Traditional Medicine Research Doi: 10.12032/TMR20200201157

Traditional Medicine Research doi: 10.12032/TMR20200201157

Traditional Chinese Medicine

Hua-Zhuo-Kai-Yu decoction inhibits apoptosis in nonalcoholic fatty liver disease

Yu-Ting Li1#, Huan-Tian Cui2#, Lu Yang3, Lu-Lu Jin3, Yu-Ming Wang3, Xue-Qian Dong3, Wei-Bo Wen4, Hong-Wu Wang1*, Zhai-Yi Zhang5*

1College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China; 2Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao 250100, China; 3Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China; 4Department of Endocrinology in Yunnan Provincial Hospital of Traditional Chinese Medicine, Kunming 650021, China; 5College of Integrated Chinese and Western Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China.

#These authors are co-first authors on this work.

*Corresponding to: Hong-Wu Wang. College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, No.10 Poyanghu Road, Jinghai District, Tianjin 301617, China. E-mail: [email protected]; Zhai-Yi Zhang. College of Integrated Chinese and Western Medicine, Tianjin University of Traditional Chinese Medicine, No.10 Poyanghu Road, Jinghai District, Tianjin 301617, China. E-mail: [email protected].

Highlights

Our study demonstrates that Hua-Zhuo-Kai-Yu decoction (HZKY), an empirical formula in traditional Chinese medicine that is derived from the classic ancient prescription Da-Chai-Hu decoction, can improve nonalcoholic fatty liver disease (NAFLD) by inhibiting apoptosis in the liver by reducing the levels of BAX, CASP3, and CASP9.

Tradition

The classic ancient prescription Da-Chai-Hu (DCH) decoction is described in the ancient book of Chinese medicine titled Shanghan Zabing Lun (Treatise on Cold Damage Diseases, 25 C.E.–220 C.E.), written by Zhang Zhongjing. According to this book, DCH is used to treat diseases of the liver, gallbladder, and pancreas. Recent studies have also demonstrated the hepatoprotective, cholagogue, anti-inflammatory, and lipid-lowering effects of DCH. In addition, clinical studies have shown that DCH improves dyslipidemia in patients with NAFLD. HZKY decoction, derived from a modification of DCH, was established by Doctor Wang Hongwu at the Tianjin University of Traditional Chinese Medicine. It has shown obvious clinical effects on NAFLD. However, the mechanisms of action of HZKY in NAFLD remain unknown.

Submit a manuscript: https://www.tmrjournals.com/tmr 1 doi: 10.12032/TMR20200201157 ARTICLE Abstract Background: Hua-Zhuo-Kai-Yu decoction (HZKY) is an empirical formula in traditional Chinese medicine that is derived from the classic ancient prescription Da-Chai-Hu decoction. It has been demonstrated to have good clinical effects on nonalcoholic fatty liver disease (NAFLD). However, the mechanism by which HZKY acts on NAFLD remains unclear. In this study, network pharmacology was used to predict the potential targets of HZKY in NAFLD. Additionally, in vivo studies were conducted to validate the crucial pathways determined using network pharmacology. Methods: Active compounds in HZKY were screened using the Traditional Chinese Medicine Systems Pharmacology and Analysis Platform and Traditional Chinese Medicine Integrated Database, and the potential targets of compounds in HZKY were predicted using Traditional Chinese Medicine Systems Pharmacology and Analysis Platform, Traditional Chinese Medicine Integrated Database, Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, and PUBCHEM. In addition, targets involved in NAFLD were obtained from the GeneCards and Online Mendelian Inheritance in Man databases, and the potential targets of HZKY in NAFLD were identified based on the common potential targets between HZKY and NAFLD. Cytoscape 3.7.2 was used to visualize crosstalk and identify the key genes from the potential targets of HZKY in NAFLD. Kyoto Encyclopedia of Genes and Genomes analysis was conducted to predict the pathways by which HZKY acts on NAFLD. Rats were fed with a high-fat diet for 12 weeks to induce NAFLD and were then orally administered HZKY. Serum lipid levels and hematoxylin and eosin and oil red O staining results were assessed to determine the effects of HZKY in NALFD. Furthermore, the mechanisms of action of HZKY in NAFLD, as determined using network pharmacology, were validated based on the inhibition of apoptosis in the liver using Western blotting. Results: A total of 269 potential targets of 130 active compounds in HZKY were identified (oral bioavailability ≥ 30% and drug-like ≥ 0.18), and 62 targets were selected after being compared with the targets of NAFLD. Bcl-2-associated X protein (BAX), caspase3 (CASP3), and caspase9 (CASP9) were the key genes with the highest values of network connectivity. In addition, 45 Kyoto Encyclopedia of Genes and Genomes pathways, including apoptosis, fatty acid synthesis, and estrogen signaling, were enriched according to the selected genes of HZKY. In vivo studies showed that the serum levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol were significantly elevated and the serum level of high-density lipoprotein cholesterol was decreased in the model group compared with those in the control group (P < 0.01 for all). The expressions of BAX, CASP9, and CASP3 were upregulated in the model group compared with those in the control group (P < 0.05, P < 0.01, and P < 0.01, respectively), while HZKY treatment decreased the body weights and serum levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol and increased the serum levels of high-density lipoprotein cholesterol in NAFLD model rats (P < 0.05, P < 0.01, P < 0.05, and P < 0.05, respectively). Hematoxylin and eosin and oil red O staining indicated that HZKY treatment reduced steatosis in the hepatocytes. Moreover, HZKY treatment inhibited apoptosis in the liver by downregulating the expressions of BAX, CASP3, and CASP9 (P < 0.05, P < 0.01, and P < 0.05, respectively). Conclusion: Our study demonstrates that HZKY improves NAFLD by inhibiting apoptosis in the liver by reducing the levels of BAX, CASP3, and CASP9. Key words: Network pharmacology, Hua-Zhuo-Kai-Yu decoction, Nonalcoholic fatty liver disease, Apoptosis

Acknowledgments: This work was performed at Tianjin University of Chinese Medicine, China, and was supported by National Natural Science Foundation of China (81560772) and Natural Science Foundation of Tianjin (17JCYBJC42800). Abbreviations: HZKY, Hua-Zhuo-Kai-Yu decoction; NAFLD, non-alcoholic fatty liver disease; TCMSP, Traditional Chinese Medicine Systems Pharmacology and Analysis Platform; TCMID, Traditional Chinese Medicine Integrated Database; H&E, ematoxylin and eosin; BAX, Bcl-2 associated X protein; CASP3, caspase3; CASP9, caspase9; TC, total cholesterol; TG, triglyceride; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein cholesterol; TCM, traditional Chinese medicine; HFD, high-fat diet; IR, insulin resistance; DCH, Da-Chai-Hu decoction. Competing interests: The authors declare that they have no conflict of interest. Citation: Li YT, Cui HT, Yang L, et al. Hua-Zhuo-Kai-Yu decoction inhibits apoptosis in nonalcoholic fatty liver disease. Tradit Med Res. 2021; 6(1): 5. doi: 10.12032/TMR20200201157. Executive editor: Nuo-Xi Pi Submitted: 5 January 2020, Accepted: 31 January 2020, Online: 8 February 2020.

© 2021 By Authors. Published by TMR Publishing Group Limited. This is an open access article under the CC-BY license (http://creativecommons.org/licenses/BY/4.0/). 2 Submit a manuscript: https://www.tmrjournals.com/tmr Traditional Medicine Research doi: 10.12032/TMR20200201157 drugs, diseases, genes, and targets based on the Background combination of subject theory, including systematic biology, pharmacology, and multiple “omics” studies Nonalcoholic fatty liver disease (NAFLD) is caused by such as genomics and proteomics [13–16]. The metabolic liver injury accompanied by extensive application of network pharmacology in studying the hepatic steatosis. Epidemiological studies have mechanisms of TCM is in line with the characteristics indicated that the morbidity of NAFLD is of TCM, including a systematic and holistic approximately 6.3%–45% in adults and 15% in perspective with multiple components. It is developing developed areas such as Beijing, Shanghai, and into an effective tool for identifying the overall Guangzhou in China [1, 2]. Long-term NAFLD can mechanisms of TCM drugs [17]. In this study, network cause chronic metabolic diseases such as diabetes, pharmacology was used to predict the potential targets liver fibrosis, liver cirrhosis, and hepatocellular of HZKY in NAFLD. Furthermore, rats were fed with carcinoma [3, 4]. Currently, clinical treatment for HFD for 12 weeks to induce NAFLD and were then NAFLD includes administration of insulin sensitizers, administered HZKY orally. Serum lipid levels and antioxidants, and lipid-lowering drugs. However, these hematoxylin and eosin (H&E) and oil red O staining drugs have been shown to have multiple side effects. results were assessed to observe the effects of HZKY Lipid-lowering drugs can trigger the dysfunction of in NALFD. Moreover, the mechanisms of action of hepatic enzymes [5]. Insulin sensitizers and HZKY in NAFLD, as determined using network antioxidants have been shown to induce water-sodium pharmacology, were validated using Western blotting, retention and increase the risk of cardiac diseases [6]. which showed apoptosis inhibition in the liver. Traditional Chinese medicine (TCM) has been demonstrated to have better treatment effects on Materials and Methods NAFLD. Ling-Gui-Zhu-Gan decoction, which is a classic ancient TCM prescription, can attenuate Network Pharmacology Study high-fat diet (HFD)-induced NAFLD by improving Identification of the main active compounds. HZKY insulin resistance (IR) and regulating lipid is composed of Chaihu 12 g (Bupleuri Radix), metabolism-related pathways [7]. The empirical Huangqin 9 g (Scutellariae Radix), Shaoyao 9 g formula of the TCM Huo-Xue-Xiao-Zhi decoction can (Paeoniae Radix Rubra), Zhishi 9 g (Aurantii Fructus decrease tumor necrosis factor-α, transforming growth Immaturus), Banxia 9 g (Pinelliae Rhizoma), Dahuang factor, and free fatty acid levels and improve hepatic 6 g (Rhei Radix et Rhizoma), Bohe 9 g (Menthae function in patients with NAFLD [8]. Additionally, the Haplocalycis Herba), Heye 9 g (Nelumbinis Folium), empirical formula of the TCM and Peilan 9 g (Eupatorii Herba). The main Yi-Qi-Huo-Xue-Qu-Tan can modulate the dysfunction compounds in HZKY were screened using the of lipid metabolism in these patients [9] . Traditional Chinese Medicine Systems Pharmacology The classic ancient prescription Da-Chai-Hu (DCH) and Analysis Platform (TCMSP) decoction (Chaihu (Bupleuri Radix), Huangqin (http://tcmspw.com/tcmsp.php) and Traditional (Scutellariae Radix), Shaoyao (Paeoniae Radix Alba), Chinese Medicine Integrated Database (TCMID) Banxia (Pinelliae Rhizoma), Shengjiang (Zingiberis (http://www.megabionet.org/tcmid). The repeated Rhizoma Recens), Zhishi (Aurantii Fructus Immaturus), compounds were counted only once. Dazao (Jujubae Fructus)) is described in the ancient Because of the oral administration of HZKY for the book on Chinese medicine titled Shanghan Zabing Lun treatment of NAFLD, oral bioavailability (OB) was (Treatise on Cold Damage Diseases, 25 C.E. – 220 regarded as the pharmacokinetic parameter for HZKY C.E.), written by Zhang Zhongjing. According to the to be considered in the network pharmacology book, DCH is used to treat diseases of the liver, evaluations. The compounds in HZKY (OB ≥ 30%), gallbladder, and pancreas. Recent studies have also which were considered to be absorbed, metabolized, demonstrated the hepatoprotective, cholagogue, and functioning in the human body, were chosen as the anti-inflammatory, and lipid-lowering effects of DCH candidate compounds [18]. Then, the structural and [10, 11]. In addition, clinical studies have shown that physiochemical properties of candidate compounds in DCH improves dyslipidemia in patients with NAFLD HZKY were identified using the DrugBank [12]. Hua-Zhuo-Kai-Yu (HZKY) decoction, derived (https://www.drugbank.ca/) database. Compounds that from a modification of DCH, was established by were drug-like (DL) ≥0.18 were identified as the main Doctor Wang Hongwu at the Tianjin University of active compounds of HZKY [19, 20]. Traditional Chinese Medicine. It has shown obvious Potential target prediction of active compounds. clinical effects on NAFLD. However, the mechanisms The potential targets for active compounds in HZKY of action of HZKY in NAFLD remain unknown. were screened using TCMSP, TCMID, Bioinformatics Network pharmacology, developed by Hopkins AL Analysis Tool for Molecular mechANism of in England, is the study of the relationships among Traditional Chinese Medicine (BATMAN-TCM), and

Submit a manuscript: https://www.tmrjournals.com/tmr 3 doi: 10.12032/TMR20200201157 ARTICLE PUBCHEM (https://pubchem.ncbi.nlm.nih.gov) Briefly, the rats were fed with HFD diet for 12 weeks. databases. The repeated targets were counted only Their body weights and serum TG, TC, LDL-C, and once. HDL-C levels were measured to evaluate the induction Potential targets for prediction of the disease. of NAFLD. In addition, H&E and oil red O staining Targets related to NAFLD were obtained from were used to observe the pathological changes in the GeneCards (https://www.genecards.org/) and online rat livers. All animal experiments were approved by Mendelian Inheritance in Man (OMIM) the Institutional Animal Care and Use Committee of (https://www.omim.org/) databases. The protein data Tianjin University of Traditional Chinese Medicine bank IDs for each target were imported into the (SYXK2019-0006) and were performed in accordance Uniprot database (http://www.uniprot.org/) for with the National Institutes of Health “Guide for the standardization, and the repeated targets were removed. Care and Use of LaboratoryAnimals”. In addition, the potential targets of HZKY in NAFLD Preparation of the HZKY Test Solution. The HZKY were identified based on the common potential targets decoction was provided by the First Affiliated Hospital between HZKY and NAFLD. of Tianjin University of Traditional Chinese Medicine Biological function and signaling pathway analysis. (Tianjin, China). The herbs included in the HZKY The Kyoto Encyclopedia of Genes and Genomes decoction were identified by the pharmacist in the (KEGG) database was used to integrate the pharmacy of the First Affiliated Hospital of Tianjin information and functions of potential targets. Briefly, University of Traditional Chinese Medicine. The water the potential targets were analyzed and enriched in the extract of HZKY was concentrated at the laboratory of KEGG database (https://www.kegg.jp/). KEGG terms Tianjin University of Traditional Chinese Medicine. with P < 0.05 and q < 0.05 were screened. Briefly, the herbs were immersed in water for 30 min Establishment and analysis of the (the surface of the water covered the herbs by 2–3 cm) compound-target-pathway network. The and boiled at 100°C for 20 min using a gallipot. The relationships among compounds, targets, and pathways liquid was then filtered through gauze. The same of HZKY in NAFLD were visualized using the volume of water and herbs were boiled for another 15 compound-target-pathway network using Cytoscape min and filtered through gauze. The two filtrates were 3.7.2 [21]. According to the compound-target-pathway mixed to obtain the water extract of HZKY [23]. The network, compounds, targets, and pathways were water extract of HZKY was concentrated to a density visualized using different colors and were combined of 0.85 g crude herb/mL under decompressed based on their potential interactions. distillation using a DZF-6050 vacuum drying box (Beijing SanBo Technology Co., Ltd., Beijing, China) Experimental Validation and SENCO R rotating evaporator (Beijing Biological Reagents. HFD (17.7% sucrose, 17.7% fructose, Innovation Technology Co., Ltd., Beijing, China). 19.4% protein, and 40% fat) was purchased from Animal grouping. After 1 week of adaptive feeding, Beijing HFK Bioscience Co., Ltd. (Beijing, China). A 30 rats were randomly classified into three groups: BCA protein assay kit and triglyceride (TG), total control, model, and HZKY groups. Rats in the control cholesterol (TC), low-density lipoprotein cholesterol group were provided standard laboratory chow (LDL-C), and high-density lipoprotein cholesterol containing 59.4% total carbohydrate, 20% protein, and (HDL-C) test kits were purchased from Nanjing 4.8% fat. An NAFLD model rat was induced using Jiancheng Biological Engineering Institute (Nanjing, HFD in the model and HZKY groups. Moreover, the China). Oil red O staining kit was obtained from HZKY group received an oral gavage of HZKY (8.5 g Solarbio Biotechnology Co., Ltd. (Beijing, China). crude herb/kg rat weight) once daily for 8 weeks after Primary antibodies against rat caspase-3 (CASP3; 4 weeks of HFD feeding. The dosage of 8.5 g crude ab13847; 1:500), caspase-9 (CASP9; ab184786; herb/kg in the HZKY group was equivalent to the daily 1:1000), BAX (Bcl-2 associated X protein; ab32503; dosage of HZKY in human adults, which was 1:10000), and β-ACTIN (ab179467 1:5000) were calculated using the following formula: equivalent purchased from Abcam, Inc. (Shanghai, China), and dosage (g/kg) = clinical dosage of crude herbs in the corresponding secondary antibodies were obtained humans (g/kg) × 6.25. Meanwhile, the control and from Abcam, Inc. (Shanghai, China). model group rats were given oral gavage of same Animals. Male Sprague–Dawley (SD) rats weighing volume of saline once daily for 8 weeks after 4 weeks 190–210 g, were purchased from Weitonglihua Animal of feeding. Co., Ltd. (Beijing, China). All animals were housed for Serum biochemical marker assays. Following HZKY 1 week in a temperature- and humidity-controlled treatment, rats were anesthetized (chloral hydrate environment (12-h light/dark cycle, 21 ± 2℃, and a intraperitoneal administration, 0.3 mL/100 g rat weight) relative humidity of 45 ± 10%) in groups of five after a 5-h period of fasting, and blood was harvested rats/cage and were allowed feed and water ad libitum. from the inner canthus using a capillary glass tube. Induction of the NAFLD model rat. The NAFLD Serum was obtained by centrifugation with a SW 60 Ti model was induced as described previously [22]. rotor (Beckman Coulter Inc., Palo Alto, CA) at 3000 4 Submit a manuscript: https://www.tmrjournals.com/tmr Traditional Medicine Research doi: 10.12032/TMR20200201157 rpm for 15 min. Serum levels of TG, TC, LDL-C, and sections were then dried at room temperature for 5 min. HDL-C were measured according to the Next, the sections were stained with oil red O manufacturer’s instructions (Nanjing Jiancheng according to the manufacturer's instructions (Solarbio Biological Engineering Institute, Nanjing, China), and Biotechnology Co., Ltd., Beijing, China). Briefly, the the absorbance was detected using a Varioskan Flash sections were stained with oil red O lipid staining microplate reader (Thermo Fisher Scientific, USA). working buffer for 45 min after fixing with 5% In a 96-well plate, 2.5 μL of distilled water, TG and formaldehyde solution, washed with 60% isopropanol TC standards, and serum samples were mixed with 250 five times, and subsequently washed with distilled μL of working solution and incubated at 37°C for 10 water for 2 min. Sections were stained again with min. The absorbance value of each pore was measured hematoxylin for 2 min, washed with distilled water for using a microplate reader at 510 nm. The derived 5 min, and dried and sealed with glycerine. They were optical density (OD) value of distilled water, TG and then immediately photographed under a light TC standards, and serum samples were denoted by microscope (BX50, Olympus America, Melville, NY, OD1, OD2, and OD3, respectively. The TG and TC USA). levels were calculated using the following formulae: Western blotting analysis. The liver samples were homogenized with whole lysis buffer to extract proteins from the liver. Then, a BCA protein assay was used to quantify and standardize the proteins. Electrophoresis (8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to isolate the proteins, which were transferred onto a In accordance with the instructions for LDL-C and polyvinylidene difluoride membrane for Western HDL-C, 2.5 μL of distilled water, LDL-C and HDL-C blotting. The membrane was then sealed with 5% milk standards, and serum samples were incubated with 180 and incubated overnight with primary antibodies μL of reagent R1 supplied by kit at 37°C for 5 min in (rabbit anti-CASP1, 1:500; rabbit anti-CASP9, 1:1000; 96-well plates. After determining the absorbance value rabbit anti-BAX, 1:10000; and rabbit anti-β-ACTIN, of a microplate reader at 546 nm, the OD values of 1:10000) at 4℃. After incubation for 1 hour, TBST distilled water, standards, and serum samples were was used to clean the membrane, and the secondary recorded as OD1, OD2 and OD3, respectively. Then, 60 antibody probe (HRP combined with goat anti-rabbit μL of reagent R2 was added into each well, repeating IgG, 1:4000) was used for detection. Blotting was the previous step, and the mixed solution was observed using chemiluminescence (TanonTM High-sig ℃ incubated at 37 for 5 min. The absorbance value was ECL, Li COR, USA). The strip strength was detected by microplate reader at 546 nm. The OD quantitatively analyzed using Image J software (NIH, values of distilled water, standards, and serum samples Bethesda, MD, USA). were recorded as OD1', OD2', and OD3', respectively. Data Analysis. All values were reported as the mean ± The LDL-C and HDL-C levels were obtained using the standard deviation (mean ± SD). SPSS software following formulae: version 20.0 was used for single factor analysis of variance, and the statistical significance standard was P < 0.05.

Results

H&E staining. Rats were sacrificed after blood Results of Network Pharmacology collection. The liver was immediately removed and Main active compounds in HZKY. The 924 fixed with 10% formalin, dehydrated, and embedded in compounds present in HZKY, including 65 from paraffin wax. It was then cut into 5-µm sections using Zhishi (Aurantii Fructus Immaturus), 60 from Peilan a slicing machine (RM2125, Leica, Buffalo Grove, (Eupatorii Herba), 58 from Huangqin (Scutellariae USA) at the laboratory at Tianjin University of Radix), 93 from Heye (Nelumbinis Folium), 92 from Traditional Chinese Medicine. Sections were stained Dahuang (Rhei Radix et Rhizoma), 75 from Shaoyao with H&E as described previously [24]. Briefly, after (Paeoniae Radix Rubra), 288 from Chaihu (Bupleuri dewaxing, rehydration, staining, dehydration, Radix), 77 from Bohe (Menthae Haplocalycis Herba), transparency, and sealing, the pathological changes in and 116 from Banxia (Pinelliae Rhizoma), were the liver were visualized under a light microscope screened using the TCMSP and TCMID. A total of 130 (BX50, Olympus America, Melville, NY, USA). active compounds, including 22 from Zhishi (Aurantii Oil red O staining. Fresh liver tissue was rapidly Fructus Immaturus), 11 from Peilan (Eupatorii Herba), frozen in liquid nitrogen and sectioned using a frozen 24 from Huangqin (Scutellariae Radix), 15 from Heye slicer (CM3050S, Leica, Buffalo Grove, USA) to (Nelumbinis Folium), 16 from Dahuang (Rhei Radix et obtain coronal cryostat sections (10-μm thick). The Rhizoma), 27 from Shaoyao (Paeoniae Radix Rubra), Submit a manuscript: https://www.tmrjournals.com/tmr 5 doi: 10.12032/TMR20200201157 ARTICLE 17 from Chaihu (Bupleuri Radix), 10 from Bohe and DL ≥ 0.18. The structure and physiochemical (Menthae Haplocalycis Herba), and 36 from Banxia properties of the compounds are listed in Table 1. (Pinelliae Rhizoma), were identified with OB ≥ 30% Table 1 List of active candidate components in HZKY No. MOL_ID Molecule_name OB/% DL Herb Node 1 MOL000006 Luteolin 36.16 0.25 AFI, MHH M1 2 MOL001798 Neohesperidin_qt 71.17 0.27 AFI M2 3 MOL001803 Sinensetin 50.56 0.45 AFI M3 4 MOL001941 Ammidin 34.55 0.22 AFI M4 5 MOL002914 Eriodyctiol (flavanone) 41.35 0.24 AFI, RS M5 6 MOL004328 Naringenin 59.29 0.21 AFI, MHH M6 7 MOL005100 5,7-Dihydroxy-2-(3-hydroxy-4-methoxyphenyl) 47.74 0.27 AFI M7 chroman-4-one 8 MOL005828 Nobiletin 61.67 0.52 AFI M8 9 MOL005849 Didymin 38.55 0.24 AFI M9 10 MOL007879 Tetramethoxyluteolin 43.68 0.37 AFI M10 11 MOL009053 4-[(2S,3R)-5-[(E)-3-hydroxyprop-1-enyl]-7- 50.76 0.39 AFI M11 5-methoxy-3-methylol-2,3-dihydrobenzofuran-2 -yl]-2-methoxy-phenol 12 MOL013276 Poncirin 36.55 0.74 AFI M12 13 MOL013277 Isosinensetin 51.15 0.44 AFI M13 14 MOL013279 5,7,4'-Trimethylapigenin 39.83 0.30 AFI M14 15 MOL013352 Obacunone 43.29 0.77 AFI M15 16 MOL013428 Isosakuranetin-7-rutinoside 41.24 0.72 AFI M16 17 MOL013430 Prangenin 43.60 0.29 AFI M17 18 MOL013433 Prangenin hydrate 72.63 0.29 AFI M18 19 MOL013435 Poncimarin 63.62 0.35 AFI M19 20 MOL013436 Isoponcimarin 63.28 0.31 AFI M20 21 MOL013437 6-Methoxy aurapten 31.24 0.30 AFI M21 22 MOL013440 Citrusin B 40.80 0.71 AFI, EH M22 23 MOL000359 Sitosterol 36.91 0.75 EH, SR, NF, PRR, M23 and MHH 24 MOL000363 Amyrin Palmitate 32.68 0.30 EH M24 25 MOL000449 Stigmasterol 43.83 0.76 EH, PRR, BR, and M25 PR 26 MOL000584 7-Acetoxy-8-hydroxy-9-isobutyryloxythymol 33.39 0.18 EH M26 27 MOL000588 9-Acetoxy-8,10-epoxy-6-hydroxythymol 61.44 0.21 EH M27 3-O-angelate 28 MOL000592 Dammaradienyl acetate 46.52 0.82 EH M28 29 MOL000595 Eupatoriopicrin 76.78 0.36 EH M29 30 MOL000596 [(3S,4aR,6aR,6aR,6bR,8aR,12S,12aR,14aR, 43.08 0.74 EH M30 14bR)-4,4,6a,6b,8a,12,14b-heptamethyl-11- methylene-1,2,3,4a,5,6,6a,7,8,9,10,12,12a,13,14, 14a-hexadecahydropicen-3-yl] acetate 31 MOL000604 Eupaformosanin 50.20 0.52 EH M31 32 MOL000605 Taraxasteryl palmitate 33.84 0.31 EH M32 33 MOL000173 Wogonin 30.68 0.23 SR M33 34 MOL000228 (2R)-7-Hydroxy-5-methoxy-2-phenylchroman-4 55.23 0.20 SR M34 -one 35 MOL000358 Beta-sitosterol 36.91 0.75 SR, RRER, PRR, and M35 PR 36 MOL000525 Norwogonin 39.40 0.21 SR M36 37 MOL000552 5,2'-Dihydroxy-6,7,8-trimethoxyflavone 31.71 0.35 SR M37 38 MOL001689 Acacetin 34.97 0.24 SR, MHH M38 39 MOL002714 Baicalein 33.52 0.21 SR, PRR, and PR M39 40 MOL002908 5,8,2'-Trihydroxy-7-methoxyflavone 37.01 0.27 SR M40

6 Submit a manuscript: https://www.tmrjournals.com/tmr Traditional Medicine Research doi: 10.12032/TMR20200201157 Table 1 List of active candidate components in HZKY (Continued) No. MOL_ID Molecule_name OB/% DL Herb Node 41 MOL002909 5,7,2,5-Tetrahydroxy-8,6-dimethoxyflavone 33.82 0.45 SR M41 42 MOL002910 Carthamidin 41.15 0.24 SR M42 43 MOL002911 2,6,2',4'-Tetrahydroxy-6'-methoxychaleone 69.04 0.22 SR M43 44 MOL002913 Dihydrobaicalin_qt 40.04 0.21 SR M44 45 MOL002915 Salvigenin 49.07 0.33 SR M45 46 MOL002917 5,2',6'-Trihydroxy-7,8-dimethoxyflavone 45.05 0.33 SR M46 47 MOL002925 5,7,2',6'-Tetrahydroxyflavone 37.01 0.24 SR M47 48 MOL002926 Dihydrooroxylin A 38.72 0.23 SR M48 49 MOL002927 Skullcapflavone II 69.51 0.44 SR M49 50 MOL002928 Oroxylin a 41.37 0.23 SR M50 51 MOL002932 Panicolin 76.26 0.29 SR M51 52 MOL002933 5,7,4'-Trihydroxy-8-methoxyflavone 36.56 0.27 SR M52 53 MOL002934 NEOBAICALEIN 104.34 0.44 SR M53 54 MOL002937 DIHYDROOROXYLIN 66.06 0.23 SR M54 55 MOL000073 Ent-Epicatechin 48.96 0.24 NF M55 56 MOL000096 (-)-Catechin 49.68 0.24 NF, RRER M56 57 MOL000098 Quercetin 46.43 0.28 NF, BR M57 58 MOL000354 Isorhamnetin 49.60 0.31 NF, BR M58 59 MOL000422 Kaempferol 41.88 0.24 NF, BR M59 60 MOL003578 Cycloartenol 38.69 0.78 NF, PR M60 61 MOL006405 (1S)-1-(4-Hydroxybenzyl)-2-methyl-3,4-dihydro 67.14 0.23 NF M61 -1H-isoquinoline-6,7-diol 62 MOL007206 Armepavine 69.31 0.29 NF M62 63 MOL007207 Machiline 79.64 0.24 NF M63 64 MOL007210 o-Nornuciferine 33.52 0.36 NF M64 65 MOL007213 Nuciferin 34.43 0.40 NF M65 66 MOL007214 (+)-Leucocyanidin 37.61 0.27 NF M66 67 MOL007217 Leucodelphinidin 30.02 0.31 NF M67 68 MOL007218 Remerin 40.75 0.52 NF M68 69 MOL000471 Aloe-emodin 83.38 0.24 RRER、MHH M69 70 MOL000554 Gallic acid-3-O-(6'-O-galloyl)-glucoside 30.25 0.67 RRER M70 71 MOL002235 EUPATIN 50.80 0.41 RRER M71 72 MOL002251 Mutatochrome 48.64 0.61 RRER M72 73 MOL002259 Physciondiglucoside 41.65 0.63 RRER M73 74 MOL002260 Procyanidin B-5,3'-O-gallate 31.99 0.32 RRER M74 75 MOL002268 Rhein 47.07 0.28 RRER M75 76 MOL002276 Sennoside E_qt 50.69 0.61 RRER M76 77 MOL002280 Torachrysone-8-O-beta-D-(6'-oxayl)-glucoside 43.02 0.74 RRER M77 78 MOL002281 Toralactone 46.46 0.24 RRER M78 79 MOL002288 Emodin-1-O-beta-D-glucopyranoside 44.81 0.80 RRER M79 80 MOL002293 Sennoside D_qt 61.06 0.61 RRER M80 81 MOL002297 Daucosterol_qt 35.89 0.70 RRER M81 82 MOL002303 Palmidin A 32.45 0.65 RRER M82 83 MOL000492 (+)-Catechin 54.83 0.24 PRR M83 84 MOL001002 Ellagic acid 43.06 0.43 PRR M84 85 MOL001918 Paeoniflorgenone 87.59 0.37 PRR M85 86 MOL001921 Lactiflorin 49.12 0.80 PRR M86 87 MOL001924 Paeoniflorin 53.87 0.79 PRR M87 88 MOL001925 Paeoniflorin_qt 68.18 0.40 PRR M88 89 MOL002776 Baicalin 40.12 0.75 PRR, BR, PR M89 90 MOL004355 Spinasterol 42.98 0.76 PRR M90 91 MOL006990 (1S,2S,4R)-Trans-2-hydroxy-1,8-cineole-B-D- 30.25 0.27 PRR M91 glucopyranoside

Submit a manuscript: https://www.tmrjournals.com/tmr 7 doi: 10.12032/TMR20200201157 ARTICLE Table 1 List of active candidate components in HZKY (Continued) No. MOL_ID Molecule_name OB/% DL Herb Node 92 MOL006992 (2R,3R)-4-Methoxyl-distylin 59.98 0.30 PRR M92 93 MOL006994 1-O-beta-D-Glucopyranosyl-8-o- 36.01 0.30 PRR M93 benzoylpaeonisuffrone_qt 94 MOL006996 1-O-beta-D-Glucopyranosylpaeonisuffrone_qt 65.08 0.35 PRR M94 95 MOL006999 Stigmast-7-en-3-ol 37.42 0.75 PRR M95 96 MOL007003 Bbenzoyl paeoniflorin 31.14 0.54 PRR M96 97 MOL007004 Albiflorin 30.25 0.77 PRR M97 98 MOL007005 Albiflorin_qt 48.70 0.33 PRR M98 99 MOL007008 4-Ethyl-paeoniflorin_qt 56.87 0.44 PRR M99 100 MOL007012 4-O-methyl-paeoniflorin_qt 56.70 0.43 PRR M100 101 MOL007014 8-Debenzoylpaeonidanin 31.74 0.45 PRR M101 102 MOL007016 Paeoniflorigenone 65.33 0.37 PRR M102 103 MOL007018 9-Ethyl-neo-paeoniaflorin A_qt 64.42 0.30 PRR M103 104 MOL007022 EvofolinB 64.74 0.22 PRR M104 105 MOL007025 Isobenzoylpaeoniflorin 31.14 0.54 PRR M105 106 MOL000490 Petunidin 30.05 0.31 BR M106 107 MOL001645 Linoleyl acetate 42.10 0.20 BR M107 108 MOL004598 3,5,6,7-Tetramethoxy-2-(3,4,5-trimethoxyphenyl 31.97 0.59 BR M108 )chromone 109 MOL004609 Areapillin 48.96 0.41 BR M109 110 MOL004624 Longikaurin A 47.72 0.53 BR M110 111 MOL004628 Octalupine 47.82 0.28 BR M111 112 MOL004644 Sainfuran 79.91 0.23 BR M112 113 MOL004648 Troxerutin 31.60 0.28 BR M113 114 MOL004653 (+)-Anomalin 46.06 0.66 BR M114 115 MOL004702 Saikosaponin c_qt 30.50 0.63 BR M115 116 MOL004718 α-Spinasterol 42.98 0.76 BR M116 117 MOL013187 Cubebin 57.13 0.64 BR M117 118 MOL001790 Linarin 39.84 0.71 MHH M118 119 MOL002881 Diosmetin 31.14 0.27 MHH M119 120 MOL005190 Eriodictyol 71.79 0.24 MHH M120 121 MOL005573 Genkwanin 37.13 0.24 MHH M121 122 MOL011616 Fortunellin 35.65 0.74 MHH M122 123 MOL000519 Coniferin 31.11 0.32 PR M123 124 MOL001755 24-Ethylcholest-4-en-3-one 36.08 0.76 PR M124 125 MOL002670 Cavidine 35.64 0.81 PR M125 126 MOL005030 Gondoic acid 30.70 0.20 PR M126 127 MOL006936 10,13-Eicosadienoic 39.99 0.20 PR M127 128 MOL006937 12,13-Epoxy-9-hydroxynonadeca-7,10- 42.15 0.24 PR M128 dienoic acid 129 MOL006957 (3S,6S)-3-(benzyl)-6-(4-hydroxybenzyl) 46.89 0.27 PR M129 piperazine-2,5-quinone 130 MOL006967 Beta-D-ribofuranoside, xanthine-9 44.72 0.21 PR M130 HZKY, Hua-Zhuo-Kai-Yu decoction ; AFI, Aurantii Fructus Immaturus (Zhishi); EH, Eupatorii Herba (Peilan); MHH, Menthae Haplocalycis Herba (Bohe); SR, Scutellariae Radix (Huangqin); NF, Nelumbinis Folium (Heye); RRER, Rhei Radix et Rhizoma (Dahuang); PRR, Paeoniae Radix Rubra (Chishao); BR, Bupleuri Radix (Chaihu); and PR, Pinelliae Rhizoma (Banxia).

Potential targets of active compounds in HZKY. A Identification of potential targets and pathways of total of 1,672 targets were obtained from 130 active HZKY in NAFLD and network analysis of compounds in HZKY based on the TCMSP, TCMID, compounds, targets, and pathways. A total of 1,334 Bioinformatics Analysis Tool for Molecular targets for NAFLD were identified from the online mechANism of Traditional Chinese Medicine, and Mendelian Inheritance in Man and GeneCards PUBCHEM databases. Repeated targets were removed, databases. After intersecting with 269 targets of HZKY, and 269 targets were identified with alignment from 62 targeted were identified as potential targets of the Uniprot database. HZKY in NAFLD. 8 Submit a manuscript: https://www.tmrjournals.com/tmr Traditional Medicine Research doi: 10.12032/TMR20200201157 The interactive relationship of 62 potential targets decreased the body weight in the HZKY group was analyzed and visualized using String 11.0. The compared with that in the model group (420.57 ± candidates with the highest values of network 15.52 vs. 467.07 ± 15.31 g, P < 0.01, Table 2). In connectivity were BAX, CASP3, CASP9, IL6, EGFR, addition, the serum levels of TG, TC, and LDL-C were MAPK8, CYCS, ESR1, VEGFA, LDLR, PPARG, significantly elevated and the serum level of HDL-C PLAU, VCAM1, CYP3A4, GSK3B, PON1, GSTP1, was decreased in the model group compared with that NQO1, CTSD, and ACACA. BAX, CASP3, and in the control group (TG: 1.70 ± 0.13 vs. 0.28 ± 0.04, CASP9 were the targets with the highest values of TC: 2.58 ± 0.34 vs. 1.40 ± 0.26, LDL-C: 1.50 ± 0.23 network connectivity. Additionally, as determined vs. 0.89 ± 0.09, and HDL-C: 1.29 ± 0.16 vs. 1.86 ± using KEGG analysis, apoptosis, fatty acid synthesis, 0.18, P < 0.01 for all, Table 2). HZKY treatment and estrogen signaling pathways were the terms with decreased the serum levels of TG, TC, and LDL-C and the highest network connectivity. increased the serum level of HDL in the HZKY group The compounds, targets, and pathways of HZKY in compared with those in the model group (TC: 2.16 ± NAFLD were imported into Cytoscape 3.7.2 to 0.22 vs. 2.58 ± 0.34, P < 0.01; TG: 1.28 ± 0.16 vs. 1.70 establish the compound-target-pathway network ± 0.13, P < 0.05; LDL-C: 1.14 ± 0.22 vs. 1.50 ± 0.23, (Figure 1). P < 0.05; and HDL-C: 1.49 ± 0.14 vs. 1.29 ± 0.16, P < According to the compound-target-pathway network, 0.05, Table 2). compounds targeted to BAX included M56, M95, and H&E staining demonstrated that the hepatic lobule M116; compounds targeted to CASP9 included M1, and nucleus were clearly visible, the sizes of the M2, M3, M5, M8, M9, M10, M13, M14, M21, M25, hepatocytes were similar, and the hepatic cord was M29, M31, M33, M34, M35, M39, M49, M50, M57, arranged neatly in the control group. Compared with M59, M62, and M125; and compounds targeted to that in the control group, in the model group, hepatic CASP3 included M1, M2, M3, M4, M5, M6, M8, M9, steatosis was obvious, indicating that there were M10, M14, M13, M19, M21, M23, M25, M33, M34, vacuolar adipose droplets of various sizes and more M35, M38, M39, M49, M50, M56, M57, M58, M59, balloon-like changes in hepatocytes, and the structure M62, M64, M65, M84, M95, M116, M125, and M130. of the hepatic tissue could not be clearly observed. Moreover, BAX, CASP9, and CASP3 are closely HZKY treatment markedly alleviated the hepatic associated with the apoptosis pathway. Therefore, the steatosis and balloon-like changes in the HZKY group apoptosis pathway was predicted to be the potential compared with that in the model group (Figure 2a). mechanism of action of HZKY in NAFLD. Similarly, oil red O staining revealed that slight lipid deposition was observed in the control group. The lipid Results of Experimental Validation droplets in the cytoplasm of hepatocytes were stained HZKY ameliorated HFD-induced NAFLD in rats. red and were of various sizes and widely distributed in Using the NAFLD model rat, we observed that 12 the cytoplasm, indicating the presence of lipid weeks of HFD led to a significant increase in body deposition in a large number of hepatocytes. However, weight in the model group compared with that in the HZKY significantly ameliorated lipid deposition and control group (467.07 ± 15.31 vs. 414.29 ± 28.93 g, P reduced the number of intracellular lipid droplets < 0.01), whereas HZKY treatment significantly (Figure 2b).

Figure 1 Compound-target-pathway network. The blue nodes represent the targets, the yellow nodes represent the active components of HZKY, and the red nodes represent pathways. Every active ingredient can act on multiple targets.

Submit a manuscript: https://www.tmrjournals.com/tmr 9 doi: 10.12032/TMR20200201157 ARTICLE Table 2 HZKY treatment decreased body weight and changed the blood lipid composition in NAFLD model rats. Group Body weight (g) TC (mmol/L) TG (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L)

Control 414.29 ± 28.93 1.40 ± 0.26 0.28 ± 0.04 0.89 ± 0.09 1.86 ± 0.18 a a a a a Model 467.07 ± 15.31 2.58 ± 0.34 1.70 ± 0.13 1.50 ± 0.23 1.29 ± 0.16 HZKY 420.57 ± 15.52c 2.16 ± 0.22b 1.28 ± 0.16c 1.14 ± 0.22b 1.49 ± 0.14b Control, model, HZKY (8.5 g crude herb/kg rat weight; n = 10 per group). Data are presented as the mean ± SD. a: P < 0.01 compared with the control group; b: P < 0.05 compared with the model group; c: P < 0.01 compared with the model group. HZKY: HZKY, Hua-Zhuo-Kai-Yu decoction; TC, total cholesterol; TG, triglyceride; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein cholesterol.

HZKY inhibited apoptosis in NAFLD model rats HZKY treatment, indicating the significant curative According to pathways identified from the network effect of HZKY in NAFLD. pharmacology, apoptosis was selected as the NAFLD is accompanied by the apoptosis of mechanism for HZKY in NAFLD. Expressions of hepatocytes [28]; electron microscopy previously BAX, CASP9, and CASP3, which were predicted as showed that the ultrastructures of mitochondria were the top targets of HZKY in NAFLD, were investigated damaged in the hepatocytes of patients with NAFLD to determine the anti-apoptotic activities of HZKY in [29]. Steatosis in hepatocytes can impair the function NAFLD. The expressions of BAX, CASP9, and and decrease the membrane potential of mitochondria. CASP3 were elevated in the model group compared A decrease in the mitochondrial membrane potential with those in the control group (for BAX, P < 0.05; for can trigger the release of cytochrome C and activate CASP9 and CASP3, P < 0.01; Figure 3), whereas caspase family proteins, ultimately inducing apoptosis HZKY treatment decreased the expressions of BAX, of hepatocytes [30]. Furthermore, dysfunction in fatty CASP9, and CASP3 in the HZKY group compared acid synthesis can cause obesity and NAFLD. The with those the model group (for BAX and CASP9, P < uptake and synthesis of fatty acids in the liver is 0.05; for CASP3, P < 0.01; Figure 3). significantly increased in the liver [31]. Excessive amounts of fatty acid can trigger IR by inducing Discussions oxidative stress and decreasing sensitivity in hepatocytes. IR can cause dysfunction of lipase activity, In this study, the potential targets of HZKY in NAFLD thereby triggering lipolysis in the peripheral organs were predicted using network pharmacology. Many and increasing the levels of fatty acids in the serum compounds in HZKY, such as baicalein, [32]. Moreover, IR induces hyperinsulinemia, and high o-nornuciferine, and quercetin, have been levels of insulin inhibit the β-oxidation of fatty acids. demonstrated to have treatment effects in NAFLD. The excess fatty acids can be transported to the liver Baicalein, derived from Banxia (Pinelliae Rhizoma), and cause steatosis in hepatocytes and NAFLD [33]. Huangqin (Scutellariae Radix), and Shaoyao Estrogen has also been demonstrated to be associated (Paeoniae Radix Alba), is shown to reduce lipid with NAFLD. Compared with premenopausal women, accumulation in NAFLD model rats by inhibiting postmenopausal women exhibit an increase in body oxidative stress [25]. O-Nornuciferine, derived from weight, lipid accumulation, hepatic inflammatory Heye (Nelumbinis Folium), decreases the serum TG responses, and oxidative stress [34]. The and TC levels in NAFLD mouse models [26]. activation of estrogen receptors can increase the Quercetin, derived from Chaihu (Bupleuri Radix) and production of bile acid in NAFLD model mice. Bile Heye (Nelumbinis Folium), improves NAFLD via acid can activate the farnesoid X receptor to inhibit downregulation of FAS and SREBP-1C expression and lipogenesis in the liver. Estrogen receptors can also upregulation of IRS1 expression in the liver [27]. In directly reduce the lipid contents and liver injury in addition, BAX, CASP9, and CASP3 were the HZKY NAFLD model mice by inhibiting the activation of targets with the highest values of network connectivity, pregnane X receptor. Estrogen can also improve and apoptosis was the main signaling pathway of NAFLD by regulating the TLR-MYD88-IL-6 pathway. HZKY in NAFLD. The effects of HZKY in NAFLD Estrogen has been demonstrated to improve hepatocyte model rats were also investigated in the present study. injury in NAFLD by inhibiting the activation of NF-κB The results showed that HZKY treatment significantly and C/EBPβ and decreasing the activity of IL-6 improved the serum levels of TG, TC, LDL-C, and promoter [35] . In addition, lack of estrogen can HDL-C in NAFLD model rats. Moreover, lipid contribute to the progression of NAFLD [36-37]. BAX, accumulation in the liver was reduced following CASP9, and CASP3 are important factors that

10 Submit a manuscript: https://www.tmrjournals.com/tmr Traditional Medicine Research doi: 10.12032/TMR20200201157 modulate apoptosis in NAFLD. Accumulation of free the anti-apoptotic activities of HZKY in NAFLD fatty acids is shown to activate and dimerize BAX in model rats. In agreement with the results of network the cytoplasm [38]. Dimerized BAX is transported to pharmacology analysis, our results show that HZKY the outer membrane of the mitochondria and activates treatment significantly decrease the levels of CASP-9 and CASP-3 to initiate apoptosis [39]. apoptosis-related proteins, including BAX, CASP9, Given the high correlation of the apoptotic pathway and CASP3, in the liver. predicted by network pharmacology, we determined

Figure 2 Hepatic steatosis was improved after HZKY treatment. a. H&E staining indicated multiple adipose droplets in the model group and only slight steatosis in the liver in the HZKY group. Black arrows indicate steatosis of hepatocytes (100×). b. Oil red O staining revealed that the number of lipid droplets was markedly decreased in the HZKY group compared with that in the model group (40×). Control, model, HZKY (8.5 g crude herb/kg rat weight; n = 6 per group). HZKY: HZKY, Hua-Zhuo-Kai-Yu decoction.

Figure 3 HZKY treatment regulated the expressions of BAX, CASP9 and CASP3 in NAFLD model rats. a. Western blotting indicated that HZKY treatment decreased the expressions of Bax, CASP9 and CASP3. b. Relative expression. Control, model, HZKY (8.5 g crude herb/kg rat weight; n = 3 per group). Data are presented as the mean ± SD. a: P < 0.05 compared with the control group; b P < 0.01 compared with the control group; c: P < 0.05 compared with the model group; d: P < 0.01 compared with the model group. BAX, Bcl-2 associated X protein; CASP3, caspase3; CASP9, caspase9; HZKY: HZKY, Hua-Zhuo-Kai-Yu decoction.

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