Anti-cancer, Anti-inflammatory, Antioxidant and Other Pharmacological Effects of Wedelactone and Their Molecular Mechanisms

Mingxinzhi WANG, Jingchao WANG, Xichun HUANG, Xinyu LIU, Quan QUAN, Chenghao JIN

College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Abstract Wedelolide is a coumarin-like active substance extracted from Ecliptae Herba. It not only has pharmacological effects of anti-pulmonary fibrosis, anti-arthritis, blood vessel protection and liver protection, but also effectively inhibits the proliferation of cancer cells and induces apoptosis of cancer cells, thereby delaying the further development of malignant tumors. In this paper, the pharmacological effects and mechanisms of wedelactone were reviewed to lay a foundation for further study and clinical application of wedelactone.

Key words Wedelactone, Anti-tumor, Anti-pulmonary fibrosis, Protection of blood vessels, Liver protection

1 Introduction

Ecliptae Herba, which is the dry terrestrial part ofEcliptaprostrata., has pharmacological effects pf hemostasis, kidney tonifying, annealing and swelling reducing. It is widely distributed in Liaoning, Hebei, Shandong and other provinces of China. Wedelolactone (WEL) is a small molecule active substance of coumarin extracted from Ecliptae Herba, and is chemically named 7-methoxy-5,11, 12-trihydroxy-coumarin, with molecular formula C16H10O7. It is white crystalline and soluble in solvents such as methanol and DMSO. WEL has a variety of biological activities, including anti-inflammation, anti-oxidation, anti-tumor and liver protection. In this paper, the pharmacological effects and molecular mechanisms of WEL in recent years will be reviewed to provide a theoretical basis for its clinical application and research.

2 Anticancer, anti-inflammatory, antioxidant and other pharmacological effects and their molecular mechanisms

2.1 Anti-cancerThe occurrence and development of tumors are closely related to the expression and regulation of intracellular proteins, cytokines and signaling pathways. For example, overexpression of apoptosis-promoting protein Bax and Caspase can increase the apoptosis rate of cancer cells, and abnormal activation of JAK2/STAT3 and PI3K/Akt signaling pathways can promote the proliferation and migration of cancer cells. Therefore, the regulation of related signaling pathways in cancer cells has become one of the hot spots in anti-tumor research. Studies have showed that WEL, as an active component of Chinese herbal extracts, can reduce the survival rate of cancer cells and induce their apoptosis by regulating cancer cell signaling pathways.

2.1.1Anti-lung cancer. Lung cancer is one of the most common malignant tumors in the world, and has a certain genetic susceptibility. Through MTT method and flow cytometry, Jiang Shanetal.[1]found that WEL can effectively reduce the viability of lung cancer A549 cells, and induce cell cycle arrest and apoptosis. TheIC50values after 24 and 48 h were 21.12 and 16.24 μmol/L, and the retarded and apoptotic rates were 47% and 30%, respectively. In order to explore the mechanism of apoptosis of A549 cells, Western Blot experiment was further conducted, and it is found that after A549 cells were treated with WEL (20 μmol/L), the expression level of anti-apoptotic protein Bcl-2 significantly decreased, and the expression level of proapoptotic protein Bax and Caspase-3 significantly increased. In addition, WEL can reduce the phosphorylation level of JAK2 and STAT3 in cancer cells, regulate the JAK2/STAT3 signaling pathway, and then improve the apoptosis-inducing effect on A549 cells, ultimately delaying and improving the development process of lung cancer.

2.1.2Anti-renal cell carcinoma. Renal cell carcinoma (RCC) originates from epithelial cells of kidney tubules, has a high metastasis rate and mortality, and is one of the main causes of neurological and digestive complications. Xian Shulietal.[2]detected the cycle, apoptosis and expression level of related proteins in ACHN and 786-O cells of renal cancer by MTT method, flow cytometry and Western Blot experiment, and found that compared with the control group, WEL can effectively reduce the viability of ACHN and 786-O cells and induce the arrest of cancer cells in G1 phase. The retardation rates were 54.14%±1.91% and 60.01%±2.61%, respectively. WEL can also down-regulate the expression level of anti-apoptotic protein Bcl-2 and its family member Bcl-xL, and up-regulate the expression level of Bax, thereby activating the mitochondrial signaling pathway of Caspase cascade, and finally inducing apoptosis of ACHN and 786-O cells.

2.1.3Anti-prostate cancer. Prostate cancer, which is the most common malignant tumour of urinary system in men, is caused by malignant hyperplasia of prostate epithelial cells. Oncogene C-myc can induce infinite proliferation of cancer cells and participate in the occurrence and metastasis of tumors. Sivalokanathanetal.[3-4]found that WEL can significantly reduce the expression level of C-myc mRNA in LNCaP, PC-3 and DU145 cells of prostate cancer, as well as the binding activity of related enzymes and DNA during transcription, and inhibit the survival rate and migration ability of cancer cells. WEL can also regulate the JNK signaling pathway in cancer cells, down-regulate the expression level of protein kinase Cε (PKCε), survivin, cyclin D1, aurora kinase and cell cycle dependent kinase 4 (CDK4), up-regulate the expression level of ATF3 gene, and then induce cycle arrest and apoptosis of cancer cells. In addition, the combination of WEL and enzalutamide can reduce the growth rate, weight and volume of transplanted tumor in nude mice.

2.1.4Anti-colon cancer. Colon cancer (CRC) is the most common malignant tumor of digestive tract, and its recurrence and metastasis is the main reason leading to the failure of resection. Li Yanxietal.[5]found that WEL can effectively reduce the viability of HCT116 cells in colon cancer, and block cell cycle and induce cell apoptosis. Western Blot and real-time quantitative PCR (RT-qPCR) showed that WEL can inhibit the proliferation of colon cancer cells and promote the apoptosis of colon cancer cells by inhibiting the expression level of β-catenin. In order to explore the effect of WEL on tumor growthinvivo, after further treatment of WEL (100 mg/kg) in transplanted nude mice, it was found that the expression level of cell-related proteins Ki67, Bcl-2, C-myc and survivin in transplanted tumor tissues significantly reduced, while the apoptosis rate of colon cancer cells significantly increased. However, WEL had no effect on normal colonic epithelial cells and vital organs such as heart and kidney in nude mice.

2.1.5Anti-retinoblastoma. Retinoblastoma (RB) is an intraocular malignancy caused by RB1 gene mutation or deletion. Jiangetal.[6]found that WEL can block RB cell cycle and induce pyroptosis and apoptosis by up-regulating the expression level of Caspase-1, Caspase-3, porin-forming proteins GSDME, GSDMD and ROS in RB cells. In order to explore the anti-tumor activity of WELinvivo, RB cells were further used to construct a nude mouse model of transplanted tumor, and it was found that WEL can effectively inhibit the growth rate, volume and weight of tumor in mice, but had no obvious toxic and side effects on normal organs and tissues. Harkinetal.[7]found that WEL can down-regulate the expression level of AIM2 mRNA in the retinal inflammasome, effectively protect the retinal neurodegeneration caused by the DNA alkylating agent N-methyl-n-nitrosourea (NMU), and improve the thickness of retina and photosensitive layer, thereby preventing the occurrence of diseases caused by retinal neurodegeneration.

2.2 Anti-inflammation and antioxidationInflammation is a dynamic expression of damage and repair of human organism, and is closely related to the occurrence of many diseases. Abnormal proliferation and apoptosis of keratinocytes (HaCaT) are the main causes of psoriasis. Xu Jingetal.[8]constructed an inflammatory injury model of HaCaT, and found that WEL can inhibit the proliferation and autophagy of HaCaT cells by down-regulating the expression level of p-mTOR protein in HaCaT after LPS stimulation, thus slowing down the development of inflammation. Lin Yuqingetal.[9]found that WEL can promote NLRP3 phosphorylation and inhibit the activation of NLRP3 inflammatorites and pyroptosis by enhancing PKA signal transduction in the G protein-coupled system, thus playing an anti-inflammatory role. Cheng Minetal.[10]used Aspergillus fumigatus spores to construct a mouse model of fungal keratitis, and through immunofluorescence, myeloperoxidase (MPO) method and Western Blot, it was found that WEL can inhibit the expression level of Caspase-1 and the secretion of interleukin IL-1β, reduce the aggregation of neutrophils in the keratitis area, and then effectively reduce the degree of inflammation of keratitis in mice. Fanetal.[11]found that WEL can up-regulate the expression level of glutathione peroxidase 4 (GPX4), down-regulate the expression level of Caspase-1 and 11, and the content of pancreatic digestive enzyme and pro-inflammatory cytokine (ferroptosis) in serum, thus inhibiting pyroptosis and ferroptosis, and ultimately slow the development of acute pancreatitis (AP).

Cellular oxidative damage can seriously affect the normal life activities of the human body. Xu Jingetal.[12]found that WEL can up-regulate the expression level of DJ-1 protein, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in human coronary endothelial cells (HCAECs), and down-regulate the content of malondialdehyde (MDA), thereby protecting HCAECs oxidative stress damage induced by hydrogen peroxide.

2.3 Induction of osteocyte differentiationCartilage injury is one of the common orthopedic diseases. Its surgical treatment is relatively difficult, and has a long recovery period. Yang Linetal.[13]found that WEL can induce cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs) through targeted binding and regulation of RNA methyltransferase 4 (Nsun4) expression. Further analysis of RT-qPCR and transcriptomics shows that WEL can improve the translation ability of Sox9, the core transcription factor of differentiation, and promote the complete chondrogenic differentiation of BMSCs. Zhuetal.[14]found that by regulating the signaling pathway of c-Jun amino terminal kinase (JNK) and external regulatory protein kinase (ERK), WEL (2 μg/mL) can up-regulate the expression level of bone cell markers (Runx2, Bglap and Sp7) and bone morph protein-2 (BMP-2) in BMSCs, and enhance bone mineralization and Smad1/5/8 phosphorylation level of BMSCs, thereby promoting the differentiation of BMSCs into osteoblasts.

2.4 Liver and blood vessel protectionThe liver plays an important role in the circulation of Qi, blood and water, and is one of the important organs of the human body. Excessive intake of antipyretic and analgesic acetaminophen (APAP) can lead to acute liver failure and even death[15]. Sun Xiaomingetal.[16]used APAP to construct a liver injury model for mice, and found that the degree of liver disease and the necrotic area of liver cells in the mice with liver injury in WEL treatment groups could be effectively improved. Further studies have shown that WEL can down-regulate the expression level of liver injury markers (ALT, AST), MDA and GSH in liver tissue homogenate and pro-inflammatory cytokines (TNF-α, IL-6) in serum of mice with liver injury, and up-regulate the expression level of SOD and GSH-PX in liver tissue homogenate, and inhibit the inflammatory response, thereby reducing the degree of liver damage and protecting the liver. Xu Jingetal.[17]found that WEL can reduce the degree of lipid peroxidation of hepatocytes by inhibiting free radical activity, thus stabilizing the biofilm structure of hepatocytes and protecting the liver from ischemia reperfusion injury. Ping Pingetal.[18]found that WEL can effectively protect liver from being damaged by carbon tetrachloride, and reduce the degree of liver tissue degeneration.

Blood vessels are important transportation channels to maintain normal life activities of the human body. Huang Chuanfengetal.[19]combined WEL with earthworm plasminase to treat the rats with hyperhomocysteinemia (HHcy), and found that it can effectively reduce the expression level of homocysteine (Hcy), interleukin 1-α and 1-β, serum cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL-C) in plasma of HHcy rats, and up-regulate the expression level of high-density lipoprotein (HDL-C), thus reducing the incidence of cardiovascular diseases, and protecting blood vessels.

2.5 Anti-rheumatoid arthritisRheumatoid arthritis (RA) is an autoimmune disease. The main symptom is joint swelling and pain, and it can induce patients to develop lung, cardiovascular, liver and kidney tissue lesions. Caoetal.[20]constructed a mouse model of arthritis (CIA) by subcutaneous injection of type II collagen, and found that compared with the control group, the expression level of pro-inflammatory factors IL-1β, IL-6, TNF-α, IL-18 and RANKL, MMP-3 and NF-κB in synovium of CIA significantly decreased after WEL treatment, and the volume of the swelling ankle joint decreased. In addition, WEL can also up-regulate the expression level of cadherin E and reduce the incidence of cardiac tissue complications caused by RA.

2.6 Anti-pulmonary fibrosis and anti-pneumoniaPulmonary fibrosis is one of the end-stage manifestations of many interstitial lung diseases, and its clinical symptoms are mostly dry cough, dizziness, dyspnea,etc.Yangetal.[21]used bleomycin (BLM) to construct a lung fiber ICR mouse model, and found that the expression levels of pro-inflammatory factors, fibrosis marker α-SMA and type I collagen in the lung tissue significantly reduced, and the degree of lung tissue injury, pulmonary fibrosis and alveolar wall thickness were effectively improved. Besides, WEL can also reduce the expression level of transforming growth factor β1 (TGF-β1) and the phosphorylation level of Smad2/3 in mouse lung tissue, and induce AMPK activation, thereby inhibiting the transformation of lung tissue cells into fibrocytes.

Pneumonia is a disease of the lungs caused by bacteria and viruses. Through RT-qPCR and Western blot experiments, Zheng Daweietal.[22]found that WEL can effectively inhibit the cellular inflammation mediated by Streptococcus pneumoniae by up-regulating the expression level of miR-142-3p, IL-10 and Bax in alveolar epithelial cells and down-regulating the expression level of IL-6 and Bcl-2.

2.7 Antineurotic diseaseCentral nervous system degenerative diseases mainly include Parkinson’s disease (PD), Alzheimer’s disease (AD),etc., and are caused by the degeneration of central nervous system tissue. Studies have shown that inhibition of phosphodiesterase (PDEs) activity can effectively delay the pathological progression of neurological diseases[23]. Mao Mingqiangetal.[24]used cortisone stimulation to construct a hippocampal mouse neuron HT-22 cell injury model, and found that the viability of damaged neurons could be effectively improved in the wedelide treatment group. Sharmaetal.[25]found that WEL can effectively inhibit α-synuclein (α-syn) aggregation, improve oxidative stress and mitochondrial dysfunction, and alleviate PD pathology.

2.8 Other pharmacological effectsDiabetes is a common metabolic disease with a high incidence, and can lead to complications of the human foot, blood vessel and nerve tissue. Shahabetal.[26]found that WEL can delay the further development of diabetes by effectively reducing the fasting blood glucose level and the expression level of glycated serum protein in mice. In addition, WEL derivative 3-butoxy-1, 8, 9-trihydroxy-6h-benzofuran[3, 2-C] benzopyrane-6-one (BTB) can play a good anti-tumor role in the treatment of breast cancer, endometrial cancer and ovarian cancer[27].

3 Outlook

WEL, an active substance isolated and purified from Ecliptae Herba, has good chemical properties, biological activity and pharmacological effects, and plays a valuable role in medical development and industrial application. Although WEL has good anti-tumor activity, the specific mechanisms and signaling pathway of its effects on tumor cells are still in the initial stage, and researchers need to conduct more comprehensive and deeper studies on WEL from the molecular, cellular and animal levels, so as to lay a foundation for accelerating the development and utilization of WEL.