Materials
Puerariae radix (PR) and Coptidis rhizoma (CR) were obtained from the First Affiliated Hospital of Jinan University (Guangzhou, China). Puerarin and L-methionine (also referred as methionine in this paper) were purchased from Aladdin Chemicals (Shanghai, China). Berberine was purchased from TopScience Co. (Shanghai, China). Dextran sulfate sodium (DSS, molecular weight of 36-50 kDa) was purchased from MP Biomedicals (Irvine, CA). Assay kits for total homocysteine (tHcy), triglyceride (TG), malondialdehyde (MDA), and myeloperoxidase (MPO) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-1β and IL-6 were purchased from Neobioscience Technology Company (Shenzhen, China).
Animals
Wild-type C57BL/6 mice (male, 5-6 weeks of age) were purchased from HFK Biotechnology (Beijing, China). Mice were maintained under a 12 h light/12 h dark cycle {lights on at 7:00 AM [=Zeitgeber time 0 (ZT0)] and lights off at 7:00 PM (=ZT12)}, with free access to food and water.
Extraction of herbal medicines
Extracts of PR and CR were prepared according to previously reported methods [27,28]. Concentrated extractions were stored at 4°C before use. Puerarin accounts for 2.69% (w/w) of total PR exact, and berberine accounts for 4.66% (w/w) of total CR exact (Fig. 1).
Sample preparation for qualitative analysis
Approximately 0.4 g of PR and CR extracts were dissolved in 10 ml 50% methanol (v/v), followed by vortex and centrifugation (13,000 g, 15 min). The supernatant was filtered through 0.22 μm membranes, and then diluted to appropriate concentration (i.e, 2 mg/ml) for analysis.
UPLC-QTOF/MS analysis
Puerarin in PR and berberine in CR were quantified as previously described [6,22]. Peak areas of puerarin and berberine were recorded with extract masses of m/z 417.05 ± 0.05 Da and 336.06 ± 0.05 Da, respectively. Representative extracted ion chromatograms are provided in Figure 1.
Methionine-induced hyperhomocysteinemia and drug treatment
Hyperhomocysteinemia was induced by feeding mice with 0.5% methionine in drinking water for eight weeks as previously described [6]. Normal control mice were provided with pure drinking water. Hyperhomocysteinemia mice were divided into eight groups randomly (n = 5 per group) to assess the effects of PR on hyperhomocysteinemia. For dose-response study, four groups of mice were respectively gavaged with 1 g/kg PR, 2 g/kg PR, 3 g/kg PR and vehicle at ZT10 once a day for 7 days. On day 8, mice were sacrificed at ZT2 to collect plasma and liver samples. For chronoefficacy study, the remaining groups of mice were gavaged with 2 g/kg PR or vehicle at ZT2 or ZT10 per day for 7 days. On day 8, mice were sacrificed at ZT2 to collect plasma and liver samples.
DSS-induced colitis and treatment
Chronic colitis was induced in mice via three cycles of 2% DSS (in drinking water) feeding as previously described [22]. Control mice were fed with pure drinking water. The colitis mice were divided into eight groups (namely, groups 1-8, n = 6 per group). Group 1, group 2 and group 3 of mice were respectively gavaged with 100, 200 and 400 mg/kg CR at ZT10 once a day for 7 days. Group 4 of mice were treated with vehicle. On day 8, CR-, vehicle-treated and normal control mice were sacrificed at ZT2 to collect plasma and colon samples. The remaining groups of mice were gavaged with 400 mg/kg CR or vehicle at ZT2 or ZT10 once a day for 7 days. On day 8, mice were sacrificed at ZT2 to collect plasma and colon samples.
Biochemical analysis
Plasma and hepatic tHcy levels were quantified by performing enzymatic cycling assays with a homocysteine kit according to the manufacturer’s protocol (Jiancheng Bioengineering Institute, Nanjing, China). Plasma and hepatic TG levels were measured using a TG assay kit according to the manufacturer’s protocol (Jiancheng Bioengineering Institute, Nanjing, China). Colonic MDA and MPO activities were measured using malondialdehyde and myeloperoxidase assay kits according to the manufacturer’s protocol (Jiancheng Bioengineering Institute, Nanjing, China). Colonic IL-1β and IL-6 levels were quantified with ELISA kits according to the manufacturer’s protocol (Neobioscience, Shenzhen, China).
Oil red O staining
Oil red O staining was performed to analyze hepatic lipid accumulation as previously described [6]. In brief, liver samples were fixed in 10% paraformaldehyde and embedded in paraffin. Paraffin sections (10 μm) were sequentially stained with oil red O and hematoxylin. Images were obtained using a Zeiss Axio Imager M1 microscope (Carl Zeiss AG, Oberkochen, Germany).
Macroscopic scoring of colitis
Changes in body weight, diarrhea and bleeding in colitis mice were recorded on a daily basis after drug treatment. Scoring was performed according to the criteria described previously [29]. Body weight changes were calculated relative to day 1. Disease activity index (DAI) is the sum of the weight loss, diarrhea, and bloody stool scores. After the mice were sacrificed, colons lengths (as an indirect marker of colonic inflammation) were immediately measured with a centimeter ruler.
H&E staining
Colon tissues were fixed in 4% paraformaldehyde and embedded in paraffin, followed by hematoxylin-eosin (H&E) staining. Histological damage was scored based on enterocyte loss, crypt inflammation, lamina propria mononuclear cells, neutrophils infiltration, and epithelial hyperplasia as previously described [30]. The total histological score ranged from 0 (no changes) to 6 (extensive cell infiltration and tissue damage).
qPCR
qPCR assays were performed as described in our previous publication [31]. Amplification reaction consisted of an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Primers are listed in Table 1.
Statistical analysis
Data are presented as mean ± SD (standard deviation). Student’s t test was used to test for statistical differences between two groups. One-way or two-way ANOVA followed by Bonferroni post hoc test was used for multiple group comparisons. The level of significance was set at p < 0.05 (*).