Abstract
The purpose of the current study was to examine the neuroprotective effect of rutin against colistin-induced neurotoxicity in rats. Thirty-five male Sprague Dawley rats were randomly divided into 5 groups. The control group (orally received physiological saline), the rutin group (orally administered 100 mg/kg body weight), the colistin group (i.p. administered 15 mg/kg body weight), the Col + Rut 50 group (i.p. administered 15 mg/kg body weight of colistin, and orally received 50 mg/kg body weight of rutin), the Col + Rut 100 group (i.p. administered 15 mg/kg body weight of colistin, and orally received 100 mg/kg body weight of rutin). Administration of colistin increased levels of glial fibrillary acidic protein and brain-derived neurotrophic factor and acetylcholinesterase and butyrylcholinesterase activities while decreasing level of cyclic AMP response element binding protein and extracellular signal regulated kinases 1 and 2 (ERK1/2) expressions. Colistin increased oxidative impairments as evidenced by a decrease in level of nuclear factor erythroid 2-related factor 2 (Nrf-2), glutathione, superoxide dismutase, glutathione peroxidase and catalase activities, and increased malondialdehyde content. Colistin also increased the levels of the apoptotic and inflammatoric parameters such as cysteine aspartate specific protease-3 (caspase-3), p53, B-cell lymphoma-2 (Bcl-2), nuclear factor kappa B (NF-κB), Bcl-2 associated X protein (Bax), tumor necrosis factor-α (TNF-α) and neuronal nitric oxide synthase (nNOS). Rutin treatment restored the brain function by attenuating colistin-induced oxidative stress, apoptosis, inflammation, histopathological and immunohistochemical alteration suggesting that rutin supplementation mitigated colistin-induced neurotoxicity in male rats.
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Introduction
Colistin (polymyxin E), a cationic polypeptide antibiotic, has been used for the treatment of infections of multidrug-resistant gram-negative bacteria such as Klebsiella pneumonia, Acinetobacter baumannii and Pseudomonas aeruginosa [1]. The main target of antimicrobial activity of colistin is the bacterial cell membrane. Colistin interacts to anionic lipopolysaccharides of Gram‐negative bacteria and causes the replacement of divalent cations such as calcium (Ca2+) and magnesium (Mg2+) in the membrane. As a result, it causes an increase in the bacterial cell permeability and subsequently, cell death [2, 3]. However, neurotoxicity and nephrotoxicity are still major adverse effects limiting its clinical uses [1, 4, 5]. The neurological symptoms such as hallucination, dizziness, confusion, seizures, vertigo and visual disturbances were recorded in colistin-treated patients [6, 7]. In previous studies, colistin has been reported to induce axonal and neuronal degeneration in brain and sciatic nerves when intravenously administrated in mice at a dose of 15 mg/kg per day for 7 days [8, 9]. In this context, the development of neuro-protective compounds that can be administered together with colistin is important to increase its clinical use.
Flavonoids, a group of natural substances with different phenolic structures, are found in fruits, vegetables, roots, grains, bark, flowers, stems, wine and tea [10, 11]. The most common native flavonoid is rutin, which is vastly present in food such as apples, onions, red wine and tea [12]. Rutin has different protective effects in vitro as well as in vivo against oxidative stress-mediated diseases and lipid peroxidation [13, 14]. It has a wide range of pharmacological and biological effects including anti-inflammatory, antioxidant, antihypertensive, antiapoptotic, antiautophagic and neuroprotective activities in several experimental models of rodents [15,16,17].
Based on these knowledge, the present study was designed to investigate the neuro-protective effects of rutin against colistin-induced neurotoxicity in rats.
Materials and methods
Drug and chemicals
Colistin (Colimycin® 150 mg/flakon, Koçak Pharma, İstanbul, Turkey) was obtained from a local pharmacy. Rutin (≥ 94%) and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals used in this study were of analytical grade.
Animals
Male Sprague Dawley rats, weighing 220–250 g (8–10 weeks of age) were used in the study following the guidelines of the Local Animal Care Committee of Ataturk University, Erzurum, Turkey (Approval No: 2019-4/56). The rats were received from Ataturk University Experimental Research and Application Center. They were housed in plastic cages under controlled environmental conditions (humidity 45 ± 5%, temperature 25 ± 1 °C and 12 h light/dark cycle) with free access to water and food.
Experimental protocol
The animals were separated into five different groups of seven rats each per plastic cages and were treated for 7 days as follows:
Group I (control): rats received normal saline (1 ml, p.o., once daily) for 7 days.
Group II (rutin): rats received rutin (100 mg/kg b.w., p.o.) for 7 days [18].
Group III (colistin): rats received colistin (15 mg/kg b. w., i.p., once daily) for 7 days [6].
Group IV (Col + Rut 50): rat received colistin (15 mg/kg b. w., i.p.) and rutin (50 mg/kg b. w., p.o.) for 7 days.
Group V (Col + Rut 100): rat received colistin (15 mg/kg b. w., i.p.) and rutin (100 mg/kg b. w., p.o.) for 7 days.
On day 8, rats were killed under mild sevoflurane anesthesia (Sevorane liquid 100%, Abbott Laboratory, İstanbul, Turkey), and brain tissues were removed immediately. Part of the brain was fixed in 10% formalin and embedded in paraffin for immunohistochemical and histopathological staining. The remained part of the brain was stored at − 20 °C for biochemical and Real-Time PCR analyses.
Evaluation of biomarkers of oxidative stress
To obtain brain tissue homogenate, the tissue was grinded using liquid nitrogen. The tissues were homogenized in 1:10 (w/v) ratio of tissue and 1.15% potassium chloride buffer. A part of the homogenate was taken and centrifuged at 10,000 rpm for 20 min at 4 °C, and the obtained supernatant was used for the measurement of glutathione peroxidase (GPx) activity and glutathione (GSH) level. The remaining part was centrifuged at 3500 rpm for 15 min and supernatants were used for the assay of catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA). The CAT, GPx and SOD activities were assayed according to the methods prescribed by Aebi [19], Matkovics [20] and Sun et al. [21], respectively. The GPx and SOD activities were expressed as U/g protein. CAT activity has been described as katal/g protein. MDA levels were estimated according to Placer et al. [22]. GSH content was determined by the method of Sedlak and Lindsay [23]. The MDA and GSH levels have been expressed as nmol/g tissue. Protein amount in the brain was measured according to Lowry et al. [24].
Evaluation of AChE and BChE enzyme activities
The activities brain tissue Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were studied as described by Ellman et al. [25]. These enzyme activities were measured spectrophotometrically at a wavelength of 412 nm (UV-1800 Shimadzu, Kyoto, Japan) and the activities were expressed as U/mg protein.
Evaluation of inflammation markers
Neuronal nitric oxide synthase (nNOS), nuclear factor kappa B (NF-κB) and tumor necrosis factor-α (TNF-α) levels in brain tissue were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (YL Biont, Shanghai, China). Brain homogenates required for all ELISA kits were obtained as described in our previous studies [18, 26].
Evaluation of apoptotic markers
p53 protein level and cysteine aspartate specific protease-3 (caspase-3) activity in the brain tissue was detected with a commercial rat ELISA kit (YL Biont) according to the manufacturer's instruction.
Evaluation of GFAP and Nrf2
Glial fibrillary acidic protein (GFAP) and nuclear factor erythroid 2-related factor 2 (Nrf2) levels were determined using a commercial rat ELISA kit purchased from YL Biont (Shangai, China) following the manufacturer's instruction.
Real time PCR assay
Total RNA was isolated from brain tissue using TRIZOL reagent (Invitrogen, Cat: 15,596,026, USA) following the manufacturer’s instructions. cDNA synthesis was performed with QuantiTect Reverse Transcription (Qiagen, Cat:330,411 Germany) from total RNA samples according to the manufacturer’s instructions [27]. Real-time quantitative PCR (RT-PCR) was performed to measure the mRNA transcript level of BDNF, Bcl-2, and Bax in the brain tissues using ROTOR-GENE Q 5plex HRM Real-Time PCR Detection System (Qiagen, Germany). GAPDH was used as internal control gene. The specificity of PCR amplification was confirmed by agarose gel electrophoresis and melting curve analysis [27, 28]. Relative folds of expressions were evaluated with the 2−ΔΔCT method [29]. All primer sequences and reaction conditions were shown in Table 1.
Histopathologic analysis
The rat brains were removed after necropsy and fixed in the 10% buffered formalin solutions for 3 days, and were then passed through alcohol-xylene processes and embedded into paraffin blocks. The 5-μm brain sections were taken into slides and rehydrated, and stained with hematoxylin at room temperature for 2 min and then with eosin for 10 s. Findings were evaluated in terms of necrotic and apoptotic cells under a light microscope (Olympus BX51-DP72) at ×20 magnification.
Immunohistochemistry staining
Following deparaffinization and hydration, brain sections were immersed to 3% H2O2 solution to inactivate endogenous peroxidase activity for 10 min. Then, the sections were boiled in sodium citrate buffer (pH 6.0) to unmask antigens for 10 min, and were cooled for 10 min. The sections were incubated with blocking solution at room temperature for 15 min to eliminate any nonspecific binding. The sections were incubated with cyclic AMP response element binding protein (CREB) Antibody (LB9) primary antibody (Novus Biologicals, Cat. No: NB100-74,393, Dilution: 1/200) and ERK 1/2 Antibody (C-9) primary antibody (Santa Cruze, Cat. No: sc-514302, Dilution: 1/250) for 30 min. Subsequently, the procedure of Expose mouse and rabbit specific HRP/DAB detection IHC kit (Abcam, No: ab80436) was followed. 3,3′-diaminobenzidine chromogen was used and counterstained with hematoxylin, dehydrated, and cover slipped. Positive cells were examined at ×20 magnification under a light microscope (Olympus BX-51, DP72). As negative controls. Sections incubated with PBS instead of the primary antibodies. ERK 1/2 and CREB immunopositivity were scored as follows: none (−), mild (+), moderate (++), and intense (+++).
Statistical analysis
The biochemical analysis was compared by one-way analysis of variance (ANOVA). Tukey’s multiple comparison test were performed. p < 0.05 were considered statistically significant. The results are represented as mean ± SEM. For RT-PCR analysis, relative mRNA fold change graphics were created by using Graph pad prism software Inc., (Version 7.0, California, USA). Results are expressed as mean ± SEM. Statistically differences were considered to be significant at *p < 0.05, **p < 0.01 and ***p < 0.001. For immunohistochemical analysis, differences were analyzed with a nonparametric test (Kruskal–Wallis) followed by Mann–Whitney U test (p < 0.05). Data are presented as means (±) standard deviations (S.D.).
Results
Effect of rutin treatment on brain oxidative stress biomarkers
Treatment with colistin significantly reduced the activities of SOD, CAT and GPx enzyme, and level of GSH (p < 0.05) by 31%, 42%, 43%, and 41%, respectively as compared to the control group (Table 2). These biochemical parameters showed a significant increase in the Col + Rut 50 and Col + Rut 100 groups as compared with the colistin-treated group. But the higher dose of rutin (100 mg/kg b. wt.) significantly increased the activities of SOD, CAT, GPx, and GSH in Col + Rut 100 group as compared to colistin group.
Colistin administration significantly increased MDA level by 49%, comparison with control group. However, MDA levels decreased to 9% in Col + Rut 50 group and 22% in Col + Rut 100 group.
Effect of rutin treatment on brain cholinergic enzymes
In this study, we measured AChE and BChE activities to assess the protective effect of rutin against colistin-induced neurotoxicity. Brain AChE and BChE enzyme activities were found to increase by 65% and 70% in colistin-treated groups compared to control groups, respectively (p < 0.05). However, rutin treatment along with colistin significantly (p < 0.05) decreased the elevation of these enyme activities in dose dependent manner (Fig. 1a, b).
Effect of rutin treatment on brain inflammation
Colistin induced a significant increase in NF-κB, TNF-α and nNOS levels as 0.42-, 0.7- and 0.47-folds respectively as compared to the control group (p < 0.05) (Fig. 2). Rutin treatment (50 and 100 mg/kg b.w.) along with colistin significantly decreased NF-κB levels by 0.10- and 0.20-fold, TNF-α levels by 0.15- and 0.29-fold and nNOS levels by 0.12- and 0.20-fold as compared with the colistin‐treated group.
Effect of rutin treatment on brain apoptosis
In the present study, the p53 level and caspase-3 activity in brain tissues were determined by ELISA. As indicated in Fig. 3, colistin significantly (p < 0.05) increased the level of p53 (+ 68%) and activity of caspase-3 (+ 70%) as compared to the control group. In contrast, rutin treatment significantly attenuated the colistin-increased level of p53 and activity of caspase-3 in a dose-dependent manner.
Effect of rutin treatment on GFAP and Nrf2 levels
The GFAP level was significantly increased (0.46-fold) in colistin-induced group compared to the control group (p < 0.05). Both doses of rutin (50 and 100 mg/kg b.w.) significantly decreased GFAP levels by 0.08- and 0.19-fold, compared to the colistin-treated group, respectively (Fig. 4a).
The Nrf2 level was significantly declined (0.29-fold) in colistin-treated group as compared to the control group (p < 0.05). The level of Nrf2 was considerably increased in Col + Rut 50 (0.13-fold) and Col + Rut 100 (0.21-fold) treated groups compared to the only colistin-treated rats (Fig. 4b).
Effect of rutin on the mRNA level of BDNF, Bax and Bcl-2
The expression levels of BDNF, Bax and Bcl-2 genes in the brains were determined by qRT-PCR. The expression level of BDNF was increased in the colistin group (p < 0.01), unchanged in rutin group (p > 0.05) in comparison to control group. BDNF expression levels were slightly up-regulated in the Col + Rutin 50 and Col + Rutin 100 groups compared to the control group (p > 0.05), but significantly down-regulated compared to the colistin group (p < 0.05) (Fig. 5a). Expression level of Bcl-2 decreased only in the colistin group (p < 0.01), almost remained unchanged (p > 0.05) in rutin group compared to the control group. Meanwhile, mRNA level of Bcl-2 was slightly down regulated in the Col + Rutin 50 and Col + Rutin 100 groups compared to the control group (p > 0.05), however it was significantly up-regulated compared to the colistin group (p < 0.05) (Fig. 5b). mRNA level of Bax was significantly up-regulated in the colistin group (p < 0.01), slightly increased in the colistin + rutin 50 and colistin + rutin 100 groups compared to the control group (p > 0.05), but it was down-regulated compared to the colistin (p < 0.05) (Fig. 5c).
Histopathology results
Figure 6a and b shows pyramidal cell layer, with normal neurons hippocampus of control and rutin-treated groups. Figure 6c shows necrotic pyramidal cell and apoptotic neurons in hippocampal area in colistin-treated group. Figure 6d shows moderate pyknotic changes and apoptotic neurons in hippocampal area of groups rutin-treated with colistin. Figure 6e also shows that the necrotic and apoptotic neurons are considerably reduced in hippocampal area.
Effects of rutin on kinases 1 and 2 (ERK1/2) and CREB immunoreactivity
Results of ERK1/2 and CREB immunopositivity in the hippocampal neurons are shown in Fig. 7a–l. Figure 7f–l shows negative control in the hippocampal neurons. ERK1/2 and CREB immunoreactivity was determined in nucleus of neuronal cells of the brain tissues of control and treatment groups. We detected an intense immunopositivity for ERK1/2 activity in a substantial number of hippocampal neurons in control and rutin treated groups (Fig. 7a, b). This immunopositivity was significantly reduced in the colistin group (Fig. 7c). It was observed that colistin combined with different doses of rutin (50 and 100 mg/kg) increased ERK 1/2 immunopositivity in hippocampal neurons (Fig. 7d, e). Also, the number of CREB-immunopositive neuronal cells in the hippocampus in the control and rutin treated groups was significantly intense compared with that in the colistin group (Fig. 7g–i). It was observed that rutin administration (50 and 100 mg/kg) with colistin increased CREB reactivity in hippocampal neurons (Fig. 7j, k).
In terms of ERK1/2 and CREB immunopositivity, no statistically remarkable difference was identified in the brains of the rats in the control, rutin and Col + Rutin 100 groups. Although there was no considerable difference between colistin and Col + Rutin 50 groups in ERK 1/2 immunopositivity, there was a significant difference between colistin and other groups in CREB immunopositivity (p < 0.05, Table 3).
Discussion
Neurotoxicity is an important undesirable effect of polymyxin therapy. Therefore, the discovery of effective neuro-protective natural compounds for co-administration during colistin treatment is critical to prolong the clinical usage of this important cationic polypeptide antibiotic. In present study, we demonstrated that rutin exhibits neuro-protective properties by mitigating oxidative stress, apoptosis and neuroinflammation in colistin-induced neurotoxicity.
The nervous system is delicate to oxidative stress owing to its high polyunsaturated fatty acid content and high oxygen demand [30]. Oxidative stress has a major role in the pathogenesis of neurodegenerative disorders [6]. Increased generation of reactive oxygen species (ROS) is a significant mechanism in colistin-induced neurotoxicity [6, 7, 9]. Notably, excessive ROS can result in the protein denaturation, peroxidation in membrane lipids, damage in nucleic acids, and they could alter neuronal function and impair nervous system [31,32,33]. Recent studies have showed ROS-mediated oxidative stress plays a major role in colistin-induced in vitro neurotoxicity [34, 35]. Nrf2 as a known transcription factor and the key regulator of the cellular response of oxidative stress has been reported to stimulate the levels of antioxidant enzymes [36]. The activation of Nrf2 reduces cellular oxidative damage by activating genes that encode antioxidant enzymes, such as heme oxygenase-1 (HO-1), CAT, SOD and GPx [37]. In our study, colistin significantly reduced SOD, CAT, GPx, GSH and Nrf2 levels in brain tissue and increased MDA content (Table 2 and Fig. 4b). Rutin works as a scavenger of ROS by donating hydrogen atoms to superoxide anions, peroxy radicals and hydroxyl radicals [18]. In present study, rutin efficiently reduced MDA level, increasing CAT, GPx, SOD, GSH and Nrf2 levels in colistin-induced neurotoxicity. Our in vivo findings were in agreement with previous studies that revealed the antioxidant properties of the rutin [17, 38].
AChE is a serine protease enzyme that hydrolyzes the neurotransmitter acetylcholine. It is mainly found in neuromuscular junctions and plays an important role in the central nervous system of cholinergic brain synapses [39, 40]. On the other hand, BChE is a serine hydrolase enzyme that hydrolyzes choline and non-choline esters and is expressed in all brain regions such as cerebellum, hippocampus and temporal cortex [41, 42]. Cholinergic neurons are essential for memory function and learning in the cerebral cortex and thus AChE and BChE play a key role in this event [6, 43]. In a previous study, it was reported that colistin increased AChE and BChE enzyme activities [6]. In present study, treatment of rutin decreased AChE and BChE enzyme activities compared with the colistin-treated group. OH groups of flavonoids have been reported to interact with the peripheral anionic region of AChE and prevents entry to the active site of the enzyme. In a study, accessible binding region of rutin was shown in AChE. The rutin-AChE interaction was stabilised through hydrogen bonds, van der Waals and hydrophobic interactions resulting a dose-dependent inhibition of the enzyme [44, 45].
Increasing evidence has showed that ROS activates pro-inflammatory mediators, such as NF-κB and TNF-α and subsequently induces neuroinflammation in brain [46]. TNF-α is a pro-inflammatory cytokine and a key mediator of inflammatory tissue damage [7]. Under pathological conditions, microglia release high levels of TNF-α, and overproduction of TNF-α is a significant component of the neuroinflammatory response related to some neurological disorders [47]. Moreover, NF-κB is a redox transcription factor, which serves as a crucial regulator of inducible expression of genes involved in inflammation and various autoimmune disease [48]. Expression of TNF‐α, inducible nitric oxide synthase (iNOS), interleukin-1β (IL‐1β) and cyclooxygenase-2 (COX-2) can be altered by the activation of NF-κB [48,49,50]. Thus, inhibition of NF-κB may provide a potential approach for the reduction of neuroinflammation. Nitric oxide (NO) is an important molecule that mediates the neurotoxicity, neurotransmission, and vasodilation [51]. However, excessive NO generation by the nNOS enzyme under neuroinflammation conditions result in the neuronal cell death and production of reactive nitrogen species [52]. In the present study, we found that rutin considerably inhibited the levels of NF-κB, TNF-α and nNOS in colistin-induced neurotoxicity. Recently, a study found that rutin reduced the TNF-α, IL-1β and IL-6 levels in sodium fluoride (NaF) induced neurotoxicity in rats [53].
It is well known that mitochondria play an important role in cellular redox homeostasis and induction of apoptotic process [54]. Colistin induced a rapid dissipation of the mitochondrial membrane potential, indicative of mitochondrial dysfunction. Pro- and anti-apoptotic Bcl-2 family proteins including Bax and Bcl-2 regulate the mitochondrial pathway [34]. Colistin increases mitochondrial outer membrane permeability by causing an increase in Bax/Bcl-2 ratios and cytochrome c release. Release of cytochrome c into the cytoplasm causes activation of casapase-3 and -9, which triggers apoptosis [34, 35]. In our study, colistin significantly increased p53 protein levels. In this context, p53 has been reported to activate Bax penetrating the mitochondria, causing mitochondrial cytochrome c release and activation of caspase enzymes, which induces apoptosis [55]. In present study, we observed the down-regulation of Bcl-2 expression as well as up-regulation of p53, caspase-3 and Bax expressions in colistin-induced brain tissues. It has been reported in a previous study that rutin ameliorates brain tissues against ethanol-induced apoptosis and causes an decrease in caspase-3, Bax, and cytochrome c expressions [56].
The ERK1/2 are members of the mitogen-activated protein kinase (MAPK) family. ERK1/2 pathway can be activated in response to oxidative stress, growth factors, cytokines, chemokines and ischemic injury [57]. Inhibition of the ERK1/2 pathway has been reported to reduce brain injury and focal infarction volume in mice after ischemia [58]. On the other hand, GFAP is known as an important component of the astrocyte cytoskeleton during brain development [59]. This component is necessary for astrocyte functioning, particularly in formation of blood–brain barrier and in regulation of central nervous system activities of the developing brain [60]. However, increased GFAP expression causes brain injury and inflammatory neurological disorders [59]. Our results showed that exposure to colistin remarkably reduced ERK1/2 expression while increased GFAP levels in brain tissue. However, rutin treatment significantly suppressed the GFAP level while increased ERK1/2 expression.
CREB is a transcription activator involved in physiological signals such as depolarization, neurotransmitters, factors controlling differentiation, mitogenic signals, synaptic activity, and stressors [61]. BDNF is an important neurotrophic factor that participate in various intracellular signaling processes, neuronal protection and survival, dendritic and axonal morphology and synaptic plasticity [62]. Transcription of BDNF is induced by activation of CREB [61]. In this study, considerable increase in the mRNA levels of BDNF and CREB expression were observed in colistin-treated rat brain tissues. However, rutin given for treatment significantly decreased expressions of CREB and BDNF. It is has been reported that rutin may protect hippocampus against beta-amyloid-induced damage via up regulation of CREB and BDNF [63].
Conclusion
In conclusion, we demonstrated that rutin supplementation alleviated colistin-induced neurotoxicity in male rats. The probable mechanisms underlying these protective effects have been demonstrated to be associated with the antioxidant, anti-apoptotic and anti-inflammatory effects of rutin in brain tissues. In addition, this study is thought to pave the way for further studies to examine other machineries underlying the neuroprotective effects of rutin.
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This work was supported by Grants from Private Buhara Hospital, Erzurum. Therefore, we are grateful to Dr. Serdar Kömeç on behalf of the Hospital.
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Çelik, H., Kandemir, F.M., Caglayan, C. et al. Neuroprotective effect of rutin against colistin-induced oxidative stress, inflammation and apoptosis in rat brain associated with the CREB/BDNF expressions. Mol Biol Rep 47, 2023–2034 (2020). https://doi.org/10.1007/s11033-020-05302-z
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DOI: https://doi.org/10.1007/s11033-020-05302-z