A clinical experience-based Chinese herbal formula improves ethanol-induced drunken behavior and hepatic steatohepatitis in mice models

BGXJW preparation

BGXJW powder was provided by Hebei Traditional Chinese medicine liver disease hospital. It is the extracts from the roots of Pueraria lobata (Willd.) Ohwi (Gegen), flos of Pueraria lobata (Willd.) Ohwi (Gehua), seeds of Hovenia Dulcis Thunb. (Zhijuzi), seeds of Cassia abbreviata Oliv. (Juemingzi), flos of Chrysanthemum abolinii (Kovalevsk.) H.Ohashi and Yonek. (Juhua), rhizomes of Alpinia officinarum Hance (Gaoliangjiang), roots of Salvia miltiorrhiza Bunge (Danshen), fruits of Morus alba L. (Sangshen), fruits of Amomum tsao-ko Crevost et Lemarie (Caoguo). The doses of these herbs are in fixed proportions.

To investigate BGXJW’s effects and the associated mechanisms, the BGXJW powder was added into 0.9% saline and ultrasounded to promote dissolution.

Reagents and instrumentsReagents

Buspirone hydrochloride (purity 98%, TRC, Toronto, Canada); phenytoin sodium(purity 99.97%, Bidepharm, Shanghai, China); Rodent Liquid Diet Lieber-DeCarli ’82, Ethanol Shake and Pour 4 Liters/Bag(Bio-serv, New Jersey, USA); Rodent Liquid Diet Lieber-DeCarli ’82, Control Shake and Pour 4 Liters/Bag( Bio-serv, New Jersey, USA); The ALT, AST, TG, T-CHO, CK-MB and CK-MM kits (96 T, Nanjing Jiancheng Bioengineering Institute, NanJing, China. TNF-α, IL-6, and IL-10 ELISA kits(96 T, CLOUD-CLONE CORP. Wuhan, China);liver tissues was isolated with FastPure Cell/Tissue Total RNA Isolation Kit (50 T, Vazyme, Nanjing, China) RIPA Lysis Buffer were purchased from Biorigin, Beijing, China; polyvinylidene fluoride membrane were purchased from MILLIPORE, Massachusetts, USA; The antibodies used against CYP2E1, SCD1, FASN, and PPAR-α were purchased from Abcam, Massachusetts, USA; antibodies against ADH1 and ALDH2 were purchased from Boster, California, USA and Cell Signaling Technology, Boston, USA separately; Antibodies against SREBP-1 (A-4) were purchased from Santa Cruz Biotechnology,Santa Cruz, USA; Antibodies against β-Actin were purchased from Bioworld, Minnesota, USA; HRP-Goat Anti-Rabbit IgG(H + L) was purchased from Biorigin, Beijing, China; Goat Anti-Mouse IgM (HRP) was purchased from Bioss, Beijing, China,. ECL kits were purchased from Biorigin, Beijing, China; RT Master Mix for qPCR II(MCE, State of New Jersey, USA); SYBR Green qPCR Master Mix (Low ROX) (MCE, State of New Jersey, USA).

Instruments

AB-SCIEX API 6500 QTRAP MS-WORKSTATION (MS BENCH) MOBILE LAB BENCH(Applied Biosystems, Foster City,USA); QuantStudio™ 6 Flex Real-Time PCR System, 384 wells(Applied Biosystems, Foster City,USA); Mini-PROTEAN® Tetra Cell, Mini Trans-Blot® Module, and PowerPac™ Basic Power Supply(Bio-Rad, State of California, USA); Pannoramic SCAN(3DHISTECH CaseViewer, Budapest, Hungary.

Quality profiling for the main components of BGXJW by UPLC-MS/MS

0.1 g BGXJW powder was accurately weighed and added to 50 mL of methanol. The sample was vortexed for 1 min and sonicated for 30 min to make the stock solution with a concentration of 2 mg/mL. Take an appropriate amount of the prepared stock solution and dilute it by 1, 10, and 100 folds with ultrapure water to form sample solutions for use respectively. Internal standard solution (500 ng/mL buspirone hydrochloride and 500 μg/mL phenytoin sodium): Accurately weigh an appropriate amount of buspirone hydrochloride (TRC, Toronto, Canada) and phenytoin sodium (Bidepharm, Shanghai, China) control samples, dissolve them in DMSO and prepare a stock solution with buspirone hydrochloride and phenytoin sodium concentrations of 1.00 mg/mL respectively. Accurately measure an appropriate amount of the stock solution and dilute it with water until the concentration of buspirone hydrochloride is 500 ng/mL and the concentration of phenytoin sodium is 500 μg/mL of mixed internal standard solution.100 µL of each sample solution was added to 100 µL of mixed internal standard solution and vortexed for 1 min, then centrifuged at 14,000 rpm for 10 min, respectively. The supernatants were taken for injection analysis. The components of BGXJW were analyzed by AB-SCIEX API 6500 QTRAP MS-WORKSTATION (MS BENCH) MOBILE LAB BENCH. UPLC-MS/MS detection of liquid chromatographic conditions is shown in Additional file 1: Table S1.

Animal experiments

Male C57BL/6 mice (SPF, SCXK [J] 2019-0010), 8 weeks old, were purchased from SPF Biotechnology Co., Ltd (Beijing, China). Mice were maintained in a controlled environment (23 ± 1 °C, lights on from 6:00 a.m. to 6:00 p.m.) with free access to water and food. All animal procedures were approved by the Institutional Animal Care and Use Committee at the Capital Medical University, Beijing, China. Approval number for ethical (IACUC-2021-0008).

To test the acute toxicity of BGXJW, mice were randomly divided into five groups: control (ctrl) group, BGXJW-treated (134, 268, 536, and 1072 mg/kg) groups. Each group has 6–10 mice. All mice were fed a control Lieber-DeCarli liquid diet for 15 days. Starting from the 5th day, mice in BGXJW-treated groups were further fed with BGXJW at 134, 268, 536 and 1072 mg/kg by oral gavage once a day for the following ten days, while mice in the ctrl group were given 0.9% saline at equal volumes daily. On the last day, all mice were sacrificed nine hours past gavage. The serum, liver, kidney, and small intestine were collected for subsequent experiments.

To exam if BGXJW could protect mice from alcohol-induced liver injury, the acute-on-chronic NIAAA model of ALD was chosen. The mice were randomly divided into five groups (6–10 mice per group): control (ctrl), ethanol (EtOH), and EtOH + BGXJW (33, 67, and 134 mg/kg) groups. All mice were fed the control Lieber-DeCarli liquid diet for the first five days to adapt liquid diet, then mice in EtOH + BGXJW groups were fed Lieber-DeCarli liquid diet containing 5%(v/v) ethanol for the next 10 days, meanwhile, BGXJW were given at indicated doses by oral gavage once a day. Mice in the ctrl group were fed a control Lieber-DeCarli liquid diet and equal volumes of 0.9% saline daily during these 10 days, while mice in the EtOH group were fed Lieber-DeCarli liquid diet containing 5%(v/v) ethanol and equal volumes of 0.9% saline daily. On the last day, all mice were administrated corresponding doses of BGXJW or equal volumes of saline. One hour later, the ctrl group was administrated with 45%(wt/v) maltodextrin solution (9 g/kg), EtOH group and EtOH + BGXJW groups were administrated with 31.5%(v/v) ethanol. All mice were sacrificed, and the serum and liver were collected for subsequent experiments after another nine hours.

To explore if BGXJW could relieve drunkenness, the alcohol binge-drinking model of ALD was used. The mice were divided into ctrl, EtOH, and EtOH + BGXJW groups. The detailed procedure could be seen in Fig. 7A. After fasting for 12 h, mice in the EtOH + BGXJW group were given 134 mg/kg BGXJW while mice in the ctrl and EtOH groups were given 0.9% saline for the corresponding volume. 30 min later, the ctrl group was given 0.9% saline, EtOH group and EtOH + BGXJW group were given 3 g/kg ethanol for the same volume. This operation was repeated 20 min later. Subsequently, the drunkenness situation of mice was detected and being recorded. The plasma samples at indicated time points were collected, centrifugated and immediately tested by gas chromatography.

Biochemical test

The serum samples were collected after centrifuging at 3000 rpm, 4 °C for 15 min and stored at − 80 °C. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), creatine kinase MB (CK-MB), creatine kinase MM(CK-MM) concentrations after indicated treatments were determined by the related kits according to the manufacturer’s instructions. The ALT, AST, TG, CK-MB and CK-MM kits were purchased from Nanjing Jiancheng Bioengineering Institute, NanJing, China.

50 mg of liver tissue was homogenized in 500 µL ethanol absolute, then centrifuged at 2500 rpm for 10 min. Triglyceride (TG), total cholesterol(T-CHO)concentrations after indicated treatments were determined by the related kits according to the manufacturer’s instructions.The TG, T-CHO kits were purchased from Nanjing Jiancheng Bioengineering Institute, NanJing, China.Hepatic triglyceride and total cholesterol levels was normalized to tissue wet weight and expressed as mg/g of liver.

ELISA assay

Frozen liver tissues were homogenized with RIPA Lysis Buffer (Biorigin, Beijing, China). TNF-α, IL-6 and IL-10 in liver lysates were determined by TNF-α, IL-6, and IL-10 ELISA kits according to the instructions. These ELISA kits were purchased from CLOUD-CLONE CORP. Wuhan, China.

Western blot

Take part of frozen liver tissues and homogenize with RIPA Lysis Buffer (Biorigin, Beijing, China). Equal amounts of protein were separated using 8–12% sodium dodecyl sulfate–polyacrylamide-gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (MILLIPORE, Massachusetts, USA). The membranes were blocked with 5% skim milk for 1 h and then incubated with indicated primary antibodies overnight at 4 °C, followed by a horseradish peroxidase-conjugated secondary antibody. The antibodies used against CYP2E1, SCD1, FASN, and PPAR-α were purchased from Abcam (Massachusetts, USA); antibodies against ADH1 and ALDH2 were purchased from Boster (California, USA) and Cell Signaling Technology (Boston, USA) separately. Antibodies against SREBP-1 (A-4) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Antibodies against β-Actin were purchased from Bioworld (Minnesota, USA). HRP-Goat Anti-Rabbit IgG (H + L) was purchased from Biorigin (Beijing, China). Goat Anti-Mouse IgM (HRP) was purchased from Bioss (Beijing, China). ECL kits were purchased from Biorigin, Beijing, China.

RT-PCR

For RT-PCR analysis, total RNA from liver tissues was isolated with FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, Nanjing, China) according to the instruction, and subsequently were reverse-transcribed to cDNA using RT Master Mix for qPCR II(MCE, State of New Jersey, USA). RT-PCR was carried out on Applied Biosystems® QuantStudio™ 6 Flex Real-Time PCR System, 384 wells using SYBR Green qPCR Master Mix (Low ROX) (MCE, State of New Jersey, USA). Target gene expression was calculated by the comparative CT method. All primers used are listed in Additional file 1: Table S5.

Histology

Liver, kidney, and small intestine collected after indicated treatments were fixed with 4% paraformaldehyde for at least 24 h, and then the sections were dehydrated, paraffin-embedded, and subjected to standard hematoxylin and eosin staining, then imaged with light microscopy (3DHISTECH CaseViewer, Pannoramic SCAN).

Hepatic steatosis was stained by oil red O staining.

Statistics

All data in experiments were expressed as mean ± standard error of the mean (SEM). The comparison between groups was evaluated by GraphPad Prism (GraphPad Software, San Diego, CA, United States). One-way analysis of variance or Tukey’s multiple comparison tests was used to perform statistical analyses. Statistically significant differences between groups were defined as p values no more than 0.05.

留言 (0)

沒有登入
gif