- Email: [email protected]
Neuroprotective effect of formononetin against TBI in rats via suppressing inflammatory reaction in cortical neurons
Biomedicine & Pharmacotherapy 106 (2018) 349–354
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Neuroprotective effect of formononetin against TBI in rats via suppressing inflammatory reaction in cortical neurons
T
Zhengzhao Lia, Guang Zenga, Xiaowen Zhenga, Wenbo Wangb, Yun Linga, Huamin Tanga, ⁎ Jianfeng Zhanga, a b
Department of Emergency, The Second Affiliated Hospital of Guangxi Medical University, Nanning, 530007, China Department of Neurosurgery, Affiliated Hospital of Guilin Medical University, Guilin, 541001, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Formononetin Traumatic brain injury Interleukin-10 Inflammation
Traumatic brain injury (TBI) refers to external force-induced brain damage, characterized with necrosis and cell loss in cerebral cortex. Interestingly, a plant-extract named formononetin (FN) is found to possess promising pharmacological activities, including cellular neuroprotection. Thus, we propose that FN may exert biological protection against TBI and discuss the underlying mechanism. In the current study, a rat TBI model was established via Feeney's classical method, followed by different concentrations of FN treatment. Nissl-special and DAPI-labeled stains were utilized to assess the proliferation of cortical neurons nearing lesioned tissue. The contents of interleukin-6 (IL6), tumor necrosis factor (TNF-α), and interleukin-10 (IL10) in serum and the cortical neurons were determined by ELISA. Further, intracephalic IL10 expression levels were detected through immunoassay and RT-PCR. Interestingly, the results exhibited within the FN-treated TBI rat model indicated elevated cortical proliferation. The levels of IL10 in serum and the cortical neurons were increased following FN treatments, while TNF-α and IL6 levels in the blood were decreased. In addition, both mRNA and protein expression levels of IL10 in the FN-treated TBI rat model were up-regulated in a dose-dependent manner. Collectively, our present findings indicate that FN provides effective neuroprotection against TBI, likely by activating IL10 expression in cortical neurons nearing lesioned tissue to inhibit neuroinflammatory reaction.
1. Introduction TBI, also referred to as intracranial injury, is a head injury that can lead to substantial adverse health outcomes [1]. Physical force is responsible for TBI occurrences and can include vehicle accidents and violent attacks [2]. TBI can lead to permanent brain dysfunction, characterized with tissue structural damage and cell death in the brain [3]. Notably, secondary injury of TBI is more serious, resulting in inflammation, oxidative stress, excitotoxicity, and mitochondrial dysfunction in the brain [4,5]. Thus, all these cascaded events that imply neural regeneration may serve as a potential strategy for combating TBI. Biologically, interleukin-10 (IL10) refers to a potential anti-inflammatory cytokine that is critical for maintaining neurogenic proliferation, growth, and survival [6,7]. In biological function, IL10 can block NF-κB activity and is involved in the regulation of the JAK-STAT signaling pathway for activating neuronal proliferation and survival [8]. Survival and programmed cell death (PCD) mechanisms are mediated through adaptor proteins binding to the death domain of the
⁎
p75NTR cytoplasmic tail. Survival occurs when recruited cytoplasmic adaptor proteins facilitate signal transduction through tumor necrosis factor receptor members such as TRAF6, which results in the release of the nuclear factor κB (NF-κB) transcription activator [9]. Thus, the induction of endogenous IL10 expression may offer potential for cell protection in the brain. In phytomedicine, the plant-isolated compound formononetin (FN) is evidenced with effective pharmacological effects, including antitumor, anti-inflammation, and anti-dyslipidemia properties [10,11]. More interestingly, our previous findings showed that FN reduced inflammatory stress in a TBI rodent model, contributing to neuronal growth [12]. However, the molecular mechanism involved in the neurogenesis needs to be further investigated. Taken together, our present study aimed to elucidate the beneficial bioeffect regarding FN-induced neuroprotection against TBI in rats and to discuss the biological mechanism involved.
Corresponding author at: Department of Emergency, The Second Affiliated Hospital of Guangxi Medical University, No. 166 Daxuedonglu Road, Nanning, 530007, China. E-mail address: [email protected] (J. Zhang).
https://doi.org/10.1016/j.biopha.2018.06.041 Received 15 April 2018; Received in revised form 6 June 2018; Accepted 12 June 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 106 (2018) 349–354
Z. Li et al.
2. Materials and methods
paraformaldehyde for 24 h, followed by paraffin embedding and 5-μm brain sectioning. After being dewaxed and rehydrated, the sections were incubated in 5% BSA working solution (Beyotime, China) at 37 °C for 1 h. After being rinsed with PBS three times, the slides were incubated in the DAPI reaction mixture for 5 min at 37 °C in a humidified chamber. Subsequently, the sections were imaged under a fluorescence microscope before data analyzing. In immunofluorescence method, the cortical sections were incubated with secondary antibody of goat antirabbit IgG H&L (1:100; Beyotime, China) for 1 h at 37 °C prior to images were captured and assayed. As results, Nissl/DAPI cell counts and IL10positive and -labeled neuronal cells were determined by mean data from five different optical views of imaged cortex, followed by statistical analysis. In ultrastructural protocol [17], the rat cortex samples (n = 5 each group) were fixed immediately with 4% PFA and 2.5% glutaraldehyde in PBS solution overnight at 4 °C. After rinsing the specimens with cold PBS, the tissue was re-fixed for 1 h with 1% osmium tetroxide and was dehydrated through different ethanol solutions. The brain samples were stained using the saturated solution of uranyl acetate in 60% ethanol and embedded in araldite. All the cortex specimens were imaged and captured under an electron microscope operating at 80 kV (Hitachi, Japan).
2.1. Materials and reagents Formononetin (FN) with purity greater than 98.0% was purchased from Sigma-Aldrich (USA). FN was dissolved in dimethyl sulfoxide (DMSO; Sigma, USA) to prepare a 200-mM stock for subsequent treatment. Other reagents used in this study were labeled as follows. 2.2. Experimental protocols Male Wistar rats, aged 9–10 weeks old, were purchased from the Experimental Animal Center of Guangxi Medical University (License No. SCXK-2009-0001). The rats were housed in a temperature-controlled (22–28 °C) environmental condition and under a 12-h light/dark cycle. All animals were allowed access to food and water freely and were acclimatized for at least 4 days prior to further experimentation. This study protocol was approved by the Animal Research Ethics Committee of Guangxi Medical University (approval No. GXMU201603001). The TBI rat model used in this study was referenced on Feeney's weight-drop method, as previously reported [12], with some modifications. Briefly, the rats were anesthetized with 2% sodium pentobarbital solution (3 ml/kg) via intraperitoneal injection before being fixed on the stereotaxic frame. The rat skull was opened by dental drill while keeping the dura mater undamaged, and then a 40-g impactor was used to bump the dura. After damaging, the scalp was sutured aseptically. The TBI rat model was identified, as previously reported [12], accompanied with visible limb convulsion, transitory apnea, and unconsciousness. Subsequently, the laboratorial rats were randomized into following groups: sham group (n = 10; surgically opened skull without the impactor hitting); TBI group (n = 10); and FN-treated TBI groups (10 and 30 mg/kg/d for 7 days via intragastrical administration after TBI establishment; n = 10 per group). As a note, any deaths occurring during the experiment were excluded from the statistical analysis.
2.7. Quantitative real RT-PCR Fresh rat cortex tissues (n = 5 each group) were homogenized to extract total RNA with TRIzol reagent (Beyotime, China). Purified RNAs were reverse-transcribed to cDNA with a commercially available cDNA synthesis kit (TIANGEN, Beijing, China) following the manufacturer's instructions. The sequences of the primers were as follows: IL10 sense primers: 5′ ACT GCT ATG TTG CCT GCT CTT 3′, anti-sense primers: 5′ ATG TGG GTC TGG CTG ACT GG 3′; β-actin sense primers: 5′ CCC ATC TAT GAG GGT TAC GC 3′, and antisense primers: 5′ TTT AAT GTC ACG CAC GAT TT 3′. Cortex IL10 mRNA expression was measured by quantitative RT-PCR by using the ABI PRISM 7500 Sequence Detector System (Applied Biosystems, Carlsbad, CA). The threshold cycle (Ct) readings were collected, and the relative expression of IL10 mRNA was calculated with the 2−Δ(ΔCT) method. The IL10 mRNA transcript level was normalized to the β-actin level in all samples [18,19].
2.3. Neurological behavior investigation Neurological signs of all rats were assessed using the neurological severity score in accordance with previous method [13].
2.8. Western blotting assay
2.4. Tissue sampling preparation
In brief, fresh rat cortex tissues (n = 3 each group) were homogenized with the radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, China). The lysates were incubated on ice for 30 min and centrifuged at 12,000 g for 15 min at 4 °C. Supernatant protein concentration was determined by using a bicinchoninic acid reagents (Beyotime, China). Brain samples with 40 μg protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After being blocked with 5% non-fat milk buffer for 1 h, the membrane was incubated overnight at 4 °C, followed by primary antibody: rabbit anti-rat IL10 antibody (Santa Cruz, CA, USA; 1:1000) and mouse anti-rat β-actin antibody (Santa Cruz, CA, USA; 1:2000). After being washed with PBST, the membrane was incubated with secondary antibodies coupled to horseradish peroxidase (1:5000) at 37 °C for 1 h. Immunoreactive bands were imaged and visualized using an enhanced chemiluminescence detection kit (Beyotime, China). The optical density of each band was quantified with the Image Analysis System (Bio-Rad, USA) and normalized to βactin protein level for further data analysis [20,21].
At the end of the study, all rats were anesthetized with ethyl carbamate solution (200 mg/kg) via intraperitoneal injection. Intracephalic perfusion was performed with 500 ml cold saline to remove the remaining blood [14]. Then, rat brain samples from ipsilateral cortex tissue nearing lesioned unit were isolated and stored immediately until further biochemical assays. 2.5. ELISA for cytokines determination The contents of interleukin-6 (IL6), tumor necrosis factor (TNF-α), and interleukin-10 (IL10) in serum (n = 8 each group) and the brain (n = 8 each group) were determined using commercially available ELISA kits (Nanjing Jiancheng Bioengineering Institute, China) according to booklet procedures [15]. 2.6. Cytohistologic stains and transmission electron microscopy (TEM) observation Cell growth count in the cortex samples (n = 5 each group) was determined by Nissl-special stain (Beyotime, China) and DAPI-labeled stain (Abcam, UK). IL10-positive neuronal cells were assessed, as previously described [16]. The rat cortex was removed and fixed with 4%
2.9. Statistical analysis Data were analyzed using Statistical Product and Service Solutions (SPSS) 19.0 software (Chicago, IL, USA). All results are expressed as the 350
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3. Results 3.1. FN improved neurological symptoms of TBI rats As results, rats in TBI group showed two deaths during development of TBI. Instead, no death was found in FN-treated groups. FN-treated TBI rats were subjected to the assessment of neurological behaviors. TBI rats showed visible neurological disorders, such as dystasia, trance and unconsciousness, and the injury-based scores were greater than that in sham rats (P < 0.05). Interestingly, FN-treated TBI rats suppressed the abnormal changes, contributing to improved mental signs and reduced injury scores (P < 0.05) (Fig. 1). 3.2. FN promoted neuronal proliferation of TBI rats As shown in Nissl-special and DAPI-labeled stains, the data exhibited that a significant reduction of neuronal number in TBI rats were observed when compared to that in the sham control (P < 0.05). In comparison with TBI rats, FN-treated rats resulted in a dose-dependent increase of nerve cell count (P < 0.05), showing elevations of Nisslstained neurons and DAPI-labeled neurocytes (Fig. 2). Further, the morphology of cortex ultrastructure was observed using TEM. In the sham control, the nucleolus and myelin sheath in selected cortical neurons exhibited a complete and organized cytoarchitecture. Instead, the myelin sheaths in TBI cortical neurons were loosened and deformed, and visible vacuolation was observed. In addition, the mitochondria in the axoplasm was swelled and lesioned. Furthermore,
Fig. 1. FN improved neurological symptoms of TBI rats. FN-treated TBI rats inhibited the abnormal alterations, contributing to improved mental signs and reduced injury scores. The results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/kg.
mean ± standard deviation (SD). Any difference between groups was processed with one-way ANOVA following by Tukey's post hoc test. A probability (P)-value < 0.05 was considered statistically significant.
Fig. 2. FN promoted neuronal proliferation of TBI rats. Both Nissl-special stain and DAPI-labeled stain of cortical neurons indicated a dose-dependent increase in FN-treated TBI cortical neurons. Results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/kg. 351
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Fig. 3. FN improved neuronal ultrastructural organization of TBI rats. FN-treated TBI rats exhibited a tight arrangement of the myelin sheath in the cortical neurons and improved organelle morphology as well as repaired and normalized cell nucleus. Note: FN = formononetin; TEM = transmission electron microscope. FN10, 30 = 10, 30 mg formononetin/kg.
Fig. 4. FN increased contents of IL10 in serum and cerebral cortex of TBI rats. FN-treated TBI rats contributed to the decrease of IL-6 and TNF-α levels in a dosedependent manner. The contents of endogenous IL10 were increased in serum and the cerebral cortex of FN-treated TBI rats. The results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/kg.
3.3. FN increased contents of IL10 in the serum and cortex of TBI rats
cortical neurons in TBI rats showed damaged morphology, including cell shrinkage and vacuolation. Following FN treatments, the myelin sheath in the brain was arranged tightly, and the organelle morphology was improved, and the cell nucleus was repaired and normalized (Fig. 3).
In our biochemical test, the data from enzyme linked immunosorbent assay (ELISA) indicated that the IL10 levels in the serum of TBI cortical neurons were significantly decreased, and cytokine 352
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dose-dependent manner (P < 0.05). In addition, neuronal expression of IL10 was reduced in TBI rats (P < 0.05). Instead, FN treatment elevated the expression of IL10 in TBI cortex neurons dose-dependently (P < 0.05) (Fig. 4).
3.4. FN up-regulated the IL10 mRNA level of TBI rats To assess the FN-mediated neuroprotection that is responsible for neurogenesis in TBI cortical neurons, neuronal IL10 expression at the gene level was assessed. The brain IL10 mRNA level in TBI rats was notably down-regulated when compared to that in the sham cortical neurons (P < 0.05). In comparison with the TBI control, FN-treated cortical neurons showed increased expression of IL10 mRNA in a dosedependent manner (P < 0.05) (Fig. 5).
3.5. FN elevated the IL10 protein level of TBI rats Fig. 5. FN up-regulated the IL10 mRNA level of TBI rats. The RNA from ipsilateral cortex tissue nearing lesioned unit of TBI rats was analyzed by RTPCR. The IL10 mRNA expression was normalized to β-actin. The results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/kg.
In immunohistochemistry analysis, the IL10-positive cells of TBI cortex were decreased when compared to those in the sham control (P < 0.05). However, the dose-dependent elevation of IL10-labeled cell count was observed in FN-treated rats (P < 0.05). Further, immunoblotting data showed that neuronal IL10 content was reduced in TBI rats when compared to that in the sham control (P < 0.05). Interestingly, the endogenous contents of IL10 were up-regulated in FNtreated TBI rats in a dose-dependent manner (P < 0.05) (Fig. 6).
contents (TNF-α and IL6) were increased compared to sham rats (P < 0.05). Interestingly, FN-treated TBI rats contributed to the increase of IL10 level and decrease of cytokine contents (IL-6, TNF-α) in a
Fig. 6. FN elevated the IL10 protein level of TBI rats. The protein extracted from ipsilateral cortex tissue nearing lesioned unit of TBI rats was analyzed by immunofluorescence and immunohistochemistry (ICH) and Western blot. The dose-dependent elevation of IL10-labeled cell counts was observed in FN-treated rats. Results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI group. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/ kg. 353
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4. Discussion
References
Increasing evidence suggests that neuronal death/loss is implicated in TBI damage pathogenesis [22]. However, our understanding is limited to effectively treat TBI patients in clinical applications. Reassuringly, the neuroprotective effects of hormone-dependent phytoestrogen have drawn great attention in managing TBI. However, the molecular mechanisms underlying these protective effects remain to be fully elucidated. In the present study, both increased Nissl-stained and DAPIlabeled nerve cells have validated the cell growth in FN-treated TBI brains, consistent with improved neurological symptoms. Thus, these initial data indicated that FN treatment ameliorated the brain function of TBI by promoting neuronal proliferation. The results also implied that FN-exerted neuronal regeneration is involved in cerebroprotective benefits against TBI. Together, these findings support that the molecular mechanism related to nerve regeneration warrants investigation in follow-up studies. Interleukin 10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is a potent cytokine primarily involved in the regulation of neuronal growth, maintenance, proliferation, and survival [23]. In biological function, IL10 is critical for the survival and maintenance of sympathetic and sensory neurons, as they undergo apoptosis when absent [24]. However, several recent studies suggest that IL10 is also involved in activating IL-10R and JAK-STAT3 pathways that regulate the life cycle of neurons [25]. Thus, we propose that intracellular IL10 expression promotes neuronal proliferation, contributing to the inhibition of damage caused by TBI. The current results indicated that neuronal loss in TBI rats correlated with reduced Nissl-stained cells and was accompanied by increased neuronal death, as shown in the DAPI assay. Interestingly, these abnormal alterations were reversed following FN treatments, yielding an increase in cell numbers, an improvement of neuronal cytoarchitecture, a reduction of IL-6 and TNF-α in serum, and an elevation of IL10 expressions in the serum and brain. Thus, we concluded that TBI resulted in significant cell necrosis or death in the cortex, thereby inducing neural impairments. In biological mechanisms, FN-mediated activation of IL10 expression promotes cell growth via combining to IL10-special receptors in cortical neurons, followed by suppression of cortical injury in TBI. In addition, we speculate that the increased serum IL10 level by FN treatment might be linked to IL10based biological pathways in peripheral cortical neurons to combat the TBI condition.
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Chen, Calycosin inhibits the in vitro and in vivo growth of breast cancer cells through WDR7-7GPR30 signaling, J. Exp. Clin. Cancer Res. 36 (November (1)) (2017) 153. [20] J. Chen, X. Zhao, X. Li, Y. Wu, Calycosin induces apoptosis by the regulation of ERβ/miR-17 signaling pathway in human colorectal cancer cells, Folia Microbiol. (Dordrecht, Netherlands) 6 (2015) 3091–3097. [21] K. Wu, C. Guo, M. Su, X. Wu, R. Li, Biocharacterization of heat shock protein 90 in acetaminophen-treated livers without conspicuous drug induced liver injury, Cell. Physiol. Biochem. 43 (2017) 1562–1570. [22] J. Larsson, A. Björkdahl, E. Esbjörnsson, K.S. Sunnerhagen, Factors affecting participation after traumatic brain injury, J. Rehabil. Med. 45 (2013) 765–770. [23] A.Y. Fouda, B. Pillai, K.M. Dhandapani, A. Ergul, S.C. Fagan, Role of interleukin-10 in the neuroprotective effect of the angiotensin type 2 receptor agonist, compound 21, after ischemia/reperfusion injury, Eur. J. 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5. Conclusion Collectively, these studies demonstrate that FN may provide neuroprotection against TBI by inducing neuronal IL10 expression. In addition, the current findings disclose that FN may serve as an alternative strategy of combating TBI. Conflict of interest The authors confirm that there are no conflicts of interest. Acknowledgments Our present study was supported by the National Natural Science Foundation of China (81460211, 81560378) and the Guangxi Emergency and Medical Rescue Talent Highland (GXJZ201506).
354
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Neuroprotective effect of formononetin against TBI in rats via suppressing inflammatory reaction in cortical neurons
T
Zhengzhao Lia, Guang Zenga, Xiaowen Zhenga, Wenbo Wangb, Yun Linga, Huamin Tanga, ⁎ Jianfeng Zhanga, a b
Department of Emergency, The Second Affiliated Hospital of Guangxi Medical University, Nanning, 530007, China Department of Neurosurgery, Affiliated Hospital of Guilin Medical University, Guilin, 541001, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Formononetin Traumatic brain injury Interleukin-10 Inflammation
Traumatic brain injury (TBI) refers to external force-induced brain damage, characterized with necrosis and cell loss in cerebral cortex. Interestingly, a plant-extract named formononetin (FN) is found to possess promising pharmacological activities, including cellular neuroprotection. Thus, we propose that FN may exert biological protection against TBI and discuss the underlying mechanism. In the current study, a rat TBI model was established via Feeney's classical method, followed by different concentrations of FN treatment. Nissl-special and DAPI-labeled stains were utilized to assess the proliferation of cortical neurons nearing lesioned tissue. The contents of interleukin-6 (IL6), tumor necrosis factor (TNF-α), and interleukin-10 (IL10) in serum and the cortical neurons were determined by ELISA. Further, intracephalic IL10 expression levels were detected through immunoassay and RT-PCR. Interestingly, the results exhibited within the FN-treated TBI rat model indicated elevated cortical proliferation. The levels of IL10 in serum and the cortical neurons were increased following FN treatments, while TNF-α and IL6 levels in the blood were decreased. In addition, both mRNA and protein expression levels of IL10 in the FN-treated TBI rat model were up-regulated in a dose-dependent manner. Collectively, our present findings indicate that FN provides effective neuroprotection against TBI, likely by activating IL10 expression in cortical neurons nearing lesioned tissue to inhibit neuroinflammatory reaction.
1. Introduction TBI, also referred to as intracranial injury, is a head injury that can lead to substantial adverse health outcomes [1]. Physical force is responsible for TBI occurrences and can include vehicle accidents and violent attacks [2]. TBI can lead to permanent brain dysfunction, characterized with tissue structural damage and cell death in the brain [3]. Notably, secondary injury of TBI is more serious, resulting in inflammation, oxidative stress, excitotoxicity, and mitochondrial dysfunction in the brain [4,5]. Thus, all these cascaded events that imply neural regeneration may serve as a potential strategy for combating TBI. Biologically, interleukin-10 (IL10) refers to a potential anti-inflammatory cytokine that is critical for maintaining neurogenic proliferation, growth, and survival [6,7]. In biological function, IL10 can block NF-κB activity and is involved in the regulation of the JAK-STAT signaling pathway for activating neuronal proliferation and survival [8]. Survival and programmed cell death (PCD) mechanisms are mediated through adaptor proteins binding to the death domain of the
⁎
p75NTR cytoplasmic tail. Survival occurs when recruited cytoplasmic adaptor proteins facilitate signal transduction through tumor necrosis factor receptor members such as TRAF6, which results in the release of the nuclear factor κB (NF-κB) transcription activator [9]. Thus, the induction of endogenous IL10 expression may offer potential for cell protection in the brain. In phytomedicine, the plant-isolated compound formononetin (FN) is evidenced with effective pharmacological effects, including antitumor, anti-inflammation, and anti-dyslipidemia properties [10,11]. More interestingly, our previous findings showed that FN reduced inflammatory stress in a TBI rodent model, contributing to neuronal growth [12]. However, the molecular mechanism involved in the neurogenesis needs to be further investigated. Taken together, our present study aimed to elucidate the beneficial bioeffect regarding FN-induced neuroprotection against TBI in rats and to discuss the biological mechanism involved.
Corresponding author at: Department of Emergency, The Second Affiliated Hospital of Guangxi Medical University, No. 166 Daxuedonglu Road, Nanning, 530007, China. E-mail address: [email protected] (J. Zhang).
https://doi.org/10.1016/j.biopha.2018.06.041 Received 15 April 2018; Received in revised form 6 June 2018; Accepted 12 June 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 106 (2018) 349–354
Z. Li et al.
2. Materials and methods
paraformaldehyde for 24 h, followed by paraffin embedding and 5-μm brain sectioning. After being dewaxed and rehydrated, the sections were incubated in 5% BSA working solution (Beyotime, China) at 37 °C for 1 h. After being rinsed with PBS three times, the slides were incubated in the DAPI reaction mixture for 5 min at 37 °C in a humidified chamber. Subsequently, the sections were imaged under a fluorescence microscope before data analyzing. In immunofluorescence method, the cortical sections were incubated with secondary antibody of goat antirabbit IgG H&L (1:100; Beyotime, China) for 1 h at 37 °C prior to images were captured and assayed. As results, Nissl/DAPI cell counts and IL10positive and -labeled neuronal cells were determined by mean data from five different optical views of imaged cortex, followed by statistical analysis. In ultrastructural protocol [17], the rat cortex samples (n = 5 each group) were fixed immediately with 4% PFA and 2.5% glutaraldehyde in PBS solution overnight at 4 °C. After rinsing the specimens with cold PBS, the tissue was re-fixed for 1 h with 1% osmium tetroxide and was dehydrated through different ethanol solutions. The brain samples were stained using the saturated solution of uranyl acetate in 60% ethanol and embedded in araldite. All the cortex specimens were imaged and captured under an electron microscope operating at 80 kV (Hitachi, Japan).
2.1. Materials and reagents Formononetin (FN) with purity greater than 98.0% was purchased from Sigma-Aldrich (USA). FN was dissolved in dimethyl sulfoxide (DMSO; Sigma, USA) to prepare a 200-mM stock for subsequent treatment. Other reagents used in this study were labeled as follows. 2.2. Experimental protocols Male Wistar rats, aged 9–10 weeks old, were purchased from the Experimental Animal Center of Guangxi Medical University (License No. SCXK-2009-0001). The rats were housed in a temperature-controlled (22–28 °C) environmental condition and under a 12-h light/dark cycle. All animals were allowed access to food and water freely and were acclimatized for at least 4 days prior to further experimentation. This study protocol was approved by the Animal Research Ethics Committee of Guangxi Medical University (approval No. GXMU201603001). The TBI rat model used in this study was referenced on Feeney's weight-drop method, as previously reported [12], with some modifications. Briefly, the rats were anesthetized with 2% sodium pentobarbital solution (3 ml/kg) via intraperitoneal injection before being fixed on the stereotaxic frame. The rat skull was opened by dental drill while keeping the dura mater undamaged, and then a 40-g impactor was used to bump the dura. After damaging, the scalp was sutured aseptically. The TBI rat model was identified, as previously reported [12], accompanied with visible limb convulsion, transitory apnea, and unconsciousness. Subsequently, the laboratorial rats were randomized into following groups: sham group (n = 10; surgically opened skull without the impactor hitting); TBI group (n = 10); and FN-treated TBI groups (10 and 30 mg/kg/d for 7 days via intragastrical administration after TBI establishment; n = 10 per group). As a note, any deaths occurring during the experiment were excluded from the statistical analysis.
2.7. Quantitative real RT-PCR Fresh rat cortex tissues (n = 5 each group) were homogenized to extract total RNA with TRIzol reagent (Beyotime, China). Purified RNAs were reverse-transcribed to cDNA with a commercially available cDNA synthesis kit (TIANGEN, Beijing, China) following the manufacturer's instructions. The sequences of the primers were as follows: IL10 sense primers: 5′ ACT GCT ATG TTG CCT GCT CTT 3′, anti-sense primers: 5′ ATG TGG GTC TGG CTG ACT GG 3′; β-actin sense primers: 5′ CCC ATC TAT GAG GGT TAC GC 3′, and antisense primers: 5′ TTT AAT GTC ACG CAC GAT TT 3′. Cortex IL10 mRNA expression was measured by quantitative RT-PCR by using the ABI PRISM 7500 Sequence Detector System (Applied Biosystems, Carlsbad, CA). The threshold cycle (Ct) readings were collected, and the relative expression of IL10 mRNA was calculated with the 2−Δ(ΔCT) method. The IL10 mRNA transcript level was normalized to the β-actin level in all samples [18,19].
2.3. Neurological behavior investigation Neurological signs of all rats were assessed using the neurological severity score in accordance with previous method [13].
2.8. Western blotting assay
2.4. Tissue sampling preparation
In brief, fresh rat cortex tissues (n = 3 each group) were homogenized with the radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, China). The lysates were incubated on ice for 30 min and centrifuged at 12,000 g for 15 min at 4 °C. Supernatant protein concentration was determined by using a bicinchoninic acid reagents (Beyotime, China). Brain samples with 40 μg protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After being blocked with 5% non-fat milk buffer for 1 h, the membrane was incubated overnight at 4 °C, followed by primary antibody: rabbit anti-rat IL10 antibody (Santa Cruz, CA, USA; 1:1000) and mouse anti-rat β-actin antibody (Santa Cruz, CA, USA; 1:2000). After being washed with PBST, the membrane was incubated with secondary antibodies coupled to horseradish peroxidase (1:5000) at 37 °C for 1 h. Immunoreactive bands were imaged and visualized using an enhanced chemiluminescence detection kit (Beyotime, China). The optical density of each band was quantified with the Image Analysis System (Bio-Rad, USA) and normalized to βactin protein level for further data analysis [20,21].
At the end of the study, all rats were anesthetized with ethyl carbamate solution (200 mg/kg) via intraperitoneal injection. Intracephalic perfusion was performed with 500 ml cold saline to remove the remaining blood [14]. Then, rat brain samples from ipsilateral cortex tissue nearing lesioned unit were isolated and stored immediately until further biochemical assays. 2.5. ELISA for cytokines determination The contents of interleukin-6 (IL6), tumor necrosis factor (TNF-α), and interleukin-10 (IL10) in serum (n = 8 each group) and the brain (n = 8 each group) were determined using commercially available ELISA kits (Nanjing Jiancheng Bioengineering Institute, China) according to booklet procedures [15]. 2.6. Cytohistologic stains and transmission electron microscopy (TEM) observation Cell growth count in the cortex samples (n = 5 each group) was determined by Nissl-special stain (Beyotime, China) and DAPI-labeled stain (Abcam, UK). IL10-positive neuronal cells were assessed, as previously described [16]. The rat cortex was removed and fixed with 4%
2.9. Statistical analysis Data were analyzed using Statistical Product and Service Solutions (SPSS) 19.0 software (Chicago, IL, USA). All results are expressed as the 350
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3. Results 3.1. FN improved neurological symptoms of TBI rats As results, rats in TBI group showed two deaths during development of TBI. Instead, no death was found in FN-treated groups. FN-treated TBI rats were subjected to the assessment of neurological behaviors. TBI rats showed visible neurological disorders, such as dystasia, trance and unconsciousness, and the injury-based scores were greater than that in sham rats (P < 0.05). Interestingly, FN-treated TBI rats suppressed the abnormal changes, contributing to improved mental signs and reduced injury scores (P < 0.05) (Fig. 1). 3.2. FN promoted neuronal proliferation of TBI rats As shown in Nissl-special and DAPI-labeled stains, the data exhibited that a significant reduction of neuronal number in TBI rats were observed when compared to that in the sham control (P < 0.05). In comparison with TBI rats, FN-treated rats resulted in a dose-dependent increase of nerve cell count (P < 0.05), showing elevations of Nisslstained neurons and DAPI-labeled neurocytes (Fig. 2). Further, the morphology of cortex ultrastructure was observed using TEM. In the sham control, the nucleolus and myelin sheath in selected cortical neurons exhibited a complete and organized cytoarchitecture. Instead, the myelin sheaths in TBI cortical neurons were loosened and deformed, and visible vacuolation was observed. In addition, the mitochondria in the axoplasm was swelled and lesioned. Furthermore,
Fig. 1. FN improved neurological symptoms of TBI rats. FN-treated TBI rats inhibited the abnormal alterations, contributing to improved mental signs and reduced injury scores. The results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/kg.
mean ± standard deviation (SD). Any difference between groups was processed with one-way ANOVA following by Tukey's post hoc test. A probability (P)-value < 0.05 was considered statistically significant.
Fig. 2. FN promoted neuronal proliferation of TBI rats. Both Nissl-special stain and DAPI-labeled stain of cortical neurons indicated a dose-dependent increase in FN-treated TBI cortical neurons. Results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/kg. 351
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Fig. 3. FN improved neuronal ultrastructural organization of TBI rats. FN-treated TBI rats exhibited a tight arrangement of the myelin sheath in the cortical neurons and improved organelle morphology as well as repaired and normalized cell nucleus. Note: FN = formononetin; TEM = transmission electron microscope. FN10, 30 = 10, 30 mg formononetin/kg.
Fig. 4. FN increased contents of IL10 in serum and cerebral cortex of TBI rats. FN-treated TBI rats contributed to the decrease of IL-6 and TNF-α levels in a dosedependent manner. The contents of endogenous IL10 were increased in serum and the cerebral cortex of FN-treated TBI rats. The results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/kg.
3.3. FN increased contents of IL10 in the serum and cortex of TBI rats
cortical neurons in TBI rats showed damaged morphology, including cell shrinkage and vacuolation. Following FN treatments, the myelin sheath in the brain was arranged tightly, and the organelle morphology was improved, and the cell nucleus was repaired and normalized (Fig. 3).
In our biochemical test, the data from enzyme linked immunosorbent assay (ELISA) indicated that the IL10 levels in the serum of TBI cortical neurons were significantly decreased, and cytokine 352
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dose-dependent manner (P < 0.05). In addition, neuronal expression of IL10 was reduced in TBI rats (P < 0.05). Instead, FN treatment elevated the expression of IL10 in TBI cortex neurons dose-dependently (P < 0.05) (Fig. 4).
3.4. FN up-regulated the IL10 mRNA level of TBI rats To assess the FN-mediated neuroprotection that is responsible for neurogenesis in TBI cortical neurons, neuronal IL10 expression at the gene level was assessed. The brain IL10 mRNA level in TBI rats was notably down-regulated when compared to that in the sham cortical neurons (P < 0.05). In comparison with the TBI control, FN-treated cortical neurons showed increased expression of IL10 mRNA in a dosedependent manner (P < 0.05) (Fig. 5).
3.5. FN elevated the IL10 protein level of TBI rats Fig. 5. FN up-regulated the IL10 mRNA level of TBI rats. The RNA from ipsilateral cortex tissue nearing lesioned unit of TBI rats was analyzed by RTPCR. The IL10 mRNA expression was normalized to β-actin. The results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/kg.
In immunohistochemistry analysis, the IL10-positive cells of TBI cortex were decreased when compared to those in the sham control (P < 0.05). However, the dose-dependent elevation of IL10-labeled cell count was observed in FN-treated rats (P < 0.05). Further, immunoblotting data showed that neuronal IL10 content was reduced in TBI rats when compared to that in the sham control (P < 0.05). Interestingly, the endogenous contents of IL10 were up-regulated in FNtreated TBI rats in a dose-dependent manner (P < 0.05) (Fig. 6).
contents (TNF-α and IL6) were increased compared to sham rats (P < 0.05). Interestingly, FN-treated TBI rats contributed to the increase of IL10 level and decrease of cytokine contents (IL-6, TNF-α) in a
Fig. 6. FN elevated the IL10 protein level of TBI rats. The protein extracted from ipsilateral cortex tissue nearing lesioned unit of TBI rats was analyzed by immunofluorescence and immunohistochemistry (ICH) and Western blot. The dose-dependent elevation of IL10-labeled cell counts was observed in FN-treated rats. Results are expressed as the mean ± SD. △P < 0.05, TBI vs. Sham group, *P < 0.05, vs. TBI group. Note: FN = formononetin. FN10, 30 = 10, 30 mg formononetin/ kg. 353
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4. Discussion
References
Increasing evidence suggests that neuronal death/loss is implicated in TBI damage pathogenesis [22]. However, our understanding is limited to effectively treat TBI patients in clinical applications. Reassuringly, the neuroprotective effects of hormone-dependent phytoestrogen have drawn great attention in managing TBI. However, the molecular mechanisms underlying these protective effects remain to be fully elucidated. In the present study, both increased Nissl-stained and DAPIlabeled nerve cells have validated the cell growth in FN-treated TBI brains, consistent with improved neurological symptoms. Thus, these initial data indicated that FN treatment ameliorated the brain function of TBI by promoting neuronal proliferation. The results also implied that FN-exerted neuronal regeneration is involved in cerebroprotective benefits against TBI. Together, these findings support that the molecular mechanism related to nerve regeneration warrants investigation in follow-up studies. Interleukin 10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is a potent cytokine primarily involved in the regulation of neuronal growth, maintenance, proliferation, and survival [23]. In biological function, IL10 is critical for the survival and maintenance of sympathetic and sensory neurons, as they undergo apoptosis when absent [24]. However, several recent studies suggest that IL10 is also involved in activating IL-10R and JAK-STAT3 pathways that regulate the life cycle of neurons [25]. Thus, we propose that intracellular IL10 expression promotes neuronal proliferation, contributing to the inhibition of damage caused by TBI. The current results indicated that neuronal loss in TBI rats correlated with reduced Nissl-stained cells and was accompanied by increased neuronal death, as shown in the DAPI assay. Interestingly, these abnormal alterations were reversed following FN treatments, yielding an increase in cell numbers, an improvement of neuronal cytoarchitecture, a reduction of IL-6 and TNF-α in serum, and an elevation of IL10 expressions in the serum and brain. Thus, we concluded that TBI resulted in significant cell necrosis or death in the cortex, thereby inducing neural impairments. In biological mechanisms, FN-mediated activation of IL10 expression promotes cell growth via combining to IL10-special receptors in cortical neurons, followed by suppression of cortical injury in TBI. In addition, we speculate that the increased serum IL10 level by FN treatment might be linked to IL10based biological pathways in peripheral cortical neurons to combat the TBI condition.
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5. Conclusion Collectively, these studies demonstrate that FN may provide neuroprotection against TBI by inducing neuronal IL10 expression. In addition, the current findings disclose that FN may serve as an alternative strategy of combating TBI. Conflict of interest The authors confirm that there are no conflicts of interest. Acknowledgments Our present study was supported by the National Natural Science Foundation of China (81460211, 81560378) and the Guangxi Emergency and Medical Rescue Talent Highland (GXJZ201506).
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