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

Table of Contents

ICH and grouped mortality

After the preliminary experiment, we performed brain dissections on the mice and observed that the ICH was primarily localized to the Willis ring and abdominal regions. Subsequently, spiral computed tomography (CT) scans and tissue slices were conducted to confirm the successful establishment of the ICH model and to identify its location within the right caudate nucleus. Measurements using Mimics software indicated that the area of the ICH region was approximately 4.68 mm² (Fig. 1a).In the Sham group, the ICH group, and the ICH + Vehicle + exercise training, no mice died (0/10). In contrast, the mortality rates in the ICH + CCCP group, ICH + Mdivi-1 + exercise training group, and ICH + RvD1 + exercise training group were 3/13, 5/15, and 1/11, respectively.

Rehabilitation training combined with RvD1 improved neurological functions after ICH

Compared with Sham group, all ICH animals showed significant neurological deficits.Compared to the Sham group, the neurological deficit in the ICH group were significantly higher on day 1 (P = 0.0004), day 5 (P < 0.0001), day 10 (P = 0.0002), day 14 (P = 0.0003), and day 21 (P = 0.0010) post-ICH (Fig. 1b). On day 1 post-surgery, there were no statistically significant differences in neurological deficit scores among the groups other than the Sham group (P > 0.05).Mice in the ICH group that began rehabilitation training on day 3 showed some improvement in neurological function compared to the ICH model group, but only on days 14 (P = 0.0183) and 21 (P = 0.0369) did the neurological deficit scores show statistical significance.The neurological deficit scores in the ICH + CCCP group decreased compared to the ICH group, with statistically significant changes on day 10 (P = 0.0059) and day 14 (P = 0.0179). The ICH + RvD1 + exercise training group showed the most significant improvement in neurological deficits, with statistically significant changes on day 5 (P = 0.0427), day 10 (P = 0.0022), day 14 (P = 0.0248), and day 21 (P = 0.0071).The ICH + Mdivi-1 + exercise training group also demonstrated improvements in neurological deficits, with statistically significant changes on day 10 (P = 0.0035), day 14 (P = 0.0149), and day 21(P = 0.0049).On day 21 post-surgery, neurological function testing showed significant improvements compared to the ICH group in the following groups: ICH + Vehicle + exercise training group(P = 0.0369), ICH + CCCP (P = 0.0407), ICH + RvD1 + exercise training group (P = 0.0071), and ICH + Mdivi-1 + exercise training group (P = 0.0274, Fig. 1c). In the muscle strength test, the ICH + RvD1 + exercise training group (P = 0.0077) and the ICH + Mdivi-1 + exercise training group (P = 0.0239) showed significant statistical differences compared to the ICH group, indicating a notable improvement in muscle strength (p < 0.05, Fig. 1d).

Rehabilitation training and RvD1 improve anxiety-like behaviors after ICH

Compared to the Sham group, mice in the ICH group exhibited significantly reduced movement trajectories in the open field(Fig. 2a). and a lower center-to-total (C/T) distance ratio (P < 0.0001,Fig. 2c), indicating anxiety-like behaviors. Mice in the groups that received rehabilitation training (including the ICH + Vehicle + exercise training group, ICH + Mdivi-1 + exercise training group, and ICH + RvD1 + exercise training group) showed increased movement distance(Fig. 2b) and a higher C/T distance ratio(Fig. 2c), along with improved speed, indicating a reduction in anxiety-like behaviors (p < 0.05, Fig. 2d). Additionally, the average speed of mice in the ICH + Vehicle + exercise training group increased compared to the ICH group (P = 0.0224), and further improvement in movement ability was observed with the addition of Mdivi-1 treatment (P = 0.0030). The ICH + RvD1 + exercise training group showed a more significant increase in average speed (P = 0.0009), leading to a marked improvement in movement ability, with almost no significant difference compared to the Sham group, demonstrating the effectiveness of RvD1 combined treatment.

RvD1 combined with rehabilitation training improves spatial learning and memory function after ICH

Representative trajectories of mice in each group during the water maze test are shown (Fig. 2f). Compared with the Sham group, the difference in the time to find the hidden platform in the ICH group gradually increased from 22 to 26 days after the operation (p < 0.05,Fig. 2i). Mice in the intervention exercise rehabilitation training showed significant improvement. Additionally, mice in the ICH + RvD1 + exercise training group, ICH + CCCP group, and ICH + Mdivi-1 + exercise training group had a significantly shorter time to find the hidden platform compared to the ICH model group (p < 0.05, Fig. 2i). After the platform was removed on day 27, mice in the RvD1 combined exercise training group and the CCCP group crossed the quadrant of the target platform and exploration time significantly more times than those in the ICH model group (p < 0.05, Fig. 2g and h). The speed of each group of mice was also prolonged, but the difference was not statistically significant (Fig. 2e).

RvD1 combined with rehabilitation training plays a protective role in neuroinflammation after ICH

To verify whether RvD1 Combined with Rehabilitation Training affects ICH-related inflammatory responses, we selected anti-inflammatory factors (CD206 and IL-10) and inflammation-related factors (IL-1β and TNF-α) for RT-qPCR analysis. Our results confirmed that, compared to mice with only cerebral hemorrhage, the expression levels of anti-inflammatory factors were significantly increased(p < 0.05, Fig. 3a and b), while the mRNA expression levels of inflammation-related factors were significantly decreased (p < 0.05, Fig. 3c and d) in mice treated with RvD1 combined with exercise rehabilitation training.To investigate the role of RvD1 in promoting inflammation resolution, we further used Mendelian randomization (MR) to examine the effects of RvD1 on circulating inflammatory factors (Table S2). The results show that RvD1 can inhibit the expression of the pro-inflammatory factor IL20RA (P < 0.05, Fig. 3e) and increase the expression of the anti-inflammatory relative factors CCL25 and LIFR (P < 0.05, Fig. 3e). In conclusion, RvD1, as a pro-resolving mediator, in combination with exercise rehabilitation training, can promote inflammation resolution following cerebral hemorrhage in mice.

Effects of RvD1 combined with rehabilitation training on the expression of BDNF, PI3K, AKT and TrkB related proteins in mice with ICH

Western blot analysis showed that the expression of AKT was relatively consistent across all six groups, without significant differences. Compared to the sham group, the levels of BDNF, PI3K, p-AKT, and TrkB protein expression were significantly reduced in the ICH model mice (p < 0.05, Fig. 4a-e). Compared to the ICH group, the expression levels of BDNF, PI3K, p-AKT, and TrkB increased after exercise rehabilitation training but did not show statistically significant differences (p > 0.05). Mice treated with intraperitoneal injections of Mdivi-1 in combination with exercise rehabilitation training also showed some increase in the expression levels of these proteins, but again without statistical significance. Mice treated with intraperitoneal injections of CCCP showed a significant increase in the expression levels of BDNF, PI3K, p-AKT, and TrkB (p < 0.05). Additionally, ICH + RvD1 + exercise training group on day 28 significantly promoted the expression levels of BDNF, PI3K, p-AKT, and TrkB, comparable to the levels seen with intraperitoneal injections of CCCP, showing statistical significance compared to the ICH group (p < 0.05).

Effects of RvD1 combined with rehabilitation training on the mRNA expression of BDNF, PI3K, AKT, and TrkB in mice with ICH

Compared with the Sham group, the mRNA expression levels of BDNF, PI3K, AKT, and TrkB in the model control group were significantly down-regulated (P < 0.05; Fig. 4f-i). Compared with the ICH group, the mRNA expression levels of BDNF, PI3K, AKT, and TrkB were up-regulated following exercise rehabilitation treatment, although the differences were not statistically significant (P > 0.05). While the mRNA expression levels of BDNF, PI3K, AKT, and TrkB were marginally elevated after intraperitoneal injection of Mdivi-1 in conjunction with exercise rehabilitation treatment, only PI3K (P = 0.0108) and AKT (P = 0.0144) showed statistical significance. When mice received intraperitoneal injections of CCCP without any additional treatments, the mRNA expression levels of BDNF, PI3K, AKT, and TrkB were significantly increased (P < 0.05). In mice that received a combination of RvD1 and exercise training, the mRNA expression levels of BDNF, PI3K, AKT, and TrkB were also significantly increased (P < 0.05).

RvD1 combined with rehabilitation training reduces neuronal apoptosis after ICH

TUNEL staining was employed to assess the changes in apoptotic neurons surrounding the injection sites across six experimental groups of mice. The results indicated that, in comparison to the Sham group, there was a significant increase in the number of TUNEL-positive apoptotic neurons in the ICH group (Fig. 5). Rehabilitation training alone led to a reduction in the number of TUNEL-positive neurons within the ICH group. Furthermore, the addition of RvD1 and Mdivi-1 resulted in an even greater decrease in TUNEL-positive neuron counts (p < 0.05). Additionally, the number of apoptotic neurons in CCCP-treated mice was also greatly improved compared with the ICH group (p < 0.05).

RvD1 combined with exercise rehabilitation training can affect mitophagy in mice

Transmission electron microscopy (TEM) was used to observe the ultrastructural changes in cells surrounding the hemorrhage 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 (Fig. 6). The formation of early autophagic mitochondrial autophagosomes can be identified by the characteristic double membrane and cristae of mitochondria, while the fusion of mitochondrial autophagosomes with lysosomes can be roughly identified by their single membrane or residuals after digestion.In all six groups, neuronal cells exhibited varying degrees of damage. The cell membranes showed different levels of rupture, and the cytoplasm exhibited edema, becoming sparse and dissolved. Organelles were notably swollen, mitochondria were enlarged with significant matrix dissolution, and the cristae showed clear breakage and disappearance, with severe cases exhibiting vacuolation. Additionally, the quantity of rough endoplasmic reticulum decreased, local membrane damage was observed, and ribosomes were visible on the surface; the Golgi apparatus showed expansion of some cisternae.

Different numbers of autophagic lysosomes were observed across the groups. Neuronal cells in the Sham group displayed only mild structural damage, with typical autophagic structures not observed. In the ICH group, neuronal cells showed more pronounced damage, with only one autophagic lysosome structure observed. The ICH + exercise training group displayed two autophagic lysosome structures. The ICH + CCCP group had a higher number of autophagic lysosomes, with four observed. In the ICH + RvD1 + exercise training group, three autophagic lysosome structures were visible, while in the ICH + Mdivi-1 + exercise training group, one autophagic lysosome structure was noted.By comparing the differences between the groups, it can be inferred that RvD1 is involved in activities related to mitochondrial autophagy.

Mendelian randomization analysis reveals the causal relationship between mitophagy-related targets in the caudate basal ganglia of the brain and ICH

We identified MR-related targets located in the Brain Caudate basal ganglia. To further explore the relationship between mitochondrial autophagy and ICH, we employed Mendelian randomization methods to analyze the causal relationship between the relevant targets and ICH. However, due to the lack of data or valid instrumental variables, this study included a total of 8 relevant targets to assess their relationship with ICH. We used the random-effects IVW (IVW-MRE) method as the primary analysis, and the causal relationships between each core target and the relevant outcomes are illustrated in the figure, with the targets supporting causal relationships including MFN2, RAB7B, and TOMM7 (Fig. 7A, p < 0.01). Subsequently, we assessed the sensitivity of the results using MR Egger, weighted median, simple mode, weighted mode, and inverse variance weighted methods. The results showed that the findings for RAB7B were consistent, while the results for TOMM7 were supported by all methods except MR Egger(Fig. 7b-d). The funnel plot of causal effects appeared largely symmetrical (Figure S1a-c). Leave-one-out analysis demonstrated that removing each single nucleotide polymorphism (SNP) still produced consistent results in the remaining Mendelian randomization analyses, thus supporting the robustness of the findings ( Figure S1 d-e). Additionally, we did not find significant heterogeneity in our analysis. MR-Egger regression analysis indicated that there was no evidence of horizontal pleiotropy (p > 0.05; Table S3). In summary, this study clarified the relationship between mitochondrial autophagy-related targets and ICH outcomes through Mendelian randomization. Interestingly, this finding is consistent with our previous experimental results, suggesting that RvD1 may influence mitochondrial autophagy, thereby alleviating neuronal damage following ICH.

Molecular docking was used to analyze the binding affinity between RvD1 and the core target of mitophagy after ICH

To assess the binding affinity between RvD1 and its core targets, we conducted molecular docking analyses using Autodock Vina v.1.2.2. Based on the positive results with P < 0.01 from the MR analysis, we selected the relevant proteins as core targets for simulated docking. The binding forms and interactions of RvD1 with MFN2 and RAB7B are shown (Fig. 8). The findings indicated that RvD1 can form stable complexes with the target proteins through hydrogen bonding. Additionally, the binding energies of MFN2, RAB7B, and TOMM7 with RvD1 were − 7.1 kcal/mol, -5.3 kcal/mol, and − 4.4 kcal/mol, respectively, indicating a certain stability of these molecular interactions.In summary, these results suggest that RvD1 may alleviate damage caused by ICH by modulating key proteins involved in mitochondrial autophagy-related pathways.

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