Normal saline

In brief, 4 × 10⁵ cells were resuspended in 0.1 mL of PBS and subcutaneously injected into the right lateral flank of C57BL/6 mice to establish the B16-F10 tumor model. Another batch of resuspended 4 × 10⁵ 4T1 cells in 0.1 mL of PBS were injected into the fourth pair of the mammary fat pad of BALB/C mice to establish the 4T1 tumor model. When the tumor sizes reached 120-180 mm³, the animals were randomly assigned into three groups (n = 6), namely, control, DOX-low, and DOX-high groups that were intragastrically administered with the vehicle (normal saline, Solarbio, Beijing, China) or 50 or 100 mg/kg/day DOX , respectively, for 14 days.

BBB permeability was assessed by Evans blue (EB) staining. Rats were anesthetized with a mixture of 2%–2.5% isoflurane and 97.5%–98% air and EB (2%, 4 ml/kg, normal saline, Solarbio) was injected into the tail vein of the stroke rat. After the EB cycle for 1 h, the left ventricle was slowly perfused with normal saline to wash out the EB. The brain tissue was cut into five pieces and photographed quickly. Left hemispheres were isolated and homogenized with 50% trichloroacetic acid (1.5 ml/g, Solarbio) for 1 min to take immune fluorescence. The tissue was kept at 4 °C overnight, centrifuged at 14 000 rpm for 15 min, and the supernatant was collected. The fluorescence intensity was detected by a full-wavelength microplate reader (excitation at 620 nm and emission at 680 nm). Data are represented as concentration of EB dye (μg)/tissue weight (g).

Before the experiments, all rats have two behavioural tests: Y‐maze (YM) and Morris Water Maze (MWM). We obtained the normal values about these behavioural tests. Rats were then randomly divided into two groups: wide type (WT) group (n = 10) and thioacetamide (TAA) group (n = 30). Liver cirrhosis was induced by intraperitoneal injection (ip) of TAA (200 mg/kg in normal saline, Solarbio) twice per week for 11 weeks.
The behavioural manifestations of hepatic encephalopathy in rats that received intraperitoneal injection of TAA evolve four stages: (a) lethargy, (b) mild ataxia, (c) lack of spontaneous movement, loss of righting reflex, but positive response to tail pinch, and (d) coma, and no response to tail pinch. If TAA‐treated rats had one of the above manifestations, it could be diagnosed as overt HE. TAA‐treated rats with no HE symptoms were then again subjected to YM and MWM behavioural tests to confirm whether or not MHE.36 MHE rats randomly assigned to two groups (n = 13 in each group), a vehicle control group and an Ori‐treated group, Ori‐treated group were intraperitoneally injected with Ori (5 mg/kg/d) (Shanghai, China) for two weeks, control and MHE groups received saline. At the end of treatment, all groups were subjected to YM and WMW test, respectively.