Formulated citrus peel extract gold lotion improves cognitive and functional recovery from traumatic brain injury (TBI) in rats

Guang Chng, Zhiwi Yu, Liwn Wang, Yutaka Miyauhi, Mihiko Suzawa,Shiming Li, Chi-Tang Ho, Hui Zhao,*, Naiyao Chn

a Department of Hematology and Tangshan Key Laboratory, Translational Medical Center, North China University of Science and Technology, Tangshan,Hebei063000,China

b Tianjin Key Laboratory of Food and Biotechnology, School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China

c Miyauchi Citrus ResearchCenter,Takasaki,Gunma,Japan

d Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources, Huanggang Normal University, Huanggang, Hubei 438000, China

e Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, United States

ABSTRACT Traumatic brain injury (TBI) is closely related to neuro-inflammatory response and causes a complex pathological process and serious consequence.In this study, we explored whether the formatted citrus peel extract gold lotion (GL) could have a therapeutic effect on rats suffered TBI.TBI rat model was prepared by the electric Cortical Contusion Impactor (eCCI) device.Prevention against TBI by GL was assessed by the behavioral, organizational of rats and the molecular markers in the brain injury area.The results showed that GL could reduce nerve damage and neurological dysfunction.The mechanisms probably due to that GL could not only reduce the associated damage markers including GFAP,OX-42, TNFα, COX-2,NF-κB and TLR4 but also promote the expression of VEGF which is responsible for protecting neuronal cells in the injury area.These findings revealed that GL could be a promising preparation for TBI prevention.

Keywords:Citrus peel Cognitive Traumatic brain injury Inflammation Gold lotion

1.Introduction

Traumatic brain injury (TBI) could trigger a series of complex inflammatory responses and cause neuronal cell death and neurological dysfunction.Of note, approximately 43% of moderate to severe patients suffer from cognitive dysfunction which brings enormous economic and mental burdens to families and societies[1].TBI-induced neurological dysfunction is caused by the primary and secondary brain injury [2].The primary injury occurs at the beginning of TBI and results from displacement of the physical structures of the brain [3,4].The secondary injury which is the leading cause of death and disability in patients, usually occurs after the primary injury and may involve serious of cascade events such as inflammatory cell necrosis and apoptosis, increased oxidative stress, cellular calcium imbalance and mitochondrial [5].Symptomatic treatment is usually used to treat TBI in clinical; however,the drugs for nerve cells protection and nerve cells death inhibition did not bring satisfactory treatment results [6-8].Therefore,further explorations of the pathogenesis of TBI and development of the effective drugs for improving the clinical treatment of TBI are important.

The innate immune response plays a key role in the pathogenesis of brain injury [9].For example, the activation of nuclear factor-κB (NF-κB) regulates a variety of inflammatory factors related to TBI pathogenesis.The inflammatory factors include cytokines, chemokines, cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and so on [10].Studies revealed that targeting the innate immune signaling, such as TLR4, can alleviate the inflammatory response and improve the neurological function in TBI rats [11-14].

Citrus peel has been well documented to provide health benefits broadly including antibacterial, antioxidant, and anti-inflammation[15,16].The active ingredient polymethoxyflavones (PMFs) were exclusively found in citrus peel.Researchers found that PMFs have anti-cancer, anti-inflammatory, anti-tumor and other biological activities [17,18].The formula product gold lotion (GL) was extracted from six kinds of citrus peel and originally used as antiultraviolet cosmetics.Afterwards, studies have confirmed that oral administration of up to 200 μL GL lasting for 5 weeks has antiinflammatory effects by down-regulating iNOS, COX-2, MMP-9, and inhibiting the activities of macrophages and dendritic cells without toxicity effect in mice [19,20].However, no studies have suggested that GL has the therapeutic potential for TBI.Therefore, we sought to investigate the efficacy of GL administrated form the first day after TBI.Also, we examined the anti-inflammatory and immuneregulatory effects of GL treatment in the presence of inflammatory response induced by TBI to determine the mechanism underlying GL in the treatment of TBI.

2.Materials and methods

2.1.Materials

Gold lotion (GL) was provided from Imperial Household Citrus Research Center.The antibodies of TLR4 and NF-κB were purchased from Santa Cruz Biotechnology, Inc.The antibodies of TNF-α and VEGF were purchased from Affinity Biosciences, Inc.The antibodies of COX-2 and Lamin A/C were purchased from Wanlei Biological Technology Co., Ltd (Shenyang, China).The antibodies of GFAP and OX-42 were purchased from BD Company.The antibody of NeuN was purchased from Cell Signaling Technology, Inc.The antibody of GAPDH was purchased from Beyotime Biotechnology.Co., Ltd.All other chemicals are chemically pure reagents.

2.2.Rats modeling and feeding

Adult male Sprague Dawley (SD) rats weighing 250-300 g (Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing,China) were used for all experiments.Rats were housed in a 12 h light, 12 h darkness cycle at a constant temperature (25±2°C),humidity (40%-50%) with food and water available and ad libitum for 7-10 days before experiments.The experiments were approved by the Animal Research Committee of Tianjin University of Commerce.

SD male rats screened out by Morris water maze platform test were randomly divided into three groups: Sham group, control group and GL group.We set day 1, 3,7, 14 and 21, as 5 observed timepoints after TBI and at each time point every eight rats from each group were disposed for evaluating.Sham rats were performed craniotomy only without hit, the other two groups of rats were prepared with controlled cortical impact (CCI) [21].

TBI was then induced using an electromagnetic CCI device(Custom Design, Houston, Tex) with a circular flat-faced, 6-mm

diameter tip, at a rate of 5 m/s to a depth of 2.51 mm for 150 ms.After injury, bleeding was stopped with sterile gauze and the incision sutured.Sham procedures included everything above with the exception of impact.10% chloral hydrate (3 μL/g) was administered for pain management before surgery.

After TBI, the rats were gavaged with intervention agent once a day, Sham group and control group rats were administrated with 1.0 mL vehicle (saline containing 15% ethanol), GL group rats were administrated with 1.0 mL GL and this dosage is no harm to rats identified by our pre-experimental test (data not shown).

2.3.Modified neurological severity score test (mNSS test)

Behavioral testing was conducted on day 1, 3, 7,14 and 21 after TBI using modified neurological severity score test (mNSS test).The mNSS test includes motor, sensory, reflex and balance tests.The mNSS test is graded on a scale of 0-18, where a total score of 18 points indicates severe neurological deficit and a score of 0 indicates normal performance; The level at 13-18 points indicates severe injury, 7-12 indicates mean-moderate injury, and 1-6 indicates mild injury.

2.4.Morris water maze (MWM)

For accessing the alterations of spatial learning and memory in TBI rats, Morris Water Maze test (MWM) was performed as previously reported with minor modifications [22].Briefly, the rats received a 4-days’ visual platform training before modeling TBI and we chose the rats with escape latent period range from 20 s to 40 s to enter the following tests.The orientation navigation tests were performed from day 15 to day 18 after TBI, the escape latency and swimming distance recorded on day 18 after TBI were statistically analyzed for detecting the spatial learning ability.Spatial exploration tests were carried out on day 19 for detecting spatial memory ability.

2.5.Brain tissue preparation

Rats were anesthetized with 10% Chloral hydrate (3 μL/g) and transcardially perfused with 100 mL cold PBS.After the skin and viscera turned pale, rats were transcardially perfused with 4%paraformaldehyde in 0.1 mol/L PBS.After the body completely stiff,the brains were removed into 4% paraformaldehyde in 0.1 mol/L PBS for fixing 4 h.The brains were then dehydrated by gradient sucrose solutions, optimal cutting temperature (OCT)-embedded.

2.6.Immunohistochemical (IHC)

IHC method was used to detect the expression of GFAP, OX-42, TNF-α and VEGF.Following slicing and fixing, sections were immersed in 3% hydrogen peroxide (5-10 min).Incubation with the primary antibodies was performed for 20 h at 4°C.A biotinylated secondary antibody was then added and visualization was achieved by using the 3,3′-diaminobenzidine (DAB) peroxidase substrate kit (Beijing Zhong Shan JinQiao Biotechnology Co., Ltd).The tissue sections counterstaining with haematoxylin were examined using light microscopy on the microscope (Olympus).The semiquantitative analysis of IHC staining of GFAP, OX-42, VEGF were analyzed by Image Pro Plus image analysis software.The results were shown as the Sum values of integral optical density (IOD).

2.7.Western blot assay

For analyzing the expression of NeuN, COX-2, NF-κB (P65), and TLR4, the injured brain tissues were collected, lysed, and centrifugated at 10000 × g, 4°C for 10 min, and the supernatant was stored at -80°C.The protein concentration was analyzed by BCA assay.Fifty micrograms of protein was loaded on 12% SDS-PAGE, and then transferred to a nitrocellulose membrane.The membrane was incubated with monoclonal antibody overnight.Being washed with PBS buffer containing 0.05% Tween-20, the membrane was probed with secondary antibody for 2 h.The immunostaining was detected by using Enhanced Chemiluminescence kit.The densities of blots were analyzed by using Imagequant software.

2.8.Statistical analysis

All values were expressed as mean±SD derived from three independent experiments.The statistical analysis was performed using the SPSS 17.0 software (Chicago, IL, USA).Value of P ＜ 0.05 was considered statistically significant.

3.Results

3.1.GL protected the cognitive functions in rats after TBI treatment

As shown in Fig.1, rats received GL treatment displayed significantly lower mNSS scores compared with the control group.Consistent with the mNSS, the escape latency and total swimming path length were significantly shorter in GL treated rats on day 18 after TBI than that in the control group (Fig.2).In addition, as shown in Fig.3, rats treated by GL exhibited the higher percentage of time staying in the target platform quadrant and the more times of the movement through the original platform area.

3.2.GL reduced the tissue damages in TBI rats

To check the pathological evidence on the cognitive protection of GL against TBI, we analyzed the brain tissue samples from the rats.As expected, the HE stained results showed that the cortical areas were widely damaged in TBI rats.The damaged areas not only included the areas which functions were associated with movement, feeling, reflection, balance, but also included the hippocampus areas which were closely related to neurogenesis and spatial learning and memory (Supplementary Fig.1).To examine the treatment effects of GL in TBI rats, the neurological damage was observed after 7 days in GL rats and TBI rats (Fig.4 and Supplementary Fig.1).The rats in control group showed more serious brain injury including the obvious lesion and congestion in the injured areas than the rats in the GL group, indicating GL probably play some role in preventing against TBI.

3.3.GL reduced the expression of GFAP and OX-42 in TBI rats and inhibited the activation of glial cells

To explore the mechanism of the protective role of GL on TBI, we examined the level of GFAP, a brain astrocyte biomarker which is usually activated after TBI [23].Compared with the GFAP-positive cells found in the cortex margin of the Sham group rats, the GFAPpositive cells were significantly increased in control rats, and the distribution of the positive cells expanded to the whole hippocampus region (Fig.5A).The IOD values of GFAP significantly increased on day 1, 3, 7 and 14 (P ＜ 0.05, control rats vs.Sham rats), and the value reached a peak on day 3 and maintained a positive expression on day 14 (Fig.5B) in control and GL rats.Whereas the values of GL group at all the monitoring points were significantly lower than that of control group (P ＜ 0.05, GL rats vs.control rats) (Fig.5B).

Fig.1.The mNSS scores were detected on day 1,3,7 and 14 of GL rats and control rats.Day 1: 6.80±1.30 vs.7.00±1.20, P ＞ 0.05; day 3: 4.40±0.89 vs.6.25±1.17, P＜ 0.01; day 7: 3.00±0.71 vs.4.38±0.92, P ＜ 0.01; day 14: 0.83±0.41 vs.1.88±0.83,P ＜ 0.01, GL vs.control.# means that there was significant difference between the two groups.

Fig.2.The results of the swimming track (A), the escape latency of place navigation(B) and the total path length of place navigation (C) of rats on day 18 after TBI.Exactly,the escape latency was 23.98±7.59 vs.32.55±7.48, P ＜ 0.05; the total swimming path length was 440.02±124.17 vs.649.48±139.91, P ＜ 0.01, GL vs.control.** means control vs.Sham, P ＜ 0.01, *** means GL control vs.Sham, P ＜ 0.005, # means GL vs.control, P ＜ 0.05.

We also tested the expression of OX-42, another activated marker of microglia.The TBI rats showed an increased level of OX-42 [24].In our study, the OX-42-positive cells of control rats were significantly increased with cell morphological changes (e.g.larger microglia cell volume, clearer cell boundaries, thicker protrusion with small visible spines of cells) (Fig.5A).The IOD values of OX-42 in control rats significantly increased from day 1 after TBI (P ＜ 0.05, control rats vs.Sham rats), on day 14 returned to the normal level (Fig.5C).In comparison with the control group, the rate and expression level of OX-42-positive cells were significantly decreased (Fig.5C).These results above showed that GL could attenuate the activation of astrocyte and microglial cells by inhibiting the expression of OX-42.

3.4.GL alleviated inflammation responses and the damage of neuronal cells

Fig.3.The results of the swimming track (A), the number of exact site crossings (B)and the time percent in the target quadrant of spatial exploration (%) (C) of rats on day 19 after TBI.(The number of exact site crossings was 5.20±1.10 vs.2.88±1.46,P ＜ 0.01; the time percent in the target quadrant of spatial exploration (%) was 34.89±4.28 vs.23.24±7.02, P ＜ 0.01, GL vs.control.* means control vs.Sham, P ＜0.05, ** means GL control vs.Sham, P ＜ 0.01, # means GL vs.control, P ＜ 0.05)

The results above suggest that GL may improve functional recovery and reduce neuronal damage following TBI.To illustrate whether GL treatment directly influenced inflammatory events, we further assessed the expression of inflammatory mediators.TNF-α is one of the main inflammatory factors at the early stage of TBI.It can reach the maximum concentration in a short time to affect the recovery of neurological function [25].The amount of TNF-α positive cells was significantly increased in the control group compared with Sham rats.Moreover, the positive cells were widely distributed around the injured area of cortex and hippocampus.Treatment with GL reduced the elevated TNF-α level in the control group (Fig.6A).In GL rats, the IOD values of TNF-α were all detected decreased on day 1, 3, 7.(P ＜ 0.05, GL rats vs.control rats) (Fig.6B).

The inflammatory factor COX-2, Toll-like receptor TLR4 and the transcription factor protein NF-κB (P65) also play important roles in inflammatory of TBI [26].The Western-blot results showed that the expression level of COX-2, TLR4 and NF-κB (P65) were all decreased in GL rats compared with control rats the first day post-TBI (Fig.7A-7D).These results showed that GL could effectively alleviate inflammatory response induced by TBI by reducing the expression of inflammatory factors.

NeuN is a marker protein of neuronal cells with the molecular weight of 46-55 kDa.The expression level of NeuN can be used to indicate the number of neuronal cells.The Western-blot result of NeuN showed that the expression level of NeuN in GL treatment rats was significantly higher than that in control rats (P ＜ 0.05) (Fig.7A,7E).This result also indicated that GL could moderate the damage of neuronal cells after TBI.

3.5.GL increased the expression of VEGF

Vascular endothelial growth factor (VEGF) is one of the most important factors that indicate the occurrence of microvessel [27].Our previous study confirmed the effect of VEGF on the occurrence of microvessel and the recovery of neurological function.IHC staining results showed basal levels of VEGF was low in Sham rats’ brain.However, the VEGF-positive cells were widely distributed in the cortex around the injured area and hippocampus area in control rats and GL rats (Fig.8A).The expression level of VEGF was also increased after TBI (P ＜ 0.05, control rats vs.Sham rats) and reached the peak on day 3 and returned to normal on day 14 in control rats (Fig.8B).GL treatment further increased VEGF expression compared with control rats at all the detection time (day 1,3, 7, 14) (P＜ 0.05).These results revealed that GL could promote the secretion of VEGF to help the brain protection from TBI.

4.Discussion

The studies of the pathological mechanism of TBI have made great progress in recent years [28].TBI is usually considered as a complex disease process causing structural damage and functional deficit.The TBI rats model in this study was established by controlled cortical impact (CCI) method.This model has the advantage of low mortality of rats with high reproducibility results [29].In our study, we found that treatment with GL significantly improved neurological function on day 1-3 after TBI by mNSS score (Fig.1).In MWM experiments, two indicators were employed in each behavior tests including positioning navigation and spatial exploration to increase the accuracy and credibility of the data [30].According to the results of MWM, GL administration could improve the spatial memory deficits on day 5-8 following TBI, supports that administration of GL in the early post-TBI period improved cognitive outcome.In addition, the brain tissue pathological analysis results revealed that GL might reduce the bleeding of the damaged brain tissue and promote the absorption of congestion (Fig.2).It is then indicated that a suitable dose of GL intervention can improve neurological dysfunction after TBI.

Fig.4.The representative brain samples from the control rat (left) and the GL treatment rat (right) on day 7 after TBI.(A) The horizontal view of rats’ brains.(B) The coronal view of rats brains.

Fig.5.The analysis of GFAP and OX-42 expression in each group.(A) IHC staining of GFAP (day 3) and OX-42 (day 1) at the region of hippocampus after TBI.(B) The semiquantitative expression analysis of GFAP in each group.(C) The semi-quantitative expression analysis of OX-42 in each groups.(*,**, or *** mean control vs.Sham, P ＜ 0.05, P＜ 0.01,or P ＜ 0.005 ; # or ## mean GL vs.control, P ＜ 0.05 or P ＜ 0.01)

Fig.6.The analysis of TNF-α expression after TBI.(A) IHC staining of TNF-α at the region of the hippocampus at the first day after TBI.(B) The semi-quantitative expression analysis of TNF-α in each group.*** means control vs.Sham, P ＜ 0.005; ### means GL vs.control, P ＜ 0.005.

TBI caused extensive damage in the cortical and hippocampus area, and astrocytes in those areas are widely activated as a result.The expression of marker protein GFAP was significantly increased after astrocytes activated in TBI rats (Fig.3A, 3B).These results were consistent with the results from the previous literature.The activated astrocytes have two sides effects on the injured neurons.On the one hand, the activated astrocytes could form the glial scars which will hamper the regeneration of neuronal axons and myelin.On the other hand, the activated astrocytes play an essential role in the development, regeneration and differentiation of neurons by secreting more than 20 cell factors including nerve growth factor (NGF), neurotrophic factors,cytokines and cell recognition factors etc.[32].We have observed that GL could reduce the amount of the activated astrocytes by decreasing the expression of GFAP (Fig.3B).Thus, we considered GL might play the protective role by reducing the formation of glial scar and promoting the regeneration of neuronal axons and myelin.

Fig.7.The Western blot analysis of COX-2, TLR-4, NF-κB (P65) and NeuN first day after TBI (A) and the relative expression analysis of COX-2 (B), TLR-4 (C), NF-κB (P65) (D)and NeuN(E).(*,**,or *** mean control vs.Sham, P ＜ 0.05, P ＜ 0.01, or P ＜ 0.005; # or ### mean GL vs.control, P ＜ 0.05 or P ＜ 0.005)

Fig.8.The analysis of VEGF expression in groups.(A) IHC staining of VEGF (400×) at the region of cortex and hippocampus on day 3 after TBI.(B) The semi-quantitative expression analysis of VEGF in the cortex.(** or *** mean control vs.Sham, P ＜ 0.01 or P ＜ 0.005; #, ## or ### mean GL vs.control, P ＜ 0.05, P ＜ 0.01 or P ＜ 0.005).

Previous research on the therapy of TBI usually focused on blocking the secondary injury.GL could be considered as a potential therapy because of its rich bioactive compounds.The total flavonoid content in GL is more than 0.45 mg/mL, as the main active ingredient, the content of the PMFs is up to 0.1 mg/mL.Previous studies have found that GL has broad anti-cancer effects, such as antimelanoma, anti-prostate cancer, anti-lung cancer and anti-liver cancer effects [19].Besides that, it has been fully confirmed that GL has the anti-inflammatory and immune-modulatory effects.Lai et al.[19] found that GL could prevent the occurrence of the inflammation-related colorectal neoplasms by down-regulating the expression of iNOS, COX-2, ornithine decarboxylase (ODC),VEGF and MMP-9 in colon.Li et al.[20] found that GL can reduce the contact allergic reaction in mice by inhibiting the action of dendritic cells.At present, there is no report on the role of GL in nerve inflammation.

Inflammatory cells from the central nervous system and peripheral blood-derived inflammatory cells directly respond to TBI and play the neuroprotective effect or participate in adverse secondary brain injury [29].Present studies have taken neuro-inflammation as the main cause of secondary brain injury [33,34].Once inflammation was initiated, many inflammatory cytokines are highly expressed and are closely related to the degree of brain damage [31].As one of the inflammatory receptors, the expression level of TLR4 was up-regulated after TBI [12,13].Studies have shown that down-regulating the TLR4 expression in mice could decrease inflammation reactions and improve neurological functions [35,36].TLR4 was able to regulate the activation of microglia and the release of inflammatory mediators by activating NF-κB [37].The activated NF-κB regulates a variety of pro-inflammatory genes,such as chemokines, COX-2 and MMP-9 which are all closely related to the pathogenesis of TBI [10].Accompanied by changes in the morphology of microglia cells which could reflect the initiation of inflammation, our results demonstrated the expression of OX-42,COX-2, NF-κB, TLR4 and TNF-α were significantly increased after TBI.Further GL intervention successfully decreased the expression of the above factors, revealing that GL probably firstly inhibited the expression of TLR4 in TBI rats, and then down-regulated the expression of NF-κB, TNF-α and COX-2.

Cerebral ischemia is an important pathophysiological basis in the secondary injury and affects the recovery after TBI [38].Several studies have found that when the oxygen concentration drops in brain tissue, hypoxia-inducible factor-1α (HIF-1α), a transcription factor involved in the regulating of oxygen balance in vivo[39], could promote the generation of microvessel by up-regulating the expression of VEGF [40].In this study, we found that GL could effectively enhance the secretion of VEGF, which has been investigated for its neuroprotective ability as a protective response to TBI.

Collectively, in the present study, we investigate the therapeutic effect and mechanism of GL for TBI treatment.The results showed that GL could significantly improve neurological damage and cognitive dysfunction in TBI rats.GL could inhibit the activation of glial cells, reduce the inflammatory response and promote the generation of microvascular.All the results reveal that GL could be used as a promising functional food for the treatment of TBI.

Appendix A.Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fshw.2020.04.012.

Declaration of Competing Interest

There is no Conflict of Interest.