clinical application. All the core components of M. (L.) Pall. were screened and docked successfully with their key targets. The core components of M. (L.) Pall. have good binding activities to their key targets, suggesting that they can effectively treat skeletal muscles. However, more Chinese medicine databases need to be used, and target prediction databases need to be improved. This study did not directly examine the chronic muscle injury dataset, but rather by examining the MTJ fibroblastic dataset, which may be biased. Furthermore, more in vivo and ex vivo clinical studies are also needed to validate the mechanisms of action M. (L.) Pall. in the treatment of chronic skeletal muscle injury.

5. Conclusion

In summary, this study demonstrates that M. (L.) Pall. can skeletal muscle injury by acting on CCL2, VEGFA, MMP2, MET, and EGFR, through luteolin and scoparone. They are the key active ingredients of M. (L.) Pall. and affect intercellular signaling, such as neuropeptide-related, tumor-related, and stress-related pathways. M. (L.) Pall. may influence the interaction of fibroblasts in muscle tendon junctions to affect muscle repair patterns. Molecular docking analysis validated some of the network pharmacology results and confirmed the multicomponent, multitarget, and multipathway characteristics of M. (L.) Pall. in the treatment of skeletal muscle injury.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Yisheng Chen, Zhiwen Luo, Jinrong Lin, and Beijie Qi contributed equally to this work.