Resolvin D1 combined with exercise rehabilitation alleviates neurological injury in mice with intracranial hemorrhage via the BDNF/TrkB/PI3K/AKT pathway

Abstract

Resolvin D1 (RvD1) is an endogenous anti-inflammatory mediator that modulates the inflammatory response and promotes inflammation resolution. RvD1 has demonstrated neuroprotective effects in various central nervous system contexts; however, its role in the pathophysiological processes of intracerebral hemorrhage (ICH) and the potential protective mechanisms when combined with exercise rehabilitation remain unclear. A mouse model of ICH was established using collagenase, and treatment with RvD1 combined with three weeks of exercise rehabilitation significantly improved neurological deficits, muscle strength, learning, and memory in ICH mice while reducing anxiety-like behavior. RvD1 combined with exercise rehabilitation upregulated anti-inflammatory factors, inhibited the inflammatory state, and activated the BDNF/TrkB/PI3K/AKT pathway. TUNEL staining confirmed a decrease in residual apoptotic neurons, while transmission electron microscopy showed an increase in mitochondrial autophagosomes with combined treatment. Mendelian randomization and molecular docking further confirmed the association of RvD1 with targets related to mitophagy and inflammatory factors, clarifying the mechanism of RvD1 involvement. In summary, RvD1 combined with exercise rehabilitation activates the BDNF/TrkB/PI3K/AKT signaling pathway, effectively reduces neuronal apoptosis and inflammatory responses following ICH in mice, and participates in mitochondrial autophagy-related states. This comprehensive therapeutic strategy promotes neurological recovery and provides insights for clinical management of this condition.

Keywords: Inflammation; Intracerebral hemorrhage; Mitophagy; Neuronal Apoptosis; RvD1; exercise rehabilitation.

Fig. 1

RvD1 combined with exercise rehabilitation training mitigates neurologic deficits and motor function after ICH. (a) Localization map of the caudate nucleus and representative tissue sections and CT scans of mice with ICH.Cerebral hemorrhage was evaluated at D1, D5, D10, D14, and D21 using the modified mNSS score(b,c) and muscle strength tests (d) to assess neurological dysfunction in each group. The results demonstrated that RvD1 combined with exercise rehabilitation training significantly improved neurological function in mice with cerebral hemorrhage at D5, D10, D14, and D21 (n = 10, data presented as mean ± SEM, *significant vs. ICH group, p < 0.05).

Fig. 2

RvD1 combined with exercise rehabilitation training improves spatial learning and memory ability and reduces anxiety in mice after ICH. The open field test and water maze test were used to study the motor activity and spatial learning and memory of the six groups of mice. (a) Representative movement trajectories of each group of mice in the open field test. The open field test showed that RvD1 combined with exercise rehabilitation training improved activity, as indicated by total distance (b), center/total(C/T) distance (c), and average speed (d). The water maze test showed that RvD1 combined with exercise rehabilitation training enhanced spatial learning and memory in mice. (f) Representative swimming trajectories of mice in each group in the water maze test. Mice were assessed for escape latency (i), speed (e), number of crossings over the target platform (g), and exploration time in the target quadrant (h). Data are presented as mean ± SEM. (* significant vs. ICH group, p < 0.05).

Fig. 3

RvD1 inhibited the mRNA expression of anti-inflammatory related factors (CD206 and IL-10) and inflammation-related states(IL-1β and TNF-α) after ICH.Expression levels of CD206 (a), IL-10 (b), IL-1β (c), and TNF-α (d) mRNAs.Data are presented as mean ± SEM. (* significant vs. ICH group, p < 0.05).(e) Volcano plot of causality between RvD1 and circulating inflammatory factors.

Fig. 4

RvD1 combined with exercise rehabilitation training can activate BDNF/TrκB/p-Akt/PI3K related pathway and increase its protein and mRNA expression. (a) Western blot analysis was used to detect the expression levels of BDNF, TrκB, PI3K proteins, and both phosphorylated and total Akt. (b-d) Quantitative analysis of BDNF, TrκB, and PI3K protein expressions in brain tissue adjacent to the injection site on the ipsilateral side after the experiment. (e) Semiquantitative measurements of the ratio of phosphorylated to total Akt. (f-i) Expression levels of BDNF (f), TrκB (g), PI3K (h), and Akt (i) mRNAs at the injection site were determined. Western blot and RT-qPCR analyses revealed that the expression levels of BDNF, TrκB, p-Akt, and PI3K in the ICH + RvD1 + exercise training group were significantly higher compared to those in the ICH group. (*Significant vs. ICH group, p < 0.05).

Fig. 5

RvD1 Combined with Exercise Rehabilitation Training Reduces Neuronal Apoptosis Post-ICH. (a) Representative TUNEL/NeuN micrographs of different groups (scale bar = 50 μm). Fluorescence colors: TUNEL (green) and NeuN (blue). The box in (b) indicates the area where apoptosis was measured. (c) Quantification of the number of TUNEL/NeuN-positive cells in each group. (*Significant vs. ICH group, p < 0.05).

Fig. 6

Effects of RvD1 combined with exercise rehabilitation training on mitophagy in mice after intracerebral hemorrhage. TEM was used to observe the mitochondrial morphology of neurons in the Sham group, ICH group, ICH + CCCP group, ICH + exercise training group, ICH + RvD1 + exercise training group, and ICH + Mdivi-1 + exercise training group. Significant changes in autophagy levels were observed in these groups. N = Nucleus, Nu = Nucleolus, M = Mitochondria, RER = Rough endoplasmic reticulum, Go = Golgi apparatus, Lib = Lipofuscin.

Fig. 7

(a)Mendelian Randomization Results for Mitophagy-Related Targets in the Brain Caudate basal ganglia on ICH. nSNP = number of SNPs included in the analysis; OR = odds ratio; CI = confidence interval; ICH = Intracranial hemorrhage.Scatter plots for the causal association between MFN2, RAB7B, and TOMM7 and ICH. SNP effects were plotted into lines for the inverse variance-weighted test (light blue line), multiplicative random effects (dark blue line), MR Egger (light green line), Simple mode (dark green line), Weighted median (light red line) and Weighted mode(dark red line).The slope of the line corresponded to the causal estimation. (b)MFN2,(c)RAB7B, (d)TOMM7.

Fig. 8

The 3D visualization of molecular docking of Mitophagy-Related Targets in the Brain Caudate basal ganglia and RvD1: (a) MFN2 ; (b) RAB7B.

Fig. 9

RvD1 combined with exercise rehabilitation training activates BDNF/TrkB/PI3K/AKT signaling pathway to reduce neuroinflammation after intracerebral hemorrhage and improve neurological prognosis.

Fig. 10

Experimental design schedule, including pre-experiment, surgery, drug injection, behavioral assessment, tissue collection, and subsequent experiments such as Western blot, RT-Qpcr, tunel staining, and mitochondrial electron microscopy autophagy.