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Liquiritinapioside – A mineralocorticoid-like substance from liquorice
Accepted Manuscript Liquiritinapioside - a mineralocorticoid-like substance from liquorice Simiao Fan, Kun Gu, Yuanyuan Wu, Houmin Luo, Yuming Wang, Tianpu Zhang, Xing Wang, Yanjun Zhang, Yubo Li PII: DOI: Reference:
S0308-8146(19)30533-3 https://doi.org/10.1016/j.foodchem.2019.03.056 FOCH 24503
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
28 October 2018 5 March 2019 11 March 2019
Please cite this article as: Fan, S., Gu, K., Wu, Y., Luo, H., Wang, Y., Zhang, T., Wang, X., Zhang, Y., Li, Y., Liquiritinapioside - a mineralocorticoid-like substance from liquorice, Food Chemistry (2019), doi: https://doi.org/ 10.1016/j.foodchem.2019.03.056
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Liquiritinapioside - a mineralocorticoid-like substance from liquorice Simiao Fan1, Kun Gu, Yuanyuan Wu, Houmin Luo, Yuming Wang, Tianpu Zhang, Xing Wang, Yanjun Zhang*, Yubo Li* Tianjin University of Traditional Chinese Medicine, 1076 North Huanan Road, Tuanbo New City, Jinghai District, Tianjin 301600, China. E-mail: [email protected] (Simiao Fan), [email protected] (Kun Gu), [email protected] (Yuanyuan Wu), [email protected] (Houmin Luo), [email protected] (Yuming Wang), [email protected] (Tianpu Zhang), [email protected] (Xing Wang) *Corresponding author: Yanjun Zhang, Yubo Li. Tianjin University of Traditional Chinese Medicine, 1076 North Huanan Road, Tuanbo New City, Jinghai District, Tianjin 301600, China. E-mail: [email protected] (Yanjun Zhang), [email protected] (Yubo Li). Tel and Fax number: +86-022-59596261.
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ABSTRACT Surface plasmon resonance (SPR) analysis of the main components of liquorice was performed and a novel strong mineralocorticoid receptor (MR) agonist, namely liquiritinapioside (LA), whose the binding constant was 1.093×10-5 M, was reported. As a supplement, LA has been further demonstrated to have a strong MR binding capacity through competitive binding experiments (the enrichment factor of LA was 10.22%). This study also validated the activity of LA on H9c2 cells. The expression of collagen I and the results of Masson staining were used to determine the ability of this substance to cause H9c2 cell fibrosis. Moreover, western blotting was used to verify the mechanism of compound-induced myocardial fibrosis. Overall, the attained results showed that LA could induce the activation of the TGF-β1/p38 MAPK signalling pathway through the MR to induce H9c2 cell fibrosis. Keywords: Liquorice; Liquiritinapioside; Myocardial fibrosis; Mineralocorticoid-like substance
1. Introduction Liquorice has great commercial as well as medicinal value and occupies a considerable share in the international and domestic markets. According to statistics, the annual global trade volume of liquorice exceeded 42.1 million US dollars as early as 2007. China is one of the world's largest exporters of liquorice, with an annual export volume of more than 30,000 tons (Li et al., 2017). As a traditional Chinese medicine, liquorice appears in approximately 2/3 of Chinese medicine prescriptions 2
due to its extensive pharmacological effects. It is mainly used for relieving coughs, asthma, resolving phlegm, invigorating qi and alleviating drug toxicity in traditional Chinese medicinal compounds (Wang et al., 2013; Yang, Wang, Yuan, & Liu, 2015). Modern clinical pharmacology studies have shown that liquorice and its bioactive compounds
have
antioxidant,
antiviral,
antitumor,
anti-inflammatory
and
immunomodulatory actions (Sil & Chakraborti, 2016; Xing, Wu, Du, Han, & Chen, 2011). In addition, liquorice is often used as a natural flavouring agent in candy, beverages and dietary supplements (Carocho, Morales, & Icfr, 2017). It is possible for people to consume excessive amounts of liquorice in their daily lives because a large number of food and health products contain liquorice. Although liquorice can provide many health benefits, the safety of liquorice intake is still controversial. According to statistics, the average annual consumption of liquorice in the world is approximately 1500 g, and some people consume as much as 100 g of liquorice per week (Bode & Dong, 2015). The use of such large or even excessive doses can cause adverse effects. Some studies have suggested that liquorice is a food related to heart problems (Brown, 2017). Animal and clinical experimental studies also confirmed that the consumption of glycyrrhizinate (GL) and glycyrrhetinic acid (GA) in large quantities will produce high mineralocorticoid hormone effects, leading to hypertension and hypokalaemia (Carmines et al., 2005; Bode et al., 2015). A recent report released by the US Food and Drug Administration showed that a large number of consecutive doses of black liquorice extract may lead to hospitalization for an arrhythmia (US FDA. 2017). 3
Mineralocorticoid receptors (MRs) combined with the corresponding hormones can mediate myocarditis and lead to myocardial fibrosis. A large number of experimental studies have shown that the use of mineralocorticoids such as aldosterone (ALD) can induce myocardial fibrosis. On the contrary, the use of MR antagonists can prevent myocardial fibrosis (Hung et al., 2016; Kimura et al., 2011; Kolkhof et al., 2014; Tn et al., 2014). The mineralocorticoid-like effect is a common factor causing adverse reactions with the use of liquorice (Bode et al., 2015; Omar et al., 2012). However, the research on mineralocorticoid-like substances in liquorice mainly focuses on GL and GA (Bode et al., 2015; Classen-Houben et al., 2009), while the strong mineralocorticoid-like components in liquorice are still unclear. In addition, there is no report on the correlation between such components in liquorice and myocardial fibrosis. Based on these findings, this study screened for strong mineralocorticoid-like compounds in liquorice using surface plasmon resonance (SPR) analysis. In addition, the binding abilities of the above components to MR protein in liquorice were observed by directly incubating the protein in liquorice solution with high performance liquid chromatography (HPLC) analysis. H9c2 cells were used as research objects, the myocardial fibrosis abilities of these substances were studied, and the potential mechanism of inducing fibrosis was verified. 2. Materials and methods 2.1. Materials, chemicals and reagents Liquorice was purchased from the Beijing Huamiao Pharmaceutical Co., Ltd. 4
(Beijing, China). Human MR (Cat.No.YB-1850P) was purchased from YBio (Shanghai, China). Spironolactone (Spi), ALD, liquiritinapioside (LA), liquiritin, glycyrrhizin, 5,7-dihydroxyflavone and isoliquiritigenin were obtained from the Sai Zhi Wei Technology Co., Ltd. (Tianjin, China). Glycyrrhizinate (GL), GA, isoliquiritinapioside (ILA) and isoliquiritin were purchased from the Shanghai Shifeng Biological Co., Ltd. (Shanghai, China). Prior to use, all solutions were filtered through a 0.22 μm membrane and degassed using ultrasound. Primary antibodies against the collagen І MMP-9 were purchased from Abcam (Cambridge, MA, UK). Antibodies for p38MAPK, p-p38MAPK and TGF-β1 were purchased from Cell Signalling Technology (Danvers, MA, USA). Antibodies for MR were purchased from OmnimAbs (USA). All other chemicals were purchased from commercial sources and were of analytical grade unless otherwise stated. Water used in the experiments was deionized, and the DMSO concentration in the article is 0.1% unless otherwise specified. 2.2. SPR screening for mineralocorticoid-like substances Based on the conditions found in the literature (Hudson, Paula, Ferreira, Ferreira, & Pires, 2018), the study optimizes the conditions according to the experimental results. (1) A Biacore T200 (Biacore Life Sciences, GE Healthcare) was used to determine the binding affinity of monomeric substances in liquorice to MR. EDC/NHS (volume ratio is 1:1, EDC is 0.4 mol/l, NHS is 0.1 mol/l) was run for 7 min to activate the carboxyl groups on the surface of the CM5 sensor (General Electric Company, USA). (2) The MR solution was dissolved in sodium acetate buffer 5
with a pH of 5.0 and injected onto the surface of the activated sensor chip at a flow rate of 7 μl/min for 20 min. The amino group in the BSA conjugate of the analyte was activated. Amino coupling reactions occur on the ester groups. (3) An ethanolamine-hydrochloric acid solution was run at a flow rate of 7 μl/min for 7 min to block any unreacted activated carboxyl functional groups. After fixing the ligand, the chip was left to stand at 4 °C. (4) The instrument setup temperature was 25 °C. After injection, the analytes were flowed through the surface of the chip at a constant flow rate of 30 μl/min, and the analyte binding to the ligand immobilized on the chip surface changes the quality of the surface material of the chip. The instrument records the change of the corresponding response value. After the injection is completed, the switching buffer flows through the surface of the chip. The process of spontaneous dissociation and association of the analyte on the ligand is monitored in real time by the response value. (5) After each measurement, the flow rate of the instrument was adjusted to 20 μl/min and eluted with 20 µl of 50 mM NaOH solution to regenerate the surface of the sensor chip. 2.3. Competitive combination of liquorice components Liquorice sample solution (100 mg/ml) was prepared, MRs were incubated with the sample solution and analysed by HPLC as described in the literature (Chen, Fan, Wu, Li, & Guo, 2019; Zhao et al., 2014; Liu et al., 2016). Information on the procedure used in this study is described in the supplementary material. 2.4. H9c2 cell culture The H9c2 cells (Saierbio, Tianjin, China) were cultured in DMEM complete 6
medium (Gibco, American) containing 10% FBS and 1% antibiotic solution containing 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere incubator with 5% CO2 (Zhang et al., 2015). 2.5. MTT assay Measurement of the cell activity using the MTT assay was carried out using a previously described method (Zhang et al., 2015). H9c2 cells were seeded into 96-well plates (Orange Scientific, Belgium) at 1.5×104 cells/well. For the completely adherent cells, grouped drug delivery culture (from 10-6 mol/l to 10-3 mol/l for ALD, ILA, LA, and GA), the DMSO vehicle control, and negative controls were applied, with 6 parallel wells per group. After treatment for 24 hours, 10 µl MTT at 5 mg/ml was added to each well. The cells were incubated at 37 °C for another 4 h, and DMSO was then added to each well. The absorbance was detected at 570 nm with a microplate reader (BIO-RAD, USA). The results were normalized against negative controls, and the results were graphically presented as the percentage of the cell inhibition rate concentration (mol/l). The IC10 and IC30 values, based on the concentration–response curves obtained for the corresponding treatments in the MTT assay, were calculated. The cells were cultured with Spi for 1 hour, and then, the cells were treated with IC10 and IC30 concentrations of the drug for 24 hours. Separately, a solvent control group and a blank control group were conducted. Three parallel wells were used in each group. The 96-well plates were placed at 37 °C with 5% CO2. Incubation was continued in the cell culture incubator for 24 h, and then, a MTT cell viability assay 7
was performed. 2.6. Masson staining Based on the conditions found in the literature (X Liu et al., 2018), the study optimizes the conditions based on the experimental results and finally obtains the best conditions. Briefly, H9c2 cells were cultured in a 24-well plate with approximately 1×105 cells per well and cultured in a 5% CO2 cell incubator at 37 ℃ for 24 hours. After the cells adhered to the well, they were replaced with the dosing medium (10-5.440 mol/l ALD, 10-5.065 mol/l LA, and 10-3.909 mol/l GA) and incubated for 24 hours. Each well was then washed twice with 1 ml of PBS and fixed with 4% paraformaldehyde for 15 min. Then, 0.5 ml of PBS was used to infiltrate the plate for 60 s. R1 nuclear dye liquor was applied for 60 s, removed, and then the plates were rinsed again for 30 s. After dyeing with R2 pulp dye liquor for 45 s, it was removed, and the wells were rinsed with rinsing liquor for 30 s. Colour separation of the R3 yellow colour separation liquid takes approximately 7 m, and then, the colour separation liquid was discarded. After directly dyeing it with the R4 blue stain solution for 1 min, it was rinsed with absolute ethanol and dried, and pictures were then taken under a microscope at 200 × magnification. 2.7. Western blotting to detect the effects of LA and GA on the fibrosis of H9c2 cells The protein samples were extracted from cells with the procedures essentially the same as described in detail elsewhere (Zhang et al., 2015). Briefly, H9c2 cells were plated in 6-well plates and treated with the indicated drugs. The drug administration groups were cultured with a specific concentration of the drug for 24 hours; in the 8
combined administration group, the cells were cultured for 1 hour with inhibitors and then cultured for 24 hours with the drugs. After treatment for 24 hours, cell lysates were prepared using RIPA buffer. The concentrations of the protein samples were determined using the BSA protein quantification kit (Nanjing Jiancheng Bioengineering Institute, China). Standard western blots were performed. Depending on the proteins of interest, antibodies against MR, TGF-β1, P38, P-P38, CTGF, collagen І and MMP-9 were used, followed by incubation with a secondary antibody conjugated to horseradish peroxidase (HRP). GAPDH was used as the internal control. The protein expression levels were determined and quantified with a LabWorksTM gel imaging and analysis system. 2.8. Statistical analysis Statistical analysis was performed using GraphPad Prism software, version 5.01, and Excel. The data were expressed as the mean ± standard error of the mean. The data were analysed using one-way analysis of variance (ANOVA), and two-tailed t-tests were performed for comparisons between two groups. Statistical significances were calculated and reported. 3. Results and discussion 3.1. Screening for mineralocorticoid-like substances in liquorice Liquorice is found in various types of food additives because of its natural and unique flavour. Furthermore, liquorice and its bioactive compounds have antioxidant (Sil et al., 2016), antiviral (Asl & Hosseinzadeh, 2010), liver-preserving, antitumor (Asl & Hosseinzadeh, 2010; Chigurupati, Auddy, Biyani, & Stohs, 2016), antibiosis, 9
antigenic, anti-inflammatory and immunomodulatory properties (Xing et al., 2011). It is widely believed that liquorice provides health benefits, but many people ignore the potential hazards of overconsumption. According to the statistics, most of the published complications are linked to the aldosterone-like action of liquorice (Nazari, Rameshrad, & Hosseinzadeh, 2017). Therefore, it is important to examine the mineralocorticoid-like compounds in liquorice to determine its safe use. In order to obtain the binding affinities of ten main components of liquorice to MRs, the binding affinities were determined by SPR in this study. According to the preliminary experimental results, the concentration values were determined, and the concentration values ranged from 0.52 ug/ml to 20 ug/ml. In addition, as a MR agonist, ALD was used as a positive control agent to determine the affinity of substances in liquorice. The sensing map shows the specific binding between the compound and MR molecule in the form of a dose-response (Fig. 1). The abscissa represents the concentration of each small molecule, and the ordinate represents the change in the refractive index. The combination ratio of the two is 1:1 through BIA evaluation software, where the vertical line represents the KD value. The value of the equilibrium dissociation constant (KD) between the compound and MR was used to describe the affinity between the molecules by BIA evaluation software. The smaller the KD value, the stronger the affinity (Canabadyrochelle et al., 2017). Table 1 shows that the affinity of ILA and MRs was the strongest, 4.917×10-7 M, that of ALD was 2.804×10-6 M, and the KD value of LA was close to that of the positive drug, which was 1.093×10-5 M. The affinities of GL and GA with the receptor were 1.452×10-5 M 10
and 1.948×10-5 M, respectively. The results showed that ILA and LA had good MR affinity, and they were stronger than GL and GA. This study examined the competitive binding ability of the above substances in the complex system of liquorice, and as a supplementary measurement, the changes of various compounds after the direct incubation of MR protein with liquorice solution were determined by HPLC. As show in Fig. S1-S3, the liquid phase conditions of liquorice components were studied by using the label map of liquorice components. The chromatographic information of each substance was determined according to the retention times of the four substances and the chromatographic results of the mixed standard in the literature. For potential active components screened out by SPR, the unique enrichment factor (EF) of each component could be used to assess the binding strength to MRs (Table 2). The results showed that the ILA peak exhibited the highest degree of binding affinity at 35.07%, followed by the GL peak at 12.09% and the LA peak at 10.22%. As expected before, the EF of each peak was different from the others, and this phenomenon might highly correlate with the competitive interactions of those components with MRs. A large number of studies have shown that it is feasible and accurate to evaluate the binding ability of candidate molecules and receptors by SPR, which has been widely used to screen small molecular drug candidates binding to therapeutic molecular biological targets (Hudson et al., 2018; Kennedy et al., 2017; Zhang et al., 2018). Although SPR technology is generally recognized as having the advantages of a fast screening speed and high accuracy, it lacks the ability to evaluate the 11
competitive combination ability of the tested components overall. In recent years, it has become a complementary method to explore the competitive binding abilities of substances by directly binding proteins to sample solutions (Cao, Jia, Shi, Xiao, & Chen, 2016; Shanshan et al., 2015). In this study, the combination of SPR technology and the direct incubation of proteins in liquorice solution showed that several substances in liquorice exhibited good binding ability with MRs, regardless of whether there was a competitive relationship or not. 3.2. LA induces the fibrosis of H9c2 cells The complications that are associated with the greatest number of fatalities due to the arrhythmogenic effect of liquorice include hypokalaemia and subsequent QT prolongation with possible torsade de pointes (Omar et al., 2012). Therefore, it is important to study the relationship between the components of liquorice and heart disease. Both the content and affinity of GL in liquorice is much higher than that of GA; however, GL almost always hydrolyses into GA when it enters the human body. Considering this, subsequent cell experiments use GA instead of GL. In this study, H9c2 cells were used to verify the myocardial fibrosis-inducing ability of the strong mineralocorticoid-like compounds in liquorice, and the above three compounds were studied. The results show that the ability of ILA to induce H9c2 cell fibrosis was weaker than those of LA and GA (Fig. S4). This finding indicates that ILA may have a strong affinity but low intrinsic activity In other words, ILA may be binding to MRs, but it does not play a role in inducing H9c2 cell fibrosis. As a result, only LA and GA were analysed using the cell verification test. 12
The toxicity ranges of the drugs in H9c2 cells were determined by MTT assays. The results of the MTT-based cytotoxicity assays showed that ALD, LA, and GA dose-dependently inhibited cell viability (Fig. 2a). Moreover, the positive drug ALD has the strongest cell inhibition, while LA was stronger than GA. The calculated results of the IC10 and IC30 concentrations of ALD, LA, and GA are shown in Table 3. In addition, to examine the role of MR activation on the LA-induced cytotoxicity, H9c2 cells were pre-treated with Spi, a MR antagonist, followed by drug exposure, and the MTT-based cytotoxicity assay demonstrated that Spi inhibition potentiated LA-induced or GA-induced cytotoxicity of H9c2 cells (Fig. 2b), suggesting the MR role of LA and GA in inducing the cytotoxicity of H9c2 cells. To determine whether it could cause H9c2 fibrosis and the necessary dose to be applied to the cells, Masson staining combined with the results of collagen І and MMP-9 expression measurements were used as the basis for judgement. The results showed that the induction of the fibrosis of H9c2 cells by the IC10 concentration is not as obvious as that for the IC30 concentration (Fig. S5). Masson staining results showed that at the IC30 concentration, compared with the normal group, the collagen fibres of the ALD, LA, and GA groups were significantly increased, the collagen volume fraction was increased, and the effect of LA was stronger than for GA (Fig. 2c). These data are also consistent with the results of the detection of collagen І, and Fig. 2d-2f shows that the expression of type І collagen is increased after treatment with these three drugs. Based on the above information, 10-5.065 mol/l LA, 10-3.909 mol/l GA, and 10-5.44 mol/l ALD were used as the treatment conditions to investigate 13
the effects of the drugs on the fibrosis of H9c2 cells in the subsequent experiment. 3.3. LA induced the fibrosis of H9c2 cells through MRs Numerous investigators have confirmed the important role of MRs in cardiovascular diseases (Lavall et al., 2014). The administration of mineralocorticoids to experimental animals for 8 weeks or more can cause cardiac hypertrophy and fibrosis (Young, 2008). Although LA and GA have a strong MR affinity, as demonstrated by the SPR technique, to further delineate whether the two compounds can act on MRs at the cellular level, H9c2 cells were cultured in groups and divided into normal, ALD, LA, GA, Spi and the Spi combined drug groups. Afterwards, proteins were extracted for western blot analysis. The blots clearly showed that the expression of MR protein was the highest after the ALD treatment; LA and GA treatments prominently induced expression level increases of the MR protein. However, after the administration of Spi, the expression levels of the MR protein were reduced and showed no significant difference between the normal groups. These results illustrate the fact that LA and GA could play a role through MRs (Fig. 2g-2j). Furthermore, we examined whether MRs are involved in the induction of the fibrosis of H9c2 cells by LA. H9c2 cells were pre-treated with Spi, followed by LA exposure. The results showed that the expression of fibrosis-related proteins was significantly reduced, along with the degree of fibrosis, in the Spi intervention group (Fig. 3a-3c). These results suggest that LA- and GA-induced H9c2 cell fibrosis is mediated through MRs, similar to ALD-induced fibrosis. 14
3.4. The TGF-β1/p38 MAPK signalling pathway is involved in the process of LA-induced fibrosis by MRs p38 MAPK regulates the cellular response to growth, apoptosis and stress signals and is involved in the fibrosis of multiple organ tissues (Yin et al., 2016). TGF-β1 alters gene expression in rat myocardium through the p38 MAPK pathway, which can cause the hypertrophy of myocardial cells (Lei et al., 2013). Numerous studies have shown that the p38 signalling pathway plays an important role in myocardial fibrosis (Arabacilar & Marber, 2015; Guo et al., 2018). In this study, western blot analysis was used to detect the expression levels of proteins closely related to the TGF-β1/p38 MAPK signalling pathway (the reasons for GA are given in Fig. S6). The results show that LA and GA regulate H9c2 cell fibrosis through this signalling pathway. To confirm whether LA induces the activation of the TGF-β1/p38 MAPK signalling pathway in cardiomyoblasts, H9c2 cells were treated with the specified concentration of LA for the indicated durations. Protein lysates were subjected to western blotting for the detection of TGF-β1, p38 MAPK, p-p38 MAPK, and CTGF protein contents. The results showed that the TGF-β1 protein was upregulated after LA administration (Fig. 3d-3e), and p38 MAPK, p-p38 MAPK, and CTGF protein levels were also significantly upregulated. After the administration of Spi, the protein expression levels of p38 MAPK, p-p38 MAPK, and CTGF were significantly downregulated compared with the LA-administered group, and the protein levels of TGF-β1 were also decreased (Fig. 3f-3h). The results in this chapter indicate that LA induced TGF-β1/p38 MAPK signalling pathway activation and modulated processes 15
related to protein expression in H9c2 cells. The next experiments, therefore, moved on to examine the role of p38 MPPK activation in the fibrosis of H9c2 cells. H9c2 cells were either given LA alone or cotreated with p38 MAPK inhibitor (SB203580) (10-5 mol/l). The results showed that SB203580 significantly inhibited the LA-mediated phosphorylation of p38 MAPK and the expression of the downstream protein CTGF (Fig. 3i-3k). In addition, the expression of fibronectin MMP-9 was also significantly downregulated, and the expression of collagen І was decreased (Fig. 3l-3m). Taken together, these results suggest that there is an association between p38 MAPK and the LA-induced fibrosis of H9c2 cells. 4. Conclusion This study found that LA may induce the activation of the TGF/p38 MAPK signalling pathway through the MR to induce the fibrosis of H9c2 cells. Therefore, patients with cardiovascular disease should be cautious in the daily consumption of food, beverages and other consumer products containing liquorice additives, and ordinary consumers should avoid long-term use. Users should also pay attention to dose control and rational drug use in the clinical use of liquorice. The findings of this study provide a reference for the evaluation of liquorice safety, enhancing the public's understanding of liquorice ingredients and their potential complications. This knowledge can prevent adverse consequences caused by the excessive use of such products. Acknowledgements The authors are grateful to the Foundation for the National Natural Science 16
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1). Omar, H. R., Komarova, I., Elghonemi, M., Fathy, A., Rashad, R., Abdelmalak, H. D., Yerramadha, M. R., Ali, Y., Helal, E., & Camporesi, E. M. (201 2). Licorice abuse: time to send a warning message. Ther Adv Endocrinol Metab, 3(4), 125–138. Shanshan, Q., Yiran, R., Xu, F., Jie, S., Xin, C., Quan, W., Xin, B., Wenjing. L., Lixin. L., & Guangxin, L. (2015). Multiple ligand detection and affinit y measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening. Analytica Chimica Acta, 886, 98–106. Sil, R., & Chakraborti, A. S. (2016). Oxidative Inactivation of Liver Mitochon dria in High Fructose Diet-Induced Metabolic Syndrome in Rats: Effect of Glycyrrhizin Treatment. Phytotherapy Research Ptr, 30(9), 1503–1512. Tn, V. D. B., Rongen, G. A., Fröhlich, G. M., Deinum, J., Hausenloy, D. J., & Riksen, N. P. (2014). The cardioprotective effects of mineralocorticoid r eceptor antagonists. Pharmacology & Therapeutics, 142(1), 72–87. Wang, X., Zhang, H., Chen, L., Shan, L., Fan, G., & Gao, X. (2013). Liquoric e, a unique “guide drug” of traditional Chinese medicine: A review of its role in drug interactions. Journal of Ethnopharmacology, 150(3), 781–790. Xing, P. P., Wu, W. H., Du, P., Han, F. M., & Chen, Y. (2011). Effects of br ucine combined with glycyrrhetinic acid or liquiritin on rat hepatic cytochr ome P450 activities in vivo. Acta Pharmaceutica Sinica, 46(46), 573–580. Yang, R., Wang, L. Q., Yuan, B. C., & Liu, Y. (2015). The Pharmacological 21
Activities of Licorice. Planta Medica, 81(18), 1654–1669. Yin, Q., Lu, H., Bai, Y., Tian, A., Yang, Q., Wu, J., Yang, C., Fan, T. P., Zha ng, Y., & Zheng, X. (2016). A metabolite of Danshen formulae attenuates cardiac fibrosis induced by isoprenaline, via a NOX2/ROS/p38 pathway. British Journal of Pharmacology, 172(23), 5573–5585. Young, M. J. (2008). Mechanisms of mineralocorticoid receptor-mediated cardia c fibrosis and vascular inflammation. Curr Opin Nephrol Hypertens, 17(17), 174–180. Zhang, L., Zhu, C., Chen, C., Zhu, S., Jie, Z., Wang, M., & Shang, P. (2018). Determination of Kanamycin Using a Molecularly Imprinted SPR Sensor. Food Chemistry, 266, 170–174. Zhang, M., Pan, X., Zou, Q., Xia, Y., Chen, J., Hao, Q ., Wang, H., & Sun, D. (2015). Notch3 Ameliorates Cardiac Fibrosis After Myocardial Infarctio n by Inhibiting the TGF-β1/Smad3 Pathway. Cardiovascular Toxicology, 1– 9. Zhao, C., Liu, Y., Cong, D., Zhang, H., Yu, J., Jiang, Y., Cui, X., & Sun, J. (2014). Screening and determination for potential α‐ glucosidase inhibitory constituents from Dalbergia odorifera T. Chen using ultrafiltration‐ LC/ESI ‐ MSn. Biomedical Chromatography Bmc, 27(12), 1621–1629. US FDA. Black Licorice: Trick or Treat? (2017). https://www.fda.gov/Forconsu mers/Consumerupdates/ucm277152.htm/ Accessed 30 October 2018.
22
Table 1 Compounds binding to MRs to obtain homeostasis KD values Compounds
KD (M)
1
ILA
4.917×10-7M
2
ALD
2.804×10-6M
3
LA
1.093×10-5M
4
GL
1.452×10-5M
5
GA
1.948×10-5M
6
Glycyrol
2.285×10-5M
7
Glycyrrhizin
2.558×10-5M
8
Isoliquiritin
4.727×10-5M
9
liquiritin
9.013×10-5M
10
isoliquiritigenin
1.489×10-4M
11
5,7-dihydroxyflavone
2.541×10-4M
K is used to describe the affinity between molecules. A smaller KD usually indicates closer binding between the ligand and the D analyte
.
Table 2 The enrichment factors (EFs) and the HPLC data of three components
Component
Λ(nm)
T(min)
A0
AT
23
AC
EF = (AT -AC) / A0 ×100％
LA
276.00
12.98 4775.68 4430.46 3942.33
10.22%
ILA
360.00
18.75 2836.61 2617.68 1622.98
35.07%
GL
248.00
37.65 11430.94 11672.65 10290.85
12.09%
Table 3 Concentrations used for evaluating the mechanisms underlying the cardiotoxicity of ALD, LA, and GA IC10(mol/l)
IC30(mol/l)
ALD
10-7.247
10-5.44
LA
10-7.530
10-5.065
GA
10-6.835
10-3.909
Figure legends: Fig. 1 SPR sensing map: a ILA; b ALD; c LA; d GA; e GL; f glycyrol; g glycyrrhizin; h isoliquiritin; i liquiritin; j isoliquiritigenin; and k 5,7-dihydroxyflavone. Fig. 2 a Cytostatic rate curve of H9c2 cells. b Inhibition of spironolactone on the fibrosis of drug-induced H9c2 cells. c Representative images of Masson's trichrome staining of H9c2 cells after the administration of the IC30 concentration to assess the degree of fibrosis of H9c2 cells. The protein lysates of H9c2 cells after treatment by ALD, LA, GA and Spi, respectively, were subjected to western blotting (d, g, h) and subsequent densitometrical analysis (e, f, i, j). All data are presented as the mean ± 24
SD (n≥3) and *P < 0.05, **P < 0.01 and ***P < 0.001 are compared to the control, respectively. Fig. 3 Effect of LA on the expression of the TGF-β/p38 MAPK signalling pathway in H9c2 cells. The protein lysates of H9c2 cells after treatment by LA, Spi and SB203580 administered alone or in combination were subjected to western blotting (a, d) and subsequent densitometrical analysis (b, c, e-j), respectively. All data are presented as the mean ± SD (n≥3) and *P < 0.05, **P < 0.01 and ***P < 0.001 are compared to the control, respectively; #P < 0.05, ##P < 0.01 and ###P < 0.001 are compared to the LA group, respectively.
25
C（μg/ml） a
3.95 2.63 1.76 1.17 0.78 0.52
b
d
g
j
20.0 13.3 8.89 3.95 2.63 0.52
h
C (μg/ml) k
8.89 5.92 3.95 2.63 1.76 0.52
8.89 5.92 3.95 1.76 1.17 0.52
C (μg/ml) f
C (μg/ml)
C (μg/ml) 13.3 8.89 5.93 3.95 2.63 0.52
c
8.89 5.92 3.95 2.63 1.76 0.52
e
C (μg/ml) 20.0 13.3 8.89 3.95 1.76 0.52
3.95 2.63 1.76 1.17 0.78 0.52
C（μg/ml） ）
C（μg/ml） 13.3 3.95 2.63 1.76 1.17 0.52
C (μg/ml)
C（μg/ml）
20.0 5.92 3.95 1.76 1.17 0.52
C (μg/ml) i
20.0 13.3 8.89 3.95 1.76 0.52
a
c
b c
Control
(μM）
e
d CON
g
CON
ALD
ALD
LA
LA
f
GA
LA+Spi
Spi
CON
h
MR
MR
GAPDH
GAPDH
i
j
ALD
GA
GA+Spi Spi
ALD
LA
GA
a
CON
ALD
LA
LA+Spi
P38 MAPK
Spi LA+SB203580 SB203580 b
c
P-P38 MAPK CTGF MMP-9 Collagen I GAPDH e
f
d CON
ALD
LA
LA+Spi
Spi β
TGF-β1 GAPDH
g
k
h
l
i
m
j
Highlights 1. Rapid screening and cell revalidation for mineralocorticoid-like substances. 2. Liquiritinapioside has a strong affinity with the mineralocorticoid receptor. 3. Liquiritinapioside regulates H9c2 cell fibrosis through the mineralocorticoid receptor.
26
S0308-8146(19)30533-3 https://doi.org/10.1016/j.foodchem.2019.03.056 FOCH 24503
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
28 October 2018 5 March 2019 11 March 2019
Please cite this article as: Fan, S., Gu, K., Wu, Y., Luo, H., Wang, Y., Zhang, T., Wang, X., Zhang, Y., Li, Y., Liquiritinapioside - a mineralocorticoid-like substance from liquorice, Food Chemistry (2019), doi: https://doi.org/ 10.1016/j.foodchem.2019.03.056
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Liquiritinapioside - a mineralocorticoid-like substance from liquorice Simiao Fan1, Kun Gu, Yuanyuan Wu, Houmin Luo, Yuming Wang, Tianpu Zhang, Xing Wang, Yanjun Zhang*, Yubo Li* Tianjin University of Traditional Chinese Medicine, 1076 North Huanan Road, Tuanbo New City, Jinghai District, Tianjin 301600, China. E-mail: [email protected] (Simiao Fan), [email protected] (Kun Gu), [email protected] (Yuanyuan Wu), [email protected] (Houmin Luo), [email protected] (Yuming Wang), [email protected] (Tianpu Zhang), [email protected] (Xing Wang) *Corresponding author: Yanjun Zhang, Yubo Li. Tianjin University of Traditional Chinese Medicine, 1076 North Huanan Road, Tuanbo New City, Jinghai District, Tianjin 301600, China. E-mail: [email protected] (Yanjun Zhang), [email protected] (Yubo Li). Tel and Fax number: +86-022-59596261.
1
ABSTRACT Surface plasmon resonance (SPR) analysis of the main components of liquorice was performed and a novel strong mineralocorticoid receptor (MR) agonist, namely liquiritinapioside (LA), whose the binding constant was 1.093×10-5 M, was reported. As a supplement, LA has been further demonstrated to have a strong MR binding capacity through competitive binding experiments (the enrichment factor of LA was 10.22%). This study also validated the activity of LA on H9c2 cells. The expression of collagen I and the results of Masson staining were used to determine the ability of this substance to cause H9c2 cell fibrosis. Moreover, western blotting was used to verify the mechanism of compound-induced myocardial fibrosis. Overall, the attained results showed that LA could induce the activation of the TGF-β1/p38 MAPK signalling pathway through the MR to induce H9c2 cell fibrosis. Keywords: Liquorice; Liquiritinapioside; Myocardial fibrosis; Mineralocorticoid-like substance
1. Introduction Liquorice has great commercial as well as medicinal value and occupies a considerable share in the international and domestic markets. According to statistics, the annual global trade volume of liquorice exceeded 42.1 million US dollars as early as 2007. China is one of the world's largest exporters of liquorice, with an annual export volume of more than 30,000 tons (Li et al., 2017). As a traditional Chinese medicine, liquorice appears in approximately 2/3 of Chinese medicine prescriptions 2
due to its extensive pharmacological effects. It is mainly used for relieving coughs, asthma, resolving phlegm, invigorating qi and alleviating drug toxicity in traditional Chinese medicinal compounds (Wang et al., 2013; Yang, Wang, Yuan, & Liu, 2015). Modern clinical pharmacology studies have shown that liquorice and its bioactive compounds
have
antioxidant,
antiviral,
antitumor,
anti-inflammatory
and
immunomodulatory actions (Sil & Chakraborti, 2016; Xing, Wu, Du, Han, & Chen, 2011). In addition, liquorice is often used as a natural flavouring agent in candy, beverages and dietary supplements (Carocho, Morales, & Icfr, 2017). It is possible for people to consume excessive amounts of liquorice in their daily lives because a large number of food and health products contain liquorice. Although liquorice can provide many health benefits, the safety of liquorice intake is still controversial. According to statistics, the average annual consumption of liquorice in the world is approximately 1500 g, and some people consume as much as 100 g of liquorice per week (Bode & Dong, 2015). The use of such large or even excessive doses can cause adverse effects. Some studies have suggested that liquorice is a food related to heart problems (Brown, 2017). Animal and clinical experimental studies also confirmed that the consumption of glycyrrhizinate (GL) and glycyrrhetinic acid (GA) in large quantities will produce high mineralocorticoid hormone effects, leading to hypertension and hypokalaemia (Carmines et al., 2005; Bode et al., 2015). A recent report released by the US Food and Drug Administration showed that a large number of consecutive doses of black liquorice extract may lead to hospitalization for an arrhythmia (US FDA. 2017). 3
Mineralocorticoid receptors (MRs) combined with the corresponding hormones can mediate myocarditis and lead to myocardial fibrosis. A large number of experimental studies have shown that the use of mineralocorticoids such as aldosterone (ALD) can induce myocardial fibrosis. On the contrary, the use of MR antagonists can prevent myocardial fibrosis (Hung et al., 2016; Kimura et al., 2011; Kolkhof et al., 2014; Tn et al., 2014). The mineralocorticoid-like effect is a common factor causing adverse reactions with the use of liquorice (Bode et al., 2015; Omar et al., 2012). However, the research on mineralocorticoid-like substances in liquorice mainly focuses on GL and GA (Bode et al., 2015; Classen-Houben et al., 2009), while the strong mineralocorticoid-like components in liquorice are still unclear. In addition, there is no report on the correlation between such components in liquorice and myocardial fibrosis. Based on these findings, this study screened for strong mineralocorticoid-like compounds in liquorice using surface plasmon resonance (SPR) analysis. In addition, the binding abilities of the above components to MR protein in liquorice were observed by directly incubating the protein in liquorice solution with high performance liquid chromatography (HPLC) analysis. H9c2 cells were used as research objects, the myocardial fibrosis abilities of these substances were studied, and the potential mechanism of inducing fibrosis was verified. 2. Materials and methods 2.1. Materials, chemicals and reagents Liquorice was purchased from the Beijing Huamiao Pharmaceutical Co., Ltd. 4
(Beijing, China). Human MR (Cat.No.YB-1850P) was purchased from YBio (Shanghai, China). Spironolactone (Spi), ALD, liquiritinapioside (LA), liquiritin, glycyrrhizin, 5,7-dihydroxyflavone and isoliquiritigenin were obtained from the Sai Zhi Wei Technology Co., Ltd. (Tianjin, China). Glycyrrhizinate (GL), GA, isoliquiritinapioside (ILA) and isoliquiritin were purchased from the Shanghai Shifeng Biological Co., Ltd. (Shanghai, China). Prior to use, all solutions were filtered through a 0.22 μm membrane and degassed using ultrasound. Primary antibodies against the collagen І MMP-9 were purchased from Abcam (Cambridge, MA, UK). Antibodies for p38MAPK, p-p38MAPK and TGF-β1 were purchased from Cell Signalling Technology (Danvers, MA, USA). Antibodies for MR were purchased from OmnimAbs (USA). All other chemicals were purchased from commercial sources and were of analytical grade unless otherwise stated. Water used in the experiments was deionized, and the DMSO concentration in the article is 0.1% unless otherwise specified. 2.2. SPR screening for mineralocorticoid-like substances Based on the conditions found in the literature (Hudson, Paula, Ferreira, Ferreira, & Pires, 2018), the study optimizes the conditions according to the experimental results. (1) A Biacore T200 (Biacore Life Sciences, GE Healthcare) was used to determine the binding affinity of monomeric substances in liquorice to MR. EDC/NHS (volume ratio is 1:1, EDC is 0.4 mol/l, NHS is 0.1 mol/l) was run for 7 min to activate the carboxyl groups on the surface of the CM5 sensor (General Electric Company, USA). (2) The MR solution was dissolved in sodium acetate buffer 5
with a pH of 5.0 and injected onto the surface of the activated sensor chip at a flow rate of 7 μl/min for 20 min. The amino group in the BSA conjugate of the analyte was activated. Amino coupling reactions occur on the ester groups. (3) An ethanolamine-hydrochloric acid solution was run at a flow rate of 7 μl/min for 7 min to block any unreacted activated carboxyl functional groups. After fixing the ligand, the chip was left to stand at 4 °C. (4) The instrument setup temperature was 25 °C. After injection, the analytes were flowed through the surface of the chip at a constant flow rate of 30 μl/min, and the analyte binding to the ligand immobilized on the chip surface changes the quality of the surface material of the chip. The instrument records the change of the corresponding response value. After the injection is completed, the switching buffer flows through the surface of the chip. The process of spontaneous dissociation and association of the analyte on the ligand is monitored in real time by the response value. (5) After each measurement, the flow rate of the instrument was adjusted to 20 μl/min and eluted with 20 µl of 50 mM NaOH solution to regenerate the surface of the sensor chip. 2.3. Competitive combination of liquorice components Liquorice sample solution (100 mg/ml) was prepared, MRs were incubated with the sample solution and analysed by HPLC as described in the literature (Chen, Fan, Wu, Li, & Guo, 2019; Zhao et al., 2014; Liu et al., 2016). Information on the procedure used in this study is described in the supplementary material. 2.4. H9c2 cell culture The H9c2 cells (Saierbio, Tianjin, China) were cultured in DMEM complete 6
medium (Gibco, American) containing 10% FBS and 1% antibiotic solution containing 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere incubator with 5% CO2 (Zhang et al., 2015). 2.5. MTT assay Measurement of the cell activity using the MTT assay was carried out using a previously described method (Zhang et al., 2015). H9c2 cells were seeded into 96-well plates (Orange Scientific, Belgium) at 1.5×104 cells/well. For the completely adherent cells, grouped drug delivery culture (from 10-6 mol/l to 10-3 mol/l for ALD, ILA, LA, and GA), the DMSO vehicle control, and negative controls were applied, with 6 parallel wells per group. After treatment for 24 hours, 10 µl MTT at 5 mg/ml was added to each well. The cells were incubated at 37 °C for another 4 h, and DMSO was then added to each well. The absorbance was detected at 570 nm with a microplate reader (BIO-RAD, USA). The results were normalized against negative controls, and the results were graphically presented as the percentage of the cell inhibition rate concentration (mol/l). The IC10 and IC30 values, based on the concentration–response curves obtained for the corresponding treatments in the MTT assay, were calculated. The cells were cultured with Spi for 1 hour, and then, the cells were treated with IC10 and IC30 concentrations of the drug for 24 hours. Separately, a solvent control group and a blank control group were conducted. Three parallel wells were used in each group. The 96-well plates were placed at 37 °C with 5% CO2. Incubation was continued in the cell culture incubator for 24 h, and then, a MTT cell viability assay 7
was performed. 2.6. Masson staining Based on the conditions found in the literature (X Liu et al., 2018), the study optimizes the conditions based on the experimental results and finally obtains the best conditions. Briefly, H9c2 cells were cultured in a 24-well plate with approximately 1×105 cells per well and cultured in a 5% CO2 cell incubator at 37 ℃ for 24 hours. After the cells adhered to the well, they were replaced with the dosing medium (10-5.440 mol/l ALD, 10-5.065 mol/l LA, and 10-3.909 mol/l GA) and incubated for 24 hours. Each well was then washed twice with 1 ml of PBS and fixed with 4% paraformaldehyde for 15 min. Then, 0.5 ml of PBS was used to infiltrate the plate for 60 s. R1 nuclear dye liquor was applied for 60 s, removed, and then the plates were rinsed again for 30 s. After dyeing with R2 pulp dye liquor for 45 s, it was removed, and the wells were rinsed with rinsing liquor for 30 s. Colour separation of the R3 yellow colour separation liquid takes approximately 7 m, and then, the colour separation liquid was discarded. After directly dyeing it with the R4 blue stain solution for 1 min, it was rinsed with absolute ethanol and dried, and pictures were then taken under a microscope at 200 × magnification. 2.7. Western blotting to detect the effects of LA and GA on the fibrosis of H9c2 cells The protein samples were extracted from cells with the procedures essentially the same as described in detail elsewhere (Zhang et al., 2015). Briefly, H9c2 cells were plated in 6-well plates and treated with the indicated drugs. The drug administration groups were cultured with a specific concentration of the drug for 24 hours; in the 8
combined administration group, the cells were cultured for 1 hour with inhibitors and then cultured for 24 hours with the drugs. After treatment for 24 hours, cell lysates were prepared using RIPA buffer. The concentrations of the protein samples were determined using the BSA protein quantification kit (Nanjing Jiancheng Bioengineering Institute, China). Standard western blots were performed. Depending on the proteins of interest, antibodies against MR, TGF-β1, P38, P-P38, CTGF, collagen І and MMP-9 were used, followed by incubation with a secondary antibody conjugated to horseradish peroxidase (HRP). GAPDH was used as the internal control. The protein expression levels were determined and quantified with a LabWorksTM gel imaging and analysis system. 2.8. Statistical analysis Statistical analysis was performed using GraphPad Prism software, version 5.01, and Excel. The data were expressed as the mean ± standard error of the mean. The data were analysed using one-way analysis of variance (ANOVA), and two-tailed t-tests were performed for comparisons between two groups. Statistical significances were calculated and reported. 3. Results and discussion 3.1. Screening for mineralocorticoid-like substances in liquorice Liquorice is found in various types of food additives because of its natural and unique flavour. Furthermore, liquorice and its bioactive compounds have antioxidant (Sil et al., 2016), antiviral (Asl & Hosseinzadeh, 2010), liver-preserving, antitumor (Asl & Hosseinzadeh, 2010; Chigurupati, Auddy, Biyani, & Stohs, 2016), antibiosis, 9
antigenic, anti-inflammatory and immunomodulatory properties (Xing et al., 2011). It is widely believed that liquorice provides health benefits, but many people ignore the potential hazards of overconsumption. According to the statistics, most of the published complications are linked to the aldosterone-like action of liquorice (Nazari, Rameshrad, & Hosseinzadeh, 2017). Therefore, it is important to examine the mineralocorticoid-like compounds in liquorice to determine its safe use. In order to obtain the binding affinities of ten main components of liquorice to MRs, the binding affinities were determined by SPR in this study. According to the preliminary experimental results, the concentration values were determined, and the concentration values ranged from 0.52 ug/ml to 20 ug/ml. In addition, as a MR agonist, ALD was used as a positive control agent to determine the affinity of substances in liquorice. The sensing map shows the specific binding between the compound and MR molecule in the form of a dose-response (Fig. 1). The abscissa represents the concentration of each small molecule, and the ordinate represents the change in the refractive index. The combination ratio of the two is 1:1 through BIA evaluation software, where the vertical line represents the KD value. The value of the equilibrium dissociation constant (KD) between the compound and MR was used to describe the affinity between the molecules by BIA evaluation software. The smaller the KD value, the stronger the affinity (Canabadyrochelle et al., 2017). Table 1 shows that the affinity of ILA and MRs was the strongest, 4.917×10-7 M, that of ALD was 2.804×10-6 M, and the KD value of LA was close to that of the positive drug, which was 1.093×10-5 M. The affinities of GL and GA with the receptor were 1.452×10-5 M 10
and 1.948×10-5 M, respectively. The results showed that ILA and LA had good MR affinity, and they were stronger than GL and GA. This study examined the competitive binding ability of the above substances in the complex system of liquorice, and as a supplementary measurement, the changes of various compounds after the direct incubation of MR protein with liquorice solution were determined by HPLC. As show in Fig. S1-S3, the liquid phase conditions of liquorice components were studied by using the label map of liquorice components. The chromatographic information of each substance was determined according to the retention times of the four substances and the chromatographic results of the mixed standard in the literature. For potential active components screened out by SPR, the unique enrichment factor (EF) of each component could be used to assess the binding strength to MRs (Table 2). The results showed that the ILA peak exhibited the highest degree of binding affinity at 35.07%, followed by the GL peak at 12.09% and the LA peak at 10.22%. As expected before, the EF of each peak was different from the others, and this phenomenon might highly correlate with the competitive interactions of those components with MRs. A large number of studies have shown that it is feasible and accurate to evaluate the binding ability of candidate molecules and receptors by SPR, which has been widely used to screen small molecular drug candidates binding to therapeutic molecular biological targets (Hudson et al., 2018; Kennedy et al., 2017; Zhang et al., 2018). Although SPR technology is generally recognized as having the advantages of a fast screening speed and high accuracy, it lacks the ability to evaluate the 11
competitive combination ability of the tested components overall. In recent years, it has become a complementary method to explore the competitive binding abilities of substances by directly binding proteins to sample solutions (Cao, Jia, Shi, Xiao, & Chen, 2016; Shanshan et al., 2015). In this study, the combination of SPR technology and the direct incubation of proteins in liquorice solution showed that several substances in liquorice exhibited good binding ability with MRs, regardless of whether there was a competitive relationship or not. 3.2. LA induces the fibrosis of H9c2 cells The complications that are associated with the greatest number of fatalities due to the arrhythmogenic effect of liquorice include hypokalaemia and subsequent QT prolongation with possible torsade de pointes (Omar et al., 2012). Therefore, it is important to study the relationship between the components of liquorice and heart disease. Both the content and affinity of GL in liquorice is much higher than that of GA; however, GL almost always hydrolyses into GA when it enters the human body. Considering this, subsequent cell experiments use GA instead of GL. In this study, H9c2 cells were used to verify the myocardial fibrosis-inducing ability of the strong mineralocorticoid-like compounds in liquorice, and the above three compounds were studied. The results show that the ability of ILA to induce H9c2 cell fibrosis was weaker than those of LA and GA (Fig. S4). This finding indicates that ILA may have a strong affinity but low intrinsic activity In other words, ILA may be binding to MRs, but it does not play a role in inducing H9c2 cell fibrosis. As a result, only LA and GA were analysed using the cell verification test. 12
The toxicity ranges of the drugs in H9c2 cells were determined by MTT assays. The results of the MTT-based cytotoxicity assays showed that ALD, LA, and GA dose-dependently inhibited cell viability (Fig. 2a). Moreover, the positive drug ALD has the strongest cell inhibition, while LA was stronger than GA. The calculated results of the IC10 and IC30 concentrations of ALD, LA, and GA are shown in Table 3. In addition, to examine the role of MR activation on the LA-induced cytotoxicity, H9c2 cells were pre-treated with Spi, a MR antagonist, followed by drug exposure, and the MTT-based cytotoxicity assay demonstrated that Spi inhibition potentiated LA-induced or GA-induced cytotoxicity of H9c2 cells (Fig. 2b), suggesting the MR role of LA and GA in inducing the cytotoxicity of H9c2 cells. To determine whether it could cause H9c2 fibrosis and the necessary dose to be applied to the cells, Masson staining combined with the results of collagen І and MMP-9 expression measurements were used as the basis for judgement. The results showed that the induction of the fibrosis of H9c2 cells by the IC10 concentration is not as obvious as that for the IC30 concentration (Fig. S5). Masson staining results showed that at the IC30 concentration, compared with the normal group, the collagen fibres of the ALD, LA, and GA groups were significantly increased, the collagen volume fraction was increased, and the effect of LA was stronger than for GA (Fig. 2c). These data are also consistent with the results of the detection of collagen І, and Fig. 2d-2f shows that the expression of type І collagen is increased after treatment with these three drugs. Based on the above information, 10-5.065 mol/l LA, 10-3.909 mol/l GA, and 10-5.44 mol/l ALD were used as the treatment conditions to investigate 13
the effects of the drugs on the fibrosis of H9c2 cells in the subsequent experiment. 3.3. LA induced the fibrosis of H9c2 cells through MRs Numerous investigators have confirmed the important role of MRs in cardiovascular diseases (Lavall et al., 2014). The administration of mineralocorticoids to experimental animals for 8 weeks or more can cause cardiac hypertrophy and fibrosis (Young, 2008). Although LA and GA have a strong MR affinity, as demonstrated by the SPR technique, to further delineate whether the two compounds can act on MRs at the cellular level, H9c2 cells were cultured in groups and divided into normal, ALD, LA, GA, Spi and the Spi combined drug groups. Afterwards, proteins were extracted for western blot analysis. The blots clearly showed that the expression of MR protein was the highest after the ALD treatment; LA and GA treatments prominently induced expression level increases of the MR protein. However, after the administration of Spi, the expression levels of the MR protein were reduced and showed no significant difference between the normal groups. These results illustrate the fact that LA and GA could play a role through MRs (Fig. 2g-2j). Furthermore, we examined whether MRs are involved in the induction of the fibrosis of H9c2 cells by LA. H9c2 cells were pre-treated with Spi, followed by LA exposure. The results showed that the expression of fibrosis-related proteins was significantly reduced, along with the degree of fibrosis, in the Spi intervention group (Fig. 3a-3c). These results suggest that LA- and GA-induced H9c2 cell fibrosis is mediated through MRs, similar to ALD-induced fibrosis. 14
3.4. The TGF-β1/p38 MAPK signalling pathway is involved in the process of LA-induced fibrosis by MRs p38 MAPK regulates the cellular response to growth, apoptosis and stress signals and is involved in the fibrosis of multiple organ tissues (Yin et al., 2016). TGF-β1 alters gene expression in rat myocardium through the p38 MAPK pathway, which can cause the hypertrophy of myocardial cells (Lei et al., 2013). Numerous studies have shown that the p38 signalling pathway plays an important role in myocardial fibrosis (Arabacilar & Marber, 2015; Guo et al., 2018). In this study, western blot analysis was used to detect the expression levels of proteins closely related to the TGF-β1/p38 MAPK signalling pathway (the reasons for GA are given in Fig. S6). The results show that LA and GA regulate H9c2 cell fibrosis through this signalling pathway. To confirm whether LA induces the activation of the TGF-β1/p38 MAPK signalling pathway in cardiomyoblasts, H9c2 cells were treated with the specified concentration of LA for the indicated durations. Protein lysates were subjected to western blotting for the detection of TGF-β1, p38 MAPK, p-p38 MAPK, and CTGF protein contents. The results showed that the TGF-β1 protein was upregulated after LA administration (Fig. 3d-3e), and p38 MAPK, p-p38 MAPK, and CTGF protein levels were also significantly upregulated. After the administration of Spi, the protein expression levels of p38 MAPK, p-p38 MAPK, and CTGF were significantly downregulated compared with the LA-administered group, and the protein levels of TGF-β1 were also decreased (Fig. 3f-3h). The results in this chapter indicate that LA induced TGF-β1/p38 MAPK signalling pathway activation and modulated processes 15
related to protein expression in H9c2 cells. The next experiments, therefore, moved on to examine the role of p38 MPPK activation in the fibrosis of H9c2 cells. H9c2 cells were either given LA alone or cotreated with p38 MAPK inhibitor (SB203580) (10-5 mol/l). The results showed that SB203580 significantly inhibited the LA-mediated phosphorylation of p38 MAPK and the expression of the downstream protein CTGF (Fig. 3i-3k). In addition, the expression of fibronectin MMP-9 was also significantly downregulated, and the expression of collagen І was decreased (Fig. 3l-3m). Taken together, these results suggest that there is an association between p38 MAPK and the LA-induced fibrosis of H9c2 cells. 4. Conclusion This study found that LA may induce the activation of the TGF/p38 MAPK signalling pathway through the MR to induce the fibrosis of H9c2 cells. Therefore, patients with cardiovascular disease should be cautious in the daily consumption of food, beverages and other consumer products containing liquorice additives, and ordinary consumers should avoid long-term use. Users should also pay attention to dose control and rational drug use in the clinical use of liquorice. The findings of this study provide a reference for the evaluation of liquorice safety, enhancing the public's understanding of liquorice ingredients and their potential complications. This knowledge can prevent adverse consequences caused by the excessive use of such products. Acknowledgements The authors are grateful to the Foundation for the National Natural Science 16
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Table 1 Compounds binding to MRs to obtain homeostasis KD values Compounds
KD (M)
1
ILA
4.917×10-7M
2
ALD
2.804×10-6M
3
LA
1.093×10-5M
4
GL
1.452×10-5M
5
GA
1.948×10-5M
6
Glycyrol
2.285×10-5M
7
Glycyrrhizin
2.558×10-5M
8
Isoliquiritin
4.727×10-5M
9
liquiritin
9.013×10-5M
10
isoliquiritigenin
1.489×10-4M
11
5,7-dihydroxyflavone
2.541×10-4M
K is used to describe the affinity between molecules. A smaller KD usually indicates closer binding between the ligand and the D analyte
.
Table 2 The enrichment factors (EFs) and the HPLC data of three components
Component
Λ(nm)
T(min)
A0
AT
23
AC
EF = (AT -AC) / A0 ×100％
LA
276.00
12.98 4775.68 4430.46 3942.33
10.22%
ILA
360.00
18.75 2836.61 2617.68 1622.98
35.07%
GL
248.00
37.65 11430.94 11672.65 10290.85
12.09%
Table 3 Concentrations used for evaluating the mechanisms underlying the cardiotoxicity of ALD, LA, and GA IC10(mol/l)
IC30(mol/l)
ALD
10-7.247
10-5.44
LA
10-7.530
10-5.065
GA
10-6.835
10-3.909
Figure legends: Fig. 1 SPR sensing map: a ILA; b ALD; c LA; d GA; e GL; f glycyrol; g glycyrrhizin; h isoliquiritin; i liquiritin; j isoliquiritigenin; and k 5,7-dihydroxyflavone. Fig. 2 a Cytostatic rate curve of H9c2 cells. b Inhibition of spironolactone on the fibrosis of drug-induced H9c2 cells. c Representative images of Masson's trichrome staining of H9c2 cells after the administration of the IC30 concentration to assess the degree of fibrosis of H9c2 cells. The protein lysates of H9c2 cells after treatment by ALD, LA, GA and Spi, respectively, were subjected to western blotting (d, g, h) and subsequent densitometrical analysis (e, f, i, j). All data are presented as the mean ± 24
SD (n≥3) and *P < 0.05, **P < 0.01 and ***P < 0.001 are compared to the control, respectively. Fig. 3 Effect of LA on the expression of the TGF-β/p38 MAPK signalling pathway in H9c2 cells. The protein lysates of H9c2 cells after treatment by LA, Spi and SB203580 administered alone or in combination were subjected to western blotting (a, d) and subsequent densitometrical analysis (b, c, e-j), respectively. All data are presented as the mean ± SD (n≥3) and *P < 0.05, **P < 0.01 and ***P < 0.001 are compared to the control, respectively; #P < 0.05, ##P < 0.01 and ###P < 0.001 are compared to the LA group, respectively.
25
C（μg/ml） a
3.95 2.63 1.76 1.17 0.78 0.52
b
d
g
j
20.0 13.3 8.89 3.95 2.63 0.52
h
C (μg/ml) k
8.89 5.92 3.95 2.63 1.76 0.52
8.89 5.92 3.95 1.76 1.17 0.52
C (μg/ml) f
C (μg/ml)
C (μg/ml) 13.3 8.89 5.93 3.95 2.63 0.52
c
8.89 5.92 3.95 2.63 1.76 0.52
e
C (μg/ml) 20.0 13.3 8.89 3.95 1.76 0.52
3.95 2.63 1.76 1.17 0.78 0.52
C（μg/ml） ）
C（μg/ml） 13.3 3.95 2.63 1.76 1.17 0.52
C (μg/ml)
C（μg/ml）
20.0 5.92 3.95 1.76 1.17 0.52
C (μg/ml) i
20.0 13.3 8.89 3.95 1.76 0.52
a
c
b c
Control
(μM）
e
d CON
g
CON
ALD
ALD
LA
LA
f
GA
LA+Spi
Spi
CON
h
MR
MR
GAPDH
GAPDH
i
j
ALD
GA
GA+Spi Spi
ALD
LA
GA
a
CON
ALD
LA
LA+Spi
P38 MAPK
Spi LA+SB203580 SB203580 b
c
P-P38 MAPK CTGF MMP-9 Collagen I GAPDH e
f
d CON
ALD
LA
LA+Spi
Spi β
TGF-β1 GAPDH
g
k
h
l
i
m
j
Highlights 1. Rapid screening and cell revalidation for mineralocorticoid-like substances. 2. Liquiritinapioside has a strong affinity with the mineralocorticoid receptor. 3. Liquiritinapioside regulates H9c2 cell fibrosis through the mineralocorticoid receptor.
26