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Introduction

Cranial nerve involvement frequently involves neuron damage, and often leads to psychiatric disorder caused by multiple inducements (1). Cranial nerve involvement affects cognitive impairments and mental health, and can lead to mental disability for patients worldwide (2). Patients with cranial nerve involvement account for >5% of the population, and is a common disabling mental disease with patients often suffering from complex psychiatric with cognitive, language, memory and behavior impairments (3,4). The key characteristics of cranial nerve involvement include neuron damage and cognitive impairment (5). However, the mechanism of multiple lower cranial nerve involvement associated with neuron damage and cognitive impairment remains to be fully elucidated. Therefore, understanding the signaling pathway between cranial nerve involvement and cognitive impairment is essential for the treatment of patients with cranial nerve involvement.

The role of histamine in cranial nerve involvement has received increasing attention in investigations. A previous study reported that the expression levels of histamine were upregulated in patients with cranial nerve involvement, formulating a stable protein complex target for histamine, which is important for maintaining the normal function of neurons, and critical for neuronal differentiation and brain development (6,7). The target for neuroprotective agents for neurocognitive repair and inhibition of neuron-function damage developed according to cognitive deficits across multiple domains in substantial intellectual impairment (8,9). In addition, although the theoretic mechanism of anti-histamine agents in cranial nerve involvement are well understood, treatment of this type of disease remains limited and lacks preclinical investigation (10–12). Therefore, investing the mechanism of anti-histamine in the treatment of cranial nerve involvement is important to better explain therapeutic effects according to observations.

Pharmaceutical studies have shown that anti-histamine drugs are efficient for the treatment of cranial nerve involvement (12,13). Lurasidone, is an azapirone derivative and a second-generation novel antipsychotic candidate, which was approved for the treatment of schizophrenia in the USA in 2010, and by the European Medicines Agency in 2014 (14,15). A previous report indicated that cranial nerve involvement was cured in some way following treatment with lurasidone in patients with schizophrenia (12). Additionally, lurasidone with lithium or valproate was identified as a therapeutic strategy for the treatment of bipolar I depression (15). The therapeutic mechanism underlying the effects of lurasidone involved decreased levels of serotonin 5-HT7, serotonin 5-HT2A, serotonin 5-HT1A and dopamine D2 by antagonist activities (16).

In this present study, the therapeutic effects of lurasidone were investigated in a cranial nerve involvement rat model. On the basis of the aforementioned evidence, the present study examined the preclinical outcomes of lurasidone for cranial nerve involvement therapy. The data obtained suggested that lurasidone repaired neuron-function loss and improved anxiety, compared with a placebo. The results also suggested that cognitive ability was improved following treatment with lurasidone.

Materials and methods

Analysis of serotonin receptors and neuroprotective protein

Serotonin receptors and neuroprotective proteins in serum were analyzed using a commercialized ELISA kit in patients with cranial nerve involvement. A total of 180 patients and 124 healthy volunteers were recruited in Sichuan People's Hospital between April 2014 and December 2014. All patients were required to sign informed consent before the examinations. Serum was collected from 10 ml blood using centrifugation at 6,000 × g for 10 min at 4°C. The ELISA assays were performed according to the manufacturer's protocols. The results were measured at 450 nm in an ELISA reader and finally converted to concentrations of serotonin 5-HT7, serotonin 5-HT2A, serotonin 5-HT1A, serotonin 5-HT6, Forkhead-Box P2 (Foxp2), SxIP and microtubule end-binding (EB) protein.

Animal experiments

Rats (n=100) of a mutant intravenously mutated cranial nerve involvement Sprague-Dawley rat model (specific pathogen-free; 6–8 weeks old) were purchased from Slack Co., Ltd. (Shanghai, China). All rats were housed under controlled conditions (temperature, 23±1°C, humidity, 55±5%) in a 12 h light/dark cycle with free access to food and water. The rats with cranial nerve involvement were randomly divided into two groups and injected intravenously with either lurasidone (0.32 mg) or placebo (PBS, 0.32 mg) as a control. The total treatment regime comprised a total of seven injections, once per day. All experimental procedures were performed according to the guidelines of Sichuan Academy of Medical Sciences, Sichuan People's Hospital (Sichuan, China). All experiments were approved by the Animal Care and Use Committee of Tianjin Medical (Tianjin, China).

Measurement of blood pressure, heart rate and blood glucose parameters

Blood levels in the rats with cranial nerve involvement were measured using a blood glucose gauge (OneTouch® VerioVue; Johnson & Johnson Medical (Shanghai) Ltd., Shanghai, China). The pressure was recorded prior to and following treatment with lurasidone or placebo. Heart rate and blood pressure parameters were measured every 2 days, as described in a previous report (17).

RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was extracted from the hippocampal cells of the rats with cortical cranial nerve involvement following treatment with lurasidone or placebo using an RNAeasy Mini kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA (1 µg) was subjected to RT into cDNA using a reverse transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.). The cDNA (10 ng) was used for qPCR analysis (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with a SYBR-Green Master Mix system (Thermo Fisher Scientific, Inc.) and a total reaction volume of 25 µl (primers, 1 µl, cDNA, 2 µl, buffer, 2 µl, reverse transcriptase, 0.5 µl, SYBR, 0.5 µl and water, 19 µl). All forward and reverse primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. (Foxp2, forward: 5′-AACAGAGACCACTGCAGGTGCC-3′; reverse: 5′-TCCCTGACGCTGAAGGCTGAG−3′; SxIP, 5′-TATGGTCTCTGCCTGTTGC-3′, 5′-TGCTACTGCCCATTACAATTCC-3′; EB, forward: 5′-GGATTTGAATCACGTTTGTGTC-3′, reverse: 5′-AACTTGCGCTCATCTTAGGC-3′; 5-HT7, forward: 5′-AATAAGGGTAAGCCAATTGTATGGA-3′, reverse: 5′-TGGTGCAAAATCAACATTCC-3′; 5-HT6, forward: 5′-TATTACGAAGGCCAACCTAT-3′, reverse: 5′-TTCTTCTTCAGGCAAATCAT-3′; 5-HT1A, forward: 5′-TCAAAAAGAAAGGAG-3′, reverse: 5′-TCATCTGAGATAAGGGCTG-3′; 5-HT2A forward: 5′-TGTTTTAACGCCATTAGGTCA-3′, reverse: 5′-TCCGAGCAACTGATAAGTCT-3′ and β-actin, forward: 5′-CGGAGTCAACGGATTTGGTC-3′, reverse: 5′-AGCCTTCTCCATGGTCGTGA-3′).

Thermocycling conditions were as follows: Pre-denaturation at 95°C for 90 sec, denaturation at 94.5°C for 30 sec and annealing at 56°C for 10 sec for 40 cycles. The alterations in relative mRNA expression levels were calculated using the 2−ΔΔCq method (18), with the results expressed as the n-fold, compared with the control.

Western blot analysis

The hippocampal cells from the rats with cranial nerve involvement treated with lurasidone or placebo were homogenized in lysate buffer containing protease inhibitor and were centrifuged at 6,000 × g at 4°C for 10 min. The supernatant was used to analyze proteins. For detection of proteins, the proteins were extracted using a protein extraction kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA) according to the manufacturer's protocol. Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples (20 µg/lane) were resolved by 15% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany) as previously described (19). For western blot analysis, primary antibodies: FoxP2 (ab16046), EB (ab157217), SxIP (ab45142), β-actin (ab8227) (all, 1:200; Abcam, Shanghai, China) were added for 12 h at 4°C following blocking in 5% skimmed milk for 1 h at 37°C. The sections were washed three times with PBS to remove primary antibodies and then incubated with HRP-labeled secondary goat anti-rabbit antibodies (ab150077; 1:2,000 dilution; Abcam) 24 h at 4°C. The results were visualized using chemiluminescence detection system.

Immunofluorescence analysis

The therapeutic effects of lurasidone on neuronal repair were evaluated using immunofluorescence staining of anti neuroprotection-associated proteins in the hippocampus of the experimental rats. Staining was performed on cerebral neurons of the hippocampus in randomly-selected animals from the lurasidone or placebo-treated groups. The immunofluorescence procedures were as previously reported and captured using a fluorescence microscope (FV3000; Olympus Corporation, Tokyo, Japan) at ×40 magnification (20).

Experiments using the elevated plus maze trial and analysis of swimming duration

The anxiety of the rats was evaluated using an elevated plus maze trial based on the hypothesis that rat experience fear of open fields. The details of the elevated plus maze trial were as described in a previous study and the space was improved to a size of 80×15×60 cm (21). The rats with cranial nerve involvement were fixed at the center of the elevated plus maze, and these rats were positioned facing an open arm for a total of 5 min. The durations spent in the open and closed arms were recorded and calculated using the following formula: D2=(B - A)/(B + A). A represents the time spent in the open arm and the B represents the time spent in the closed arm. The anxious behavior was measured using the above formula and the path efficacy was calculated. The behavioral capacity of the rats with cranial nerve involvement was analyzed by swimming duration according to the method described in a previous study (22).

Efficacy and safety assessment

Assessments of efficacy and dose-limiting toxicity in the presence of lurasidone were performed in the present study. Safety assessments included the incidence rates (≥10%) of the most frequent treatment-emergent adverse events in a 30-day treatment period in the experimental and control groups. The efficacy and safety data included all rats in the cranial nerve involvement, therapeutic drug and control groups.

Statistical analysis

All data are presented as the mean ± standard error of the mean of triplicate experiments. All data were analyzed using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA). Unpaired data was determined using Student's t test and comparisons of data between multiple groups were analyzed using one-way analysis of variance. Kaplan-Meier analysis was used to estimate the risk of relapse and re-treatment during the 368-day treatment period. P<0.05 was considered to indicate a statistically significant difference.

Results

Characteristics of the cranial nerve involvement rat model

Adult Sprague-Dawley (6–10 weeks old) rats with cranial nerve involvement were subjected to cerebral artery occlusion and reperfusion and examined in the designed experiments. The rats in the cranial nerve involvement model received lurasidone treatment or placebo treatment as a control. The preclinical parameters of cranial nerve involvement, including body temperatures, body weight, blood pressure, heart rate and blood glucose, were recorded prior to and following treatment. The characteristics of the rats and patients with cranial nerve involvement are summarized in Table I. The data indicated that the states of the rats with cranial nerve involvement, determined using the Positive and Negative Syndrome Scale (PANSS) were improved following lurasidone treatment, compared with the placebo. In addition, physiological parameters exhibited significant differences between the lurasidone and placebo groups. Of note, the mean blood pressure and heart rate during the experiment were significantly different between the lurasidone and placebo groups. In addition, the MTD was identified and the most common treatment-associated adverse events were hypertension, diarrhea, lethargy, rash, proteinuria and vomiting (Table II; ≥10%). A dose of 0.32 mg of lurasidone met criteria for further preclinical experiments in terms of the tolerability and therapeutic effects.

Table I. Characteristics of patients and rats with cranial nerve involvement.

Table I.

Characteristics of patients and rats with cranial nerve involvement.

Parameter	n	%
Total healthy volunteers	180	100
Men	85	47
Women	95	53
Total patients	124	100
Men	61	49
Women	63	51
Total rats with cranial nerve involvement	100	100
Male	50	50
Female	50	50
Positive and negative syndrome scale score	121.4±6.2	−
Body weight (g)	132±10.5	−
Blood glucose (mmol/l)	8.3±2.5	−
Blood pressure (mm Hg)	143±12	−
Heart rate	370±26	−
Drug therapy	80	100
Placebo	40	50
Lurasidone	40	50

Table II. Treatment-associated adverse effects of lurasidone with overall incidence ≥10%.

Table II.

Treatment-associated adverse effects of lurasidone with overall incidence ≥10%.

	Lurasidone (n=20 per group)
	Total	0.08–0.16	0.25–0.32
Adverse effect	(n=60)	mg	mg	0.40 mg
Hypertension	12	3	4	5
Diarrhea	7	1	3	3
Proteinuria	9	2	3	4
Vomiting	10	2	3	5
Lethargy	8	2	3	3
Rash	9	2	3	4

Detection of serum histamine and neuroprotective proteins in patients with cranial nerve involvement

A previous study indicated that histamine chemical compounds were upregulated in patients with cranial nerve involvement (23). In the present study, histamine chemical compounds, including serotonin 5-HT7, serotonin 5-HT2A, serotonin 5-HT1A, serotonin 5-HT6, and the neuroprotective protein, Foxp2, SxIP motif and EB protein, were detected in sera of patients with schizophrenia. The concentrations of histamine chemical compound in the serum were elevated in patients with cranial nerve involvement, compared with healthy volunteers. The results, as shown in Fig. 1A-D, showed that plasma concentration levels of serotonin 5-HT7, serotonin 5-HT2A, serotonin 5-HT1A and serotonin 5-HT6 were downregulated, as determined using ELISA, compared with those of healthy volunteers. In addition, plasma concentration levels of three important neuroprotective protein, Foxp2, SxIP motifi and EB protein, were decreased in patients with cranial nerve involvement, compared with healthy volunteers (Fig. 1E-G). These results suggested that the expression levels of histamine were downregulated and may be associated with the expression of neuroprotective proteins.

Figure 1. Expression of serotonin-media serotonergic neurons receptors and neuroprotective protein in patients with cranial nerve involvement and healthy volunteers. Differences in levels of (A) serotonin 5-HT7, (B) serotonin 5-HT2A, (C) serotonin 5-HT1A and (D) serotonin 5-HT6 between patients with cranial nerve involvement and healthy volunteers. Differences in levels of neuroprotective protein (E) Foxp2, (F) SxIP motif and (G) EB between patients with cranial nerve involvement and healthy volunteers. Data are presented as the mean ± standard of the mean. **P<0.01 (determined using one-way analysis of variance). Foxp2, Forkhead-Box P2; EB, microtubule end-binding protein.

Therapeutic effects of lurasidone in the cranial nerve involvement rat model

To investigate the beneficial preclinical outcomes of lurasidone, a rat model of cranial nerve involvement model was established. The results (Fig. 2A) indicated that the total PANSS score was significantly reduced in the rats with cranial nerve involvement treated with lurasidone compared with those treated with placebo. In addition, neuron impairment was analyzed on day 30 following lurasidone or placebo treatment. The results (Fig. 2B) showed that neuron impairment was significantly reduced in the hippocampus of lurasidone-treated mice. In addition, the risks of relapse and re-treatment in a 180-day observation period were analyzed following treatment with lurasidone, compared with the placebo. P-values were 0.00063 and 0.00048 for relapse rate and re-treatment, respectively (Fig. 2C and D). Furthermore, as shown in Fig. 2E and F, the lurasidone-treated mice had significantly higher rates of remission and reduced anxiety, compared with the placebo (P=0.0075 and P=0.0086, vs. placebo, respectively).

Figure 2. Short-term and long-term therapeutic effects of lurasidone on cranial nerve involvement in rats. (A) Total PANSS scores of rats with cranial nerve involvement treated with lurasidone or placebo in a 30-day short observation period. (B) Immunofluorescence staining showing numbers of neurons between the lurasidone- and placebo-treated rats with cranial nerve involvement. Scale bar=70 µm; arrows indicate neurons. (C) Kaplan-Meier estimated the probability of relapse during treatment with lurasidone or placebo. (D) Cumulative retreatment was evaluated by long-term observation. (E) Cumulative remission was evaluated by long-term observation between rats treated with lurasidone or placebo. (F) Anxiety in rats with cranial nerve involvement was evaluated using a plus-maze test by long-term observation between the lurasidone and placebo treatment groups. Student's t test and one way analysis of variance showed a statistically significant effect of treatment with cooperative therapy Data are presented as the mean ± standard error of the mean. **P<0.01 and ***P<0.001 between drug-treated and placebo groups. PANSS, Positive and Negative Syndrome Scale.

Neuroprotective effect of lurasidone in rats with cranial nerve involvement

The short-term and long-term effects of lurasidone were apparent in mice with cranial nerve involvement. In order to obtain further insight into the primary mechanism of lurasidone efficacy, the expression levels of Foxp2, SxIP and EB, and the distribution of neurons in the hippocampus were analyzed using RT-qPCR and immunohistochemcal analyses. The results (Fig. 3A) showed that the Foxp2 gene, associated with language competence, was increased in the hippocampus of the cranial nerve involvement rats following treatment with lurasidone (P=0.00044), compared with those in the placebo-treated group. In addition, the expression levels of neuroprotective SxIP motif and EB proteins were also upregulated following treatment with lurasidone, which led to neuroprotection and synaptic plasticity (Fig. 3B and C; P=0.0052 and P=0.0066, vs. placebo, respectively). As shown in Fig. 3D, the immunohistology revealed that neuron distributions were homogeneous in the hippocampus following treatment with lurasidone, compared with the placebo.

Figure 3. Therapeutic effects of lurasidone by increasing neuroprotective protein levels in the hippocampus. (A) Relative mRNA and protein expression of Foxp2 in the hippocampus. (B) Expression of SxIP motif in the hippocampus of rats with cranial nerve involvement following long-term treatment with lurasidone or placebo. (C) Expression of EB in the hippocampus of rats with cranial nerve involvement rat following long-term treatment with lurasidone or placebo. (D) Immunofluorescence staining analysis of the expression of Foxp2, SxIP motif and EB in the hippocampus of rats with cranial nerve involvement rat following long-term treatment with lurasidone or placebo. Magnification, ×40. Data are presented as the mean ± standard error of the mean. **P<0.01 and ***P<0.001 between drug-treated and placebo groups.

Antagonistic effect of lurasidone on histamine in rats with cranial nerve involvement

An important characteristic of cranial nerve involvement is anxiety. It was concluded that anxiety was in remission according to the experimental data. The behavior of rats with cranial nerve involvement was assessed using an improved water maze and recognition test. The results (Fig. 4A) indicated that the behavior of the rats with cranial nerve involvement improved following treatment with lurasidone, compared with the placebo (P=0.0059, vs. placebo). In addition, the data showed that lurasidone significantly improved cognitive impairment in rats with cranial nerve involvement (P=0.0059, vs. placebo; (Fig. 4B). Changes of histamine, including serotonin 5-HT7, serotonin 5-HT2A, serotonin 5-HT1A, and serotonin 5-HT6 in the serotonin systematic signaling pathway were also examined. The results (Fig. 4C) showed that the concentration levels of serotonin were downregulated in the cerebrospinal fluid of the lurasidone-treated rats, compared with the placebo-treated rats. The same observations were found in the hippocampus of cranial nerve involvement mice between the lurasidone and placebo groups (Fig. 4D). These data indicated that the serotonin systematic signaling pathway was inhibited by the antagonistic effect of lurasidone, which led to beneficial outcomes in behavior and cognitive improvements in the rats with cranial nerve involvement.

Figure 4. Efficacy of lurasidone by targeting antagonistic molecules. (A) Behavior capacity was analyzed by swimming following treatment with lurasidone or placebo. (B) Object recognition and short retention memory were assessed in rats with cranial nerve involvement following long-term treatment with lurasidone or placebo. Data are expressed as the mean ± standard error of the mean and total duration spent exploring all objects, determined by: Relative discrimination index=(duration in closed arms-duration in open arms)/(duration in closed arms + duration in open arms). (C) Differences in the mRNA expression levels of serotonin 5-HT7, serotonin 5-HT2A, serotonin 5-HT1A and serotonin 5-HT6 between the lurasidone and placebo-treated rats with cranial nerve involvement. (D) Expression levels of serotonin 5-HT7, serotonin 5-HT2A, serotonin 5-HT1A and serotonin 5-HT6, analyzed using immunofluorescence staining. **P<0.01 (determined using one-way analysis of variance).

Discussion

Currently, although several factors have been reported to exhibit correlation with the initiation and development of cranial nerve involvement, the mechanism of interactions between cranial nerve involvement and affecting factors remain to be fully elucidated (24). Several potentially neuroprotective agents function to upregulate gene transcription at the onset of brain ischemia, which may be an effective approach for limiting brain tissue damage (25). Studies on the mechanisms of cranial nerve involvement have shown that the serotonin systematic signaling pathway shows high correlation with the initiation, development, treatment and prognosis of cranial nerve involvement (26,27). In addition, an increasing number of studies have reported that the overexpression of histamine in the cerebrospinal fluid is a potential therapeutic target in patients with cranial nerve involvement and has been found be important in the pathogenesis of cranial nerve involvement (28–31).

Targeted therapy for cranial nerve involvement is a novel concept and the rationale for the trial in a mouse model of cranial nerve involvement, which was performed in a previous study (32). Serotonin is one of the most important predisposing and aggravating factors, which orchestrate the responses to neuron impairment and other exogenous insults (33). A previous study reported that serotonin was significantly correlated with neuron loss and continuation of non-motor symptoms in patients with Parkinson's disease treated with dopamine grafts (34). Serotonin has been shown to stimulate GnRH neuron excitability to exert biphasic actions through 5-HT1 and 5-HT2 receptors in the mouse indicated that 5-HT1 and 5-HT2 receptors were indicated as potential targets in the treatment of neuron impairment (31). Serotonergic neuron dysfunction in the hippocampal area is regulated by extracellular serotonin receptors, leading to hyperexcitability and discharge in the neurons (35). These findings suggest that the inhibition activity of the serotonin serotonergic neurons signaling pathway may benefit patients with cranial nerve involvement in clinical treatment.

A second antipsychotic agent, lurasidone, has been approved for the treatment of patients with schizophrenia and was identified as an adjunctive therapy with lithium or valproate for the treatment of bipolar I depression (15). In the present study, lurasidone was selected to investigate therapeutic efficacy in mice with cranial nerve involvement. Lurasidone is similar to the majority of antipsychotic drugs, in that it possesses an antagonist at serotonin 5-HT7, serotonin 5-HT2A, serotonin 5-HT1A and serotonin 5-HT6, respectively (12). However, reports on the clinical use of antipsychotic treatment for cranial nerve involvement are limited and primary mechanism of lurasidone as a long-term medicine remains to be fully elucidated.

In the present study, the efficacy of lurasidone in a rat model of cranial nerve involvement was evaluated. The antagonist of lurasidone for serotonin receptors was confirmed and demonstrated promising pharmacological function for the treatment of patients with cranial nerve involvement. The data demonstrated that 0.32 mg of lurasidone once a day was beneficial in the treatment of cranial nerve involvement. The body weight of the rats was minimally affected, and hypertension and heart rate parameters were low risk in terms of clinically meaningful alterations. Of note, cognitive competence was significantly improved following treatment with lurasidone, and lurasidone downregulated histamine receptors via the serotonin serotonergic neuron signaling pathway and increased the expression of neuroprotective proteins by impaired neurons. In conclusion, lurasidone showed efficacy in a rat model of cranial nerve involvement, and indicated that lurasidone may offer potential for application in the treatment of cranial nerve involvement.

References