NDP-MSH binding melanocortin-1 receptor ameliorates neuroinflammation and BBB disruption through CREB/Nr4a1/NF-κB pathway after intracerebral hemorrhage in mice

All experimental protocols for this study were approved by the Animal Ethics Committee of Chongqing Medical University. The study complied with the National Institutes of Health guide for the care and use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. A total of 218 C57BL/6 mice (male, weight about 25 g) were purchased from and bred at the Animal Center of Chongqing Medical University. All mice were housed in a light- and temperature-controlled room with free access to food and water.

Four separate experiments were designed as follows (Fig. 1). A total of 218 mice were used (Additional file 1: Table S1).

The ICH model was induced by autologous blood injection as previously described [22]. Briefly, the mice were anesthetized and fixed prone in a stereotaxic frame. Drill a small hole about 1 mm in diameter at 2 mm to the right of the bregma. Then 30 μl autologous arterial blood without anticoagulation was drawn from the central artery of the tail and delivered into the basal ganglion (stereotaxic coordinates: 0.2 mm anterior, 2.3 mm right lateral to the bregma, and 3.5 mm ventral to the skull). Firstly, 5 μl of blood was injected at 0.7 mm above the target position. Five minutes later, the remaining 25 μl blood was delivered at 3.5 mm depth. The needle was left for 10 min more after injection and withdrawn slowly at a rate of 1 mm/min. Bone wax was then applied to cover the drilled hole. The sham-operated animals were delivered an equal volume of sterile saline at the same position.

Intracerebroventricular injection was performed as previously described [23]. Briefly, mice were anesthetized and placed in a stereotactic head frame in the prone position. A longitudinal incision was made along the midline and a burr hole was drilled to the right of the bregma (1.0 mm lateral of the bregma). Following the manufacturer’s instructions, Mc1r siRNA (Thermo Fisher Scientific, USA, MSS275666, GCG AUU CUG UAU GCC CAC AUG UUC A, UGA ACA UGU GGG CAU ACA GAA UCG C), Nr4a1 siRNA (Thermo Fisher Scientific, USA, MSS205160, GAA GAU GCC GGU GAC GUG CAA CAA U, AUU GUU GCA CGU CAC CGG CAU CUU C), or scramble siRNA was dissolved in sterile RNase-free water. Mc1r siRNA mixture or scramble siRNA (100 pmol/2 μl) was delivered into the ipsilateral ventricle at the depth of 2.5 mm. The needle was left for an additional 5 min after injection to avert possible leakage and was slowly withdrawn at a rate of 1 mm/min. The burr hole was sealed with bone wax, and the incision was closed with sutures. Mice were placed in an individual recovery cage.

Neurobehavioral functions were evaluated using the modified Garcia test and corner turn test at 24 or 72 h following ICH by a blinded investigator as previously described [24]. In the modified Garcia test, seven items including spontaneous activity, axial sensation, vibrissae touch, limb symmetry, lateral turning, forelimb walking, and climbing were tested. In the corner turn test, mice were allowed to approach a 30° corner. The mice exited the corner with either a right turn or left turn. Ten trials were performed, with at least a 30-s break between the trials. The percentage of a right turn to 10 trials was then calculated.

To evaluate BBB permeability, Evans blue (Aladdin, China) was injected intraperitoneally (100 μl of 4% solution in saline) as previously described with a slight modification [25]. After 3 h circulation, mice were transcardially perfused with cold phosphate-buffered saline (0.1 M, PBS, pH 7.4) under deep anesthesia. Afterwards, the brain was removed and divided into left and right hemispheres and stored at − 80 °C immediately. The right part of the brain was homogenized in 1100 μl PBS, sonicated, and centrifuged (12,000 g, 4 °C, 30 min). The supernatant was collected and added an equal amount of trichloroacetic acid (TCA) to incubate overnight by 4 °C. After centrifugation (12,000 g, 4 °C, 30 min), Evans blue stain was measured by spectrophotometer (Thermo Fisher Scientific, USA) at 610 nm.

Brain water content was measured at 24 h and 72 h after ICH by an investigator blind to group information as previously described [26]. In short, mice were sacrificed under deep anesthesia. The brain was immediately removed and cut into 4 mm coronal slice. The brain slice was separated into five parts: ipsilateral and contralateral basal ganglia, ipsilateral and contralateral cortex, and cerebellum. The cerebellum was retained as an internal control. Each part was immediately weighed on an electronic analytical balance (FA2204B, Techcomp, USA) to determine the wet weight (WW) and then dried at 100 °C for 72 h to determine the dry weight (DW). Brain water content (percentage) was calculated as [(WW − DW)/WW] × 100%.

Double fluorescence staining was performed as described previously [27]. The mice were deeply anesthetized and were transcardially perfused with 20 ml ice-cold PBS followed by 20 ml of 4% paraformaldehyde at 24 h post-ICH. The whole brain was collected and then fixed in 4% paraformaldehyde for another 24 h. Afterwards, the brain was fixed in 20% sucrose solution until the tissue sink to the bottom followed by 30% sucrose solution for another 24 h. After being frozen at − 25 °C, the brain was cut into 10-μm-thick coronal sections using a cryostat (CM1860; Leica Microsystems, Germany). To conduct double immunohistochemistry staining, the brain sections were incubated with primary antibody of anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:100, Abcam, ab153696), anti-glial fibrillary acidic protein (GFAP, 1:200, CST, 3670, AB_561049), anti-von Willebrand factor (vWF, 1:50, Santa Cruz, sc-365712, AB_10842026), anti-NeuN (1:100, Abcam, ab104224, AB_10711040), and anti-Mc1r (1:50, Genetex, GTX108190) overnight at 4 °C. After being incubated with the appropriate secondary antibody (1:200, Bioss) at 37 °C for 1 h, the sections were visualized and photographed with a fluorescence microscope (U-HGLGPS, OLYMPUS, Japan). Microphotographs were analyzed with cellSens Standard software. The numbers of Iba-1-positive cells were identified and counted in three different fields in peri-hematoma area from five random coronal sections per brain, and data were expressed as cells/field.

After mice were perfused with ice-cold PBS (0.1 M, pH 7.4) at 24 h post-operation, the peri-hematoma tissues were collected and stored in − 80 °C freezer until use. Western blotting was performed as previously described [28]. After sample preparation, equal amounts of protein were loaded onto an SDS-PAGE gel. After being electrophoresed and transferred to a PVDF membrane, the membrane was blocked 2 h at 37 °C followed by incubated with the primary antibody overnight at 4 °C. The primary antibodies were anti-Mc1r (1:1000, Abcam, ab180776), anti-Nr4a1 (1:500, Abcam, ab13851, AB_300679), anti-phospho-CREB (1:1000, cell signaling, 9198, Ser133, AB_2561044), anti-CREB (1:1000, cell signaling, 9197, AB_331277), anti-phospho-NF-κB p65 (1:1000, cell signaling, 3033, AB_331284), anti-NF-κB p65 (1:1000, cell signaling, 8242, AB_10859369), anti-IL-1β (1:1000, cell signaling, 31202), anti-TNF-α (1:1000, cell signaling, 11948, AB_2687962), anti-MMP-9 (1:500, Abcam, ab38898, AB_776512), anti-occludin (1:50000, abcam, ab167161, AB_2756463), anti-ZO-1 (1:1000, affinity, AF5145), anti-Lama5 (1:1000, abcam, ab184330), and anti-β-actin (1:5000, proteintech, 60008-1-Ig). The secondary antibodies (ZSGB-BIO) were incubated for 1 h at 37 °C. Immunoblots were then probed with an ECL Plus chemiluminescence reagent kit (4A Biotech) and visualized with the image system (Bio-Rad, Universal Hood III). All data were analyzed using the software ImageJ.

All data were expressed as mean and standard deviation (mean ± SD). All analyses were performed using SigmaPlot 11.0 and GraphPad Prism 6 (GraphPad software, San Diego, CA, USA). Firstly, Shapiro-Wilk normality test was implemented in determining data normality. For the data that conformed to normal distribution, one-way ANOVA analysis followed by Tukey’s post hoc test was used for multiple-group comparisons. For the data that failed the normality test, Kruskal-Wallis one-way ANOVA on ranks, followed by Tukey’s multiple comparison post hoc analysis was performed. Statistical differences between two groups were analyzed using Student’s unpaired, two-tailed t test. P value of less than 0.05 was defined statistically significant (Additional file 2).

The total mortality of ICH mice was 9.34% (17/182) in this study. None of the sham group mice died. There was no significant difference in mortality rate among the experimental groups. Six mice were ruled out from this study due to no hemorrhage (Additional file 1: Table S1).

As shown in Fig. 2a, the Mc1r expression in the peri-hematoma tissue was significantly increased at 24 h and reached its peak at 72 h after ICH, when compared to the sham group. Double immunofluorescence staining showed that Mc1r was mainly expressed in the microglia, astrocytes, and endothelial cells in the peri-hematoma tissue at 24 h after ICH (Fig. 2c).

The neurological deficits and brain edema were evidently worse at 24 and 72 h post-ICH in the ICH + vehicle and ICH + NDP-MSH (1.5 μg/mouse) groups, when compared with sham group. However, the administration of NDP-MSH (5 μg/mouse) and NDP-MSH (15 μg/mouse) significantly improved the neurological deficits (Fig. 3a, b) and reduced brain edema in ipsilateral basal ganglion (Fig. 3c). Based on these results, the optimal dose of NDP-MSH was 5 μg/mouse, which was used for the rest of the experiments. BBB permeability was assessed by EB extravasation in the right cerebral hemispheres. EB extravasation in the ICH + vehicle group was significantly increased at 24 h after ICH, whereas NDP-MSH treatment (5 μg/mouse) prominently decreased EB dye leakage compared with the ICH + vehicle group (Fig. 3d).

To further investigate, the protective role of NDP-MSH and Mc1r siRNA was administered by i.c.v injection to knockdown the expression of endogenous Mc1r. Western blot showed that the Mc1r expression was inhibited by Mc1r siRNA at 72 h after injection (Fig. 4a). The knockdown of Mc1r abolished the protective effect of NDP-MSH on neurological functions (Fig. 4b, c), brain edema (Fig. 4d), and BBB integrity (Fig. 4e) at 24 h post-ICH.

Treatment with NDP-MSH increased phospho-CREB (p-CREB) expression in the peri-hematoma tissue at 24 h post-ICH, which increased the expression of downstream molecules including Nr4a1, ZO-1, occludin, and laminin-α5 (Lama5) and inhibited the expression of downstream inflammation-related proteins and MMP-9 (Fig. 5a-j), compared with ICH + vehicle group. In contrast, the knockdown of Mc1r using specific siRNA got opposite changes on the expression of downstream signaling molecules (Fig. 5a–j), compared with the ICH + NDP-MSH group.

We investigated whether the anti-inflammatory function of NDP-MSH was associated with the decrease in the numbers of microglia in peri-hematoma tissue. As presented in Fig. 6, the numbers of Iba-1-positive cells were dramatically increased in ICH + vehicle group at 24 h post-ICH. The administration of NDP-MSH significantly reduced the number of Iba-1-positive cells, whereas the knockdown of Mc1r abolished this effect.

To further determine whether the neuroprotective effects of NDP-MSH were regulated by Nr4a1, Nr4a1 siRNA was administered by i.c.v injection at 48 h before ICH induction and treated with NDP-MSH at 24 h post-ICH. Nr4a1 siRNA significantly decreased Nr4a1 expression at 72 h after injection (Fig. 7a). The knockdown of Nr4a1 exacerbated neurological impairments (Fig. 7b, c) and increased brain water content (Fig. 7d) at 24 h after ICH. Furthermore, the knockdown of Nr4a1 significantly increased the expression of p-NF-κB p65, IL-1β, TNF-α, and MMP-9 with a decrease of ZO-1, occludin, and Lama5 in the peri-hematoma tissue (Fig. 7e–l).