reperfusion injury in rats

European Journal of Pharmacology 877 (2020) 173066

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Preventive effect of silymarin-loaded chitosan nanoparticles against global cerebral ischemia/reperfusion injury in rats

T

Akbar Hajizadeh Moghaddama,∗, Seyed Reza Mokhtari Sangdehia, Mojtaba Ranjbarb, Vahid Hasantabarc a

Department of Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran Faculty of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran c Department of Organic Polymer Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Ischemia/reperfusion Silymarin-loaded chitosan nanoparticles Depression Oxidative stress Inflammation Bioavailability

Chitosan-based polymeric nanocarrier has been utilized as a novel drug delivery device in recent years due to its prominent role in the treatment of central nervous system disorders. The aim of this study was to investigate the anti-oxidative and anti-inflammatory effects of silymarin-loaded chitosan nanoparticles (SM–CS–NPs) on rat model of global cerebral ischemia/reperfusion (I/R). All rats were randomly distributed into four groups: Control, I/R, SM and SM–CS–NPs. Oral administration of SM and SM–CS–NPs was started 14 days prior to bilateral common carotid artery occlusion (BCCAO). Depressive-like behaviors, infarct volume, some oxidative stress markers and inflammatory factors were assessed after induction of I/R. SM–CS–NPs pretreatment significantly ameliorated depressive-like behaviors and infarct volume after I/R. SM–CS–NPs also significantly decreased the levels of malondialdehyde (MDA), and expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and significantly increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRx), and glutathione (GSH) levels in I/R brain. The current study demonstrated that SM–CS–NPs pretreatment effectively prevents oxidative and inflammatory damage in the brain caused by I/R, and it can be considered as a useful pretreatment to attenuate the negative effects of I/R.

1. Introduction Brain ischemic disease is one of the primary causes of disability and mortality, which is a major challenge to public health (Godinho et al., 2018). This developing disorder is usually caused by a transient or permanent reduction in cerebral blood flow (Xue et al., 2017). Studies have shown that there is a link between oxidative stress and the production of reactive oxygen species acting as a source of injury (Allen and Bayraktutan, 2009). Patients who survive I/R or hypoxia commonly develop cognitive deficit and depression. The impairments are strongly correlated with damage to various cortical regions including disturbances of attention deficits, disability, social problems and poststroke depression (Godinho et al., 2018). Although the exact mechanisms of behavioral disorders remain unclear, IR-induced oxidative stress in the brain has been shown to play a critical role in the progression of cognitive impairment. In addition, the oxidative stress, as a source of injury to biological molecules that leads to toxicity and degeneration of

the neuron, is one of the major contributing factors to the development of depressive disorders (Nabavi et al., 2018). In addition to oxidative stress, inflammation can play a pivotal role in damaging brain tissue. The inflammatory response following I/R is a well-known event that involves the activation of astrocyte and microglial which react by secreting cytokines, such as TNF-α and IL-6 (Liu et al., 2017). However, there are very limited clinically effective therapies to improve functional outcomes associated with I/R. Consequently, there is a pressing need to identify new safe and effective drugs to treat I/R (Godinho et al., 2018). Flavonoids and polyphenols are one of the most important groups of natural antioxidants available in human diets. SM is composed of several isomer flavonolignans. Silybin is the chief active constituent in SM mixture that shows anti-oxidant activity and exerts anti-inflammatory effect (Surai, 2015; Turgut et al., 2008). Previous studies showed that inhibition of TNF-α and IL-6 mediates the antiinflammatory effect of SM (Hou et al., 2010; Stolf et al., 2017). Nevertheless, poor bioavailability of SM is the main factor limiting its

∗

Corresponding author. E-mail addresses: [email protected] (A.H. Moghaddam), [email protected] (S.R. Mokhtari Sangdehi), [email protected] (M. Ranjbar), [email protected] (V. Hasantabar). https://doi.org/10.1016/j.ejphar.2020.173066 Received 13 December 2019; Received in revised form 1 March 2020; Accepted 10 March 2020 0014-2999/ © 2020 Elsevier B.V. All rights reserved.

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Fig. 1. Timeline of the experimental design.

performed from 09:00 to 15:00. The animal experiments were approved by the Institutional Animal Ethics Committee (IR.UMZ.REC.1397.024); the experiments were done according to the codes of the University of Mazandaran for the Care and Use of Animals.

use. It depends on factors such as poor water solubility (Khalkhali et al., 2015), extensive phase II metabolism (Calani et al., 2012), improper tissue distribution, and degradation in the gastrointestinal tract and rapid excretion in bile and urine (Javed et al., 2011). Pharmacokinetic investigations have demonstrated that only 23%–47% of SM reaches the systemic circulation and peak plasma concentrations are achieved within 6 h (Taleb et al., 2018; Yang et al., 2013). Hence, high dose of SM is required to achieve therapeutic plasma levels. This leads to the development of a novel drug delivery system to improve its solubility and thereby bioavailability (Yang et al., 2013). These systems include complexation with cyclodextrin and phospholipids (Arcari et al., 1992), incorporation in solid dispersion (Yanyu et al., 2006) and formulation of a self-emulsifying drug delivery system (Woo et al., 2007; Wu et al., 2006). CS is obtained by the deacetylation of chitin (Quiñones et al., 2018). CS has been used significantly in the pharmaceutical and biomedical industry due to excellent properties such as wide availability, inherent pharmacological activity, biocompatibility, biodegradability, negligible toxicity, and immunogenicity (Park et al., 2010). The properties and functionality of CS are greatly influenced by its molecular weight, the degree of acetylation, and viscosity that could be effective in using CS as a matrix molecule for drug delivery (Ahsan et al., 2018). Thus, the aim of this study was to investigate the Preventive effect of SM–CS–NPs against cerebral I/R injury in rat brain with biochemical and behavioral analysis.

2.4. Stroke inducing For the induction of I/R, rats were anaesthetized with chloral hydrate (350–400 mg/kg, i.p). BCCAO was subsequently induced following the standard experimental animal model of I/R (Aggarwal et al., 2010; Nabavi et al., 2018). Briefly, carotid arteries on both sides were chosen and vascular clamps were used for ligating them for 5 min (time of ischemia). After 10 min reperfusion, the vascular clamps were detached for the next 10 min and visual inspection of the carotid arteries for reperfusion was performed; afterwards, both carotid arteries were ligated for 5 min. The vascular clamps were finally detached and arteries were inspected for blood reflow. The surgical incisions were sutured and the sutured area was rinsed with 70% ethanol and sprayed with an anti-septic solution. Ultimately, all rats were kept in separate cages under standard conditions to recover normal body temperature. Rectal temperature was checked daily, and rats with normal body temperature (37 ± 1 °C) were selected for investigation. 2.5. Experimental design The rats were randomly divided into 4 groups (10 rats per group): control, I/R, SM, and SM–CS–NPs. SM and SM–CS–NPs (15 mg/kg) were orally administrated to the rats 14 days before I/R induction. Following 24 h of I/R induction, the forced swimming and tail suspension tests were applied to study all the animals in terms of depressive-like behaviors (Fig. 1).

2. Material and methods 2.1. SM–CS–NPs syntheses At first, in a beaker, 5 gr of choline chloride was mixed with 2.85 gr of glucose and heating was applied to 80 °C for 1 h. This process was continued until clear a solution was obtained. Then, 1 gr SM added to it within 1 h. The mixture was stirred for an extra 30 min at the same temperature to reach to a light-brown mixture that was honey-like. Afterwards, 15 gr of CS was added into the above mixture while stirring was done at 80 °C. The obtained mixture was stirred under the same condition for 3 h to form SM–CS–NPs composite as a wet-like solid (42 mg SM per gr composite).

2.6. Forced swimming test (FST) FST is a test that assesses depressive-like behaviors. FST was measured according to the method of Dalvi & Lucki (Dalvi and Lucki, 1999). In brief, rats were individually forced to swim inside a vertical plastic cylinder (40 cm in height and 25 cm in diameter) containing 27 cm of fresh water maintained at 23–25 °C. A rat was placed in the cylinder and was forced to swim for 6 min. The immobility and climbing times were then recorded in the last 4 min of the test.

2.2. Characterization Fourier-transform infrared spectra (FT-IR) of CS, SM and SM–CS–NPs were obtained using a (Bruker Tensor 27 Fourier transform, Karlsruhe, Germany) FT-IR spectrometer with aid of KBr pellets within the wave number range of 500–4000 cm−1. The surface morphology of CS, SM, and SM–CS–NPs were characterized by Field Emission scanning electron microscopy (FE-SEM) (TESCAN, MIRA III).

2.7. The tail suspension test (TST) TST was measured according to the method of Cryan (Cryan et al., 2005). In brief, the rat was suspended at a height of 60 cm above the floor by adhesive tape placed 1 cm from the tail tip of each animal. They were allowed to hang for 6 min, and immobility time was recorded in the last 4 min of the test.

2.3. Animals 2.8. Analysis of infarction volume Male Wistar rats (8 weeks, 250–300 g) were purchased from the Laboratory Animal Center of Baqiyatallah University of Iran. They were kept in the animal house at room temperature under a 12 h light–dark cycle and 65% ± 5% humidity. Rats were accustomed to the test area for 24 h prior to behavioral tests and all the experiments were

Twenty-four h after behavioral tests, the rats were killed immediately after anesthesia. The brains were then removed and sectioned into five coronal slices (2-mm thick) via sharp blades for staining with 2% 2, 3, 5-triphenyltetrazolium chloride stain (TTC, Sigma, USA). 2

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of NADPH was accelerated by GRx and NADP was obtained through oxidation. The reduced absorbance at 340 nm was measured. Rotruck method was implemented to assay GPx activity (Rotruck et al., 1973). In short, reaction mixture included phosphate buffer containing GSH and brain tissue supernatant pre-incubated at 37 °C for 5 min (recorded at 340 nm) (Fukuzawa and Tokumurai, 1976).

Table 1 Sequences of primers used in qRT-PCR. Gene

Primer

Sequence

Amplicon lengths (bp)

GAPDH

forward reverse Forward reverse forward reverse

5′-ATCCTGCACCACCAACTGC-3′ 5′-ACGCCACAGCTTTCCAGAG-3′ 5′-GGAGGAGCAGCTGGAGTG-3′ 5′-CCTTGAAGAGAACCTGGGAGTAGA-3′ 5′-TCACAGAGGATACCACCCACAA-3′ 5′-CAGTGCATCATCGCTGTTCATAC-3′

129

TNF-α IL-6

131

2.14. Estimation of IL-6 and TNF-α

146

The expression of IL-6 and TNF-α mRNA were determined by qRTPCR amplification. The total RNA from the cerebral tissue of rats was isolated using the RNeasy Mini Kit (QIAGEN) according to the instructions of the manufacturer. To specify the RNA concentration and purity, a UV spectrophotometer was applied to measure the absorbance at 260 and 280 nm. To remove genomic DNA from RNA samples, 1 μg of the total RNA was treated with RNase-free DNase I (Fermentas) according to the protocol of the manufacturer. Superscript III Reverse Transcriptase (Fermentas) was applied following the manufacturer's instructions for the first-strand cDNA synthesis. Poly-dT primer was applied for the cDNA synthesis of the mRNAs. The reactions were incubated at 42 °C for 60 min and 70 °C for 10 min. The cDNA samples were diluted to 1/20 and the PCRs were performed using Corbett RotorGene 6000 real-time thermal cycler using the SYBR Green kit (Applied Biosystems, Foster City, CA). The primer sequences for the three genes studied are given in Table 1. The reaction mixture included 10 μl of 2x SYBR Green qPCR Mix, 0.8 μl of 10 μM primer mix (forward and reverse primers) and 5 ml of diluted cDNA. Incubation was done at 95 °C for 3 min to initiate the PCR reactions followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 40 s. A melting curve was performed at the end of the PCR within the range of 55–95 °C, and the temperature was increased step by step by 0.5 °C every 2 s. The 2−ΔΔCT method has been extensively used as a relative quantification strategy for quantitative real-time polymerase chain reaction (qPCR) data analysis.

The slices were put in TTC solution at 37 °C for 30 min in a container. The slices were photographed using a digital camera for analyses using Image j 1.8.0 software. The infarct area was expressed as a percentage of the total brain volume (Li et al., 2016). 2.9. Preparation of tissue homogenate 150–200 mg of cortex tissue of each rat was homogenized in 1 ml of buffer (10 nmol/l Tris-HCl, pH = 7.4, 1 mmol/l EDTA, and 0.32 mol/l sucrose), and then centrifuged (13,600 g, 4 °C, 30 min). The supernatant was taken and used for measuring the MDA and GSH levels, activities of SOD, CAT, GPx and GRx and protein content. 2.10. Measurement of protein content The Bradford assay was applied for measuring the protein concentrations of the homogenates of cerebral tissue. In this test, bovine serum albumin was used as the standard (Bradford, 1976). 2.11. Determination of reduced GSH level The reduction in GSH levels was measured by the method of Fukazawa & Tokumura (Fukuzawa and Tokumurai, 1976). Briefly, 20 μl of the supernatant was mixed with 1.1 ml of 0.25 M sodium phosphate buffer pH 7.4 and 130 μl of DTNB (0.04%). Afterwards, distilled water was added to have a final volume of 1.5 ml and the absorbance of the reaction mixture was read at λ = 412 nm. Finally, results were expressed as mg GSH/gr protein.

2.15. Statistical analysis All statistical analyses were done using GraphPad 8.0.2, GraphPad Software, Inc.). The data were Mean ± S.D. Statistical differences were performed ANOVA followed by Tukey's test. In all cases, a P < sidered statistically significant.

2.12. Estimation of lipid peroxidation MDA, a marker for lipid peroxidation, was measured by the method of Esterbauer and Cheeseman (1990). In brief, about 1 ml of thiobarbituric acid (0.67%) and 0.5 ml of trichloroacetic acid (20%) were added to the supernatant containing 1 mg protein. Then, the mixture was incubated in a boiling water bath for 1 h. After cooling, centrifuging was done for removing the precipitate. The absorbance of the reaction mixture was read at λ = 535 nm against a blank.

Prism (version expressed as using one-way 0.05 was con-

3. Results 3.1. FE-SEM FE-SEM was to investigate the reactant for topographical and surface morphology and introduced materials. Microscopic images of CS showed a smooth surface, which was due to the nature of the natural polymers (Fig. 2A). When CS was mixed with eutectic solvent (Eu), superficial nature changing was observed due to the hydrogen bonding and ionic interactions between Eu and polymeric chain that result in the formation of the granular shapes (Fig. 2B). To determine the effect of Eu on SM nature, FE-SEM images of the mixture were investigated, and results showed that SM nanosize particles have been distributed homogenously in the solvent matrix (Fig. 2C). Therefore, interesting results were observed in the FE-SEM of the final mixture of SM-Eu with CS. Images showed the unique collapsed strings which are the consequent interactions among the three components, namely Eu, SM, and CS, which resulted in the separation of the polymeric chains of CS and loading of SM on them by Eu. Based on the diameter of the strings, it was concluded that loading on the polymeric chains occurred successfully, otherwise, polymeric chains would not have been formed and the diameter of the free polymeric chains would not have been in this range (Fig. 2D).

2.13. Measurements of antioxidant enzymes activities Genet method was implemented for measuring the CAT and SOD activities (Genet et al., 2002). In brief, 20 μl of the supernatant was mixed with 0.48 mM pyrogallol, 0.1 mM EDTA and 50 mM sodium phosphate buffer pH 7.0. The absorbance of the reaction mixture was read at λ = 420 nm for 2 min at 25 °C against a blank. The enzyme activity was expressed as the amount of enzyme inhibiting pyrogallol autoxidation half-maximally. For determination of CAT activity, 20 μl of the supernatant was mixed with 10 mM hydrogen peroxide and 50 mM sodium phosphate buffer pH 7.0. The absorbance of the reaction mixture was read at λ = 240 nm for 5 min at 25 °C against a blank. The CAT activity was expressed as μmole of H2O2 decomposed per minute per mg protein. Pinto method was implemented for measuring the GRx activity (Pinto and Bartley, 1969). The reduction of GSH in the presence 3

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Fig. 2. FE-SEM images of CS (A), CS-Eu (B), SM-Eu (C) and SM–CS–NPs (D).

C]C peaked at 1512 and 1638 cm−1. The absorption bands at about 1737 cm−1 assigned to C]O were related to ketone group, and the ones attributed to stretching vibration of aliphatic C–H groups peaked at 2849, 2928 cm−1, and finally, those related to stretching vibration of

3.2. FT-IR The FT-IR spectrum of pure SM (Fig. 3) showed characteristic stretching vibration peak of C–O bond at 1161 and 1273 cm−1, and for 4

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Fig. 3. FT-IR spectra of CS, SM and SM–CS–NPs.

numerous O–H groups peaked at 3449 cm−1. Also, the FT-IR spectrum of CS has the characteristic peaks at the 1025 and 1076 attributed to stretching vibration peak of C–O–C group. A broad peak at 1600 to 1650 were related to C–N and amidic non-hydrolyzed group, and with respect to stretching vibration of the C–H bonds, they peaked at 2871 cm−1. A broad peak at 3441 were assigned to numerous O–H bonds in CS polymeric chain. Furthermore, SM–CS–NPs composite spectrum which was prepared using melting method showed that broader peaks at 1050 to 1250, 1481, 1632, a small peak at 1730, and broad peaks at 2920 and 3390 related to different etheric bonds, C]C, C]O (amidic), C]O (ketonic), aliphatic C–H and hydrogenic bonds, respectively. In brief, considering FT-IR spectrum of SM–CS–NPs and the fact that characteristic peaks of SM and CS are present in the introduced composite, it is concluded that physical blending was effective in order to form the above-mentioned composite via hydrogenic bonds and electrostatic attraction. The physicochemical properties of SM–CS–NPs with respect to the FE-SEM images and the FT-IR spectrum are summarized in Table 2.

Fig. 4. Effect of SM and SM–CS–NPs pretreatments on immobility time in forced swimming test (A) and the tail suspension test (B). Data are reported as the mean ± S.D. of ten rat in each group. I/R, SM and SM–CS–NPs mean ischemia/reperfusion, silymarin and silymarin-loaded chitosan Nanoparticles, respectively. **P < 0.01, ***P < 0.001 as compared to control group. + + P < 0.01, +++P < 0.001 as compared to I/R group.

3.3. Effects of SM and SM–CS–NPs on immobility time in FST and TST The results depicted in Fig. 4A and B shows that I/R induction significantly increased immobility time in FST and TST compared with the control group. SM and SM–CS–NPs pretreatments significantly reduced (P < 0.01 and P < 0.001) immobility time compared with I/R group in FST and TST. SM–CS–NPs pretreatment caused potentiation in the protective effect of SM (decrease in total immobility time) compared with their effect alone.

Fig. 5. Effect of SM and SM–CS–NPs pretreatments on climbing times in the forced swimming test. Data are reported as the mean ± S.D. of ten rat in each group. I/R, SM and SM–CS–NPs mean ischemia/reperfusion, silymarin and silymarin-loaded chitosan Nanoparticles, respectively. ***P < 0.001 as compared to control group. +++P < 0.001 as compared to I/R group.

3.4. Effects of SM and SM–CS–NPs on climbing time in FST The results depicted in Fig. 5 show that I/R induction significantly decreased climbing time in FST compared with the control group. SM and SM–CS–NPs pretreatments significantly increased the climbing time in FST compared to I/R group (P < 0.001). Table 2 Physicochemical properties of the SM–CS–NPs. Loading capacity or dosage of SM in the SM–CS–NPs

Size of SM–CS–NPs according to SEM images

Functional group of the SM–CS–NPs based on FT-IR data

Weight of introduced SM–CS–NPs = 5 gr Choline+ 2.85 gr glucose+ 1 gr SM+ 15 gr CS = 23.85 gr So dosage is: 1 gr SM/23.85 gr SM–CS–NPs = 0.042 mg SM/gr SM–CS–NPs

Strings diameter were about 87, 90, 103 and so on.

1050 1481 1632 1730 2920 3390

5

to 1250 (C–O) (C]C) (C]O of amidic group) (C]O of ketonic group) (C–H) (O–H)

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Fig. 6. Effect of SM and SM–CS–NPs pretreatments on the infarction volume (%) after I/R injury. The infarction volume is represented as the unstained area. Data are reported as the mean ± S.D. of ten two in each group. I/R, SM and SM–CS–NPs mean ischemia/reperfusion, silymarin and silymarin-loaded chitosan Nanoparticles, respectively. ***P < 0.001 as compared to control group. +++P < 0.001 as compared to I/R group. $$P < 0.01 as compared to SM group.

group (P < 0.001); whereas SM and SM–CS–NPs pretreatments significantly decreased this enhancement expression compared to I/R group (P < 0.001). Also, SM–CS–NPs pretreatment produced significant decrease in both factors compared to SM (P < 0.001).

3.5. Effects of SM and SM–CS–NPs on infarction volume The results depicted in Fig. 6 show that no infarction volume was observed in the control group, while an obviously enhanced lesion area (unstaining area) was observed in I/R group; SM and SM–CS–NPs pretreatments resulted in a significantly decreased (P < 0.001 and P < 0.001) infarction volume compared to I/R group. Moreover, SM–CS–NPs pretreatment significantly decreased infarction volume compared to SM groups (P < 0.01).

4. Discussion Herein, we compared the efficiency of SM and SM–CS–NPs in search of a better therapeutic approach in combating I/R-induced oxidative stress and inflammation in rat brain. In most of the previous studies on I/R, SM has been administered at unusually higher doses because of its poor bioavailability in the brain (Ghosh et al., 2010; Javed et al., 2011). Numerous researches also reported that CS, a novel drug delivery system, could be utilized for efficiently improving oral bioavailability (Fazil et al., 2012; Paolicelli et al., 2009). Moreover, it has been reported that particle size of formulated NPs plays an important role in absorption, passive drug targeting and loading capacity (Ahmad et al., 2016; Gupta et al., 2014; Mukerjee and McBreen, 1998). Thus, we prepared and evaluated SM–CS–NPs in I/R injury in rats. The FE-SEM image confirmed that the size of SM–CS–NPs was optimized at less than 200 nm, and FT-IR confirmed that SM–CS–NPs was successfully prepared. In this study, the therapeutic potency of SM–CS–NPs was evaluated in rats using I/R induced brain injury as an experimental model. BCCAO, which is a well-known experimental model to investigate the complications after I/R (Aggarwal et al., 2010; Cai et al., 2011; Muley et al., 2013; Schmidt-Kastner et al., 2001; Yanpallewar et al., 2005), was used to induce I/R brain injury into rats. An increase in infarction volume and depressive-like behaviors has been reported after I/R. Wei et al. (2015) reported that the risk of depression after stroke is related to lesion location. It has been shown that SM–CS–NPs pretreatment reduced infarction volume in I/R showing protection against I/R-induced damage. In the present study, The results of FST and TST showed that induction of I/R causes depressive-like behaviors. In FST, significantly increased immobility time was observed in I/R injury rats in comparison with the control group. Administration of SM–CS–NPs decreased the immobility time indicating the production of anti-depressant effects in pretreated rats, while weaker effects were observed in SM group. The findings of TST verified the FST results further. Some studies reported increased generation of free radicals and oxidative damage in the rat brain after I/R injury (Chamorro et al., 2016). The protective effect of SM is reported in the model of I/R in various researches. SM has exerted antioxidant effects in renal ischemia/reperfusion injury-induced morphological changes in rat (Turgut et al., 2008). Hou et al. showed the inhibitory activity of SM on oxidative stress and inflammation, induced by the activation of NF-κB and STAT1 (Hou et al., 2010). It has been shown that the activation of antioxidant enzymes is increased by SM, which reportedly shows a membrane stabilizing activity as well (Gaur and Kumar, 2010). In the current paper, oxidative damage significantly resulted in I/R as shown by increased MDA levels besides a noticeable reduction in the activity of

3.6. Effects of SM and SM–CS–NPs on cerebral GSH and MDA levels As shown in Table 3, Induction of I/R caused a significant increase (P < 0.001) in MDA levels and a significant decrease (P < 0.05) in GSH levels compared to the control group. SM and SM–CS–NPs pretreatments showed a significant decrease (P < 0.001) in MDA levels compared to I/R group. On the other hand, SM–CS–NPs pretreatment showed a significant increase (P < 0.001) in GSH levels. 3.7. Effects of SM and SM–CS–NPs on cerebral antioxidant enzymes activities The activities of SOD, CAT, GPx and GRx were significantly reduced in I/R rats compared to the control group. SM–CS–NPs pretreatment significantly enhanced these activities in I/R rats. However, SM pretreatment did not exert any significant effects on CAT, GPx and GRx activities compared to I/R group. SM–CS–NPs group significantly elevated SOD (P < 0.05), CAT (P < 0.05) and GRx (P < 0.01) activities in the cortex compared to SM group, except for one instance; SM–CS–NPs group had no significant effect on GPx activity in the cortex compared to SM group (Table 4). 3.8. Effects of SM and SM–CS–NPs on the expression of inflammatory factors As shown in Figs. 7 and 8, induction of I/R significantly increased the expression of IL-6 and TNF-α in the cortex compared to the control Table 3 Effect of SM and SM–CS–NPs pretreatments on cerebral GSH and MDA levels after I/R injury. Groups

MDA (μg/mg protein)

GSH (mg GSH/gr protein)

Control I/R SM SM–CS–NPs

0.06 0.25 0.11 0.06

0.46 0.16 0.40 0.60

± ± ± ±

0.00 0.01b 0.02c 0.00c

± ± ± ±

0.07 0.02a 0.06 0.06c

Data are reported as the mean ± S.D. of five rat in each group. I/R, SM and SM–CS–NPs mean ischemia/reperfusion, silymarin and silymarin-loaded chitosan Nanoparticles, respectively. a P < 0.05, b P < 0.001 as compared to control group. c P < 0.001 as compared to I/R group. 6

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Table 4 Effect of SM and SM–CS–NPs pretreatments on cerebral anti-oxidant enzymes activities after I/R injury. Groups

CAT (U/mg protein)

SOD (% inhibition)

GPx (U/mg protein)

GRx (U/mg protein)

Control I/R SM SM–CS–NPs

260.20 ± 30.51 51.84 ± 1.88b 100.08 ± 13.27 214.04 ± 29.10ce

88.13 40.13 69.01 86.02

138.17 ± 27.43 66.48 ± 10.80a 111.02 ± 14.92 156.32 ± 21.81c

64.30 16.19 24.43 95.16

± ± ± ±

1.31 3.29b 4.43d 1.24de

± ± ± ±

16.70 2.09a 2.86 21.30cf

Data are reported as the mean ± S.D. of five rat in each group. I/R, SM and SM–CS–NPs mean ischemia/reperfusion, silymarin and silymarin-loaded chitosan Nanoparticles, respectively. a P < 0.05, b P < 0.001 as compared to the control group. c P < 0.01, d P < 0.001 as compared to I/R group. e P < 0.05, f P < 0.01 as compared to SM group.

protect cells from hydroxyl radicals, singlet oxygen, and superoxide radical damage (Sinha et al., 2001). SOD acts as the first line of defense against oxidative stress in catalyzing the dismutation reaction of superoxide anions to hydrogen peroxide and oxygen. As such, SOD, CAT, GPx and GRx, are the known antioxidant enzymes involved in the defense systems of cells exposed to oxygen (Zhan and Yang, 2006). CAT has one of the highest turnover numbers among all enzymes, and any molecule of CAT can convert millions of molecules of hydrogen peroxide/oxygen to water per second (Aguilera et al., 2010). In the current paper, a reduced activity of SOD, CAT, GPx, GRx and GSH levels was observed following I/R injury. Thus, an antioxidant agent would be able to scavenge the excess reactive oxygen species efficiently to prevent this reduced enzyme activity and GSH levels. According to our findings, administrating SM–CS–NPs (but not the free silymarin, SM) significantly prevented decreased SOD, CAT, and GRx activities. The increased SOD, CAT, and GRx activities could be linked with the increased expression of their gene product by SM pretreatment. Studies show that the SOD gene expression is up regulated by SM in liver diseases (Lang et al., 1993; Müzes et al., 1991). Moreover, decreased GSH levels were reversed by SM–CS–NPs (but not the free silymarin, SM) pretreatment. Some studies argue that SM induces GPx (Taleb et al., 2018). According to previous studies (Muley et al., 2012; Raza et al., 2011; Thakare et al., 2016; Turgut et al., 2008), due to the low bioavailability of SM, the antioxidant effects of SM directly correlate with its use at high doses. Therefore, since we used SM at a low does (i.e., 15 mg/kg), it produced almost no significant antioxidant effect, While SM–CS–NPs had significant antioxidant effects than SM at the same dose level. Together with oxidative stress, the inflammatory response is a prime pathological factor in I/R leading to cell death (Ji et al., 2011). Several experimental studies have shown that reperfusion is associated with an inflammatory response leading to TNF-α release and IL-6 induction. In addition, new evidence indicates that increased inflammatory factors contribute to the progression of I/R (Kim et al., 2016; Liu et al., 2017). Microglia and macrophages are believed to be the major production sources of TNF-α in I/R (Zhu et al., 2012). Based on the evidence in this study, I/R injury increased the mRNA expression of TNF-α and IL-6. SM–CS–NPs pretreatment prevented I/R-induced up-regulation of TNFα and IL-6. The distinctive result of this study was that a significant anti-inflammatory effect for SM–CS–NPs was found compared to SM. One possible explanation for the decrease in TNF-α and IL-6 expression may be due to its ability to inhibit the activation of microglia by SM (Jin et al., 2016; Wang et al., 2002).

Fig. 7. Effect of SM and SM–CS–NPs pretreatments on the expression of TNF-α after I/R injury. Data are reported as the mean ± S.D. of three rat in each group. I/R, SM and SM–CS–NPs mean ischemia/reperfusion, silymarin and silymarin-loaded chitosan Nanoparticles, respectively. ***P < 0.001 as compared to control group. +++P < 0.001 as compared to I/R group. $$ $ P < 0.001 as compared to SM group.

Fig. 8. Effect of SM and SM–CS–NPs pretreatments on the expression of IL-6 after I/R injury. Data are reported as the mean ± S.D. of three rat in each group. I/R, SM and SM–CS–NPs mean ischemia/reperfusion, silymarin and silymarin-loaded chitosan Nanoparticles, respectively. ***P < 0.001 as compared to control group. +++P < 0.001 as compared to I/R group. $$ $ P < 0.001 as compared to SM group.

antioxidant enzymes in I/R brain. Pretreatment with SM–CS–NPs reduced the extent of MDA production. Substantial evidence in the literature has demonstrated the potentiality of SM and its metabolites to diminish oxidative stress-induced lipid peroxidation reaction. The decrease in brain MDA levels in this study in SM–CS–NPs pretreatment is possibly due to antioxidant property of SM (Muriel et al., 1992). Normally, the reactive oxygen species generation and the endogenous scavenging system are balanced, which detoxifies the reactive oxygen species. During IR, a sudden increase of reactive oxygen species cannot be handled effectively by the endogenous system that protects neurons in the normal conditions. As a result, the highly reactive oxygen species damages the components of neurons (Sims et al., 2000). GSH is an endogenous antioxidant that can react with free radicals to

5. Conclusion We designed a combined powerful formulation, prepared with silymarin and chitosan nanoparticle, SM–CS–NPs, to prevent I/R injury in rats. The results of the study suggest that SM–CS–NPs exerted its preventive effects more effectively compared to SM. This effect would be due to the increased bioavailability in SM–CS–NPs compared to SM. Thus, SM–CS–NPs could be developed as a potential preventive agent against I/R. It might be worthy of notice that other promising approaches for the 7

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application exist but have not been extensively studied and developed. The results of our study have prompted us to make further investigations to ascertain the in vivo capacity of formulations based on CS-NPs as a drug carrier to improve the low bioavailability of polyphenols.

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