Fasudil attenuates Neuro-Inflammation and Oxidative Stress via Rhoa/Rock Pathways in Delayed Neuropsychologic Sequelae Mice Model


 Background

Delayed neuropsychologic sequelae is common in patients after carbon monoxide poisoning without effective methods worldwide. Fasudil exerts neuroprotective effect and alleviates oxidative stress in some neurodegenerative disorders. However, the mechanism between DNS and FS remains unclear. The study aims to explore the efficacy and mechanism of Fasudil in DNS mice model.
Objective

The delayed neuropsychologic sequelae model was induced with a hyperbaric oxygen chamber. All rats were randomly assigned to three groups (n=10): air control group (AC), CO poisoning group (CO), and CO poisoning +Fasudil group (CO+FS). Rats in the CO+FS group were given Fasudil (10 mg/kg/day, ip). The morris water maze was documented to estimate spatial learning and memory of mice. The demyelination state in brain was observed through LFB staining. The protein of MBP was examined with immunofluorescence staining. The levels of IL-6, TNF-α, TGF-β, SOD, and MDA were examined by ELISA. The mRNA levels of Rho, ROCK2, MLC1 and MYPT1 were analyzed by rt-PCR.
Result

The cognitive impairment in the CO+FS group were significantly reduced than those of the CO group (P<0.05). LFB staining and immunofluorescence staining of MBP results showed that FS significantly treatment attenuated demyelination (P<0.05). Compared with the CO group, the levels of TNF-α, IL-6, MDA, ROCK2, MLC1, and MYPT1 significantly decreased (P<0.05), and the levels of SOD were significantly increased in the CO+FS group (P<0.05).
Conclusion

In a word, Fasudil attenuated delayed neuropsychologic sequelae by inhibiting inflammation, oxidative stress and downregulating Rho/ROCK pathway in DNS mice model. We conclude that Fasudil may be a novel treatment for delayed neuropsychologic sequelae.


Result
The cognitive impairment in the CO+FS group were signi cantly reduced than those of the CO group (P<0.05). LFB staining and immuno uorescence staining of MBP results showed that FS signi cantly treatment attenuated demyelination (P<0.05). Compared with the CO group, the levels of TNF-α, IL-6, MDA, ROCK2, MLC1, and MYPT1 signi cantly decreased (P<0.05), and the levels of SOD were signi cantly increased in the CO+FS group (P<0.05).

Conclusion
In a word, Fasudil attenuated delayed neuropsychologic sequelae by inhibiting in ammation, oxidative stress and downregulating Rho/ROCK pathway in DNS mice model. We conclude that Fasudil may be a novel treatment for delayed neuropsychologic sequelae.

Background
Acute CO poisoning is one of the common and fatal cause of poisoning and its manifestations include mild headache, nausea, fatigue, dizziness, syncope, coma, seizures and even respiratory and circulatory failure and death [1]. DNS, also known as delayed encephalopathy, occurs after a 2-60 days period of remission which is called the "lucid interval" in about 2-30% patients who recover from acute CO poisoning [2]. Delayed neuropsychologic sequelae refers to symptoms such as impaired memory, cognitive dysfunction, depression, anxiety, vestibular and motor de cits [3]. At present, hyperbaric oxygen (HBO) therapies is considered as an effective treatment to incidence of DNS [4], but other researchers disputed the effectiveness of HBO therapies [5]. Thus, there is still a lack of effective treatment for delayed neuropsychologic sequelae [6]. The pathogenesis of DNS is complex and not completely clear. It is reported that in ammatory response and oxidative is closely related to the pathophysiological mechanism of CO poisoning [7]; thus, immunomodulatory therapy and antioxidant therapy are essential for patients after CO poisoning [8].
Fasudil, a Rho kinase inhibitor, has been extensively reported to relieve vasospasm in patients with subarachnoid hemorrhage (SAH) by inhibiting the phosphorylation of myosin light chain (MLC) [9,10]. In addition, several studies revealed that Fasudil has other effects, including anti-in ammatory, antiapoptosis, reducing oxidative stress and ischemia-reperfusion [11,12].Fasudil also exert neuroprotective effect and attenuate cognitive function in some neurodegenerative diseases [13]. For example, Kumar et.
al [14] reported that Fasudil can alleviate cognitive de cit via inhibiting expression of eNOS and activity of NF-κB mediated by PI3-kianse in STZ-ICV rat model of AD. Huang et. al [15] found that Fasudil can attenuate permeability damage of blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in a model of experimental autoimmune encephalomyelitis (EAE). These studies indicated the important role of Fasudil on neuroprotective effect, but it is rarely reported in DNS model, and its potential mechanism has not been clari ed. This study focuses on the effect and mechanism of Fasudil in DNS rat model from the perspective of regulating immunity and oxidative stress.

Animals
Male Sprague-Dawley (SD) rats weighting 200-250g were obtained from the Experimental Animal Center of Southwest Medical University and placed in a thermostatic speci c pathogen-free laboratory animal room with free access to feeds and water. Animal procedures were conducted in accordance with the guidelines of the European Directive 2010/63/EU [16] and was approved by the Ethics Committee of Southwest Medical University (Approval No.20210006).

DNS model
The DNS model was established by hyperbaric oxygen chamber according to published protocol [17]. Brie y rats were exposed to CO (1,000 ppm) for 40 min and then 3,000 ppm for 20 min, until they lost consciousness, and then they were removed to breathe room air and regain consciousness.

Experimental Design and Fasudil administration
In this experiment, DNS model rats were randomly assigned to the CO group, which received intraperitoneal injection of phosphate-buffered saline (PBS), or the CO+FS group, which received intraperitoneal injection of Fasudil. Each group had 10 mice (n=10). And ten mice were exposed to air as the AC group, which received intraperitoneal injection of PBS. Rats were injected with PBS or fasudil (10 mg/kg) (Sigma Chemical)[18] once daily for 14 consecutive days following model building as shown in Fig.1. Rats were sacri ced for brain tissue collection under deep anesthesia by 10% pentobarbital sodium (40mg/kg) [19].

Morris water maze test (MWM)
The cognitive function of the rats was evaluated by the Morris water maze test as previously described [19]. In the visible platform trial, put a black ag on the platform and release each mouse into the water from one of the four start locations facing the tank wall. Give each mouse 60 s to search for the platform and allow it to stay on it for 10-30 s. Record the time the mouse nds the platform as escape latency, and analyze the swimming speed. Place each mouse into the pool at each of the four different starting quadrants for four trials, moving the platform to a different location with each subsequent trial.
In the hidden platform trial, place the same platform without a ag in the SE quadrant. Randomly place the mouse into the pool from each of the four quadrants for four trials and record the escape latency of each trial for subsequent analysis for 5 consecutive days, with the platform and the visual cues at constant positions. If the rats could not nd the platform after 2 min, they were pulled onto the platform and the time was recorded as 120 s. In the probe trial, remove the platform and locate each mouse in the NW quadrant, which is the quadrant furthest away from the SE quadrant. Record the time spent in the SW quadrant and the platform crossover number in the maze. The general steps are showed in Fig.1 Luxol fast blue (LFB) The sections were depara nized, rehydrated, and stained with LFB to assess the demyelinated area. Furthermore, white matter demyelination was scored in LFB-stained sections as 1 for normal myelination, 2 for mild or minor demyelination (> 50% myelin staining preserved), or 3 for moderate to severe demyelination (< 50% myelin staining preserved), as reported before [20].

Immuno uorescence analysis
Immuno uorescence staining was carried out as previously described [22]. Brains were isolated and xed in 4% PFA for 48 h and then dehydrated with a 10% sucrose formaldehyde solution and a 20% sucrose formaldehyde solution for 24 h. Brain tissue at 2 mm thick was cut near the cerebral white matter and was embedded in para n. The tissue was then cut into 4-µm-thick continuous brain slices. After dehydration with gradient alcohol and xylene, then reduce endogenous peroxidase activity with 3% H 2 O 2 and blocking with 5% bovine serum for 30 min at room temperature; the tissues were then subsequently incubated with the primary antibodies for anti-MBP (1:200; BA0094; Boster, USA) at 4 °C overnight. Then, uorescent dye-conjugated secondary antibody for goat-anti-rabbit (1:50; AS-1110; Aspen, USA) were added for 30 min at 37 °C in the dark. Finally, the nuclei were stained with DAPI solution (1:1000, AS1075; Aspen, USA) for 10 min at room temperature, in darkness. Images were scanned by a Nikon reverse laserscanning confocal microscope (Nikon, CI-S, Japan). Imaging of the stained sections was photographed using confocal microscopy (Germany). The uorescence quantitative analysis was performed by ImageJ.
Quantitative Real-Time polymerase chain reaction (QRT-PCR) Sample collection, RNA isolations and qPCR were carried out as previously described [23]. Total RNA was extracted from cerebral white matter by ExTrizol Reagent (Life Technologies) and then reversed transcribed into cDNA by a cDNA Synthesis Kit (CW2569, Cwbiotech, China) according to the manufacturers' protocols. The primer sequences are designed as following as RhoA forward, TGACACCAGGCGCTAATTCA, RhoA reverse, GCTCTGGCAATGGAGTCTGT; ROCK2 forward, CTGGGAAGAGGCAGCTTCAA, ROCK2 reverse, CTCTGCTGTGGGGTTAAGCA; MLC1 forward, CCAGTCCCATGTTACGGTCC, MLC1 reverse, CTAATCCTGACGCATGGCCT; MYPT1 forward, AGCTGAAGCGCTGGATCG, MYPT1 reverse, CAGGCGGCGAGGAAGAC; GAPDH forward, ACTGTGCCGTTGAATTTGCC, GAPDH reverse, TTGCAGTGGCAAAGTGGAGA. Qt-PCR conditions consisted of an initial denaturing step of 3 min at 95•C followed by 40 cycles of 3 s denaturation at 95•C, 20 s annealing at 60•C, and 20 s extension at 72•C. Then, the plates were read with a StepOnePlus Real-Time PCR system (4376600, ABI, USA). qPCR was performed with SYBR Green (Biotool, Houston). The mRNA expression was normalized to the mRNA expression of GAPDH, and the results were calculated using the comparative cycle threshold (∆∆Ct) method.

Statistical analysis
All value was analyzed using GraphPad Prism v 7.01. The data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by LSD multiple-range test or two-way repeated measures analysis of variance (ANOVA) was used to determine the statistical differences. A p-value<0.05 was considered signi cant.

FS treatment attenuated Spatial Learning and Memory De cits induced by CO-poisoning
The results of MWM test presented in Figure 2. In the visible platform trial, no signi cant differences were observed in the escape latency or swimming speed among the groups on the 15th day (Fig.2a, 2b). In the hidden platform trial, the escape latency of three groups all decreased gradually. The escape latency from days 17−20 was longer in the CO group than in the AC group (p< 0.01) (Fig.2c), and in the CO+FS group was shorter from days 18−20 than in the AC group (p< 0.01) (Fig.2c). In the probe trial, the platform crossover number and the percentage of time in the SW quadrant in the CO group was signi cantly lower than in the AC group (p < 0.01), whereas a signi cant reduction of these two parameters were obtained in the CO+FS group in comparison with the CO group (p < 0.01) (Fig.2d, 2e). Thus, these results showed that FS attenuated spatial learning and memory de cits induced by CO-poisoning.
FS treatment alleviated the demyelination induced by CO-poisoning LFB staining and immuno uorescence staining of MBP protein were performed to evaluate demyelination at 21 days post CO-poisoning. As shown in Fig.3a, the LFB staining of white matter from the CO group exhibited irregular vacuoles and broken myelin patches, while this was relatively not obvious in the CO +FS group. Assigning a score to demyelination intensity, CO poisoning signi cantly increased the demyelination score compared to the AC group (p <0.05) (Fig.3b). Furthermore, the lower protein levels of MBP in the CO group also indicated that CO-poisoning could cause evident MBP degradation in the brain tissues (P< 0.05) (Fig. 3c, 3d). When Fasudil treatment substantially promoted the remodeling of myelin sheath and alleviated impairment of MBP (P< 0.05) (Fig. 3).

FS treatment reduce in ammatory cytokines induced by CO-poisoning
The serum TNF-α, IL-6, and TGF-β levels in the white matter were determined by ELISA on days 21 following CO poisoning. Compared with the AC groups, rats in the CO group exhibited signi cantly higher levels of TNF-α (P< 0.01) (Figure 4a). While the level of IL-6 was slightly increased with no difference (P>0.05) (Figure 4b). Treatment with Fasudil signi cantly decreased the expression of TNF-α and IL-6 (P< 0.05) (Figure 4a, 4c). TGF-β levels in the CO group were not signi cantly different from those in the CO/FS group and AC groups (P > 0.05) (Figure 4b). These results indicated that FS could alleviated in ammatory responses on days 21 following CO poisoning.

FS treatment attenuate oxidative stress induced by CO poisoning
To determine the oxidative stress, MDA contents and SOD activity in the hippocampus and white matter were measured. Compared with those in the AC group, the MDA content in the hippocampus and the white matter were obviously increased after CO exposure (p<0.05 or p<0.01), while FS treatment signi cantly decreased the production of MDA (p<0.05 vs. CO group) (Fig. 5a,5c). The levels of SOD activity were signi cantly decreased in the hippocampus and the white matter of CO group (p<0.05 or p<0.01 vs. AC group) (Fig. 5b, 5d). Also, FS administration induced the increase production of SOD (p<0.05 vs. CO group). These results indicated that Fasudil could attenuate oxidative stress levels induced by CO poisoning.

Fs treatment inhibit the Rho-Kinase Pathway in DNS rats
Rt-PCR was conducted to measure the expression of Rho, ROCK2, MYPT1 and MLC. Compared with the AC groups, the levels of ROCK2 and MYPT1 mRNA in the CO group were signi cantly increased after 21 days (P<0.05) (Fig.6b, 6d). While the mRNA expression levels of RhoA and MLC were not signi cantly changed after CO poisoning(P>0.05) (Fig.6a, 6c). Fasudil treatment signi cantly lowered the levels of ROCK2, MYPT1, and MLC1 expression (P<0.01) (Fig.6b, 6c, 6d) and partially lowered RhoA expression (P> 0.05) (Fig.6a). These results suggest that Fasudil alleviates the RhoA/ROCK-mediated signaling pathway in DNS model.

Discussion
DNS is common in patients after acute CO poisoning [24]. Multiple mechanisms collectively induced disease onset and severity [25]. In the acute stage of carbon monoxide poisoning, the direct toxic effect of CO and its mediated hypoxia leads to mitochondrial dysfunction, oxidative damage, cell apoptosis, activation of in ammatory cascade, demyelination of white matter, immune system dysfunction, especially in organs sensitive to ischemia [26]. It is speculated that MBP was modi ed by MDA which is ultimate product of membrane lipid peroxidation; thus, MBP lost its normal cationic properties resulting in recognition by an antibody and activating the adaptive immunological response [27,28]. Therefore, when the progressing injuries of MBP exceed an unknown tripping point, pathophysiological variations in brain tissues and a succession of neurodegenerative symptoms could occur in some patients [29].
The RhoA/ROCK pathway participates in diverse neuronal biological activities, such as migration, dendrite development, and axonal extension [13]. And some studies have con rmed that RhoA / ROCK pathway is related to the pathogenesis of Alzheimer's disease (AD) [30], spinal cord injury (SCI) [31], stroke [32] and experimental autoimmune encephalomyelitis [33]. Rho-associated protein kinase (ROCK) is a downstream effector of RhoA [34], and the most promising drug targets [35]. Fasudil (hexahydro-1-(5isoquinolylsulfonyl)-1H-1,4-di-azepime), also known as HA-1077, which has the isoquinoline and the homopiperazine ring, is widely used as a ROCK inhibitor [36]. Fasudil has been used in the treatment of neurodegenerative diseases. Study has demonstrated that the learning and memory function of amyloid precursor protein/presenilin-1 transgenic (APP/PS1) mice is signi cantly enhanced after treatment with Fusudil by modulating gut microbiota and metabolites [37]. Another study about APP/PS1 mice reported that Fasudil increased antioxidative substances, decreased lipid peroxides, and inhibited neuronal apoptosis by inhibiting ROCK/MAPK and activating Nrf2 signaling pathways. Research focusing on the ischemic stroke illustrated that Fasudil combined with bone marrow mesenchymal stem cells (BMSCs) synergistically promotes neurovascular remodeling and neurological function recovery in rat model [38]. In seizure-induced neurite injury, fasudil can increase neurite outgrowth and alleviated spine loss both in Neuro-2a cells and in cultured hippocampal neurons via inhibiting RhoA/Rho kinase pathway [39]. These studies show that fasudil has protective effects on cognition and cerebrovascular function. Therefore, in the current study, we explored the role of Fusudil in delayed encephalopathy after CO poisoning, and the results showed that Fusudil indeed alleviate CO-mediated behavioral disorders and damage of myelin as we expected.
RhoA/ROCK pathway is one of the key regulators of cellular oxidative stress and in ammatory damage [40,41]. Some studies reported that Rho-kinase (ROCK) inhibitor can downregulate NADPH oxidase 2 (NOX2) expression which promoted by the phosphorylation level of myosin light chain (p-MLC) in rat ischemia/reperfusion (I/R) model [42]. And p-MLC are subjected to MLC kinases, type 1 Ser/Thr phosphatase, and MLC phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP depends on its associated regulatory subunit, namely myosin PPase targeting subunit 1 (MYPT1) [43]. Previously published research suggested that chronic stress promotes cognitive impairment and dendritic spine loss in hippocampal neurons possibly by through phosphorylation of the MYPT1 and inactivates MLCP [44]. In addition, researchers found that RhoA/ROCK pathway is an important mediator of the crosstalk between hematopoietic and non-hematopoietic cells which is necessary to e ciently clear pathogens while preventing the emergence of autoimmunity [45]. These results preliminarily elucidated the protective mechanism of RhoA/ROCK pathway. In the study the in ammatory response and oxidative stress increased signi cantly in DNS model after CO poisoning, as well as the expression of ROCK2 and MYPT1 were markedly increased. In addition, the trend is reversed by treatment of fasudil, which was attributed to the protective effect of fasudil on CO poisoning.

Conclusion
In short, our research shows that Fasudil can alleviate delayed neuropsychologic sequelae by reducing in ammation and oxidative stress mediated by Rho/ROCK2 pathway. This study will provide new ideas for the treatment of CO poisoning.

Availability of data and materials
All data generated or analysed during the current study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was nancially supported by the Luzhou People's Government-Southwest Medical University science and technology strategic cooperation project (Grant No.2017LZXNYD-J36).
Authors' contributions YX and LJ designed the work, and wrote the manuscript. YX, FM, LS, XH, GY, and HQ assisted to performed the experiments and analyzed the data. All authors have reviewed and approved the nal version of the manuscript, and agree to be accountable for all aspects of the work.    score of brain sections from rats in the AC, CO, CO+FS group, Scale bar =200 μm. (d) Immuno uorescence images and (c) quanti cation showed the expression of MBP (red) and nuclear (blue) in the AC, CO, CO+FS groups, Scale bar =100 μm. The data in each group are shown as mean ± SD (n=10). The symbol * indicates p< 0.05 vs.AC group and # indicates p<0.05 vs. the CO group.