KD is a systemic inflammatory syndrome of small and medium vessels. In recent years, more and more evidences have shown that the pathogenesis of this condition is related to infectious factors, susceptibility genes and autoimmune activation. Traditional Chinese medicine is suitable for treating diseases with complex mechanisms and, because of its multiple curative effect and small side effects. The effects of BBR on KD in clinical practice were tested based on some previous findings regarding this topic(13). The present study can be considered the first clinical trial to evaluate the effect of BBR in patients with KD. In study, network pharmacology method was used to analyze the possible molecular mechanisms of CR in the treatment of KD.
In the clinical trial stage, C-reactive protein (CRP) and peripheral blood cell parameters were also used as markers of systemic inflammation. Among them, CRP plays a role in promoting phagocytosis and immune regulation. Data have shown that during the course of KD, the increase of CRP level is related to coronary artery dilatation and is a high risk factor of complication with coronary artery injury for KD(26). NLR and PLR are markers of the balance between inflammatory response and immune regulation and are associated with cardiovascular adverse events. Studies have shown that NLR is directly proportional to the intensity of inflammatory response(27). At the same time, Turkmen et al. indicated that PLR might be more effective than NLR in predicting the severity of systemic inflammatory response(28). The present study indicated that the BBR treatment accelerate the reduction of CRP, NLR and PLR, which means that BBR can alleviate the inflammatory response in patients with KD. The rate of IVIG-resistance in BBR treatment group was significantly lower than that of control group, indicating that BBR increases the therapeutic efficiency of routine therapy in KD.
In order to investigate the mechanisms of BBR on KD, network pharmacology was used. 9 target compounds of CR were collected from TCMSP, 369 target genes of CR were collected from TCMSP, SWISS, SEA and STITCH database and a total of 624 target genes of CR were obtained by searching related databases together with 41 target genes obtained by the intersection of CR and KD. By further screening this sample of genes, AKT1、CASP3、TP53、MAPK1/3/8/14、PTGS2、SRC、RELA、MTOR、NOS2/3、ICAM1 were found and set as target genes as well.
AKT1, a serine/threonine protein kinase, is widely expressed in various tissues. It is known that activated AKT plays a regulatory role in cell cycle, apoptosis and proliferation by activating downstream factors. Also, the specific activation of AKT1 in vascular endothelial cells can alleviate the injury after carotid artery ligation by increasing the expression of nitric oxide and protecting the function of endovascular cortex(29). By initializing AKT1 in vascular smooth muscle cells it is possible to effectively inhibit the apoptosis and negative remodeling of vascular smooth muscle cells after carotid artery ligation, highlighting the protective role of AKT1 in vascular remodeling(30). In addition, study have confirmed that p21 phosphorylated by AKT1 in endothelial cells may promote angiogenesis and metastasis, suggesting that p21 phosphorylation may play an important role in KD coronary artery abnormalities(31).
MAPK, an intracellular serine/threonine protein kinase, is an important signaling system for cell-mediated extracellular signals to intracellular responses, and plays a key role in cell proliferation, apoptosis, inflammation, immunity and angiogenesis (32). Studies have found that inhibiting MAPK signaling pathway activation can reduce the occurrence of inflammatory response(33). So, this research postulated that MAPK might play an important regulatory role in the occurrence and development of vasculitis in KD.
SRC encodes tyrosine protein kinase also have to be mentioned, since it is a member of the non-receptor protein tyrosine kinase family. This protein is constantly associated with multiple signaling pathways in cells and its related genes are involved in important biological processes such as growth, differentiation, adhesion and transcription. Studies have confirmed that SRC-1 gene is related to the susceptibility to coronary artery aneurysm of KD complications(34), indicating that SRC gene is linked to the regulatory mechanism of Kawasaki disease.
Additionally, CASP3 is a key apoptotic protease in the final pathway of apoptotic cell death, mediating exogenous and endogenous cell death signaling pathways. CASP3 leads to transcriptional activation of inflammatory genes by activating the NF-κB pathway in the mechanism of KD. As genetic variation of genes may also cause damage and remodeling of vascular structures, TP53 is considered to be an important tumor suppressor gene that can affect cell cycle, DNA repair, apoptosis, signal transduction, transcription and autophagy, as well as regulate the growth, differentiation and senescence of cells. PTGS2 contains 10 exons and 9 introns, encoding cyclooxygenase 2(Cox-2). Cox-2, in this case, is expressed in vascular smooth muscle, monocytes and fibroblasts, and is a cardinal inflammatory mediator in the process of atherosclerosis(35). In addition, overexpression of COX-2 may cause inflammation of the vascular wall, plaque instability and intimal hyperplasia(36). It is believed that the occurrence of this vascular wall inflammation is closely related to the mechanism of KD, and this hypothesis is consistent with the results of the network pharmacology processes here conducted.
RELA is a member of the NF-κB family. NF-κB plays a key role in inflammatory and immune responses in cells. Studies have shown that in the pathological process of KD, the NF-κB signaling system regulates transcription of almost all genes involved in inflammatory mediators and cell proliferation and activation. Activation of NF-κB signaling pathway is linked to the occurrence of KD vasculitis in the acute phase, which is likely to aggravate KD vasculitis response and participate in the formation of coronary artery injury(37). ICAM-1 is considered an initiator of inflammatory cell adhesion and is also closely related to endothelial dysfunction(38), playing an important role in the development of cardiovascular disease(39, 40). After endothelial injury, ICAM-1 expression increases, aggravating vascular injury by releasing more cytokines and chemokines(41). These findings are consistent with the network-pharmacologic outcomes of this study and these target genes could be potential candidates for KD.
In addition, the GO enrichment analysis in this study showed that reactions to bacteria-derived molecules, reactions to lipopolysaccharides, and apoptotic processes are the major biological processes for CR treatment of KD. The disturbance of these biological processes is likely the cause of KD vasculitis and suggests that CR may play a protective role in blood vessels by improving these biological processes. Accordingly, CR may then be important in the regulation of different biological functions of KD.
Finally, the enrichment analysis of KEGG pathway found that the four signaling pathways closely related to CR and KD prevention and treatment included AGE-RAGE signaling pathway, fluid shear stress and atherosclerosis, TNF-signaling pathway and Toll1-like receptor signaling pathway. Since RAGE is a cell surface molecule of the immunoglobulin superfamily, a high expression of it on the surface of circulating endothelial cells in KD children is associated with the occurrence of coronary complications(42). Also, Toll1-like receptor signaling pathway is a family of receptors composed of members of multiple receptors and has a key function when it comes to inflammatory and immune injuries of endothelial cells caused by pathogen infection and immune response(43). Clinical studies have been helpful to confirm the correlation between TLR signaling pathway and inflammatory immune injuries of vascular endothelial cells in KD(44).
This study systematically explored the putative bioactive compounds in CR and pharmacological targets of CR for KD prevention and treatment through network pharmacology. All the ingredients of CR were extracted edible plants, and they were all claimed safe in previous applications. Therefore, this study provides a new way to explore the mechanism of CR in treating KD. However, the examinations here conducted are mainly based on the network pharmacology and bioinformatics database data, and has certain limitations. It is suggested for the future that relevant experiments are carried out to better explain the mechanisms of CR in the treatment of KD.