4.1 Analysis of critical targets of TMP for the treatment of OA
We screened ten potential core targets of TMP associated with OA by network pharmacology screening: ALB, ESR1, IL10, CAT, F2, C3, MPO, CYP3A4, CYP2C9, and ANXA1, and the results of molecular docking simulation also showed that TMP could present better binding with most of the potential core targets, with binding energies of less than − 5Kcal/ mol. However, we found that only the target genes of ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1, whose mRNA expression was increased in the TMP group(P < 0.05), showed statistically significant differences between the TMP group and OA group. This suggests that ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1 are most likely to be the pharmacological basis of TMP in treating OA.
In human articular chondrocytes, ESR binding to estradiol can inhibit NF-κB pathway activation and thus exert anti-inflammatory effects [16, 17]. CAT is an essential member of the antioxidant enzyme system in living organisms, which can break down H2O2 into H2O and O2, preventing the toxic effects of excessive H2O2 on cells [18]. C3 is a central component of the complement system, which interacts with tumor necrosis factor α (TNF-α), platelet-activating factor (PAF), interleukin 1 (IL-1), interleukin 6 (IL-6), and other factors. C3 is a central component of the complement system, which can synergize with or constrain various cytokines, such as tumor necrosis factor α (TNF-α), platelet-activating factor (PAF), interleukin 1 (IL-1), interleukin 6 (IL-6), and interleukin 8 (IL-8), and plays a vital role in inflammatory response, tissue and organogenesis, modulation of host immune function, mediation of apoptotic cell clearance, and facilitation of tissue repair after injury [19, 20]. There is also a close relationship between C3 and bone metabolism [21], and C3 can influence osteoclast differentiation through the OPG/RANKL/RANK signaling pathway [22]. CYP3A4 and CYP2C9 are members of the cytochrome P450 enzyme family, which can catalyze the synthesis of steroid hormones, regulate the metabolism of liposoluble hormones, and regulate the metabolism of therapeutic drugs [23–25]. ANXA1 (membrane-associated protein A1) can bind reversibly to phospholipid membranes and calcium ions and can inhibit the migration and adhesion of inflammatory cells [26], inhibit the synthesis and release of inflammatory mediators [27], promote apoptosis, and facilitate phagocytosis by macrophages [28], and is associated with NF-κB signaling pathway, serine/threonine protein kinase (Akt), and mitogen-activated protein kinase (MAPK) [29]. The above suggests that TMP is likely to participate in the body's oxidative stress, immunoinflammation, drug metabolism, cartilage and bone metabolism through its action on ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1, and thus play a role in treating OA.
4.2 Analysis of signaling pathways associated with TMP therapy for OA
From KEGG enrichment analysis, we found that neutrophil extracellular trap formation is an important signaling pathway for TMP to treat OA. Neutrophil extracellular traps (NETs) are large meshworks composed of DNA, histones, and granule proteins released by neutrophils [30], which can trap and kill pathogens and induce thrombosis [31]. In the microcirculation within the bone, thrombus can cause venous stasis and form intraosseous hypertension, which in turn alters the structure and function of the subchondral bone and articular cartilage tissues, induces an inflammatory response in the synovial membrane, disrupts the dynamic equilibrium between synthesis and degradation of cartilage matrix, and induces biomechanical alterations of articular cartilage, leading to degenerative changes in articular cartilage [32]. Therefore, we believe that inhibiting the release of NETs or effectively improving stasis in OA patients may be one of the effective methods to delay the degeneration of articular cartilage. This animal experiment showed that knee joint effusion was significantly reduced in the TMP group compared with the OA group (P < 0.05).KEGG results also showed that some of the potential core target proteins of TMP, such as C3, HDAC8, MPO, ELANE, HDAC7, etc., were enriched in the signaling pathway of NET formation. The above suggests that TMP is likely to affect the NETs signaling pathway by acting on the key targets such as C3 and MPO, thus exerting the effects of anti-thrombosis, improving the joint microenvironment, and lowering the intraosseous pressure in the treatment of OA.
We found that the actin cytoskeleton regulatory signaling pathway is also one of the critical signaling pathways for TMP treatment of OA. The regulatory signaling pathway of the actin cytoskeleton is closely related to endothelial cell (EC) permeability.
When the EC actin skeleton changes, it will lead to EC contraction and increased permeability, leading to massive plasma extravasation, resulting in tissue edema and interstitial fluid accumulation [33]. This suggests that inhibiting the alteration of EC actin skeleton may be one of the effective methods to reduce joint effusion in OA. In addition, the regulatory signaling pathway of the actin cytoskeleton is also closely related to the phagocytosis of macrophages and the formation of immune synapses in T cells [34]. Macrophages can phagocytose foreign particulate matter and pathogenic microorganisms. Actin can be involved in the regulation of phagocytosis in macrophages. When the dynamic changes of the macrophage actin cytoskeleton are inhibited, the phagocytosis function can be reduced[35]. T-cell immune synapse (IS) is a specific inter-cellular connectivity structure. Actin remodeling is involved in forming T-cell IS, which enhances the T-cell responses and affects the killing function of cytotoxic T lymphocytes [35, 36]. If actin remodeling is inhibited, T-cell responses can be reduced. This study showed that the potential core targets of TMP, such as BDKRB2, MSN, F2, and SLC9A1, were all enriched in the regulatory signal pathway of the actin cytoskeleton and participated in regulating the actin cytoskeleton. Animal experiments also showed that knee joint effusion was significantly reduced in the TMP group (P < 0.05). The above suggests that TMP is likely to affect the regulatory signaling pathway of the actin cytoskeleton by acting on the core target proteins, such as F2 and CDC42, and then play the roles of decreasing EC permeability, inhibiting the immune function of macrophages and T cells, and reducing the degree of joint effusion and inflammation in OA.
Based on KEGG results, we found that complement and coagulation cascade signaling pathways are also crucial for TMP treatment of OA. The coagulation cascade reaction consists of a series of enzymatic reactions that form a thrombus and stop bleeding, preventing bleeding after vessel rupture [37]. However, if the coagulation cascade reaction is overactivated or abnormal, it may lead to thrombosis and thromboembolism [38]. Therefore, preventing overactivation of the coagulation cascade reaction is essential to prevent the progression of OA. Complement plays a vital role in the immune defense of the body. Some studies have shown that the expression of complement proteins is increased in both synovial tissue and synovial fluid in OA. At the same time, the membrane attack complex (MAC) content is positively correlated with the degree of synovial inflammation [39]. In addition, chondrocytes also secrete complement proteins in response to IL-1β and TNF-α stimulation, promoting OA inflammation [40]. This suggests that targeted blockade of the complement system can effectively prevent OA progression and reduce OA inflammation. The results of this study showed that the essential target proteins of TMP, C3, BDKRB2, F2, and SERPINA5, were mainly enriched in the complement and coagulation cascade signaling pathway, and the molecular docking simulation also showed that TMP could be firmly bound to C3 and F2. The above suggests that TMP may influence the complement and coagulation cascade signaling pathways through essential target proteins, such as C3 and F2, and then regulate inflammation and thrombosis in OA.