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Abstract

Silymarin (SMN) is used as an antioxidant complex to attenuate the pro-oxidant effects of toxic agents. This study was designed to investigate the impact of a long-term administration of SMN on proinflammatory mediators, oxidative stress biomarkers and on the levels of interleukin-1β (IL-1β) transcript in the hippocampus. A total of 40 adult male Wistar rats were assigned into control and test groups. Animals in the test group were subdivided into four subgroups according to the following treatment profile: carbon tetrachloride (CCl₄, 0.5 ml/kg), SMN 25, SMN 50 and SMN 100 (mg/kg). The animals received the compounds by gastric gavage. Following the 8-week treatment period, animals in the CCl₄ group showed body weight loss, while the test groups except SMN 100 revealed a significant (p < 0.05) positive body weight gain. The levels of nitric oxide (NO) and malondialdehyde (MDA) as pro-oxidant and lipid peroxidation index, respectively, increased in CCl₄- and SMN 100-treated groups, while SMN at lower dose levels did not alter the NO and MDA content. The concentration of total thiol molecules increased in the SMN 50 group and showed a remarkable decrease in CCl₄ and SMN 100 groups. Animals treated with CCl₄ or SMN 100 showed an upregulation of IL-1β, while animals in SMN 25 and SMN 50 groups showed a slight downregulation of expression of IL-1β at the messenger RNA level. These findings suggest that SMN at higher dosage level might exert pro-oxidant effect as an increase in the level of MDA and proinflammatory mediators such as NO, and upregulation of IL-1β in the hippocampus were shown.

Keywords

antioxidant interleukin-1β pro-oxidant proinflammatory mediators silymarin

Introduction

Silymarin (SMN) is a flavonoids complex extracted from the seeds of milk thistle (Silybum marianum). SMN was used traditionally as a raw extract of ripe seeds of S. marianum, and it is currently used as a standardized mixture of silidianin, isosilibinin and silichristin.¹ As silibinin represents up to 80% of the standard formulation of SMN, the pharmacological properties of SMN and SMN-containing products are mainly attributed to silibinin as the most active compound of the mixture. Traditionally, SMN has been used as a natural remedy for digestive problems and in particular for diseases of the liver and the biliary tract, for menstrual disorders and varicose veins.² Other potential indications of SMN are related to its anticancer and anti-inflammatory properties demonstrated predominantly in in vitro assays.^3
–5 For example, it has been reported that a 7-day pretreatment with SMN at a dose of 50 mg/kg body weight (bw) prevents the adriamycin-induced cardiotoxicity and nephrotoxicity in rats, presumably due to an inhibition of lipid peroxidation and protection against glutathione (GSH) depletion.⁶

Various beneficial effects of SMN in humans and animals in both in vivo and in vitro systems mainly attribute to its antioxidant capability. However, there are increasing number of reports indicating that certain flavonoids can act as both antioxidants and pro-oxidants. For example, the antioxidant and pro-oxidant effects of green tea polyphenols and quercetin in a dose- and concentration-dependent manner have been reported.^7,8

Since oxidative stress is one of the main mechanisms in the pathogenesis of neurodegenerative diseases, the growing interest to use the antioxidant compounds and in particular SMN for the prevention and treatment of neurodegenerative diseases would be reasonable.^9–12 Moreover, as the hippocampus is an important part of the brain, which has been implicated in many neurodegenerative diseases including Alzheimer’s disease and schizophrenia,¹³ the current study aimed to clarify the possible antioxidant and pro-oxidant effects of SMN on hippocampus at various dosage levels and relatively long-term administration. Additionally, there is evidence indicating that oxidative stress that is characterized by increase in pro-oxidant and depletion of GSH may result in the initiation of inflammatory reactions.¹⁴ Thus, the second goal of this investigation was to explore the possible effect of long-term administration of SMN on the levels of interleukin-1β (IL-1β) transcript in the hippocampus region. As a positive control, carbon tetrachloride (CCl₄) treatment was selected. The reason for the selection of CCl₄ as a reference pro-oxidant is that not only its hepatotoxicity has long been known but also very recently its oxidative effect in the brain has been reported.¹⁵

Materials and methods

Chemicals

SMN standard (S 0292) and 5.5′-dithiobis-2-nitrobenzoic acid (DTNB) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). Thiobarbituric acid (TBA), phosphoric acid (85%), dimethyl sulfoxide and ethanol were obtained from Merck (Germany). N-butanol was obtained from Carl Roth, GmbH Co. (Germany). TRI reagent was purchased from Applied Biosystems, by life technologies (Nieuwerkerk, The Netherlands). Commercially available standard kits were used for the determination of alkaline phosphatase (ALP, 744, Man Inc. Tehran, Iran) and alanine aminotransaminase (ALT, 10-513, Zist Shimi, Inc. Tehran, Iran). All other chemicals were commercial products of analytical grade. SMN was dissolved in ethanol (10 mg/ml) and diluted in normal saline to obtain the appropriate dosage levels, which are stated below.

Animals and experimental design

A total of 40 adult male Wistar rats (200–220 g) were obtained from the animal resource of the Faculty of Veterinary Medicine, Urmia University. The rats were in good health conditions. The animals were acclimatized for 1 week and both during adaptation and experimental periods the animals had free access to food and water. The experimental protocols were approved by the ethical committee of Urmia University in accordance with the principles of laboratory animal care (AECUU/123/2011). Animals were assigned into control and test groups (n = 8). Animals in the test group are subdivided into the following groups:

CCl₄ group: the animals in this group received CCl₄ (0.5 ml/kg bw);

SMN 25 group: the rats were treated with SMN at dosage levels of 25 mg/kg bw for 8 weeks by gastric gavage;

SMN 50 group: the rats were treated with SMN at dosage levels of 50 mg/kg bw for 8 weeks by gastric gavage and

SMN 100 group: the rats were treated with SMN at dosage levels of 100 mg/kg bw for 8 weeks by gastric gavage.¹⁶

The control group received only normal saline (0.9%, 5 ml/kg) containing the same amount of the test compound solvent during the experiment. Before the experimental procedures, all animals were weighed individually, and this procedure was repeated at the end of the study to evaluate any treatment-related changes in body weight gain.

Serum preparation and tissue sample collection

On day 57, blood samples were obtained by cardiac puncture under light anesthesia, which was provided using diethyl ether. After 1 hour at room temperature, the samples were centrifuged at 3000 r/min for 10 min to obtain the serum. The serum samples were then stored at −20°C until further analyses.

The anesthetized animals were finally euthanized using CO₂ gas. Brain specimen were immediately removed and rinsed with chilled normal saline. The samples were then snap frozen in liquid nitrogen and maintained at −70°C until further biochemical analyses. The hippocampus region of the brain was isolated according to Paxinos and Watson.¹⁷

Determination of the serum levels of ALP and ALT

Serum level of ALP and ALT was measured using commercially available standard kits according to manufacturer’s instructions.¹⁸

NO measurement

The total nitric oxide (NO) content of the hippocampus region of the animals from all the groups was measured according to the Griess reaction.¹⁹ In the Griess reaction, NO is rapidly converted into the more stable nitrite, which in an acidic environment is converted into HNO₂. In reaction with sulphanilamide, HNO₂ forms a diazonium salt, which reacts with N-(1-naphthyl) ethylenediamineċ2HCl to form an azo dye that can be detected by the absorbance at a wavelength of 540 nm. The NO content of the examined organs was expressed as nanomoles per milligram of protein in samples.

Measurement of TTMs

Total levels of sulfhydryl in the hippocampus region of rats were measured as described previously.²⁰ Briefly, 0.3–0.4 g of the hippocampus samples were homogenized in ice-cold potassium chloride (KCl; 150 mM) and the mixture centrifuged at 3000 r/min for 10 min; to 0.2 ml of the supernatant of the tissue homogenate 0.6 ml Tris-ethylendiaminetetraaceticacid (EDTA) buffer (Tris base 0.25 M, EDTA 20 mM, pH 8.2) was added and thereafter 40 μl DTNB (10 mM in pure methanol) were added to the 10-ml glass test tube. The final volume of this mixture was made up to 4.0 ml by an extra addition of methanol. After 15 min of incubation at room temperature, the samples were centrifuged at 3000 r/min for 10 min and ultimately the absorbance of the supernatant was measured at 412 nm. The total thiol molecule (TTM) capacity was expressed as nanomoles per milligram of protein in samples. The protein content of the samples was measured according to Lowry’s method.²¹

MDA determination

To determine the lipid peroxidation rate, malondialdehyde (MDA) content of the collected tissue samples was measured using the TBA reaction as described previously.²² In short, 0.3–0.4 g of the hippocampus samples were homogenized in ice-cold KCl (150 mM) and then the mixture was centrifuged at 3000 r/min for 10 min; 0.5 ml of the supernatant was mixed with 3 ml phosphoric acid (1% v/v) and then following vortex mixing, 2 ml of 6.7 g/l TBA was added to the samples. The samples were heated at 100°C for 45 min and chilled on ice. Finally, 3 ml N-butanol was added and the samples were further centrifuged at 3000 r/min for 10 min. The absorbance of supernatant was measured spectrophotometerically at 532 nm and the concentration of MDA is calculated according to the simultaneously prepared calibration curves using MDA standards. The amount of MDA was expressed as nanomoles per milligram protein of the samples. The protein content of the samples was measured according to Lowry’s method.¹⁸

RNA isolation and RT-PCR

To evaluate the effect of test compounds on the level of messenger RNA (mRNA) of IL-1β in the hippocampus region of brain, total RNA was isolated using the standard TRIZOL method.²³ To avoid genomic DNA contamination, extra care was taken when the colorless aqueous phase is collected after chloroform extraction. The RNA amount was determined spectrophotometrically (260 nm and A260/280 = 1.8–2.0), and the samples were stored at −70°C. For reverse transcriptase–polymerase chain reaction (RT-PCR), complementary DNA (cDNA) was synthesized in a 20-μl reaction mixture containing 1 µg RNA, oligo(dT) primer (1 µl), 5× reaction buffer (4 µl), RNAse inhibitor (1 µl), 10 mM dNTP mix (2 µl) and M-MuLV reverse transcriptase (1 µl) according to the manufacturer’s protocol (Fermentas GMBH, Germany). The cycling protocol for 20 μl reaction mix was 5 min at 65°C, followed by 60 min at 42°C and 5 min at 70°C to terminate the reaction.

Second strand cDNA synthesis

The RT-PCR reaction was carried out in a total volume of 25 µl containing PCR master mix (12.5 μl), forward- and reverse-specific primers (each 0.5 μl), cDNA as a template (1 µl) and nuclease free water (10.5 µl). PCR conditions were run as follows: (a) general denaturation at 95°C for 3 min as 1 cycle, followed by 40 cycles at 95°C for 20 s; (b) annealing temperature (62°C for β-actin and 60°C for IL-1β) for 30 s; (c) elongation: 72°C for 1 min and 72°C for 5 min for β-actin and for IL-1β, respectively. The products of RT-PCR were separated on 1.5% agarose gels containing ethidium bromide and visualized using Gel Doc 2000 system (Bio-Rad).

The specific primers for rat’s IL-1β and β-actin were designed and manufactured by CinnaGen (CinnaGen Co. Tehran, Iran).²⁴ Primer pairs for RT-PCR were as follows:

IL-1β:

Forward primer 5′-GACCTGTTCTTTGAGGCTGAC-3′ and

Reverse primer 5′-TCCATCTTCTTCTTTGGGTATTGTT-3′

β-actin:

Forward primer 5′-CTGACCGAGCGTGGCTACAG-3′ and

Reverse primer 5′-GGTGCTAGGAGCCAGGGCAG-3′

Histopathological examinations

The collected tissue samples from the liver and brain that previously had been stored in 10% buffered formaldehyde were embedded in paraffin and 5–6 µm sections were cut using a rotary microtome and stained with hematoxylin and eosin for light microscopic examinations. To evaluate the level of damages following exposure to CCl₄ and SMN (100 mg/kg), indexes such as fibrosis, edema and infiltration of inflammatory cells were considered.

Statistical analyses

For all results, numerical mean and standard deviation of the measured parameters were calculated. The results of three independent experiments for each assessment were analyzed using Graph Pad Prism software (version 2.01. Graph Pad software Inc. San Diego, California, USA). The comparisons between the groups were made by the analysis of variance followed by Bonferroni post hoc test.

Results

SMN altered the body weight gain and serum level of ALP at lower dose levels

The body weight gain in animals that received CCl₄ in comparison with control group showed a significant (p < 0.05) decline, while the other groups except the rats which received 100 mg/kg of SMN showed a significant (p < 0.05) increase in body weight gain (Table 1). Measuring the levels of ALP and ALT in serum revealed that only CCl₄ could elevate the levels of ALP and ALT and by contrast the serum level of ALP in animals that were treated with 25 mg/kg of SMN showed a slight but significant (p < 0.05) reduction. The serum levels of ALP and ALT in other test groups were not found to be significantly (p > 0.05) different from that in the control group (Table 1).

Table 1.

Effect of SMN on BWG and the serum level of ALP and ALT

Groups	BWG (g)	ALP (U/l)	ALT (U/l)
C	52.7 ± 5.7	1803.9 ± 125.5	41.7 ± 4.6
CCl₄	40.7 ± 4.9^a	3817.4 ± 87.6^a	62.9 ± 8.9^a
SMN 25	76.4 ± 4.5^a	1642.2 ± 25.1^a	37.1 ± 3.4^NS
SMN 50	63.3 ± 4.1^a	1701.7 ± 25.6^NS	35.3 ± 5.2^NS
SMN 100	56.0 ± 6.6^NS	1842.0 ± 22.2^NS	47.9 ± 2.8^NS

BWG: body weight gain; ALP: alkaline phosphatase; ALT: alanine aminotransaminase; NS: not significant; C: control, CCL₄: rats that received CCl₄; SMN: silymarin; SMN 25: rats that received 25 mg/kg SMN; SMN 50: rats that received 50 mg/kg SMN; SMN 100: rats that received 100 mg/kg SMN.

^aSignificant differences (p < 0.05) between control and test groups at the same column.

SMN enhanced the oxidative stress biomarkers at high dosage level

The NO content of the hippocampus in CCl₄-exposed animals and in the rats that were treated with 100 mg/kg of SMN showed a significant (p < 0.01) enhancement, while the levels of NO in other groups of the study either remained the same as in the control group or showed a slight and nonsignificant (p > 0.05) reduction (Figure 1(a)).

Figure 1.

Effect of CCl₄ and various doses of SMN on the levels of (a) NO and (b) TTM (nmol/mg of protein) in the hippocampus; bars represent mean ± SD; n = 8 in each group. Asterisks indicate significant differences (asterisks in NO content indicates p < 0.01 for differences between the control and highlighted groups and asterisks in the level of TTM of the hippocampus indicates p < 0.05 for differences between the control and groups) between control and test groups. Bars represent averaged results from three independent experiments and SD. C: control; CCL₄: rats that received CCl₄; SMN 25: animals that received 25 mg/kg of SMN; SMN 50: animals that received 25 mg/kg of SMN; SMN 100: animals that received 100 mg/kg of SMN; NS: non-significant; SMN: silymarin; NO: nitric oxide; TTM: total thiol molecules; SD: standard deviation.

Comparing the concentration of TTM in the hippocampus of rats revealed that CCl₄ exposure resulted in a nonsignificant (p > 0.05) reduction of TTM content. At the same time, administration of 50 mg/kg of SMN for 8 weeks resulted in a significant (p < 0.05) increase in the level of TTM of the hippocampus. SMN of 100 mg/kg bw during the 8 weeks of treatment period resulted in a slight but significant (p < 0.05) decrease in TTM content of the hippocampus in the rats (Figure 1(b)).

The level of MDA produced in the control and test groups was determined. The MDA content of the hippocampus was only increased significantly (p < 0.05) in the CCl₄ group and in the test group of rats, which were treated with 100 mg/kg of SMN (Figure 2).

Figure 2.

Effect of CCl₄ and different dose levels of SMN on MDA content (nmol/mg of protein) in the hippocampus; bars represent mean ± SD; n = 8 in each group. Asterisks indicate significant differences (p < 0.05) between control and test groups. Bars show averaged results from three independent experiments and SD. C: control; CCl₄: rats that received CCl₄; SMN 25: animals that received 25 mg/kg of SMN; SMN 50: animals that received 25 mg/kg of SMN; SMN 100: animals that received 100 mg/kg of SMN; NS: nonsignificant; SMN: silymarin; MDA: malondialdehyde; SD: standard deviation.

SMN increased the level of mRNA of IL-1β in the hippocampus

The level of mRNA of proinflammatory cytokine IL-1β was determined using real-time PCR technique and the results were normalized against the level of mRNA of β-actin serving as a housekeeping gene. The results of RT-PCR represent a significant (p < 0.01) upregulation of IL-1β in the animals that were exposed to CCl₄ or were treated with 100 mg/kg of SMN for 8 weeks (Figure 3(a)). Figure 3(b) shows the results of the densitometric analyses, which were performed using the software of Molecular Analyst (Bio-Rad, The Netherlands).

Figure 3.

Effect of CCl₄ and SMN on the expression of IL-1β in the hippocampus; (a) represent the level of mRNA of IL-1β and β-actin from corresponding animals and (b) demonstrates the IL-1β mRNA levels that were measured by densitometry and normalized to the expression level of β-actin mRNA. Relative normalized transcript level of the IL-1β in the hippocampus of rat. The values were normalized to β-actin values. Asterisks indicate significant differences (p < 0.01) between control and test groups. Error bars represent the standard deviation of the mean of the measured mRNA levels. C: control, CCl₄: rats that received CCl₄; SMN 25: animals that received 25 mg/kg of SMN; SMN 50: animals that received 25 mg/kg of SMN; SMN 100: animals that received 100 mg/kg of SMN; NS: nonsignificant; SMN: silymarin; IL-1β: interleukin-1β; mRNA: messenger RNA.

Histopathological findings

The normal histological features of the liver and brain from the control rats are demonstrated in Figures 4(a) and (d), respectively. Histopathological findings in the liver of CCl₄-exposed animals represented severe fibrosis, marked edema and remarkable infiltration of inflammatory cells around the vessels (Figure 4(b)). At the same time, CCl₄ exposure resulted in infiltration of inflammatory cells in the brain tissue along with remarkable edema (Figure 4(e)). Histopathological findings in the liver and brain of the animals which received SMN (100 mg/kg) indicate that although there are mild infiltration of inflammatory cells and edema, the severity of the histopathological symptoms, however in comparison with CCl₄-exposed animals, relatively declined (Figure 4(c) and (f)).

Figure 4.

Photomicrograph of rat’s liver and brain; (a) and (d) represent the normal liver and brain tissues, respectively, (b) and (e) are showing the liver and brain tissues from animals that are exposed to CCl₄. Arrows indicate severe edema in both tissues and head arrows show the remarkable fibrosis in the liver. (c) and (f) are showing a normal liver and moderately inflamed brain tissue, respectively, in the animals that received silymarin (SMN) at dose level of 100 mg/kg. Hematoxylin and eosin staining, (magnification: (a), (b), and (c): ×400; (d), (e) and (f): ×100) and scale bar is 0.2 mm.

Discussion

Our results showed that the administration of SMN over a period of 8 weeks at various dose levels resulted in different antioxidant and pro-oxidant effects in the hippocampus. Moreover, SMN at high dosage level (100 mg/kg), similar as CCl₄, upregulated the level of mRNA of IL-1β in the hippocampus region of the brain.

SMN at the levels of 25 and 50 mg/kg bw dose, after 8 weeks resulted in a significant increase in body weight gain in comparison with the control group, while at 100 mg/kg of SMN, the body weight did not change significantly (p > 0.05). At the same time, CCl₄ at 0.5 ml/kg dose level also resulted in body weight loss. It seems that CCl₄ either with inflammation induction and hepatic fibrosis resulted in anorexia or with malabsorption and maldigestion related to hepatic disorders caused negative body weight gain.²⁵ The reason for different effects of SMN at various dosage levels on body weight gain might attribute to its dose-dependent ability, further biochemical and molecular analyses confirmed this hypothesis.

Since CCl₄ but not SMN (100 mg/kg) could remarkably elevate the levels of ALP and ALT, tissue-specific effect of SMN might explain this finding. Although the activity of ALP and ALT is a useful biochemical indicator of the liver diseases, an increase in serum level of ALP may reflect physiologic or pathologic changes beyond those of the hepatic origin.²⁶

SMN at low dose levels (25 and 50 mg/kg) either did not alter the oxidative stress markers such as MDA content or enhanced the level of TTM in hippocampus, indicating its antioxidant effect. SMN also showed the same profile on NO content of the hippocampus at lower dose levels. There are enormous published data indicating that SMN in both in vitro and in vivo systems exerts antioxidant effects.^27,28 Interestingly, SMN at lower dosage level did not change the expression of IL-1β at mRNA level as there was no challenge of inflammatory pathways. This finding is in accordance with previous studies as the anti-inflammatory and antioxidant effects of SMN have been shown in the prevention of sepsis-induced brain damages. SMN at a dosage level of 50 mg/kg reduced the sepsis-induced level of MDA, NO, IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) in the brain of rats.²⁹

The most remarkable finding of the present study is the impact of long-term administration of SMN at high dosage level (100 mg/kg) on oxidant and proinflammatory factors in the hippocampus. A marked increase in NO and MDA content and decrease in the level of TTM of the hippocampus in CCl₄ and SMN 100 groups unlike the lower dose levels of SMN indicate the pro-oxidant effects of CCl₄ and SMN. It has been reported that CCl₄ due to its conversion to CCL₃ ⁻ and acting as a free radical results in a significant reduction in hepatic GSH and therefore acts as a potent pro-oxidant.³⁰ The biochemical changes due to the exposure to CCl₄ were later confirmed by significant increase in the level of mRNA of IL-1β as a well-established proinflammatory marker. Our finding about SMN (100 mg/kg) is against the anti-inflammatory and antioxidative effects of SMN. To explain this, however, one should note that most of the reports about the beneficial antioxidative and anti-inflammatory properties of SMN are associated with its short-term usage and there is indeed lack of knowledge about the impact of long-term administration of SMN in animals and humans. Moreover, it has been documented that most of the phytochemicals, which are used as antioxidant agents, may exert pro-oxidant properties too. Besides, any antioxidant or pro-oxidant effects of phytochemicals at least in in vitro studies could be cell type- and concentration-dependent.³¹ Previous studies indicate that flavonoids that possess multiple hydroxyl groups may exert pro-oxidant effect.³² The obtained pro-oxidant properties of SMN at high given dose, which is characterized by a significant increase in NO and MDA and remarkable decrease in TTM in the hippocampous region of brain, may be explained by various dose-dependent effects of SMN. It is interesting to note that different compounds of SMN mixture possess more than four hydroxyl groups that may explain the pro-oxidant effect of SMN at higher dose levels.

Recently, it has also been reported that SMN exerts different effects in various regions of the brain because SMN increased the reactive oxygen species content of the hippocampus and declined them in the cortex region of the aged rats. Moreover, previous studies demonstrated that the protective effect of SMN is varied between aged and young animals.³³

It has been shown that SMN exerts an anti-inflammatory effect by the downregulation of inflammation-associated proteins such as inducible NO synthase, cyclooxygenase-2 and myeloperoxidase, transcriptional factors including nuclear factor-κβ and signal transducer and activator of transcription-1, and the prevention of proinflammatory cytokines (e.g. IL-1β and TNF-α) production.³⁴ Our results concerning the level of mRNA of IL-1β in the hippocampus of the animals, which received 100 mg/kg of SMN, are not in agreement with previous data as the expression of IL-1β as a proinflammatory cytokine gene was markedly upregulated. The reason for this controversy may be related to huge variation between the dosage level and also the treatment period.

Our histopathological findings support the molecular results; as the infiltration and edema of inflammatory cells were the dominant features of the liver and brain tissues in animals, which received CCl₄, indicating that CCl₄ is able to induce inflammation not only in the liver but also in the brain too. On the other hand, the protective property of SMN (100 mg/kg) in the liver but not in the brain suggests that SMN might have tissue specific effects with different pathways.

Since the administration of SMN often takes place for a long period of time for various purposes such as the improvement of high-fat diet–induced hepatic injuries and oxidative stress³⁵ and to decrease the chronic alcohol-induced injuries³⁶; thus, it would be noteworthy that in case of the long-term administration of SMN at least at higher doses (100 mg/kg), the biomarkers such as level of proinflammatory mediators in serum and some vital organs be monitored.

Taken together, this study showed that unlike the lower dosage levels, long-time administration of high dosage (100 mg/kg) of SMN might cause pro-oxidant effects on organs such as hippocampus. Moreover, biochemical and molecular changes due to long-term usage of high-dose SMN might attribute to the augmentation of NO and MDA content and equally to the upregulation of proinflammatory mediators such as IL-1β at mRNA level.

Footnotes

Acknowledgement

The authors thank Mr SH Tabatabaie and Mr F Farhangpajoh for their valuable technical assistances in this study.

Authors' Note

This study was conducted in the Faculty of Veterinary Medicine of Urmia University.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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Long-term administration of Silymarin augments proinflammatory mediators in the hippocampus of rats: Evidence for antioxidant and pro-oxidant effects

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Animals and experimental design

Serum preparation and tissue sample collection

Determination of the serum levels of ALP and ALT

NO measurement

Measurement of TTMs

MDA determination

RNA isolation and RT-PCR

Second strand cDNA synthesis

Histopathological examinations

Statistical analyses

Results

SMN altered the body weight gain and serum level of ALP at lower dose levels

SMN enhanced the oxidative stress biomarkers at high dosage level

SMN increased the level of mRNA of IL-1β in the hippocampus

Histopathological findings

Discussion

Footnotes

Acknowledgement

Authors' Note

Funding

References