The Novel Chinese Medicine JY5 Formula Alleviates Hepatic Fibrosis by Inhibiting the Notch Signaling Pathway

Advanced liver fibrosis can lead to cirrhosis, resulting in an accelerated risk of hepatocellular carcinoma and liver failure. Fuzheng Huayu formula (FZHY) is a traditional Chinese medicine formula treated liver fibrosis in China approved by a Chinese State Food and Drug Administration (NO: Z20050546), composed of Salvia Miltiorrhiza bge., Prunus davidiana (Carr.) Franch., cultured Cordyceps sinensis (BerK.) Sacc. Mycelia, Schisandra chinensis (Turcz.) Baill., Pinus massoniana Lamb., and Gynostemma pentaphyllum (Thunb.) Makino. However, the main active substances and mechanism of FZHY are unclear. The aim of this study is to identify a novel anti-fibrotic compound, which consists of the main active ingredients of FZHY, and investigate its mechanism of pharmacological action. The main active ingredients of FZHY were investigated by quantitative analysis of FZHY extracts and FZHY-treated plasma and liver in rats. The anti-fibrotic composition of the main active ingredients was studied through uniform design in vivo, and its mechanism was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced liver fibrosis models in rats and mice, and transforming growth factor beta 1-induced LX-2 cell activation model in vitro. A novel Chinese medicine, namely JY5 formula, consisting of salvianolic acid B, schisantherin A, and amygdalin, the main active ingredients of FZHY, significantly alleviated hepatic hydroxyproline content and collagen deposition in CCl4-and BDL-induced fibrotic liver in rats and mice. In addition, JY5 inhibited the activation of hepatic stellate cells (HSCs) by inactivating Notch signaling in vitro and in vivo. In this study, we found a novel JY5 formula, which exerted anti-hepatic fibrotic effects by inhibiting the Notch signaling pathway, consequently suppressing HSCs activation. These results provide an adequate scientific basis for clinical research and application of the JY5 formula, which may be a potential novel therapeutic candidate for liver fibrosis.

Before the experiment, the rats were fasted overnight but with free access to water. After oral administration of FZHY decoction at 20g raw drug /kg, the rats (n=5 for each time point) were anesthetized and sampled at 0.167 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 11 h, 24 h and 48 h (1.4 g/kg). The hepatic portal vein blood (2 mL) and systemic blood (6-8 mL) were obtained from the portal vein and abdominal aorta, respectively, and the right liver lobe was excised. After centrifuged for 5 min at 10000 rpm, the plasma was obtained. After acetonitrile precipitation, the processed plasma and liver samples were injected to the mass instrument for analysis.

Experimental liver fibrosis models
CCl4-induced liver fibrosis rat model: Wistar rats were randomly divided into the control (Oil) group (n=8) and the CCl4 group (n=24). The model group rats were injected subcutaneously with 50% CCl4 solution in olive oil at a 1mL/kg dose, twice a week for 9 weeks. The control rats were injected subcutaneously with equivalent volume olive oil at a 1mL/kg dose. At the first day of the 7 th week, the CCl4 rats were randomly divided into 3 groups: the CCl4 group (n=8), the CCl4 plus JY5 group (n=8) and the CCl4 plus SORA group (n=8). Rats were respectively administered intragastrically with JY5 (salvianolic acid B: 16mg/kg, amygdalin: 0.5mg/kg, schisantherin A: 2mg/kg) and SORA (5mg/kg) added to 0.3% CMC-Na suspension at a 10mL/kg dose in drug-treated groups, once a day for 3 weeks. In the control group and CCl4 group, rats were administered intragastrically with equivalent volume 0.3% CMC-Na suspension at a 10mL/kg dose.
BDL-induced liver fibrosis rat model: SD rats were randomly divided into the control (Sham) group (n=8) and the BDL group (n=24). BDL surgery was performed as previously described (Zhang et al., 2017). At the first day of the second week after BDL, the BDL rats were randomly divided into 3 groups: the BDL group (n=8), the JY5 group (n=8) and the DAPT group (n=8). Rats were administered intragastrically with JY5 (salvianolic acid B: 16mg/kg, amygdalin: 0.5mg/kg, schisantherin A: 2mg/kg) added to 0.3% CMC-Na suspension at a 10mL/kg dose in the JY5 group. The DAPT group rats were injected intraperitoneally with DAPT (30mg/kg) dissolved in DMSO at a dose of 30mg/kg. In Sham group and BDL group, rats were administered intragastrically with equivalent volume 0.3% CMC-Na suspension at a 10mL/kg dose. The rats were treated with drugs once a day for three weeks.
CCl4-induced liver fibrosis mice model: C57/ BL6 mice were randomly divided into the control (Oil) group (n=8) and the CCl4 group (n=24). The CCl4 mice were injected intraperitoneally with 15% CCl4 solution in olive oil at a 2mL/kg dose, three times a week for 6 weeks. The control mice were injected intraperitoneally with equivalent volume olive oil at a 2mL/kg dose. At the first day of the 4 th week, the CCl4 mice were randomly divided into 3 groups: the CCl4 group (n=8), the CCl4 plus JY5 group (n=8) and the CCl4 plus SORA group (n=8). Mice were respectively administered intragastrically with JY5 (salvianolic acid B: 22.4mg/kg, amygdalin: 0.7mg/kg, schisantherin A: 2.8mg/kg) and SORA (7mg/kg) added to 0.3% CMC-Na suspension at a 10mL/kg dose in drug-treated groups, once a day for 3 weeks. In the control group and CCl4 group, mice were administered intragastrically with equivalent volume 0.3% CMC-Na suspension at a 10mL/kg dose.
All rats and mice were anaesthetized under sodium pentobarbital i.p. injection, then blood and liver samples were collected, and stored at −80°C for subsequent testing. Partial liver tissues were fixed in 10% neutral-buffered formalin for pathological analysis.

Histopathological and immunohistochemical analysis
According to corresponding standard protocol, liver sections were stained with Hematoxylin & Eosin (H&E, lot. 20161225, NJBI, Nanjing, China) and Sirius Red (SR). All slides were scanned using the Leica SCN400 slide scanner (Leica Microsystems Ltd., Mannheim, Germany). The whole SR-stained liver sections were analyzed using ImageScope software to calculate the percentage of collagen positive areas (Percent Total Positive × Total Stained Area / Total Analysis Area), which could semi-quantitatively evaluate liver fibrosis better.
IHC staining was performed using the GTVision TM Ⅲ Detection Kit with peroxidase/DAB, rabbit/mouse (Cat No. GK500705, Gene Tech Co., Ltd, Shanghai, China). Paraffin-embedded sections were dewaxed and rehydrated, subsequently subjected to heat-mediated antigen retrieval in 0.01M sodium citrate buffer, and rinsed in diH2O prior to staining. After cooling to room temperature, the sections were incubated in 3% H2O2-methanol mixture solution for 10 minutes to block endogenous peroxidase. Blocking the samples was performed by incubation with 10% goat serum at room temperature for 30 min, before incubation with primary antibody overnight at 4°C in a wet box. The next day, incubation with the second antibody and chromogen detection was performed using the DAB chromogen by light microscopy. After nuclear counterstaining by hematoxylin, the sections were differentiated, dehydrated and transparentized. Finally, the sections were sealed with neutral gum and scanned using the Leica SCN400 slide scanner to further analysis.

Luciferase reporter assay
The transcriptional activity of Notch was measured using RBP-кB luciferase reporter plasmid constructed by Shanghai Jikai Gene Chemical Technology Co. Ltd. following the supplier's instructions for use. The transiently co-transfected LX-2 cells with firefly luciferase and renilla luciferase plasmid using Lip8000TM (C0533, Beyotime Biotechnology, China) were treated with TGF-β1(5ng/mL), and simultaneously treated with salvianolic acid B (32μM), amygdalin (1μM), schisantherin A (4μM) or JY5 (37μM) for 24 hours, respectively. RBP-кB luciferase activity was detected by Dual-Lumi™ luciferase reporter gene assay kit (RG088S, Beyotime Biotechnology, China) following the manufacturer's instructions. With renilla luciferase a the internal control in each transfection, the relative luciferase activity was calculated as the ratio of fireflyto-renilla luciferase activity.