Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats

Abstract

Background: Microglia-mediated neuroinflammation plays a crucial role in the pathogenesis of hypoxic-ischemic (HI)-induced brain injury. Activation of melanocortin-1 receptor (MC1R) has been shown to exert anti-inflammatory and neuroprotective effects in several neurological diseases. In the present study, we have explored the role of MC1R activation on neuroinflammation and the potential underlying mechanisms after neonatal hypoxic-ischemic brain injury in rats.

Methods: A total of 169 post-natal day 10 unsexed rat pups were used. HI was induced by right common carotid artery ligation followed by 2.5 h of hypoxia. BMS-470539, a specific selective MC1R agonist, was administered intranasally at 1 h after HI induction. To elucidate the potential underlying mechanism, MC1R CRISPR KO plasmid or Nurr1 CRISPR KO plasmid was administered via intracerebroventricular injection at 48 h before HI induction. Percent brain infarct area, short- and long-term neurobehavioral tests, Nissl staining, immunofluorescence staining, and Western blot were conducted.

Results: The expression levels of MC1R and Nurr1 increased over time post-HI. MC1R and Nurr1 were expressed on microglia at 48 h post-HI. Activation of MC1R with BMS-470539 significantly reduced the percent infarct area, brain atrophy, and inflammation, and improved short- and long-term neurological deficits at 48 h and 28 days post-HI. MC1R activation increased the expression of CD206 (a microglial M2 marker) and reduced the expression of MPO. Moreover, activation of MC1R with BMS-470539 significantly increased the expression levels of MC1R, cAMP, p-PKA, and Nurr1, while downregulating the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1β) at 48 h post-HI. However, knockout of MC1R or Nurr1 by specific CRISPR reversed the neuroprotective effects of MC1R activation post-HI.

Conclusions: Our study demonstrated that activation of MC1R with BMS-470539 attenuated neuroinflammation, and improved neurological deficits after neonatal hypoxic-ischemic brain injury in rats. Such anti-inflammatory and neuroprotective effects were mediated, at least in part, via the cAMP/PKA/Nurr1 signaling pathway. Therefore, MC1R activation might be a promising therapeutic target for infants with hypoxic-ischemic encephalopathy (HIE).

Keywords: BMS-470539; Melanocortin-1 receptor; Microglial polarization; Neonatal hypoxia-ischemia; Neuroinflammation; Nurr1.

Fig. 1

Expression levels of α-MSH, MC1R, and Nurr1 post-HI. a Representative pictures of Western blot data. b Western blot data showed that the expression levels of α-MSH significantly increased at 12 h post-HI, and peaked at 24 h. c, d The endogenous expression levels of MC1R and Nurr1 increased over time and peaked at 48 h post-HI. Data were represented as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc test (*p < 0.05 versus sham, ^#p < 0.05 versus 6 h HI, ^@p < 0.05 versus 24 h HI, ^&p < 0.05 versus 48 h HI; n = 4 per group). e Representative microphotographs of double immunofluorescence of α-MSH (green) with microglia (Iba-1, red), astrocyte (GFAP, red), and neuron (NeuN, red) in the peri-infarcted area at 24 h post-HI. DAPI was stained blue. Merged images showed that α-MSH was colocalized with microglia, astrocytes, and neurons. n = 2 per group. Scale bar = 100 μm

Fig. 2

Effect of MC1R activation on brain infarct area, body weight, and short-term neurological function at 48 h post-HI. a, b TTC staining showed that medium (160 μg/kg) and high (500 μg/kg) doses of BMS-470539 treatment significantly reduced brain infarct area compared to the vehicle. c Rats in the vehicle group showed to lose significant weight compared to the sham group, medium (160 μg/kg) and high (500 μg/kg) doses of BMS-470539 treatment groups. d Geotaxis reflex showed that medium (160 μg/kg) and high (500 μg/kg) doses of BMS-470539 significantly improved neurological function compared to the vehicle group. Data were represented as mean ± SD. *p < 0.05 versus sham, ^#p < 0.05 versus vehicle, ^@p < 0.05 versus BMS-470539 (50 μg/kg). n = 6 per group

Fig. 3

Immunofluorescence staining of MC1R and Nurr1 with microglia in the brain at 48 h post-HI. Immunofluorescence staining showed an increase expression of MC1R (a) and Nurr1 (b) on microglia in the vehicle group when compared with the sham group, and further increased expression of MC1R (a) and Nurr1 (b) in the BMS-470359 treatment group. Merge showed the colocalization of MC1R and Nurr1 on microglia. Microglia were stained red. MC1R and Nurr1 were stained green. DAPI was stained blue. n = 2 per group. Scale bar = 100 μm

Fig. 4

Effects of MC1R activation on brain atrophy and long-term neurological function at 28 days post-HI. a–c Representative pictures of Nissl staining the brain slices showing tissue loss in the ipsilateral hemisphere. The BMS-470539 treatment group significantly reduced the percentage of tissue loss and brain atrophy compared to the vehicle group. d, e Activation of MC1R with BMS-470539 significantly improved sensorimotor function as shown by foot-fault and rotarod tests. f–i The BMS-470539 treatment group significantly improved in spatial memory and learning abilities compared to the vehicle group, as demonstrated by less swim distance to find the platform (f), less escape latency (g), and more time spent in the target quadrant during the probe test (h, i). h Representative image of swim track in probe trial. Data was represented as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA or two-way ANOVA followed by Tukey multiple-comparison post hoc analysis. *p < 0.05 versus sham; ^#p < 0.05 versus HI + vehicle; n = 8 per group

Fig. 5

Effects of knockout MC1R and Nurr1 on infarct area, body weight, and neurological function at 48 h post-HI. a, b The infarct area was significantly increased in both treatment groups with CRISPR compared to the HI + BMS-470539 group or HI + BMS-470539 + control CRISPR group. c Activation of MC1R with BMS-470539 significantly reduced body weight loss compared to the vehicle. However, both treatment groups with CRISPR interventions significantly reversed these effects compared to the HI + BMS-470539 group or HI + BMS-470539 + control CRISPR group. d The geotaxis test showed that the rat pups treated with BMS-470539 and either MC1R CRISPR or Nurr1 CRISPR had significantly impaired neurological function compared to the corresponding controls. Data was represented as mean ± SD. *p < 0.05 versus sham, ^#p < 0.05 versus HI + vehicle, ^@p < 0.05 HI + BMS-470539 or HI + BMS-470539 + control CRISPR. n = 6 per group

Fig. 6

MC1R activation increased the expression of CD206 at 48 h post-HI. Representative microphotographs of immunofluorescence staining and quantification of CD206 with microglia (CD11 b/c) at 48 h post-HI. a, b The number of CD206⁺ CD11 b/c⁺ cells significantly increased in the vehicle group compared to the sham group. Activation of MC1R with BMS-470539 further increased the number of CD206⁺ CD11 b/c⁺ cells compared to the vehicle group, while knockout CRISPR interventions significantly reversed these effects. CD206 was green. CD11 b/c was red. Blue was for DAPI. Scale bar = 100 μm. Data was represented as mean ± SD. The one-way ANOVA was followed by Tukey’s post hoc test (*p < 0.05 versus sham; ^#p < 0.05 versus HI + vehicle; ^@p < 0.05 HI + BMS-470539 or HI + BMS-470539 + control CRISPR; n = 6 per group)

Fig. 7

MC1R activation on immunofluorescence staining of IL-1β and MPO at 48 h post-HI. Representative microphotographs of immunofluorescence staining and quantification of a, c IL-1β-positive cells and b, d MPO-positive cells. The number of IL-1β-positive cells or MPO-positive cells was significantly increased in the vehicle group compared to the sham group. BMS-470539 treatment significantly suppressed neuroinflammation, while these effects were reversed by knockout of MC1R or Nurr1 with CRISPR. IL-1β and MPO were green. Blue was for DAPI. Scale bar = 100 μm. Data was represented as mean ± SD. The one-way ANOVA was followed by Tukey’s post hoc test (*p < 0.05 versus sham; ^#p < 0.05 versus HI + vehicle; ^@p < 0.05 HI + BMS-470539 or HI + BMS-470539 + control CRISPR; n = 6 per group)

Fig. 8

MC1R activation on neuroinflammation via cAMP/PKA/Nurr1 signaling pathway at 48 h post-HI. a Representative picture of Western blot data showing bands of the expression levels of MC1R, cAMP, p-PKA, Nurr1, IL-1β, TNFα, and IL-6 either with BMS-470539 treatment alone, BMS-470539 + MC1R KO CRISPR, BMS-470539 + Nurr1 KO CRISPR, and BMS-470539 + control CRISPR groups. b–h Western blot data quantification of bands showed that BMS-470539 treatment significantly increased the expression of MC1R, cAMP, p-PKA, and Nurr1 compared to the HI + vehicle group. Knockout of MC1R using CRISPR significantly decreased MC1R, cAMP, p-PKA, and Nurr1 expression levels compared to the HI + BMS-470539 group or HI + BMS-470539 + control CRISPR group. Furthermore, knockout of Nurr1 using CRISPR significantly decreased the levels of Nurr1, but did not affect MC1R, cAMP, and p-PKA expression compared to the corresponding controls. Activation of MC1R with BMS-470539 showed significantly decreased levels of IL-1β, TNFα, and IL-6, while both treatment groups with CRISPR interventions significantly reversed these effects. Data was represented as mean ± SD. The one-way ANOVA was followed by Tukey’s post hoc test (*p < 0.05 versus sham; ^#p < 0.05 versus HI + vehicle; ^@p < 0.05 HI + BMS-470539 or HI + BMS-470539 + control CRISPR; n = 6 per group)

Fig. 9

The potential molecular mechanisms of anti-inflammatory and neuroprotective effects through the MC1R/cAMP/PKA/Nurr1 signaling pathway post-HI