Rat HAPI microglial cell lines were purchased from Shenzhen HaodiHuatuo Biological Technology Co., Ltd., and by DNA species identification it was confirmed that the present HAPI cell line was derived from rats. Cells were cultured in DMEM (cat. no. 11965092; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS at 37˚C with 5% CO₂. The cells were split into five groups as follows: Control group; IFN-β-induced inflammation group; and three physcion groups. The control group did not contain any treatment except for DMEM. The inflammatory group cells were treated with rat IFN-β recombinant protein (250 pg/ml; Thermo Fisher Scientific, Inc.) for 24 h at 37˚C, andthe treatment group cells were incubated with 50, 100 or 200 µmol/l physcion (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h at 37˚C following pretreatment with IFN-β.

The cells were seeded into a 96-well plate at 5,000 cells/well and incubated overnight at 37˚C with 5% CO₂. Cells were then treated with different concentrations of physcion for 24 h, as previously described. Subsequently, 10 µl CCK-8 reagent (Dojindo Molecular Technologies, Inc.) was added into each well for incubation according to the manufacturer's instructions, followed by assessment of the optical density value at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

The cells were seeded into a 96-well plate at 5,000 cells/well and incubated overnight at 37˚C with 5% CO₂. When the cells had grown to 80-90% confluence, they were washed with PBS and 0.05% trypsin was added for digestion. After 1 min, the digestion was stopped and centrifugation was performed at 200 x g for 3 min at room temperature. After removal of the supernatant, the cells were washed with PBS, 2X EdU (dissolved in DMEM; Beyotime Institute of Biotechnology) was added at room temperature for 30 min, and then fixation was performed with 4% polyoxymethylene for 15 min at room temperature. Finally, the cells were washed in PBS with 3% BSA and treated with Triton-X-100 (1%) for 5 min at room temperature. Subsequently, the cells were incubated with DAPI (10 µg/ml; Beyotime Institute of Biotechnology) for 10 min in the dark. Finally, a fluorescence microscope was utilized to capture the images. Five randomly selected fields were imaged and Image J (Version 1.45; National Institutes of Health) was applied for quantification.

Total RNA was extracted from HAPI cells (1x10⁷) using TRIzol^® reagent, and its concentration and quality were determined using the NanoDrop^™ 2000 (both from Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Subsequently, total RNA was reverse transcribed into cDNA according to the instructions of the PrimeScript^™ RT Master Mix (Perfect Real Time) Kit (Takara Biotechnology Co., Ltd.). The TB Green^® Fast qPCR Mix (Takara Biotechnology Co., Ltd.) was used to perform the RT-PCR procedure. The primer was synthesized by Shanghai Sangong Biotech. The primer sequences were as follows: JAK2 forward, 5'-TGGGAATGGCTTGCCTTACA-3' and reverse, 5'-TTGGGTGGATACCAGATCCTT-3'; and STAT3 forward, 5'-CTGAGGTACAATCCCGCTCG-3' and reverse, 5'-TGGCTGCATATGCCCAATCT-3'. Relative expression of mRNA was normalized to that of U6 and GAPDH, respectively, using the 2^-ΔΔCq method (17). The primer sequences were as follows: U6 forward, 5'-TGCTGGCATTGGCAGTACAT-3' and reverse, 5'-AAACATGGAACGCCTCATGATTTG-3'; and GAPDH forward, 5'-GCATCTTCTTGTGCAGTGCC-3' and reverse, 5'-GATGGTGATGGGTTTCCCGT-3'.

Cell apoptosis was examined using flow cytometry methods. Cells from the logarithmic growth phase were seeded into 6-well plates overnight and then treated separately with different reagents. For each group, 3x10⁵ cells were centrifuged at 200 x g for 5 min at room temperature. The cells were washed and stained with PI and Annexin V-FITC (Annexin V-FITC Apoptosis Detection Kit; cat. no. C1062S; Beyotime Institute of Biotechnology). Cell apoptosis was examined by FACSCaliber (BD Biosciences) and analyzed by FlowJo software (version 10.0; Treestar, Inc.).

Cells were incubated overnight and treated with different reagents and then further incubated for 48 h. Cells were collected by centrifugation at 200 x g for 5 min at room temperature, and total protein was extracted using RIPA buffer (BestBio). Total protein (30 µg/lane) was quantified with a BCA Kit (Beyotime Institute of Biotechnology) and separated by SDS-PAGE on a 10% gel. The separated proteins were subsequently transferred onto a polyvinylidene membrane (MilliporeSigma). Subsequently, the membranes were blocked with 5% BSA for 60 min at room temperature and incubated overnight at 4˚C with the following primary antibodies: Bcl-2 (1:1,000; cat. no. ab32124; Abcam); Bax (1:1,000; cat. no. ab32503; Abcam); JAK2 (1:1,000; cat. no. ab108596; Abcam); p-JAK2 (1:1,000; cat. no. ab32101; Abcam); STAT3 (1:1,000; cat. no. ab68153; Abcam); p-STAT3 (1:1,000; cat. no. ab267373; Abcam); and GAPDH (1:2,000; cat. no. ab9485; Abcam). Following primary incubation, the membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:2,000; cat no. ab150077; Abcam) for 1 h at room temperature. Protein bands were visualized using Novex^® ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific, Inc.) and protein expression was quantified by Gel-Pro analyzer 4.0 (Media Cybernetics, Inc.). GAPDH was used as the loading control.

According to the instructions provided by the manufacturer, the expression levels of IL-6, TNF-α, IL-1β, monocyte chemoattractant protein-1 (MCP-1), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and the concentration levels of malondialdehyde (MDA) and nitric oxide (NO) in the cell (1x10⁴) supernatant were analyzed by measuring the absorbance value at 450 nm using a microplate reader. The expression levels of IL-6 were examined by Rat IL-6 ELISA Kit (cat. no. PI328; Beyotime Institute of Biotechnology), TNF-α by Rat TNF-α ELISA Kit (cat. no. PT516; Beyotime Institute of Biotechnology), IL-1β by Rat IL-1β ELISA Kit (cat. no. PI303; Beyotime Institute of Biotechnology), MCP-1 by Rat MCP-1 ELISA Kit (cat. no. PC128; Beyotime Institute of Biotechnology), SOD by Rat SOD ELISA Kit (cat. no. DS-496; Shanghai Guangrui Biological Technology Co., Ltd.), POD by Rat POD ELISA Kit (cat. no. CR102665; YCEXTRACT Biotechnology; Wuxi Yuncui Biotechnology Co., Ltd.), CAT by Rat CAT ELISA kit (cat. no. CSB-E13439r; Cusabio Technology, LLC), MDA by Rat MDA ELISA Kit (cat. no. NDC-EKX-A1T9QG-96; Nordic BioSite AB) and NO by NO Assay Kit (cat. no. S0021S; Beyotime Institute of Biotechnology).

Cells were cultured at a density of 1.8x10⁵ cells/ml, and then incubated for 24 h. The cells with different treatments were then treated with 2',7'-dichlorodihydrofluorescein diacetate (10 µM; Beyotime Institute of Biotechnology) in the dark, the cells were resuspended in PBS, followed by an examination of the fluorescence intensity using a microscope. Image J was applied for quantification.

The data are expressed as mean ± standard deviation. SPSS version 19.0 (IBM Corp.) was implemented to analyze the data. One-way ANOVA with Turkey's post hoc test was used for comparison among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

The molecular structure of physcion is shown in Fig. 1A. The CCK-8 assay indicated that IFN-β significantly increased the viability of HAPI cells; however, this phenomenon was inhibited following treatment of the cells with physcion (50, 100 or 200 µmol/l) in a dose-dependent manner (Fig. 1B). Similarly, the results of the EdU incorporation assay indicated that IFN-β induced the proliferation of HAPI cells. Nevertheless, IFN-β-induced proliferation in HAPI cells was reversed by physcion (50, 100 or 200 µmol/l) addition (Fig. 1C and D).

Subsequently, the effects of physcion on the induction of apoptosis in IFN-β-treated HAPI cells were investigated. Flow cytometry analysis demonstrated that IFN-β reduced the extent of HAPI cell apoptosis, while physcion (50, 100 or 200 µmol/l) treatment could increase the proportion of apoptotic cells (Fig. 2A). Concomitantly, the apoptotic effect of physcion was confirmed using western blot analysis. It was shown that IFN-β increased Bcl-2 levels and decreased Bax levels; however, this effect was counteracted following physcion (50, 100 or 200 µmol/l) treatment (Fig. 2B).

The effects of physcion on the IFN-β-induced inflammatory response were assessed in HAPI cells. As shown in Fig. 3A-D, the production of inflammatory factors (IL-6, TNF-α, IL-1β and MCP-1) was enhanced in HAPI cells following induction of IFN-β expression. Nonetheless, treatment of the cells with physcion (50, 100 or 200 µmol/l) restrained the production of these inflammatory factors.

Furthermore, the effects of physcion were assessed in HAPI cells following oxidative stress induced by IFN-β. The activity levels of oxidant and antioxidant enzymes were evaluated. The results indicated that IFN-β increased the activity levels of SOD, POD and CAT and the concentration of MDA, respectively, compared with those in the control cells. In addition, physcion (50, 100 or 200 µmol/l) treatment counterbalanced the oxidative stress induced by IFN-β as manifested by the decreased levels of SOD, POD, CAT and MDA (Fig. 4A-D). The data indicated that IFN-β significantly reduced oxygen-glucose deprivation/reperfusion-induced NO content and ROS production in HAPI cells; however, these changes were offset following physcion treatment (Fig. 5A-C).

A previous study showed that inhibition of the JAK2/STAT3 pathway could improve central nervous system injury (18). Therefore, to investigate if physcion could improve IFN-β-induced HAPI cell injury via the JAK2/STAT3 pathway, the mRNA and protein levels of key genes in this pathway were detected. The results indicated that both IFN-β and physcion did not affect the mRNA levels of JAK2 and STAT3 (Fig. 6A). However, the protein levels of p-JAK2 and p-STAT3 were increased in HAPI cells treated with IFN-β. These changes were counteracted following physcion treatment (Fig. 6B).