Page 101 - GTM-4-1

Global Translational Medicine Comparative study of Schisandra species on VCI

platform location and other behavioral data were analyzed Corporation, United States). Major constituents were
using SuperMaze software. separated using an ACQUITY UPLC BEH C column
18
(2.1 × 100 mm, 1.7 μm) at a flow rate of 0.3 mL/min and
2.10. Full-field laser perfusion imaging a temperature of 23°C, with an injection volume of 2 μL.
One hour after final administration, anesthetized The elution gradient was performed using 0.1% formic
mice were subjected to craniotomy for full-field laser acid water (A) and acetonitrile (B) as follows: 0 – 1 min,
perfusion imaging (FLPI) (FLPI2, Moor Instruments, 5% – 30% B; 1 – 2 min, 30% – 40% B; 2 – 4 min, 40% – 52%
United Kingdom). The FLPI system was configured to a B; 4 – 6 min, 52% – 60% B; 6 – 9 min, 60% – 62% B;
low-resolution, high-speed imaging mode with a display 9 – 10 min, 62% B; 10 – 13 min, 62% – 66% B; 13 – 15 min,
rate of 25 Hz, a time constant of 1.0 s, and an exposure time 66% – 70%. Mass weight was detected in centroid mode
of 20 ms. Every measurement lasted 1 min, generating six using an electrospray ionization source over a mass
stabilized blood flow images with the camera positioned range of 50 – 1700 Da, with a scan time of 0.3 s. The MS
10 cm above the brain. detector was set with a capillary voltage of 3 kV, a sampling
cone voltage of 40 V, a source temperature of 100°C, a
2.11. Luxol fast blue (LFB) staining desolvation temperature of 350°C, and collision energy of
On day 40, anesthetized mice received transcardial 6 kV. Data were collected using MassLynx 4.1 software and
perfusion with saline followed by paraformaldehyde. Brains profiled using Progenesis QI 2.3 software, with assistance
were post-fixed, embedded in paraffin, sectioned into from a self-constructed Schisandra compound database.
4-μm slices, and stained with 0.1% LFB, followed by 0.05% 2.15. Evaluation of drug-likeness
lithium carbonate. The sections were then sealed with a
coverslip and examined under a light microscope. The For compounds identified using UPLC-Q-TOF/MS,
severity of demyelination was scored as follows: 0: normal; six physicochemical properties, including lipophilicity,
1: disarrangement of nerve fibers; 2: obvious vacuoles; and size, polarity, solubility, saturation, and flexibility, were
3: injured and disappearance of myelinated fibers. evaluated for bioavailability. Predictions of passive human
gastrointestinal absorption (HIA), blood-brain barrier
2.12. Immunohistochemistry staining (BBB) permeability, and interactions with permeability
Paraffin sections underwent antigen retrieval, treatment glycoprotein (P-gp) for active efflux were conducted using
with 3% H O , and blocking with 3% bovine serum the SwissADME web tool (http://www.swissadme.ch).
2
2
albumin. Sections were incubated overnight at 4°C
with a rabbit anti-myelin basic protein (MBP) antibody 2.16. Statistical analysis
(1:1000; Cat. no. K002910P, Solarbio, China), followed by Raw data were presented as mean ± SEM. One-way
incubation with horseradish peroxidase-labeled anti-rabbit ANOVA was used for multiple group comparisons in
secondary antibody (1:200; Cat. no. PV-9000, Solarbio, GraphPad Prism 8.0, followed by Fisher’s least significant
China) and diaminobenzidine staining. The sections were difference test for normally distributed data, or the
counterstained with hematoxylin and examined under a Kruskal–Wallis H-test for non-normally distributed data.
light microscope. The degree of demyelination was semi- Statistical significance was set at P < 0.05 or P < 0.01.
quantified using Image-Pro Plus software.
3. Results
2.13. Ethanol extract preparation for SCF and SSF
3.1. SCF exhibits stronger anti-neuroinflammatory
SCF and SSF fruits were harvested from Huanren (Jilin activity than SSF in LPS-induced BV2 cells model
province) and Shangluo (Shaanxi province), China,
respectively, in September 2020. The samples were Microglial activation is a prominent feature following
23
identified by Dr. Yaodong Qi (Institute of Medicinal Plant chronic cerebral hypoperfusion. To evaluate and compare
Development, Chinese Academy of Medical Sciences), and the anti-neuroinflammation effects of SCF and SSF, an LPS-
voucher specimens were stored in the authors’ laboratory. induced BV2 cell model was established. A pilot study (data
The dried fruit (500 g) was powdered, extracted using not shown) confirmed that SCF and SSF had no significant
gradient ethanol (1:10 v/v), and enriched to obtain an effects on BV2 cell viability at concentrations below
ethanol extract. 80 μg/mL. Based on preliminary experimental results, 40,
16, 6.4, and 2.56 μg/mL were selected for further study.
2.14. Chemical constituent analysis of SCF and SSF As shown in Figure 1, LPS stimulation significantly
SCF and SSF extracts were analyzed using ACQUITY increased the production of NO, TNF-α, and IL-6 in
UPLC coupled with a Xevo G2 QTOF detector (Waters BV2 cells compared to the control group. Treatment with

Volume 4 Issue 1 (2025) 93 doi: 10.36922/gtm.6879