The animal experiments adhered to the protocols outlined in the National Institutes of Health’s “Guide for the Care and Use of Laboratory Animals” and received approval from the Animal Care and Use Committee at Jinzhou Medical University (2022022701). A total of 75 clean, healthy male Sprague Dawley (SD) rats weighing between 250 and 280 grams and aged 8 to 9 weeks, were supplied by the Experimental Animal Center at Jinzhou Medical University (Jinzhou, China). On day 3 after adaptive feeding, rats were randomly allocated into different groups (n = 15): (1) sham group, (2) middle cerebral artery occlusion/reperfusion (MCAO/R) surgery group, (3) fingolimod low-dose (F-L) group (0.5 mg/kg), (4) fingolimod medium-dose (F-M) group (1.0 mg/kg), (5) fingolimod high-dose (F-H) group (2.0 mg/kg).

Fingolimod (Cat #HY-11063, MedChemExpress, Monmouth Junction, NJ, USA) was prepared in dimethylsulfoxide (DMSO, D8371, Beijing Solebao Technology Co., Ltd., Beijing, China) and diluted in a 0.9% NaCl saline solution to final concentrations (0.5, 1.0, and 2.0 mg/kg). On days 0 (immediately after MCAO) and 1 (24 h) after MCAO, rats in the MCAO/R group were intraperitoneally administered normal saline containing DMSO, whereas rats in the F-L, F-M, and F-H groups were intraperitoneally administered the same volume of fingolimod at different concentrations (0.5, 1.0, and 2.0 mg/kg).

MCAO procedure was executed using an adapted thread embolism method, as outlined in prior research [9]. The rats were anesthetized by placing them in a chamber containing a mixture of 4.0–5.0% isoflurane (Cat# R510, RWD Life Science, Shenzhen, Guangdong, China) and oxygen and then maintained during MCAO surgery with 1.5–2.0% isoflurane in a mixture of 70% N${}_2$O and 30% O${}_2$. Once anesthesia was stable, the rats were positioned on the surgical operating table with their limbs secured. Before surgery, the fur around the neck was shaved with a blade and disinfected using povidone-iodine and 70% ethanol.

A median incision approximately 4 cm in length was made in the middle of the neck. The subcutaneous fat and surrounding connective tissue were carefully stripped to expose the carotid sheath and dissected to expose the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). The superior thyroid and the occipital arteries were isolated and electrocoagulated. The CCA and ICA were temporarily clamped, and the ECA was ligated with a nylon monofilament at the distal end. A V-shaped incision was made at the distal end of the ECA at a 45${}^{\circ }$ angle. Subsequently, a monofilament nylon suture (Beijing Xinong Biotechnology Co., Ltd., Beijing, China) was gently inserted into the ICA via the incision until mild resistance was felt (approximately 18–20 mm distal to the carotid bifurcation).

The filament was removed 2 h after MCAO to restore cerebral blood flow. Hemostasis was checked, and the wound was closed using nylon sutures. For rats in the sham group, the CCA was isolated without ligation or hypoxia. The rats’ rectal temperature was maintained at approximately 37.0 $\pm$ 0.5 °C throughout the MCAO surgical process. After surgery, all rats were allowed to move freely, eat, and drink in their cages.

The Zea-Longa score was used to evaluate neurological recovery following the method described in a previous study [10]. The scoring system ranges from 0 to 4, with the following interpretations: 0 = no apparent neurological deficits, 1 = incomplete extension of the left forelimb, 2 = a leftward circling motion, 3 = left-sided weakness, and 4 = the absence of spontaneous walking and a lack of consciousness.

The rats were humanely sacrificed 2 h after the second administration of the drug by administering excessive anesthesia (amobarbital; Cat #17784, Cayman Chemical Company, Ann Arbor, MA, USA, 10%, 0.5 mL/100 g). After cardiac perfusion, rat brains were excised through decapitation and then cut into 2 mm thick coronal sections. These slices were then placed in a 2% 2,3,5-Triphenyltetrazolium Chloride (TTC, T8877, Sigma, Burlington, MA, USA) solution and incubated at 37 °C for 10 min. Subsequently, slices were fixed with 4% paraformaldehyde. The infarct volume was assessed using ImageJ software (version 1.5, NIH, Bethesda, MD, USA). To calculate the percentage of the ischemic lesion area, we used the following equation: infarct volume (%) = (volume of contralateral hemisphere – volume of non-infarcted ipsilateral hemisphere)/volume of contralateral hemisphere $\times$ 100%.

The rats were euthanized 2 h after the second administration of the drug under excessive anesthesia (amobarbital; Cat# 17784, Cayman Chemical Company, Ann Arbor, MA, USA, 10%, 0.5 mL/100 g). Subsequently, cerebral tissue was derived from rats without myocardial perfusion. The humidity of the cerebral tissue was assessed immediately after the rats were sacrificed. The cerebral tissue was microwave-dried in a microwave oven at 100 °C until the cerebral weight remained constant, known as dry weight. Brain water content was assessed using the following formula: brain water content (%) = [(humid brain weight – dry brain weight)/humid brain weight] $\times$ 100.

Pro-inflammatory factors TNF-$\alpha$ (Cat# ml003057), IL-1$\beta$ (Cat# ml064292), and IL-6 (Cat# ml002859) levels were assessed via commercially available kits purchased from Shanghai Enzyme-linked Biotechnology Limited Company (Shanghai, China). The rats were euthanized 2 h after administration of the second drug under excessive anesthesia (amobarbital; Cat# 17784, Cayman Chemical Company, Ann Arbor, MA, USA, 10%, 0.5 mL/100 g). Blood samples were collected from the heart via needle aspiration and transferred to Rapid Serum Tubes for serum sample collection. The samples were then centrifuged at a speed of 3000 rpm for 10 min. Following this, the serum samples were carefully transferred into new cryovials for preservation at –80 °C for subsequent enzyme-linked immunosorbent assays (ELISA). Prior to the ELISA, the samples were thawed at room temperature. We performed ELISA according to the manufacturer’s protocol using an ELISA kit (Shanghai Enzyme-linked Biotechnology Limited Company, Shanghai, China). The steps involved in the ELISA procedure were as follows: (1) Adding standard: 50 µL standards with different concentration gradients were added in different holes. (2) Adding samples: 40 µL of sample dilution was added to the holes, then 10 µL of the sample fluid was added. The contents were then mixed gently. (3) Adding enzyme: 100 µL of horseradish peroxidase (HRP)-conjugate reagent was added to the hole designated for the standard solutions and samples, excluding the blank hole. (4) Incubation: The plates secured with a seal and then placed in an incubator set to a temperature of 37 °C for a period of 60 min. (5) Washing: The sealing membrane was carefully taken off, and the liquid was subsequently was discarded. Washing liquid was poured into each hole, left for 30 s, and then poured out, and this step was repeated a total of five times. The 96-well plate was carefully patted and dried. (6) Color development: 50 µL of color developer A was carefully dispensed into each hole, after which an equal volume of color developer B, also 50 µL, was added. The mixture in each hole was then gently stirred to ensure thorough blending. Subsequently, the 96-well plate was placed in an incubator set to 37 °C and incubated for a duration of 15 min in a dark environment. (7) Stopping reaction: 50 µL of stop solution was poured into the hole, which halted the reaction and caused the color of each hole to change from blue to yellow. (8) Assay: The optical density (OD) for each well was determined using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), with the readings taken at a wavelength of 450 nm. The blank well was set to zero, and readings were taken within 15 min of adding the stop solution.

Immunohistochemical staining was carefully conducted throughout the entire process according to a previously mentioned protocol [11]. Rat cerebral tissues were completely immersed in formalin, fixed, dehydrated, and embedded in paraffin wax. The paraffin-embedded cerebral tissue blocks mentioned above were then cut into 5 µm thick slices. The tissue slices were placed in fixation boxes containing citrate antigen retrieval buffer and fixed in a microwave oven. Once the slices had reached room temperature, they were immersed in a solution containing 3% hydrogen peroxide to block the activity of endogenous peroxidase and then immersed in phosphate-buffered saline (PBS) wash buffer three times. The slices were then incubated at 4 °C in the refrigerator with various primary antibodies: rabbit anti-high mobility group box 1 (HMGB1) antibody (1:500; Cat# ET1601-2, HuaAn Biotechnology, Hangzhou, Zhengjiang, China), rabbit toll-like receptor 4 (TLR4) antibody (1:400; Cat# ab22048, Abcam, Cambridge, MA, USA), and rabbit anti-nuclear factor kappa-B p65 (NF-$\kappa$Bp65) antibody (1:400; Cat# ab86299, Abcam, Cambridge, MA, USA). The next day, the slices were immersed in PBS wash buffer thrice for 5 min each. The sections were subsequently treated with horseradish peroxidase-labeled goat anti-rabbit/mouse secondary antibody for a duration of 20 min. After another three 5-minute washes with PBS wash buffer, the slices were subjected to color development using diaminobenzidine chromogenic solution. The sections were carefully washed with tap water, counterstained with hematoxylin, dehydrated, and made transparent. Finally, the slides were covered with neutral balsam and examined under a microscope. ImageJ software (version 1.5, NIH, Bethesda, MD, USA) was used to count the positive cells.

The infarcted rat hippocampus was lysed in radioimmunoprecipitation assay (RIPA) buffer (Cat #P0013B, Beyotime, Shanghai, China) containing a mixture of protease and phosphatase inhibitors. The mixture containing the lysate and tissue was subjected to centrifugation at a speed of 12,000 rpm for a duration of 30 min, and the supernatant was carefully poured into a new tube. The supernatant was gently mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and then heated at 95 °C for 5 min in a water bath. About 20 µg of protein was segregated and subsequently moved onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) with a pore size of either 0.22 micrometers or 0.45 micrometers. To block nonspecific protein binding, the PVDF membrane was soaked in a solution containing 5% skim milk. After blocking, the membrane was then immersed in the following primary antibodies overnight at a controlled temperature of 4 °C within a refrigerated environment: anti-HMGB1 (1:1000; Cat# ET1601-2, HuaAn Biotecchnology, Hangzhou, Zhengjiang, China), anti-TLR4 (1:500, Cat# WL00196, WanLei Biotechnology, Shenyang, Liaoning, China), anti-NF-$\kappa$Bp65 (1:500, Cat# WL02169, WanLei Biotechnology, Shenyang, Liaoning, China), and anti-$\beta$-actin (1:10,000; Cat# AMC0001, ABclonal Technology, Wuhan, Hubei, China). After incubation with the primary antibodies, the membranes were then treated with secondary antibodies that had been conjugated with horseradish peroxidase (HRP): goat anti-rabbit IgG (1:10,000, Cat #7074P2, Cell Signaling Technology, Danvers, MA, USA) and goat anti-mouse IgG (1:10,000, Cat #7076P2, Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. Finally, the immunoblots were visualized using an enhanced luminescence kit (Cat #BL520A, Biosharp, Hefei, Anhui, China), and the intensity of the immunoblot bands was quantified using the ImageJ software. The original western blot figures are presented in the Supplementary Material.

Experimental data are presented as mean $\pm$ standard deviation (SD) and median with minimum to maximum values. Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Quantitative data were analyzed using unpaired parametric tests, whereas rank data were analyzed using the non-parametric Wilcoxon’s rank sum test. For data from multiple groups, a one-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used. Statistical significance was set at p $\le$ 0.05.

TTC staining revealed no apparent damage to cerebral tissue in the sham group (Fig. 1A). On the contrary, rats in the MCAO/R group exhibited significant brain tissue lesions 1 d after cerebral ischemia or reperfusion injury (Fig. 1A). However, treatment with various fingolimod doses significantly reduced the volume of the cerebral infarct area in rats subjected to MCAO/R (Fig. 1A,B; p 0.001). These results indicate that fingolimod treatment reduces the cerebral infarction volume on day 1 after cerebral ischemia or reperfusion.

Fig. 1.

Fingolimod effectively reduces cerebral infarction volume, alleviates cerebral edema, and improves neurological function in MCAO/R rats. (A) On the first day post-MCAO/R procedure, TTC staining images were captured, wherein the infarcted regions of the brain were depicted in white and the healthy regions in red. (B) Quantification of TTC staining (Data presented as mean $\pm$ SD, and analyzed using one-way ANOVA with Tukey’s post-hoc test; N = 3). (C) Brain water content on day 1 after MCAO/R (Data presented as mean $\pm$ SD and analyzed using one-way ANOVA with Tukey’s post-hoc test; N = 4). (D) The Zea-Longa score, indicative of neurological deficit, was evaluated one day post-MCAO/R, with data presented as the median along with the minimum and maximum values, and statistical analysis conducted using Wilcoxon’s rank sum test (N = 15). **p 0.01 and ***p 0.001 vs. sham group. #p 0.05 and ###p 0.001 vs. MCAO/R group. $$p 0.01 and $$$p 0.001 vs. F-L group. &p 0.05 and &&&p 0.001 vs. F-M group. MCAO/R, middle cerebral artery occlusion/reperfusion; TTC, 2,3,5-Triphenyltetrazolium Chloride; ANOVA, analysis of variance; F-L, fingolimod low-dose; F-M, fingolimod medium-dose; F-H, fingolimod high-dose; SD, standard deviation.

To assess the effectiveness of fingolimod therapy in treating cerebral edema, the brain water content was determined one day post-reperfusion. The findings indicated that the rats subjected to MCAO/R had elevated water content in their cerebral tissues when compared to the rats in the sham group (Fig. 1C, p 0.001). Nevertheless, the cerebral water content of MCAO/R rats treated with high-dose fingolimod (2.0 mg/kg) was markedly reduced when compared to MCAO/R group (Fig. 1C, p 0.001). These findings indicate that a high dose of fingolimod alleviated cerebral edema induced by cerebral ischemia.

Additionally, to examine the effects of different dosages of fingolimod on the restoration of neurological function, we used the Zea-Longa Scale neurological score for each group. The results demonstrated that high-dose fingolimod administration (2.0 mg/kg) resulted in a statistically significant reduction in the neural function score compared to that in the MCAO/R group (Fig. 1D, p 0.05). The findings suggest that treatment with fingolimod improves the neurological impairment induced by cerebral ischemia in rats after MCAO/R.

The levels of pro-inflammatory cytokines in serum, including TNF-$\alpha$, IL-1$\beta$, and IL-6, were measured using ELISA to evaluate the potential anti-inflammatory efficacy of fingolimod in cerebral ischemia. The findings revealed that the serum levels of TNF-$\alpha$, IL-1$\beta$, and IL-6 proteins were significantly higher in rats that underwent MCAO/R when compared to the levels observed in the sham-operated control group (Fig. 2A–C, p 0.001). Nevertheless, the administration of fingolimod at multiple dosages resulted in a marked reduction in the serum IL-1$\beta$ and IL-6 protein levels when compared to the MCAO/R group without treatment (Fig. 2A,B, p 0.01, p 0.001). Moreover, treatment with median and high doses of fingolimod led to an evident decrease in TNF-$\alpha$ levels compared to the MCAO/R group without treatment (Fig. 2C, p 0.001). These findings imply that fingolimod protects against MCAO/R-caused brain injury by exerting its anti-inflammatory properties.

Fig. 2.

Fingolimod inhibits inflammation in rats after MCAO/R. (A) Enzyme-Linked Immunosorbent Assay (ELISA) was employed to measure the serum concentrations of interleukin-1 beta (IL-1$\beta$) in rats, with data expressed as the mean $\pm$ SD and analyzed utilizing one-way ANOVA with Tukey’s post-hoc test for multiple comparisons (N = 4). (B) Similarly, ELISA was utilized to ascertain the serum levels of interleukin-6 (IL-6) in rats, presenting data as mean $\pm$ SD and conducting analysis via one-way ANOVA with Tukey’s post-hoc test (N = 4). (C) ELISA was also applied to quantify the serum levels of tumor necrosis factor alpha (TNF-$\alpha$) in rats, with results depicted as mean $\pm$ standard deviation (SD), and statistical analysis performed using one-way ANOVA with Tukey’s post-hoc test (N = 4). **p 0.01 and ***p 0.001 vs. sham group. ##p 0.01 and ###p 0.001 vs. MCAO/R group. $$$p 0.001 vs. F-L group. &&p 0.01 vs. F-M group.

The expression levels of HMGB1 following MCAO/R injury in rats were measured via western blot assays and immunohistochemical staining. Western blot analysis demonstrated an apparent increase in the levels of HMGB1 protein in the MCAO/R group when compared to the sham group (Fig. 3A,B; p 0.01). In contrast, the expression levels of HMGB1 protein were considerably reduced in both the F-M and F-H groups as compared to the MCAO/R group (Fig. 3A,B; p 0.05).

Fig. 3.

Fingolimod down-regulates the expression of HMGB1 in MCAO/R rats. (A) The expression levels of HMGB1 within the hippocampal region of these rats were measured using the technique of western blot analysis, with the assessment being conducted day 1 post the MCAO/R procedure. (B) Quantification analysis of HMGB1 by western blot (Data presented as mean $\pm$ SD and analyzed using one-way ANOVA with Tukey’s post-hoc test; N = 4). (C) Positive immunohistochemical staining for HMGB1. (D) Quantification of immunohistochemical staining for HMGB1 (Data presented as mean $\pm$ SD and analyzed using one-way ANOVA with Tukey’s post-hoc test; N = 4). Scale bar = 50 µm. **p 0.01 and ***p 0.001 vs. sham group. #p 0.05 and ##p 0.01 vs. MCAO/R group. $$p 0.01 vs. F-L group. HMGB1, high mobility group box 1.

Additionally, we performed immunohistochemical analysis of HMGB1 in hippocampal slices from rats in the different groups. Immunohistochemistry revealed HMGB1-positive protein deposition in both the cell nucleus and cytoplasm. The number of cytoplasm-positive HMGB1 staining cells was significantly higher in the MCAO/R group than in the sham group (Fig. 3C,D; p 0.001). However, the quantity of cells exhibiting positive staining for HMGB1 in the cytoplasm was found to be reduced in both the F-M and F-H groups when compared to the MCAO/R group (Fig. 3C,D; p 0.05, p 0.01).

These results suggest that fingolimod treatment downregulated HMGB1 protein expression in MCAO/R rats. Additionally, fingolimod treatment inhibited the translocation of HMGB1 from the cell nucleus to cytoplasm to induced by cerebral ischemic injury.

The effect of various doses of fingolimod on TLR4 expression following MCAO/R injury in rats was evaluated using western blot assays and immunohistochemical staining.

Western blot analysis demonstrated that ischemic brain injury increased the expression level of TLR4 (Fig. 4A,B; p 0.05). However, the medium and high doses of fingolimod treatment led to an apparent decrease in TLR4 levels (Fig. 4A,B; p 0.05, and p 0.01, respectively) compared to those in the MCAO/R group. These observations were supported by the immunohistochemical staining results. The count of TLR4-positive cells within the MCAO/R group was considerably greater than that observed in the sham group (Fig. 4C,D; p 0.001). On the contrary, a significant reduction in the number of TLR4-positive cells was noted in both the F-M and F-H groups when compared to the MCAO/R group (Fig. 4C,D, p 0.001).

Fig. 4.

Fingolimod inhibits TLR4 expression in MCAO/R rats. (A) The expression of TLR4 in the hippocampus of rats was detected using western blot analysis on day 1 after MCAO/R. (B) Quantification of TLR4 expression via western blot analysis (Data presented as mean $\pm$ SD and analyzed using one-way ANOVA with Tukey’s post-hoc test; N = 4). (C) Immunohistochemical staining showing positive TLR4 expression. (D) Quantification of immunohistochemical staining for TLR4 (Data presented as mean $\pm$ S.D., and analyzed using one-way ANOVA with Tukey’s post-hoc test; N = 4). Scale bar = 50 µm. *p 0.05 and ***p 0.001 vs. sham group. #p 0.05, ##p 0.01 and ###p 0.001 vs. MCAO/R group. $p 0.05 and $$$p 0.001 vs. F-L group. &p 0.05 vs. F-M group. TLR4, Toll-like receptor 4.

In summary, these results demonstrate that fingolimod treatment can effectively suppress the expression levels of TLR4 in rats that have undergone MCAO/R.

To assess the impact of fingolimod treatment on NF-$\kappa$Bp65 protein expression following ischemic brain injury in rats, both western blotting and immunohistochemical staining techniques were utilized. The findings from the western blot analysis indicated a significant elevation in the levels of NF-$\kappa$Bp65 protein within the MCAO/R group when contrasted with the levels observed in the sham-surgery group (Fig. 5A,B; p 0.001). Nevertheless, a notable reduction in the levels of NF-$\kappa$Bp65 protein was observed in both the F-M and F-H groups when compared to the levels found in the MCAO/R group. (Fig. 5A,B; p 0.01, p 0.001). These observations were supported by the immunohistochemical staining results. Immunohistochemistry revealed NF-$\kappa$Bp65-positive protein primarily deposition in the cell nucleus. The number of nuclear NF-$\kappa$Bp65-positive staining cells was significantly higher in the MCAO/R group than in the sham group (Fig. 5C,D; p 0.001). Conversely, there were fewer nuclear NF-$\kappa$Bp65-positive staining cells in the F-M and F-H groups than in the MCAO/R group (Fig. 5C,D; p 0.05, p 0.01).

Fig. 5.

Fingolimod treatment reduces NF-$\kappa$Bp65 expression in MCAO/R rats. (A) The expression of NF-$\kappa$Bp65 in the hippocampus of rats was detected using western blot analysis on day 1 after MCAO/R. (B) Quantification analysis of NF-$\kappa$Bp65 via western blot (Data presented as mean $\pm$ SD and analyzed using one-way ANOVA with Tukey’s post-hoc test; N = 4). (C) Positive immunohistochemical staining for NF-$\kappa$Bp65. (D) Quantification of immunohistochemical staining for NF-$\kappa$Bp65 (Data presented as mean $\pm$ SD and analyzed using one-way ANOVA with Tukey’s post-hoc test; N = 4). Scale bar = 50 µm. **p 0.01 and ***p 0.001 vs. sham group. #p 0.05, ##p 0.01 and ###p 0.001 vs. MCAO/R group. $p 0.05 vs. F-L group. NF-$\kappa$Bp65, nuclear factor kappa-B p65.

These findings provide compelling evidence that fingolimod treatment reduces the NF-$\kappa$Bp65 expression level in rats after ischemic brain injury. Additionally, treatment with fingolimod can inhibit the translocation of NF-$\kappa$Bp65 into the nucleus caused by cerebral ischemic/reperfusion injury.