The results of our study showed that there are 24 compound targets of AM in LN. After PPI network analysis, we obtained 10 core targets according to degree value. Further, we performed ROC analysis to validate the core targets internally, and we found that the 10 core genes had high diagnostic values for LN, among which four key target genes, namely CASP3, IL-1β, STAT1, and PPARγ, had the highest diagnostic values. We also performed differential gene expression analysis on the external dataset GSE99339, which showed that the 10 core genes had significant differential expression levels in LN. Further, molecular docking revealed that four key targets in LN had the highest binding properties to quercetin, the first bioactive component of AM. We performed GO and KEGG analyses on the 24 compound targets separately and found that the key target genes CASP3 and IL-1β were enriched in the MAPK signaling pathway. Therefore, we speculated that AM suppresses LN by regulating inflammation at least through the MAPK signaling pathway.
Autophagy is an intracellular catabolic modality that maintains cellular homeostasis. Defects in autophagy are involved in the pathogenesis of SLE 7, 11, 12. Studies have shown that autophagy is involved in the process of foot-cell injury in LN 13. The MAPK cascade is a key signaling pathway regulating autophagy, cell proliferation and differentiation, apoptosis, and cellular stress response 14, 15. This cascade inhibits autophagy by activating mTOR 16, 17. Our findings revealed that the key target genes of AM in LN are enriched in the MAPK signaling pathway, and therefore we hypothesized that AM suppresses LN by regulating the MAPK signaling pathway. Indeed, autophagy is generally accompanied by apoptosis.
Caspase-3 is an essential member of the caspase family, an executor of the apoptotic pathway 18. Several studies have shown activated caspase-3 in glomerular cells and infiltrated inflammatory cells (neutrophils and macrophages) in LN, and the increase in the number of apoptotic cells correlates with the severity of the glomerular lesions, demonstrating the involvement of apoptosis in the associated kidney injury 19. Similarly, our analysis of the differentially expressed genes in LN revealed that caspase-3 expression increases in the kidneys of LN patients, and external datasets confirmed this result. We, therefore, hypothesize that regulation of caspase-3 expression may reduce apoptosis and thus treat LN.
In addition, our results showed increased expression of IL-1β in LN. Studies have demonstrated that the development of LN is closely related to inflammation 20. IL-1β is involved in the pathogenesis of autoimmune diseases 21, and it acts as a pro-inflammatory cytokine and coordinates the inflammatory response by activating the NF-κB signaling pathway and promoting the release of cyclooxygenase-2 and interferon-γ 22. In patients with LN, renal tubular epithelial cells play an essential role in the tubular interstitial inflammation by producing several inflammatory mediators, including interleukin (IL)-6 and IL-1 22–25. Additionally, Castejon et al. have found that the active form of IL-1β directly contributes to the inflammatory damage in LN 26. The p38MAPK signaling pathway promotes the development of LN by activating lymphocytes through the production and activation of inflammatory factors, such as monocyte chemotactic factor (MCP-1) and IL-10. A study by Liu et al. found that AM reduces the production of pro-inflammatory markers, such as IL-1β, and significantly inhibits the activation of STAT1, which in turn inactivates inflammatory cytokines 27. Inflammation is closely related to autophagy; inflammatory mediators can inhibit autophagy, and impaired autophagy can also induce inflammatory responses. Therefore, we hypothesized that AM plays a role in regulating the expression of the key target gene IL-1β, which may be associated with the regulation of autophagy, to attenuate the inflammatory response and thereby improves the prognosis of LN.
Our study found that PPARγ and STAT1 were also significantly upregulated and had diagnostic values for LN. Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear transcription factor, can regulate thylakoid cell proliferation, prevent trans-differentiation, inhibit fibroblast proliferation and macrophage infiltration, and suppress inflammatory mediators. Activated PPARγ can ameliorate renal inflammation and fibrosis by inhibiting the TLR4/MyD88/NF-κB pathway 28. Studies have shown that STAT1 can accelerate the progression of LN by activating interferons, regulating the expansion and differentiation of T and B cells, and disrupting immune tolerance. Furthermore, the STAT1 expression in peripheral blood mononuclear cells was elevated in patients with SLE, and this elevation correlates with disease activity 29. STAT1 has good binding properties for the first bioactive ingredient of AM. Therefore, we speculate that AM may also suppress LN by interfering with PPARγ and STAT1. However, the exact mechanism needs to be further explored.
In this study, we found that the key target genes of AM that are involved in the treatment of LN are all associated with quercetin, which is a vital bioactive component of AM and has anti-oxidative, anti-apoptotic, and anti-inflammatory properties 30, 31. Quercetin can suppress hepatic fibrosis and atherosclerosis through signaling pathways, such as the MAPK and SIRT1 pathways, in hepatic and cardiovascular diseases 32, 33. Studies have demonstrated that quercetin can play a therapeutic role in LN by inhibiting the activation of the nuclear factor-κB signaling pathway and suppressing the excessive proliferation of thylakoid cells. Accordingly, our results indicate that quercetin regulates the expression of caspase-3 and IL-1β through the MAPK signaling pathway, consequently regulating autophagy and suppressing apoptosis and cellular inflammatory response to treat LN.
This study has some limitations. First, the small sample size of the microarray data may have had some influence on the results. Second, although we performed clinical validation, the results of this study should be verified via extensive clinical studies and experimental validation.