The National Institutes of Health Guide for the Care and Use of Laboratory Animals was followed for all experiments and treatments on animals. And all animal experiments were sanctified and supervised by the Ethics Committee of the Chongqing Southwest Hospital (approval no. AMUWEC20224059). A total of 118 adult C57BL/6J mice (male, 23–26 g, aged between 8–11 weeks, 112 were enrolled in experiments and 6 died) were acquired from the Southwest Hospital experiments animal center. Throughout the experiment, food and water were freely available to the mice. The feeding room has a constant temperature of 22–25 °C and humidity of 55–60%, and 12 hours dark/12 hours light circle.

The mice were stabilized on the stereotactic instrument (No.68001, RWD Life Science Corporation, Shenzhen, Guangdong, China) after proper anesthetization by a mixed 2% isoflurane/air (2–3 L/min). The mice ICH model was constructed according to the previous description [26]. Briefly, after performing a scalp incision and drilling a cranial hole, 16 µL autologous blood from the tail vein was harvested after tail shearing and pumped into the basal ganglia (from bregma: 2.1 mm lateral, 0.9 mm anterior, and 3.0 mm ventral) by a sterilized syringe (No.7000, Hamilton, Bonaduz, Glaubenden, Switzerland) at 2 µL/min. After injecting the blood, the syringe needle was fixed for five min to prevent blood back-flow. An animal surgery temperature control device (No.78600 Zhongshi Corporation, Beijing, China) was used to control mice’s body temperatures during surgery. Then, the weight of each mouse was measured. Mice experiment groups were randomly divided by a mice breeder who is blind to this study by means of a serious of random number. Needle insertion was the only procedure performed on the Sham mice. In the ICH group, 16 µL autologous blood from the tail vein was injected as mentioned. In the ICH + PAP-1 group, mice were further randomly divided by the dosage of PAP-1 (No.HY-10015, MedChemExpress, Monmouth Junction, NJ, USA) (20, 40, and 60 mg/kg/d). The PAP-1 was administrated via intraperitoneal injections immediately after ICH. PAP-1 was dissolved in dimethyl sulfoxide (DMSO) and corn oil with 0.05:0.95 before injection. Each mouse received a 2 mL mixture in one shot daily, and the injections continued for 3 or 7 days according to the experimental design. The ICH group only received the same volume of 2 mL mixed dimethyl sulfoxide and corn oil intraperitoneal injections.

Open field test (OFT) was used for mice behavioral changes evaluation, as described before [27]. Briefly, a field box was evenly divided into four sections adjacent to each other but isolated by a board. Each section was 50 cm square and 50 cm high. Each mouse was placed in one of the chambers for five min allowing free movement. Their activities were video recorded. Then, the chamber was cleaned with purified water and ready for the next test. The resultant videos were then analyzed by a software (VideoTrack, ViewPoint Behaviour Technology, Lyon, Rhone, France). The mean velocity of the movement was then calculated from the software by two examiners blinded to the group’s design.

Basso mouse score (BMS) is a scoring system used to evaluate mice’s joint movement and coordination [28, 29]. Each mouse was placed on a broad experimental table one by one for free movement under the observation of two investigators. After one min of observation, the score from each observer blind to group design was recorded. The scoring scale ranges from 0 to 9, and the significance of each point is described previously [28, 29].

Transmission electron microscopy (TEM) was performed to detect the integrity of the white matter bundle as described [30]. Briefly, after perfusion, samples (1 mm${}^3$) were incubated overnight in 1.25% glutaraldehyde and then post-fixed for three days at 4 °C in 1.25% glutaraldehyde. Then, the samples were rinsed thrice and fixed by 1% osmium tetroxide (OsO${}_4$) for 2 h. After re-dyeing with uranyl acetate, dehydration was conducted using gradient acetone, followed by infiltration with propylene oxide. Thereafter, the samples were embedded in epoxy resin, sliced by an ultra-microtome (EM UC7, Leica, Wetzlar, Hessian, Germany), and exposed to TEM (HT7700, Hitachi, Tokyo, Japan). G-ratio was measured by ImageJ (V.1.8, NIH, Bethesda, Rockville, MD, USA) for evaluating demyelination per field that was randomly selected. At least 25 white matter bundles in each sample were taken into statistics. Three independent samples per group were taken into the experiment.

Briefly, a double antibody sandwich method was used in this study. The following enzyme-linked immune-sorbent assay (ELISA) kits were used: Murine TNF-$\alpha$ (No.BGK06804, Biogems, Los Angeles, CA, USA); Murine IL-1$\beta$ (No.BGK10749, Biogems, Los Angeles, CA, USA); Murine IL-4 (No.BGK07750, Biogems, Los Angeles, CA, USA); Murine IL-10 (No.BGK18893, Biogems, Los Angeles, CA, USA). After isolating ipsilateral brain tissue, the homogenate was centrifuged (4 °C, 12000 rpm, 30 min) and the concentration of the total protein was detected by a Bicinchoninic Acid kit (No.P0010, Beyotime, Shanghai, China). 100 µL supernate or standard was incubated with a monoclonal antibody pre-coated ELISA kit for 120 min at room temperature (RT). Then, after washing with PBS thrice, the biotinylated antibody was added for 90 min incubation at RT, followed by washing. Afterwards, 100 µL prepared 1$\times$ Avidin-Biotin-Peroxidase complex was added to each well for 40 min incubation at RT. Next, 90 µL color-developing reagent was added and incubated for 30 min in the dark at RT. 100 µL stop solution was added, followed by a 30 min reaction. Finally, the optical density (OD) value was measured at 450 nm by a spectrophotometer (Varioskan Flash, Thermo Scientific, Waltham, MA, USA). The concentration of inflammatory factors was calculated by comparing the OD value with the standard curve and recorded as pg/mg total protein after standardization. Each sample consisted of at least two repeat wells, and each group comprised at least three independent samples.

Brain sections were obtained from the aforementioned freezing microtome and then perforated with 0.3% Triton-X 100 (No.P0090, Beyotime, Shanghai, China)for 30 min. Then, the 5% bovine serum album (BSA) was added for 2 h blocking at RT. Thereafter, the following primary antibodies were incubated at 4 °C overnight: Mouse anti-Iba-1 (1:200, No.ab283319, Abcam, Cambridge, Cambridgeshire, UK); Rabbit anti-CD16 (1:100, No.16559-1-AP, Proteintech, Philadelphia, PA, USA); Rabbit anti-CD206 (1:100, No.18704-1-AP, Proteintech, Philadelphia, PA, USA); Rabbit anti-iNOS (1:100, No.22226-1-AP, Proteintech, Philadelphia, PA, USA); Rabbit anti-Arg-1 (1:100, No.16001-1-AP, Proteintech, Philadelphia, PA, USA); Rabbit anti-Kv1.3 (1:200, No.14079-1-AP, Proteintech, Philadelphia, PA, USA); Mouse anti-MBP (1:200, No.sc-365701, Santa Cruz, Dallas, TX, USA); Rabbit anti-NFH (1:200, No.BM0100, Boster, Wuhan, Hubei, China). After incubation, the sections were washed thrice with PBS; each sample was washed for at least 5 min. Then, the following secondary antibodies were incubated for 2 h at RT: Goat anti-Mouse 488; Goat anti-Mouse 555; Goat anti-Rabbit 488; Goat anti-Rabbit 555. Then, brain sections were washed with PBS and DAPI (4’,6-diamidino-2-phenylindole) was used for nuclear staining for 5 min at RT. Afterwards, the neutral resin was administrated for mounting. A fluorescent microscope (Axioskop2, Carl ZEISS, Weimar, German) was then taken to record the images of the resultant samples. At least 3 independent samples were studied in each group. At least 3 sections were prepared for the evaluation of each sample. The images were investigated by Image J (V.1.8, NIH, Bethesda, MA, USA). At least 3 images of the peri-hematoma area were taken from each section for statistics.

Mouse brain peri-hematoma tissue was extracted from homogenate and centrifuged at 4 °C, 12000 rpm 30 min after sacrifice. The protein concentration was also detected by the Bicinchoninic Acid kit above. Then, the protein samples were mixed with 5$\times$ loading buffer (No.P0015, Beyotime, Shanghai, China) and denaturation for 5 min at 95 °C. After calculation, a total of 50 µg protein was separated by gel electrophoresis using 10 or 12.5% SDS- polyacrylamide gel electrophoresis (SDS-PAGE) at a constant 80 V and electro-blotted to polyvinylidene difluoride (PVDF, Roche, Indianapolis, IN, USA) membranes under a constant current passage of 200 mA. Thereafter, the membranes were blocked by 5% BSA for 2 h at RT. Then the sample was incubated separately with the following primary antibodies overnight at 4 °C: Rabbit anti-Kv1.3 (1:1000, No.14079-1-AP, Proteintech, Philadelphia, PA, USA); Rabbit anti-MBP (1:1000, No.BA0094, Boster, Wuhan, Hubei, China); Rabbit anti-CD16 (1:1000, No.16559-1-AP, Proteintech, Philadelphia, PA, USA); Rabbit anti-CD206 (1:1000, No.18704-1-AP Proteintech, Philadelphia, PA, USA); Rabbit anti-iNOS (1:1000, No.22226-1-AP, Proteintech, Philadelphia, PA, USA); Rabbit anti-Arg-1 (1:1000, No.16001-1-AP, Proteintech, Philadelphia, PA, USA); Mouse anti-TNF-$\alpha$ (1:1000, No.sc-52746, Santa Cruz, Dallas, TX, USA); Mouse anti-IL-1$\beta$ (1:1000, No.sc-32294, Santa Cruz, Dallas, TX, USA); Rabbit anti-IL-4 (1:1000, No.66142-1-Ig, Proteintech, Philadelphia, PA, USA); Rabbit anti-IL-10 (1:1000, No.60269-1-Ig, Proteintech, Philadelphia, PA, USA); Mouse anti-NF-$\kappa$B p65 (1:200, No.sc-8008, Santa Cruz, Dallas, TX, USA); Mouse anti-NF-$\kappa$B p50 (1:200, No.sc-8414, Santa Cruz, Dallas, TX, USA); Mouse anti-p-NF-$\kappa$B p65 (1:200, No.sc-136548, Santa Cruz, Dallas, TX, USA); Mouse anti-p-NF-$\kappa$B p50 (1:200, No.sc-271908, Santa Cruz, Dallas, TX, USA); Mouse anti-$\beta$-Tubulin (1:5000, No.T200608, Zen-bio, Chengdu, Sichuan, China); Mouse anti-GAPDH (1:5000, No.250133, Zen-bio, Chengdu, Sichuan, China); Mouse anti-$\beta$-Actin (1:5000, No.700068, Zen-bio, Chengdu, Sichuan, China). After washing thrice with Tris-HCl buffer solution + Tween (TBST) post-incubation, the membranes were incubated with the secondary antibodies Goat anti-Rabbit or Goat anti-Mouse for 2 h at RT. The resultant band OD of membranes was detected by an imaging system (Evolution-Capt Edge, Vilber, Paris, Evelyn, France) with a western blotting chemiluminescence kit (No.WP20005, Thermo Fisher Scientific, Waltham, MA, USA). Images were analyzed by the aforementioned system (Evolution-Capt Edge, Vilber, France). At least 3 samples were there in each group and at least 3 times exposure was made in each sample.

After liquid nitrogen milling, the total RNA of mice hematoma ipsilateral brain was extracted by Trizol reagent (No.DP451, Tiangen, Beijing, China). Then, an extraction procedure was performed, followed by an extraction kit manual (No.DP451, Tiangen, Beijing, China). After RNA concentration measurement and dilution, RNA reverse-transcription was made by a reverse-transcription kit (No.RR047A, Takara Bio Inc., Shiga, Japan) on a PCR system (V.1.5, Bio-Rad CFX manager, Hercules, CA, USA). Thereafter, real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was conducted using SYBR-green Premix Ex Taq™ (No.RR820A, Takara Bio Inc., Shiga, Japan) on the system. In brief, a total of 25 µL reaction system was added to the 96-well plates followed by the manual (No.RR820A, Takara Bio Inc., Shiga, Japan) and then cycled for 41 rounds under conditions prescribed in the manual. Each sample was studied in triplicate. The threshold cycle (CT) values were collected, and the mRNA level was calculated by 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{CT}}$. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was set as an internal control. Relative gene expression was normalized to the Sham group and the primers used in the present study are listed in Table 1.

All data in this study were presented as mean $\pm$ SEM. SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) was used for the statistic. For the data obtained from the RT-qPCR of the ion channel, multiple t-tests were performed. For comparison of Kv1.3 immunoblot and immunostaining, the Student’s t-test was deployed. Behavioral data collected were analyzed by a two-way analysis of variance (ANOVA), followed by Turkey’s post-hoc test. One-way ANOVA was used for other data analyses. Furthermore, multiple comparisons were performed using Turkey’s post-hoc test only if the data lived up to the normal distribution checked by Shapiro-Wilk normality test. In this study, p 0.05 was considered to be statistically significant.

To determine which subunit of voltage-gated potassium channel plays the dominant role in enhancing neuroinflammation around the post-ICH hemorrhage, the mRNA expression of several subunits belonging to voltage-gated potassium channel was investigated using RT-qPCR (Fig. 1). Results demonstrated an increased expression of Kv1.3 mRNA from day 1 to day 7, especially on day 3, while the Kv1.3 mRNA expression exhibited no notable difference on day 14 between the Sham group and the ICH group (Fig. 2A). Moreover, the expression of other potassium channels exhibited no significant difference, such as Kv1.1, Kv1.5, and Kca3.1, between the two groups (Fig. 2A). Subsequently, the immunoblot bands indicated that in the ICH group, Kv1.3 protein expression was higher than that in the Sham group on day 3 (Fig. 2B,C). Next, the immunostaining images revealed higher optical densities of Kv1.3 in the ICH group than that in the Sham group around hematoma (Fig. 2D,E). Collectively, these results demonstrated that Kv1.3’s expression was markedly enhanced around the hematoma following ICH. The highest expression of Kv1.3 is on day 3 of ICH among the checkpoints of days 1, 3, 7, and 14.

Fig. 1.

Schematic illustration showing the experimental design and timeline in the current study. ICH, intracerebral hemorrhage; PAP-1, 5-(4- Phenoxybutoxy) psoralen.

Fig. 2.

Kv1.3 expression was significantly increased after ICH. (A) Bar charts illustrating the Kv1.1, Kv1.3, Kv1.5, and Kca3.1 mRNA expressions in Sham and ICH groups at different time points. n = 3 each group, ${}^{**}$p 0.01, ${}^{***}$p 0.001; multiple t-tests. (B) Immunoblot bands showing the Kv1.3 protein expression in Sham and ICH groups on day 3 after ICH. (C) Summarized bar graph depicting Kv1.3 expression from (B). n = 3 each group, ${}^{*}$p 0.05; Student’s t-test. (D) Immunostaining of Iba-1 (red) and Kv1.3 (green) in Sham and ICH groups on day 3 after ICH. Scale bars: 50 µm. (E) Summarized bar graph depicting the optical density of Kv1.3 from (D). n = 3 each group, ${}^{**}$p 0.01; Student’s t-test. ICH, intracerebral hemorrhage; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DAPI, 4’,6-diamidino-2-phenylindole.

The aforementioned results revealed that Kv1.3 expression was enhanced in microglia in ICH mice. Considering that Kv1.3 blockage using PAP-1 exerts a neuroprotective effect in neurodegenerative diseases and ischemic stroke [15, 16, 19], the different dosages (20, 40, and 60 mg/kg) of PAP-1 were used to test the neuroprotective effect of Kv1.3 blockage on ICH mice. The weight measurement and behavioral tests were conducted to validate the time-point and dosage for further research on days 1, 3, 7, and 14 (Fig. 1). Then the various examinations were performed on day 3 in the current study (Fig. 1).

First, the mice in the ICH + PAP-1 group weighed more than those in the ICH group, and the dosages of 40 and 60 mg/kg showed a better weight gain effect than the dosage of 20 mg/kg from day 3 to 14 (Fig. 3A). Next, the curves collected from BMS indicated that mice that received PAP-1 treatment exhibited higher scores than that in the ICH group, and the scores in ICH + PAP-1 40 mg/kg and ICH + PAP-1 60 mg/kg groups were higher than that in ICH + PAP-1 20 mg/kg group from day 3 to 14 (Fig. 3B). Subsequently, the results obtained from OFT revealed that the mean velocity of mice in OFT in the ICH group decreased significantly after ICH, whereas the same was considerably increased with the application of PAP-1 (Fig. 3C,D). Meanwhile, mice in ICH + PAP-1 40 mg/kg and ICH + PAP-1 60 mg/kg groups showed increased mean velocity than that in ICH + PAP-1 20 mg/kg group (Fig. 3C,D). Collectively, these results indicated that PAP-1 administration improved neurological deficits after ICH. The dosage was 40 mg/kg, and the checkpoint was day 3 for future experiments in the present experiments.

Fig. 3.

Application of PAP-1 facilitated neurological outcomes after ICH. (A) Weight variation curves of mice in each group. n = 8 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (B) Basso mouse score in each group. n = 8 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (C) Representative mice locomotion trajectory in the open field test. Blue square: the starting point; Yellow circle: the ending point; Red trajectory: velocity $>$25 cm/s; Green trajectory: velocity $>$7 but 25 cm/s; Black trajectory: velocity 7 cm/s. (D) Open field test showing each group’s mean velocity of mice. n = 8 each group, ${}^{***}$p 0.001 ICH vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 ICH + PAP-1 (40 mg/kg) vs. ICH group; ${}^{\$}$p 0.05 ICH + PAP-1 (20 mg/kg) vs. ICH group; p 0.001 ICH + PAP-1 (60 mg/kg) vs. ICH group.

The integrity of white matter bundles contributes greatly to neurological outcomes after ICH. Results indicated that the effect of PAP-1 administration on WMI was elucidated on day 3 following ICH. The immunostaining results of MBP and NF200 depicted that the optical density of MBP and NF200 decreased around hematoma on day 3 after ICH, while the treatment of PAP-1 partially ameliorated this phenomenon (Fig. 4A–C). Next, the immunoblot results elucidated a downregulated myelin basic protein (MBP) expression in the ICH group, whereas the application of PAP-1 abrogated this effect (Fig. 4D,E). Furthermore, the integrity of the white matter bundle was determined by TEM. As a result, the ICH group had a much greater G-ratio for the myelin sheath. In contrast, PAP-1 treatment markedly decreased this phenomenon (Fig. 4F,G). Combinedly, these results implied that ICH caused WMI, and the application of PAP-1 alleviated ICH-induced WMI in mice.

Fig. 4.

Administration of PAP-1 reduced ICH-induced white matter injury. (A) Immunostaining of MBP (red) and NF200 (green) in each group on day 3 after ICH. Scale bars: 50 µm. (B) Summarized bar graph depicting MBP expression from (A). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. (C) Summarized bar graph depicting NF-200 expression from (A). n = 3 each group, ${}^{**}$p 0.01 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. (D) Immunoblot bands illustrating MBP expression in each group on day 3 after ICH. (E) Summarized bar graph depicting MBP expression from (D). n = 3 each group, ${}^{**}$p 0.01 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. ICH group. (F) TEM images showing white matter bundles in each group. Orange arrows indicate demyelination. Scale bars: 2 µm. (G) Summarized bar graph depicting the white matter bundles’ G-ratio in each group. At least 70 white matter bundles in each group were considered in statistics, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. ICH group. MBP, Myelin Basic Protein; NF, Neurofilament Protein; TEM, Transmission electron microscopy.

To decipher why PAP-1 administration reduced WMI, a further investigation of anti-inflammatory and pro-inflammatory mediator levels was conducted. Initially, the results obtained using RT-qPCR assays on day 3 after ICH corroborated that Tnf-$\alpha$, Il-1$\beta$, and Il-6 mRNA expressions were significantly increased, whereas the elevated mRNA expression above was prominently decreased with the treatment of PAP-1 (Fig. 5A). Next, the immunoblot bands indicated that TNF-$\alpha$ and IL-1$\beta$ protein expressions were prominently increased, while this effect was abrogated with the application of PAP-1 on day 3 after ICH (Fig. 5B–D). Subsequently, the ELISA results showed that the concentration of TNF-$\alpha$ and IL-1$\beta$ were considerably increased, but this phenomenon was markedly compromised with the application of PAP-1 (Fig. 5E,F). Collectively, these results indicated that PAP-1 reduced the accumulation of pro-inflammatory factors on day 3 after ICH.

Fig. 5.

Administration of PAP-1 decreased the pro-inflammatory factors around hematoma after ICH. (A) Bar charts illustrating Tnf-$\alpha$, Il-1$\beta$, and Il-6 mRNA expression in each group on day 3 after ICH. n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (B) Immunoblot bands demonstrating TNF-$\alpha$ and IL-1$\beta$ expression in each group. (C) Summarized bar graph depicting TNF-$\alpha$ expression from (B). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (D) Summarized bar graph depicting IL-1$\beta$ expression from (B). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. ICH group. (E) ELISA results representing the concentration of TNF-$\alpha$ in each group on day 3 after ICH. n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. (F) ELISA results representing the concentration of IL-1$\beta$ in each group on day 3 after ICH. n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; ELISA, enzyme-linked immunosorbent assay .

Subsequently, the bar charts showed that the mRNA expressions of anti-inflammatory cytokines, including nerve growth factor (Ngf), Il-10, and brain-derived neurotrophic factor (Bdnf), were increased after ICH, and their expressions were notably enhanced with PAP-1 administration (Fig. 6A). Furthermore, immunoblot shows elevated IL-4 and IL-10 protein expressions, and this effect was markedly enhanced with the PAP-1 application on day 3 after ICH (Fig. 6B–D). Thereafter, the ELISA findings demonstrated that the content of IL-4 and IL-10 were prominently increased, and this effect was reinforced with the administration of PAP-1 (Fig. 6E,F). Taken together, these results verified that PAP-1 improved the neuro-inflammatory microenvironment by decreasing the accumulation of pro-inflammatory factors and enhancing the deposition of anti-inflammatory factors on day 3 after ICH.

Fig. 6.

The application of PAP-1 increased the anti-inflammatory cytokines around hematoma after ICH. (A) Bar charts illustrating Ngf, Il-10, and Bdnf mRNA expression in each group on day 3 after ICH. n = 3 each group, ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (B) Immunoblot bands illustrating IL-4 and IL-10 expression in each group on day 3 after ICH. (C) Summarized bar graph depicting IL-4 expression from (B). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. (D) Summarized bar graph depicting IL-10 expression from (B). n = 3 each group, ${}^{*}$p 0.05 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (E) ELISA results representing the concentration of IL-4 in each group on day 3 after ICH. n = 3 each group, ${}^{**}$p 0.01 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. ICH group. (F) ELISA results representing the concentration of IL-10 in each group on day 3 after ICH. n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. IL-4, interleukin-4; IL-10, interleukin-10.

The above findings confirmed that the application of PAP-1 improved the post-ICH neuro-inflammatory niche. Because the microglia transformation of M1/M2 phenotypes exerted the aforementioned effect, the polarization of microglia was investigated. First, the results indicated that the M1-microglia markers Cd16, Cd32, and Inos mRNA expression were substantially upregulated after ICH, and PAP-1 abrogated this effect on day 3 (Fig. 7A). Then, the immunoblot implied that CD16 and iNOS protein expression exhibited the same tendency as the mRNA expression level (Fig. 7B–D). Afterwards, the immunostaining images indicated that the Iba1${}^{+}$CD16${}^{+}$ cells’ percentage was significantly increased after ICH, while this effect was partly abrogated by the application of PAP-1 on day 3 after ICH (Fig. 7E,F). Concurrently, the proportion of Iba1${}^{+}$iNOS${}^{+}$ cells showed the same trend as Iba1${}^{+}$CD16${}^{+}$ cells (Fig. 7G,H).

Fig. 7.

Application of PAP-1 reduced the M1-like microglia around the hematoma after ICH. (A) Bar charts illustrating Cd16, Cd32, and Inos mRNA expression in each group on day 3 after ICH. n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (B) Immunoblot bands illustrating CD16 and iNOS expression in each group on day 3 after ICH. (C) Summarized bar graph depicting CD16 expression from (B). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. ICH group. (D) Summarized bar graph depicting iNOS expression from (B). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. (E) Immunostaining of CD16 (red) and Iba-1 (green) in each group on day 3 after ICH. Scale bars: 50 µm. (F) Summarized bar graph depicting Iba1${}^{+}$CD16${}^{+}$ cells percentage from (E). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (G) Immunostaining of iNOS (red) and Iba-1 (green) in each group on day 3 after ICH. Scale bars: 50 µm. (H) Summarized bar graph depicting Iba1${}^{+}$iNOS${}^{+}$ cells percentage from (G). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. ICH group. iNOS, inducible nitric oxide synthase.

Next, the bar graphs implied that the M2-like microglia markers’ mRNA expression levels, including Cd206, Arg-1, and Tgf-$\beta$ were elevated after ICH, and the administration of PAP-1 significantly enhanced this effect on day 3 after ICH (Fig. 8A). Thereafter, the immunoblot bands confirmed that the CD206 and Arg-1 protein expression was increased after ICH, and this phenomenon was intensified with the PAP-1 treatment on day 3 (Fig. 8B–D). Subsequently, the immunostaining images reported that the proportion of Iba1${}^{+}$CD206${}^{+}$ cells increased after ICH, and this effect was prominently expedited by PAP-1 application on day 3 after ICH (Fig. 8E,F). Meanwhile, the percentage of Iba1${}^{+}$Arg-1${}^{+}$ cells revealed the same tendency as Iba1${}^{+}$CD206${}^{+}$ cells on day 3 after ICH (Fig. 8G,H). Collectively, these results indicated that the usage of PAP-1 facilitated M1-like microglia shift into M2-like microglia, therefore improving the neuroinflammatory microenvironment through declining the accumulation of pro-inflammatory factors and enhancing the deposition of anti-inflammatory factors after ICH.

Fig. 8.

Application of PAP-1 increased the M2-like microglia around the hematoma after ICH. (A) Bar charts showcasing Cd206, Arg-1, and Tgf-$\beta$ mRNA expression in each group on day 3 after ICH. n = 3 each group, ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (B) Immunoblot bands illustrating CD206 and Arg-1 expression in each group on day 3 after ICH. (C) Summarized bar graph depicting CD206 expression from (B). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (D) Summarized bar graph depicting Arg-1 expression from (B). n = 3 each group, ${}^{*}$p 0.05 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (E) Immunostaining of CD206 (red) and Iba-1 (green) in each group on day 3 after ICH. Scale bars: 50 µm. (F) Summarized bar graph depicting Iba1${}^{+}$CD206${}^{+}$ cells percentage from (E). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. (G) Immunostaining of Arg-1 (red) and Iba-1 (green) in each group on day 3 after ICH. Scale bars: 50 µm. (H) Summarized bar graph depicting Iba1${}^{+}$Arg-1${}^{+}$ cells percentage from (G). n = 3 each group, ${}^{*}$p 0.05 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group.

In previous studies, the NF-$\kappa$B signaling pathway activation was primarily detected by the quantity and function of p65 and p50, subunits of NF-$\kappa$B [31, 32, 33]. First, RT-qPCR was used to evaluate the P65 and P50 mRNA levels. Results indicated that the P65 and P50 mRNA expression significantly increased after ICH, while PAP-1 administration abrogated this phenomenon (Fig. 9A, B). Immunoblot results showed that the phosphorylated-p65 expression level, indicating the functional form of p65, was increased after ICH. While compared with those in the ICH group, PAP-1 decreased the phosphorylated-p65 expression (Fig. 9C). NF-$\kappa$B subunit p50 showed the same trends as that in p65 (Fig. 9C). Compared with the Sham group, the ICH group has increased ratios of both phosphorylated-p65/p65 and phosphorylated-p50/p50 (Fig. 9D,E). In comparison with the ICH group, the aforementioned ratios were significantly decreased in the ICH + PAP-1 group (Fig. 9D,E). Collectively, these results indicated that NF-$\kappa$B was activated after ICH in mice, which was counterbalanced by PAP-1 administration.

Fig. 9.

Application of PAP-1 inhibiting NF-$\kappa$B activation after ICH. (A) Bar charts showing NF-$\kappa$B P65 mRNA expression in each group on day 3 after ICH. n = 3 each group, ${}^{**}$p 0.01 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. (B) Bar charts showing NF-$\kappa$B P50 mRNA expression in each group on day 3 after ICH. n = 3 each group ${}^{**}$p 0.01 vs. Sham group; ${}^{\mathrm{\#}}$p 0.05 vs. ICH group. (C) Immunoblot bands illustrating phosphorylated-NF-$\kappa$B p65, NF-$\kappa$B p65, phosphorylated-NF-$\kappa$B p50, and NF-$\kappa$B p50 expression in each group on day 3 after ICH. (D) Summarized bar graph depicting phosphorylated-NF-$\kappa$B p65 expression from (C). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. ICH group. (E) Summarized bar graph depicting phosphorylated-NF-$\kappa$B p50 expression from (C). n = 3 each group, ${}^{***}$p 0.001 vs. Sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ICH group. NF-$\kappa$B, Nuclear factor kappa B.