Drugs and main agents
Drugs: rivaroxaban, an oral direct inhibitor of factor Xa, which is a potential coagulation factor for thrombin generation and clot formation, was purchased from ChemBest Research Laboratories Ltd. (CAS No. 366789-02-8).
Biochemical Reagents: a Glutathione Transaminase Test Kit (Cat No. C009-1) and a Glutathione Transaminase Test Kit (Cat No. C010-1) were purchased from Nanjing Jiancheng Institute of Biological Engineering. The hydroxyproline standard (trans-4-hydroxy-L-proline, Cat No. H54409) was purchased from Sigma.
Pathology-related testing reagents: SABC Immunohistochemical Staining Kit (Cat No. SA1028-Rabbit IgG), anti-PAI1 antibody (Cat No. ab66705), anti-fibrinogen rabbit monoclonal antibody (Cat No. ab189490), and anti-von Willebrand factor antibody (Cat No. ab6994) were all purchased from Sigma.
Establishment of a PVT model with cirrhosis in rats
Forty-one male Sprague‒Dawley rats weighing 160–180 g were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. We performed PPVL [8] and intraperitoneal injection of CCl4 to establish a rat model of cirrhosis complicated with PVT, as shown in the flow chart in Fig. 1B. Briefly, the skin of the rats was disinfected (Fig. 1-A1), after which a 1.5-2 cm longitudinal incision was made in the upper abdomen (Fig. 1-A2). After the main portal vein was exposed and isolated, a 20G needle was inserted into the portal vein, after which the portal vein was ligated with silk thread (Fig. 1-A3). At this time, the small intestine and stagnant mesenterial veins became stabile, and the color of these veins turned dark blue or violet (Fig. 1-A4). Then, the needle was removed slowly, which partially restored the portal vein blood flow. Although the original color of the intestines returned after removal, the vessel diameter of the portal vein became much narrower than that before ligation. After penicillin was injected intraperitoneally (Fig. 1-A5), the peritoneum and the skin were closed separately (Fig. 1-A6). After one week, the rats that received PPVL were injected intraperitoneally with different doses of CCl4 from week 1 to week 10, as shown in Fig. 1B.
Experimental design
The rats were divided into 7 groups: the control group (n = 7), the PPVL group (receiving PPVL but not CCl4 administration; n = 6), the week 4 model group (n = 6), the week 6 model group (n = 5), the week 8 model group (n = 6), the week 10 model group (n = 8) and the rivaroxaban-treated group (n = 10). The rats in the week 4 to week 10 model groups underwent the PPVL operation and were then subcutaneously injected with 25%-40% CCl4 dissolved in olive oil twice a week for 4 to 10 weeks. Beginning in the fifth week, the rats in the rivaroxaban group were orally administered rivaroxaban (20 mg/kg body weight [9]) for 6 weeks. All animal experiments were approved by the institutional animal ethics committees of the Laboratory Animal Center at Shanghai University of Traditional Chinese Medicine, Shanghai, China (Ethics Number: SZY201804011). All protocols and experimental procedures were conducted in accordance with the relevant institutional guidelines and regulations.
Biochemical tests
The sera were collected from the rats and assayed for alanine transaminase (ALT) and aspartate transaminase (AST) levels with kits. In addition, 4 ml of plasma was collected from each rat treated with anticoagulation agents, aliquoted into 2 tubes, and applied for the detection of PLT, Fibrinogen (FIB), D-dimer and AT III by the Laboratory Center of Shuguang Hospital Affiliated with the Shanghai University of Traditional Chinese Medicine.
Hepatic Hyp content assay
The hydroxyproline (Hyp) content in the tissue was measured with Jamall’s method [10]. Briefly, 100 mg of liver sample was homogenized and hydrolyzed in 12 M HCl at 110°C for 18 h. After filtration of the hydrolysate, chloramine T was added to a final concentration of 2.5 mM for 10 min at room temperature. The mixture was then treated with 25% (w/v) p-dimethylaminobenzaldehyde and 27.3% (v/v) perchloric acid in isopropanol and incubated at 50°C for 90 min. After cooling to room temperature, the samples were examined at 558 nm against a reagent blank that contained the solutions without tissue. The concentration of Hyp in each sample was determined from a standard curve, which was generated from a series of known quantities of Hyp ranging from 0.2 to 1.6 µg (Peptide Co., Japan). The Hyp concentration is expressed as µg/g of liver weight.
Histological examinations
Collected liver tissues were fixed in 4% formalin and embedded in paraffin. Sections (4 µm) were stained with hematoxylin-eosin (HE) and Sirius red. A grading system previously described was used to evaluate the stages of fibrosis [11].
Immunohistochemistry
Paraffin-embedded slices (4 µm) were subjected to immunohistochemical staining. Endogenous peroxidase activity was blocked by methanol with 3% H2O2 and bovine serum albumin (BSA). After washing with PBS, the sections were incubated with the following primary antibodies at 4°C overnight: anti-fibrinogen antibody (ab189490, 1/1500) and anti-PAI1 antibody (ab66705, 1/200). On the second day, the sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 37°C. Diaminobenzidine (DAB) was used as a chromogen, followed by hematoxylin counterstaining.
Immunofluorescence
The collected liver tissues were put into Tissue-Tek OCT embedding medium and snap-frozen in liquid nitrogen. Then, the tissues were fixed with acetone for 10 min, washed with PBS, and blocked with 0.5% BSA for 1 h at 37°C. The tissues were incubated with the primary antibody Anti-Von Willebrand Factor (ab6994, 1/200) at 4°C overnight. The next day, fluorescein isothiocyanate-labeled secondary antibodies were added to the samples, which were incubated for 1 h at 37°C. Subsequently, DAPI (ab228549, 1/1000) was used to stain the nucleus. The tissues were observed under a confocal microscope for imaging.
Western blot analysis
Liver lysates were separated on 10% SDS‒PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA and incubated with different primary antibodies: anti-fibrinogen antibody (ab92572, 1/1000) or anti-PAI1 antibody (ab66705, 1/1000) at 4°C overnight. After three washes with PBS, the membranes were incubated with secondary antibodies at room temperature for 1 hr. After an additional three washes, the membranes were scanned and imaged with Li-Cor odyssey.
Portal vein ultrasonic detection
An experienced B-mode ultrasound doctor used vascular Doppler ultrasound to observe portal vein thrombosis, portal vein blood flow velocity, and portal vein diameter. The vascular ultrasound instrument used was a Philips Lu22, the probe model was L12-5, the ultrasound frequency was set to 11 M, and the blood flow angle was < 60°.
Statistical analysis
All the data were analyzed by using SPSS software version 26.0. Differences between the groups were assessed by nonparametric one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc test. Quantitative data are expressed as the mean ± standard deviation (SD). A P value < 0.05 was considered to indicate statistical significance.