Inflammation plays a protective role when the body is infected or injured, but excessive inflammatory response will cause a wide range of diseases. Currently, more and more researches have confirmed that inflammation is closely related to the occurrence of various chronic or malignant diseases such as type 2 diabetes, rheumatoid arthritis, asthma, cancer and so on[1, 2]. Therefore, there is an urgent need to develop comprehensive strategies for inflammatory disease. A certain amount of studies have been conducted on the anti-inflammatory effect of luteolin, and shown that luteolin exhibits significant anti-inflammatory effect in a variety of cell and animal models28, 29. However, the detailed and systematic molecular mechanism by which luteolin inhibits inflammation remains uncertain.
Evaluation of the ADME properties is the first step in digging for a drug. Studies have shown that one of the most important reasons for the failure of drug development are poor pharmacokinetics and toxic properties19. Early evaluation of ADME properties of drugs can significantly improve the success rate, and reduce the cost and the occurrence of drug toxicity and side effects of drug development19. Obviously, evaluation of ADME properties is of great significance to simplify and accelerate the drug discovery process. DL is designed to evaluate how “drug-like” a compound is and the potential of a compound to develop into a drug. The criterion of DL ≥ 0.18 has been widely used to filter out compounds with undesirable ADME-relatedproperties18. OB is one of the most critical parameter of oral drugs and the threshold of OB ≥ 30% represents a good indicator of the promising effectiveness of drug delivery to the blood circulation18. Moreover, molecular weight (MW) < 500 Daltons, the lipid-water partition coefficient (ALogP) < 5, the number of hydrogen bond donors (Hdon) < 5 and the number of bond acceptors (Hacc)༜10 are called “Lipinski's Rule of Five”. A compound that complies with “Lipinski's Rule of Five” means that it will have better pharmacokinetic properties and higher bioavailability, and therefore more likely to become a drug19. As shown in Table 2, luteolin was satisfied with DL ≥ 0.18, OB ≥ 30% and “Lipinski's Rule of Five” indicating that luteolin has great potential to be developed into a promising drug.
Target fishing is the second step in drug mining. The results of target genes of luteolin against inflammation network showed that 226 targets of luteolin against inflammation were obtained. Furthermore, 9 core genes were screened including MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1. The main function of MMP9 is to degrade and reshape the homeostasis of the extracellular matrix, and plays a critical role in inflammatory response, tissue configuration, regulating matrix-bound growth factor and cytokine expression and cancer30. Moreover, research shows that luteolin decrease MMP9 expression to treat ischemic stroke, colon cancer and diabetes31, 32. MAPK pathway is the intersection of signal pathways such as cell proliferation, inflammation, differentiation, functional synchronization, transformation and apoptosis, and MAPK pathway participates in cell proliferation, differentiation, canceration, metastasis, apoptosis and so on33. Luteolin decreases inflammation through inhibiting MAPK1 pathway and thus performs a beneficial treatment in atherosclerosis34. CASP3 is the main terminal cleaving enzyme in the process of apoptosis and activation of CASP3 causes apoptosis and inflammation35. Studies prove that luteolin can effectively increase CASP3 expression to induced apoptosis in HaCaT cells and cancer cells36, 37. EGFR, a member of the epidermal growth factor receptor family, plays an important role in tumor cell proliferation, angiogenesis, tumor invasion and metastasis. Moreover, a decease in EGFR expression exerts anti-inflammatory activity in inflammatory diseases such as asthma38. It has been confirmed that luteolin inhibits the activation of EGFR to manage glioblastoma, lung cancer, pancreatic cancer and so on38–41. SRC can interact with phosphorylate STAT3, regulate TLR4-induced inflammatory response, regulate tumorigenesis of cancer cells and HIF1α expression42, 43. Canarium subulatum and boerhavia diffusa L with high content of luteolin perform anti-inflammatory activities by decreasing SRC expression44, 45. HRAS mutations are more common in bladder cancer and head and neck cancer, and luteolin increases HRAS expression to regulate cell cycle progression, which may be involved in decreasing inflammation response46, 47. ESR1 is essential for sexual development and reproductive function and also responsible for bone growth and maintaining normal functions of the cardiovascular and nervous systems. ESR1 mutations or abnormal expression are associated with tumor onset and excessive inflammatory response, and study suggests that luteolin might regulate acute inflammation in renal injury through affecting the expression of ESR148. It is worth noting that the CRP/ALB ratio is a relevant biomarker that reflects microvascular permeability, and CRP/ALB ratio has a strong correlation in assessing cohn's disease activity, analyzing the risk of acute myocardial infarction and predicting mortality in hemodialysis patients49–52. Furthermore, monitoring CRP and ALB levels together can better assess the prognosis of bacterial infectious diseases, and CRP/ALB ratio can be used as a new inflammatory prognostic indicator to predict outcomes in patients with hepatocellular carcinoma53, 54. Interestingly, ALB was confirmed as a potential core gene in the target genes of luteolin against inflammation network. The above results suggest that MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1 may be critical target genes of luteolin against inflammation.
Based on the analysis of gene function, the results revealed that luteolin may regulate the metabolism, energy pathways, signal transduction and activity of receptor protein and series of proteinase to defend inflammation. KEGG pathways suggested that the 31 critical signaling pathways might be the mechanism of luteolin against inflammation, including pathways in cancer, metabolic pathways, PI3K-AKT signaling pathway, Ras signaling pathway, Rap1 signaling pathway and so on. The results of GO and KEGG analysis are in line with our prediction that core genes were mainly involved in anti-inflammatory biological processes and signaling pathways. Finally, we used RT-qPCR and ELISA to further confirm the accuracy of predicted anti-inflammatory targets of luteolin. Surprisingly, the results of RT-qPCR and ELISA showed that luteolin markedly inhibited MAPK1, EGFR, HRAS, HSP90AA1, MMP9 and SRC mRNA production at protein and mRNA levels. However, we found that the levels of ALB, CASP3 and ESR1 did not increase significantly after LPS stimulation, which indicated that we need to further analyze the effect of luteolin on predicted targets expression in vivo and in vitro in proteomics and proteomics studies in the future.