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Neuroprotective effect of cerium oxide nanoparticles in a rat model of experimental diabetic neuropathy
Accepted Manuscript Title: Neuroprotective effect of cerium oxide nanoparticles in a rat model of experimental diabetic neuropathy Authors: Rezvan Najafi, Asieh Hosseini, Habib Ghaznavi, Saeed Mehrzadi, Ali M. Sharifi PII: DOI: Reference:
S0361-9230(17)30056-4 http://dx.doi.org/doi:10.1016/j.brainresbull.2017.03.013 BRB 9192
To appear in:
Brain Research Bulletin
Received date: Accepted date:
2-2-2017 29-3-2017
Please cite this article as: Rezvan Najafi, Asieh Hosseini, Habib Ghaznavi, Saeed Mehrzadi, Ali M.Sharifi, Neuroprotective effect of cerium oxide nanoparticles in a rat model of experimental diabetic neuropathy, Brain Research Bulletinhttp://dx.doi.org/10.1016/j.brainresbull.2017.03.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Neuroprotective effect of cerium oxide nanoparticles in a rat model of experimental diabetic neuropathy
Rezvan Najafi1, Asieh Hosseini2, Habib Ghaznavi2, Saeed Mehrzadi2, , Ali M. Sharifi2,3*
1. Research Center for Molecular Medicine, Hamadan University of Medical Sciences, Hamadan, Iran 2. Razi Drug Research Center, and department of pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. 3. Endocrine and Metabolism Research Institute, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.
Word count: 2487
Correspondence to: Dr Ali M. Sharifi, Dept. of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. Telefax: (+98) 021 88622523 E-mail: [email protected]
Highlights:
CeO2 nanoparticles increased body weight, total thiol molecules, total antioxidant power and ADP/ATP ratio in diabetic rat.
CeO2 nanoparticles decreased lipid peroxidation and nociception latency in STZ-treated rats.
CeO2 nanoparticles improved the histopathology and morphological abnormalities of DRG neurons. 1
Abstract Objective: Neuropathies are a nerve disorders that caused by diabetes. Neuropathy affects over 50 percent of diabetic patients. High blood glucose and their toxic byproducts are the main causes for nerve dysfunction. In the present study, we examined the neroprotective effects of cerium oxide (CeO2) nanoparticles in diabetic rats. Method: Rats divided into four groups: control group, diabetic group, the diabetic group treated with CeO2 nanoparticle at a dose of 65 mg/kg and diabetic group received CeO2 nanoparticle at a dose of 85 mg/kg. Diabetes was induced by single intraperitoneal injection of 65 mg/kg streptozotocin (STZ). 8 weeks after the induction of diabetes, body weight and pain sensitivity in all groups were measured. The blood sample was collected for biochemical analysis. The dorsal root ganglion (DRG) neurons were isolated for histopathological stain and morphometric parameters studies. Results: Reduction of body weight, total thiol molecules (TTM), total antioxidant power (TAP) and ADP/ATP ratio in diabetic rat was reversed by CeO2 nanoparticles administration. We showed that lipid peroxidation (LPO) and nociception latency were significantly increased in STZ-treated rats and decreased after CeO2 nanoparticles administration. DRG neurons showed obvious vacuole and various changes in diameter, area and the count of A and B cells in STZ-diabetic rat. CeO2 nanoparticles improved the histopathology and morphological abnormalities of DRG neurons. Conclusion: Our study concluded the CeO2 nanoparticles have a protective effect against the development of DN.
Keywords: Dorsal Root Ganglion; CeO2 nanoparticles; Diabetic Neuropathy; High Glucose; Oxidative Stress
2
1. Introduction Diabetes mellitus (DM) is a major health problem characterized by defects in insulin secretion, insulin resistance, or both (Whalen et al., 2015). DM is a metabolic disorders that induces microvascular complication such as nephropathy, retinopathy and neuropathy and macrovascular complication including ischemic heart disease (Sayin et al., 2015). High glucose - induced oxidative stress has been shown as one of the main links between diabetes and its complication. In chronic hyperglycemia increases the generation of reactive oxygen species (ROS) through glucose auto- oxidation and protein glycosylation (Mrowicka, 2005). Once increase the production of ROS, they react with macromolecules including carbohydrate, lipid, protein and DNA leading to the loss of their function (Sheikh et al., 2010). In addition, ROS induce various cellular signaling pathways that lead to development of complication of diabetes. NF-kB in response to an increase in ROS generation, enhance the transcription of pro-inflammatory cytokines and chemokines such as MCP-1, IL-1β, IL-6 and TNF-α (Oyenihi et al., 2014). Diabetic neuropathy (DN) may be the most common complication that present in 60-70% of diabetic patients. DN occurs when there is an imbalance between nerve damage and repair. The mechanism responsible for DN includes oxidative stress, polyol pathway, nonenzymatic glycolation and the protein kinase C pathway .Therefore, it is seen that antioxidant therapy may help prevent or delay neuropathy as well as other diabetes complication (Vincent et al., 2004). Nanoparticls due to having unique mechanical, electrical, chemical, optical and biological properties are an ideal therapeutic agent for treatment of various diseases (Bodapati, 2011). Some nanoparticles have antioxidant properties and act by scavenging free radicals. Among them, CeO2 nanoparticles play a major role because of the high potential in free radical scavenging (Mohammad et al., 2008). CeO2 nanoparticles present in both trivalent (Ce3+) and tetravalent (Ce4+) state that allowing the CeO2 nanoparticles release and store oxygen.
In surface of CeO2 nanoparticle, oxygen vacancies interact with free radical,
mimicking the antioxidant enzymes activities including catalase and super oxide dismutase (Dunnick et al., 2015; Rubio et al., 2015). 3
The present study was designed to examine the protective effects of CeO2 nanoparticles on diabetic neuropathy in rats.
2. Materials and methods 2.1. Experimental animals A total of 28 Male Wistar rats with 2-3 months of age and weighing 180 - 250 g were obtained from the animal facility of the Iran University of Medical Science. Rats were housed in individual stainless steel cages at a 45 - 55% humidity and temperature of 20 - 22°C under a 12 h light/dark cycle. All rats were fed with standard rodent water and diet. This study was approved by the Ethical Committee of Iran University of Medical Sciences base on National Institutes of Health Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985).
2.2. Experimental design Diabetes induced by a single intraperitoneal injection of 65 mg/kg STZ in citrate buffer. The control group received citrate. Blood samples were obtained 3 and 7 days after the STZ injection via tail vein and glucose levels were measured using a glucometer. Rats with a blood glucose level of >200 mg/dl was considered diabetic. The fasting blood glucose level in the nondiabetic rats was 85 ±5 mg/dl. Diabetic rats were randomized into 3 groups (n=7): group 1, diabetic control; group 2, treated with 65 mg CeO2 nanoparticles /kg body weight orally; group 3, treated with 85 mg CeO2 nanoparticles / kg body weight orally. Group 2 and 3 received CeO2 nanoparticles for one week before and one week after of STZ injection. 8 weeks after conformation of diabetes, DN occurred (Hosseini et al., 2011). All experiments were performed 8 weeks after injection of STZ. Blood glucose and body weight were examined before and at the end of the experimental period.
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2.3. CeO2 nanoparticles Characterization CeO2 nanoparticles was obtained from Navarrean Nenoproducts Technology (Spain). Field emission scanning electron microscopy was performed by FE-SEM, Hitachi, Japan S-4160. The zeta potential and particle size distribution were measured by a Nano Z-Sizer (Malvern Instrument Zen 3600).
2.4. Hot plate test A hot plate test used to measure pain sensitivity (Socrel Hot plate model DS37, Ugo Basile, Italy). Rats were placed on the plate with the diameter of 19 cm, height of 30 cm and temperature of 52°C ± 2°C. Response time to thermal pain was calculated from the onset of the test and front legs licking or jumping. Maximum time was considered 60 Sec (Karami et al., 2011).
2.5. Sample preparation The animals were anesthetized with intraperitoneal injection of ketamine and xylazin and blood sample were collected from the heart and centrifuged at 1200 g for 10 min at 4 °C for separating the plasma. The plasma samples were frozen at -80 °C for biochemical analysis. For tissue preparation the DRG was isolated and fixed into 10% paraformaldehid and then embedded in paraffin. The 40 μm sections were stained with Hematoxylin-Eosin (H&E) and assessed by light microscope. We used four sections per sample.
2.6. Morphometry of DRG neurons DRG neurons were defined as A or B cell (Hosseini et al., 2011). A cell has a light nucleus with one large nucleolus in central and granular cytoplasm. The nucleus of B cell is light and contains multiple nucleolus that peripherally located. The cytoplasm of B cell is dark, more homogenous and intensively stained compare to A cell. In this study we determined the diameter, number and area of A and B cells in all groups.
5
2.7. Measurement of intracellular ADP/ATP ratio The frozen DRG was homogenized in ice and centrifuged at 12,000g for 15 min at 4 °C. The supernatant was neutralized with 3M KOH in 1.5M Tris, and centrifuged. The supernatants were separated using reverse-phase HPLC. Chromatographic separation was done by column (SUPELCO- SILTMLC-18-T) with a flow rate of 1.2 ml/min and ammonium dihydrogen phosphate (75 mM) as mobile phase. The adenine nucleotides were assessment at 254 nm (µg/ml per mg of tissue) and changes in energy was reported as the ADP/ATP ratio (Najafi et al., 2015).
2.8. Measurement of lipid peroxidation (LPO) LPO was assessed by the reaction of thiobarbituric acid with malondialdehyde (MDA). The sample was mixed with Trichloroacetic Acid (20%), centrifuged at 1500 g for 10 min. the precipitate was dissolved in H2SO4 (0.05 M). TBA (0.2% in sodium sulfate) was added and the sample was incubated in a boiling water bath for 30 minutes. At the end, LPO was extracted by n-butanol and its absorbance was measured at a wavelength of 532 nm (Ghaznavi et al., 2015).
2.9. Measurement of total thiol molecules (TTM): TTM was measured by the reaction of DTNB with thiol molecules that forms a yellow complex. Samples were mixed with Tris-EDTA buffer and subsequent mixed with DTNB (10 mM). After 20 min the absorbance of samples were determined at 412 nm (Hosseini et al., 2015).
2.10. Measurement of total antioxidant power (TAP) Reduction of ferric ions (Fe+3) to ferrous ions (Fe+2), by the samples represents of antioxidant power. TAP was assessed by FRAP test. In this method, reaction of Fe+2 and 2, 4, 6-tris (2-pyridyl)-1, 3, 5-triazine (TPTZ) generates a blue color with a maximum absorbance at 593 nm (Najafi et al., 2015).
2.11. Statistical Analysis 6
Data were presented as the mean ± standard error of the mean (SEM). The significance of the differences between groups was analyzed using one-way ANOVA and Tukey’s posthoc tests by Stats Direct version 2.7.8. A P <0.05 was considered to be statistically significant.
3. Results 3.1. CeO2 nanoparticles characterization The Result from Dynamic light scattering (DLS) confirmed size distribution of CeO2 nanoparticles were ranged from 18.2 to 50.7 (Fig. 1a). Zeta potentials of CeO2 nanoparticles were 6.25. SEM imaging confirmed poly- dispersion of these nanoparticles (Fig. 1b).
3.2. Effect of CeO2 nanoparticles on Body Weight 8 weeks after STZ injection, diabetic rat exhibited significant loss of weight as compared with non diabetic rats in diabetic rats (p< 0.01). Treatment with CeO2 nanoparticles at a concentration of 65 mg/kg improved the body weight (p< 0.05). There is no significant changes in group received 85 mg/kg CeO2 nanoparticles compare to diabetic rat (Fig. 2).
3.3. The effect of CeO2 nanoparticles on the nociceptive threshold in Hot plate examination At the end of 8 the week, diabetic rats exhibited significant increase in pain threshold as compared with the control group (p<0.001). As shown in Fig. 3, CeO2 nanoparticles administration at the 65mg/kg and 85 mg/kg concentration significantly decreased the latency time as compared to untreated diabetic rat (p<0.001 and p< 0.05 respectively)
3.4. The effect of CeO2 nanoparticles on of DRG neurons observations As shown in Fig. 4, the DRG neurons are smaller, more basophilic and have prominent vacuoles in DN rats compared to control group. Changes in DRG neurons were reversed by CeO2 nanoparticles administration. 7
3.5. The effect of CeO2 nanoparticles on morphometric parameters of A and B neurons In DN rat the, A cells count was significantly decreased while B cell count had increased compared to non diabetic rats (p< 0.05). Diameter and area of A cells and B cells were markedly decreased in DN compared to control group. The effect of diabetes was inhibited by CeO2 nanoparticles, but we did not observe any significance difference in diameter of B cell in CeO2 nanoparticles (85 mg/kg) in comparison with DN rats (Table 1).
3.6. Effect of CeO2 nanoparticles on the plasma LPO, TAP and TTM level: In control diabetic rat the MDA levels were increased in compare to control group (p< 0.001), whereas the level of TTP and FRAP decreased in these conditions (p< 0.001). Treatment of rats with CeO2 nanoparticles, markedly reduced lipid peroxidation, and reversed high glucose-induced increase in TTM and TAP compared to control diabetic rat (Table 2).
3.7. Effect of CeO2 nanoparticles on intracellular ADP/ATP ratio: A marked decrease in the ADP / ATP ratio was observed in diabetic rat as compared with non diabetic rat (p< 0.001). Administration of CeO2 nanoparticles for 8 weeks resulted in a significant recovery in the ADP/ATP level in comparison to the control diabetic rats (Fig. 5).
4. Discussion DN as the most common complication of diabetes is a type of nerve injury caused by diabetes. Several causative factors involved in pathogenesis of DN such as hyperglycemia and oxidative stress (Vinik, 2008). Glucose metabolism pathway is responsible for the development of microvascular complications. These include: 1) nonenzymatic glycation of protein 2) increased the activity of polyol pathway 3) activation of PKC signaling pathway 4) increased hexosamin pathway (Feldman, 2003) . Thus increasing of these pathways linked to hyperglycemia- mediated ROS over production through the mitochondrial electron transport chain which have delicious effects on the nerve (Greene et al., 2000). It seems that 8
sensory neuron is a primary target for neural complication of diabetes. Oxidative stress exerts more severe effect at the DRG neuron that as the nerve (Schmeichel et al., 2003). Previous studies indicated that the STZ- injected diabetes rat model used in our study revealed morphological and functional features of the DN (Hosseini et al., 2011). In this research, we examined the DRG morphometry, sensory nerve function and antioxidant status in diabetic rats. Our results showed a toxic effect of high glucose leading to neurotoxicity. Note after the administration of CeO2 nanoparticles these factors were considerably improved. Indeed, this study indicated for the first time that CeO2 nanoparticles can be used as an effective therapeutic regenerative agent which inhibits the nerve damage caused by diabetes. CeO2 nanoparticles have unique advantages compare to other antioxidants including: the ability of regeneration of free radicals scavenging function through spontaneously moving between reduction and oxidation status, having a large number of active sites that used for scavenging of free radical and the ability to pass cross the blood - brain barrier due to their small size thereby make CeO2 nanoparticles an appropriate option for treatment of neural injury (Korsvik et al., 2007; Rzigalinski et al., 2006). Protective effects of CeO2 nanoparticles have been investigated in several studies in vitro and invivo. The results have confirmed the idea that CeO2 nanoparticles play an effective role in the attenuation of oxidative damage in biological tissue (Amin et al., 2011; Dowding et al., 2014; Estevez et al., 2011; Ghaznavi et al., 2015; Hosseini et al., 2013; Hosseini et al., 2014). In the present study, the nocieptive threshold was markedly higher than the control group. Ceo2 treatment restores pain threshold. DN is associated with elevated thermal perception thresholds that eventually lead to the sensory loss and degeneration of fibers in the peripheral nerve. Several pathogenesis mechanisms involved in this phenomenon such as increased aldose reductase, PKC, PARP, oxidative stress and downstream effectors of ROS and oxidative stress (Calcutt et al., 2004; Obrosova, 2009). All these pathways results in TNF-α and NF-KB activation and COX2 induction. COX2 expression leads to change in prostaglandin profile with enhanced production of vasoconstricting PGH2, PGF2α and thromboxane and increased production of vasodilatory prostacyclin. COX2 also increases the ROS generation that 9
exacerbated oxidative stress. On the other hand, hyperglycemia-increased TNF-α increased the permeability of microvascular and nerve damage (Kuhad et al., 2008; Pop‐Busui et al., 2006). Sensory loss in diabetic rat can be prevented not only by several antioxidants such as alpha lipoic acid, but also by agents that able to counteracting downstream effectors such as PARP (Cameron et al., 2001; Li et al., 2005). Therefore free radical scavengers such as CeO2 nanoparticles can be act as a powerful analgesic agent for neuropathy pain. The other finding of this study is an increase of B cell number and reduction of A cell count and volume of the DRG neuron. Myelinated fibers connect mostly to type A (The large neurons) and unmyelinated fibers connect mostly to B type (the small neurons) of DRG neuron. conduction velocity has a direct relation to the size of neurons (Kishi et al., 2002). Modification of protein by ROS decrease protein function, slowed axonal transport, decrease intermediate growth factor delivery from the synapse to the cell body, leading to induction of apoptosis (Metodiewa and Kośka, 1999). In addition, oxidative stress leads to activation of poly ADP –ribos polymers pathway which control the expression of genes involved in neuronal dysfunction (Kuhad and Chopra, 2009). In the current study, we evaluated the biochemical parameters of oxidative stress. STZ- injected rat had a significantly higher MDA level compare to non diabetic rat. Administration of CeO2 nanoparticles caused a considerable reduction in the MDA concentration. Polyunsaturated fatty acids located in the plasma membrane as well as proteins and DNA, are major targets for peroxidation by ROS. MDA as a major end product of lipid peroxidation uses to assess the tissue injury by free radicals (Pizzimenti et al., 2010). Previous studies showed that the increased MDA concentration in diabetic rat reduced by CeO2 nanoparticles (Pourkhalili et al., 2011) . Moreover, we observed that CeO2 nanoparticles at varying doses increase the level of TTM and TAP. It seems that the ROS scavenging effect of CeO2 nanoparticles is responsible for the improvement of TTM. Thiol molecule contains a sulphydryl group. Thiols are the major compound of the total body antioxidants include glutathione, cystein, taurine and methionine that play an essential role in defense against free radicals (Mungli et al., 2009). 10
In this study, we observed that ADP/ATP ratio increased in diabetic rat, whereas CeO2 nanoparticles administration restored this change. Previous studies reveal the ability of CeO2 nanoparticles in increasing the level of ATP (Hosseini et al., 2014; Pourkhalili et al., 2012). The ADP/ATP ratio is a authentic index for the determination of cell death and cell survival (Schütt et al., 2012). hyperglycemia increased mitochondrial ROS generation and subsequent decreased mitochondrial membrane potential ultimately leading to reduced ATP synthesis (Herrera et al., 2001). It seems that CeO2 nanoparticles increase ATP levels by limiting the mitochondrial ROS generation and oxidative stress. Based on the present study, we conclude that CeO2 nanoparticles as an antioxidant agent can be used for treatment of diabetic neuropathy. However, further research is required to explore.
Acknowledgment This study was supported by a grant from Iran University of Medical Sciences
Conflict of interest: None.
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Whalen, K., Miller, S., Onge, E.S., 2015. The Role of Sodium-Glucose Co-Transporter 2 Inhibitors in the Treatment of Type 2 Diabetes. Clinical therapeutics.
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(a)
(b)
Fig.1. CeO2 nanoparticles characterization. (a) Histogram of CeO2nanoparticles size distribution. (b) SEM image of CeO2nanoparticles.
17
Weight (g)
400
†
**
300 200 100
)
rt ic le
(8 5
m
g/ kg
) g/ kg m pa no na
eO 2 C
C
eO 2
na
no
pa
D
rt ic le
(6 5
ia be tic
C
co
on tr ol
nt ro l
0
Fig. 2. Protective effects of CeO2 nanoparticles on weight of DN rats after two months. Values are given by mean ± S.E.M of seven animals. **P < 0.01 vs control group; †P < 0.05 vs diabetic group.
18
25
***
Latency (S)
20
†
††
15 10 5
) g/ kg m
rt ic le s
(8 5
m pa no na 2 eO C
C
eO
2
na
no
pa
D
rt ic le s
(6 5
ia be tic
C
co
g/ kg
)
nt ro l
on tr ol
0
Fig. 3. Effects of CeO2 nanoparticles on latency in diabetic rats after two months Values are given by .
mean ± S.E.M of six animals. ***P < 0.001 vs control group; †P < 0.05, ††P < 0.01 vs diabetic group.
19
(A)
(B)
20 µm
20 µm
(C)
(D)
20 µm
20 µm
Fig. 4. H&E-stained DRG neurons. (A) Control group, (B) diabetic neuropathy group, (C) Nanocerium oxide (65 mg/kg) + STZ and (D) Nanocerium oxide (85 mg/kg) + STZ groups. (A cell= red arrow, B cell= yellow arrow).
20
ADP/ATP ratio
10
*** ††
8 6
†††
4 2
) g/ kg
)
m
g/ kg
(8 5
m rt ic le s
2 eO C
C
eO
2
na
na
no
no
pa
pa
D
rt ic le (6 5
ia be tic
co
co nt ro l
nt ro l
0
Fig. 5. Effects of CeO2 nanoparticles on ADP/ATP level in diabetic rats after two months Values are .
given by mean ± S.E.M of six animals. ***P < 0.001 vs control group; †††P < 0.001, ††P < 0.01 vs diabetic group.
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Table 1. The effect of CeO2 nanoparticles on morphometric parameters of A and B neurons
control Number of A cells
Diabetic control ***
22.71 ± 0.82
11.71 ± 0.55
Number of B cells
5.786 ± 0.4824
14.93 ± 0.5393
Diameter of A cells (µm)
181.4 ± 2.251
117.5 ± 3.582
Diameter of B cells (µm)
84.69 ± 2.4
61.57 ± 2.54
Area of A cells (µm2) Area of B cells (µm2)
20290 ± 7552 1150 ± 28.65
***
***
***
13160 ± 477.8 829.5 ± 25.38
***
***
CeO2 nanoparticles (65 mg/kg) †††
19.57 ± 0.7
†††
8.500 ± 0.3743 165.0 ± 4.028
†††
72.98 ± 1.62
††
18010 ± 476.7
†††
†††
1079 ± 42.38
CeO2 nanoparticles (85 mg/kg) ††
15.21 ± 0.52
††
12.14 ± 0.6535 134.9 ± 2.184
64.81 ± 1.3 15700 ± 466.4
†
†
956.9 ± 24.54
Data are represented as mean ± SEM (n = 4). ***P < 0.05 vs control group; †P< 0.05, ††P< 0.01, 0.001 vs diabetic neuropathy group.
22
††
†††
P<
Table 2. Effect of CeO2 nanoparticles on the plasma LPO, TAP and TTM level
Groups
LPO (µM)
TAP (mM)
TTM (nM)
control
2.594±0.25
725.0±10.41
0.7960± 0.04
Diabetic control
5.445±0.13
550.0±12.91***
0.3928± 0.02***
Nanocerium oxide (65 mg/kg)
4.001±0.1
672.3±8.27†
0.6845± 0.02††
Nanocerium oxide (85 mg/kg)
4.344±0.21
669.0±13.91†
0.6283± 0.07†
***
†††
††
Data are represented as mean ± SEM (n = 4). ***P < 0.001 vs control group; †P< 0.05; ††P < 0.01 , †††P< 0.001 vs group was treated only glucose
23
S0361-9230(17)30056-4 http://dx.doi.org/doi:10.1016/j.brainresbull.2017.03.013 BRB 9192
To appear in:
Brain Research Bulletin
Received date: Accepted date:
2-2-2017 29-3-2017
Please cite this article as: Rezvan Najafi, Asieh Hosseini, Habib Ghaznavi, Saeed Mehrzadi, Ali M.Sharifi, Neuroprotective effect of cerium oxide nanoparticles in a rat model of experimental diabetic neuropathy, Brain Research Bulletinhttp://dx.doi.org/10.1016/j.brainresbull.2017.03.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Neuroprotective effect of cerium oxide nanoparticles in a rat model of experimental diabetic neuropathy
Rezvan Najafi1, Asieh Hosseini2, Habib Ghaznavi2, Saeed Mehrzadi2, , Ali M. Sharifi2,3*
1. Research Center for Molecular Medicine, Hamadan University of Medical Sciences, Hamadan, Iran 2. Razi Drug Research Center, and department of pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. 3. Endocrine and Metabolism Research Institute, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.
Word count: 2487
Correspondence to: Dr Ali M. Sharifi, Dept. of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. Telefax: (+98) 021 88622523 E-mail: [email protected]
Highlights:
CeO2 nanoparticles increased body weight, total thiol molecules, total antioxidant power and ADP/ATP ratio in diabetic rat.
CeO2 nanoparticles decreased lipid peroxidation and nociception latency in STZ-treated rats.
CeO2 nanoparticles improved the histopathology and morphological abnormalities of DRG neurons. 1
Abstract Objective: Neuropathies are a nerve disorders that caused by diabetes. Neuropathy affects over 50 percent of diabetic patients. High blood glucose and their toxic byproducts are the main causes for nerve dysfunction. In the present study, we examined the neroprotective effects of cerium oxide (CeO2) nanoparticles in diabetic rats. Method: Rats divided into four groups: control group, diabetic group, the diabetic group treated with CeO2 nanoparticle at a dose of 65 mg/kg and diabetic group received CeO2 nanoparticle at a dose of 85 mg/kg. Diabetes was induced by single intraperitoneal injection of 65 mg/kg streptozotocin (STZ). 8 weeks after the induction of diabetes, body weight and pain sensitivity in all groups were measured. The blood sample was collected for biochemical analysis. The dorsal root ganglion (DRG) neurons were isolated for histopathological stain and morphometric parameters studies. Results: Reduction of body weight, total thiol molecules (TTM), total antioxidant power (TAP) and ADP/ATP ratio in diabetic rat was reversed by CeO2 nanoparticles administration. We showed that lipid peroxidation (LPO) and nociception latency were significantly increased in STZ-treated rats and decreased after CeO2 nanoparticles administration. DRG neurons showed obvious vacuole and various changes in diameter, area and the count of A and B cells in STZ-diabetic rat. CeO2 nanoparticles improved the histopathology and morphological abnormalities of DRG neurons. Conclusion: Our study concluded the CeO2 nanoparticles have a protective effect against the development of DN.
Keywords: Dorsal Root Ganglion; CeO2 nanoparticles; Diabetic Neuropathy; High Glucose; Oxidative Stress
2
1. Introduction Diabetes mellitus (DM) is a major health problem characterized by defects in insulin secretion, insulin resistance, or both (Whalen et al., 2015). DM is a metabolic disorders that induces microvascular complication such as nephropathy, retinopathy and neuropathy and macrovascular complication including ischemic heart disease (Sayin et al., 2015). High glucose - induced oxidative stress has been shown as one of the main links between diabetes and its complication. In chronic hyperglycemia increases the generation of reactive oxygen species (ROS) through glucose auto- oxidation and protein glycosylation (Mrowicka, 2005). Once increase the production of ROS, they react with macromolecules including carbohydrate, lipid, protein and DNA leading to the loss of their function (Sheikh et al., 2010). In addition, ROS induce various cellular signaling pathways that lead to development of complication of diabetes. NF-kB in response to an increase in ROS generation, enhance the transcription of pro-inflammatory cytokines and chemokines such as MCP-1, IL-1β, IL-6 and TNF-α (Oyenihi et al., 2014). Diabetic neuropathy (DN) may be the most common complication that present in 60-70% of diabetic patients. DN occurs when there is an imbalance between nerve damage and repair. The mechanism responsible for DN includes oxidative stress, polyol pathway, nonenzymatic glycolation and the protein kinase C pathway .Therefore, it is seen that antioxidant therapy may help prevent or delay neuropathy as well as other diabetes complication (Vincent et al., 2004). Nanoparticls due to having unique mechanical, electrical, chemical, optical and biological properties are an ideal therapeutic agent for treatment of various diseases (Bodapati, 2011). Some nanoparticles have antioxidant properties and act by scavenging free radicals. Among them, CeO2 nanoparticles play a major role because of the high potential in free radical scavenging (Mohammad et al., 2008). CeO2 nanoparticles present in both trivalent (Ce3+) and tetravalent (Ce4+) state that allowing the CeO2 nanoparticles release and store oxygen.
In surface of CeO2 nanoparticle, oxygen vacancies interact with free radical,
mimicking the antioxidant enzymes activities including catalase and super oxide dismutase (Dunnick et al., 2015; Rubio et al., 2015). 3
The present study was designed to examine the protective effects of CeO2 nanoparticles on diabetic neuropathy in rats.
2. Materials and methods 2.1. Experimental animals A total of 28 Male Wistar rats with 2-3 months of age and weighing 180 - 250 g were obtained from the animal facility of the Iran University of Medical Science. Rats were housed in individual stainless steel cages at a 45 - 55% humidity and temperature of 20 - 22°C under a 12 h light/dark cycle. All rats were fed with standard rodent water and diet. This study was approved by the Ethical Committee of Iran University of Medical Sciences base on National Institutes of Health Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985).
2.2. Experimental design Diabetes induced by a single intraperitoneal injection of 65 mg/kg STZ in citrate buffer. The control group received citrate. Blood samples were obtained 3 and 7 days after the STZ injection via tail vein and glucose levels were measured using a glucometer. Rats with a blood glucose level of >200 mg/dl was considered diabetic. The fasting blood glucose level in the nondiabetic rats was 85 ±5 mg/dl. Diabetic rats were randomized into 3 groups (n=7): group 1, diabetic control; group 2, treated with 65 mg CeO2 nanoparticles /kg body weight orally; group 3, treated with 85 mg CeO2 nanoparticles / kg body weight orally. Group 2 and 3 received CeO2 nanoparticles for one week before and one week after of STZ injection. 8 weeks after conformation of diabetes, DN occurred (Hosseini et al., 2011). All experiments were performed 8 weeks after injection of STZ. Blood glucose and body weight were examined before and at the end of the experimental period.
4
2.3. CeO2 nanoparticles Characterization CeO2 nanoparticles was obtained from Navarrean Nenoproducts Technology (Spain). Field emission scanning electron microscopy was performed by FE-SEM, Hitachi, Japan S-4160. The zeta potential and particle size distribution were measured by a Nano Z-Sizer (Malvern Instrument Zen 3600).
2.4. Hot plate test A hot plate test used to measure pain sensitivity (Socrel Hot plate model DS37, Ugo Basile, Italy). Rats were placed on the plate with the diameter of 19 cm, height of 30 cm and temperature of 52°C ± 2°C. Response time to thermal pain was calculated from the onset of the test and front legs licking or jumping. Maximum time was considered 60 Sec (Karami et al., 2011).
2.5. Sample preparation The animals were anesthetized with intraperitoneal injection of ketamine and xylazin and blood sample were collected from the heart and centrifuged at 1200 g for 10 min at 4 °C for separating the plasma. The plasma samples were frozen at -80 °C for biochemical analysis. For tissue preparation the DRG was isolated and fixed into 10% paraformaldehid and then embedded in paraffin. The 40 μm sections were stained with Hematoxylin-Eosin (H&E) and assessed by light microscope. We used four sections per sample.
2.6. Morphometry of DRG neurons DRG neurons were defined as A or B cell (Hosseini et al., 2011). A cell has a light nucleus with one large nucleolus in central and granular cytoplasm. The nucleus of B cell is light and contains multiple nucleolus that peripherally located. The cytoplasm of B cell is dark, more homogenous and intensively stained compare to A cell. In this study we determined the diameter, number and area of A and B cells in all groups.
5
2.7. Measurement of intracellular ADP/ATP ratio The frozen DRG was homogenized in ice and centrifuged at 12,000g for 15 min at 4 °C. The supernatant was neutralized with 3M KOH in 1.5M Tris, and centrifuged. The supernatants were separated using reverse-phase HPLC. Chromatographic separation was done by column (SUPELCO- SILTMLC-18-T) with a flow rate of 1.2 ml/min and ammonium dihydrogen phosphate (75 mM) as mobile phase. The adenine nucleotides were assessment at 254 nm (µg/ml per mg of tissue) and changes in energy was reported as the ADP/ATP ratio (Najafi et al., 2015).
2.8. Measurement of lipid peroxidation (LPO) LPO was assessed by the reaction of thiobarbituric acid with malondialdehyde (MDA). The sample was mixed with Trichloroacetic Acid (20%), centrifuged at 1500 g for 10 min. the precipitate was dissolved in H2SO4 (0.05 M). TBA (0.2% in sodium sulfate) was added and the sample was incubated in a boiling water bath for 30 minutes. At the end, LPO was extracted by n-butanol and its absorbance was measured at a wavelength of 532 nm (Ghaznavi et al., 2015).
2.9. Measurement of total thiol molecules (TTM): TTM was measured by the reaction of DTNB with thiol molecules that forms a yellow complex. Samples were mixed with Tris-EDTA buffer and subsequent mixed with DTNB (10 mM). After 20 min the absorbance of samples were determined at 412 nm (Hosseini et al., 2015).
2.10. Measurement of total antioxidant power (TAP) Reduction of ferric ions (Fe+3) to ferrous ions (Fe+2), by the samples represents of antioxidant power. TAP was assessed by FRAP test. In this method, reaction of Fe+2 and 2, 4, 6-tris (2-pyridyl)-1, 3, 5-triazine (TPTZ) generates a blue color with a maximum absorbance at 593 nm (Najafi et al., 2015).
2.11. Statistical Analysis 6
Data were presented as the mean ± standard error of the mean (SEM). The significance of the differences between groups was analyzed using one-way ANOVA and Tukey’s posthoc tests by Stats Direct version 2.7.8. A P <0.05 was considered to be statistically significant.
3. Results 3.1. CeO2 nanoparticles characterization The Result from Dynamic light scattering (DLS) confirmed size distribution of CeO2 nanoparticles were ranged from 18.2 to 50.7 (Fig. 1a). Zeta potentials of CeO2 nanoparticles were 6.25. SEM imaging confirmed poly- dispersion of these nanoparticles (Fig. 1b).
3.2. Effect of CeO2 nanoparticles on Body Weight 8 weeks after STZ injection, diabetic rat exhibited significant loss of weight as compared with non diabetic rats in diabetic rats (p< 0.01). Treatment with CeO2 nanoparticles at a concentration of 65 mg/kg improved the body weight (p< 0.05). There is no significant changes in group received 85 mg/kg CeO2 nanoparticles compare to diabetic rat (Fig. 2).
3.3. The effect of CeO2 nanoparticles on the nociceptive threshold in Hot plate examination At the end of 8 the week, diabetic rats exhibited significant increase in pain threshold as compared with the control group (p<0.001). As shown in Fig. 3, CeO2 nanoparticles administration at the 65mg/kg and 85 mg/kg concentration significantly decreased the latency time as compared to untreated diabetic rat (p<0.001 and p< 0.05 respectively)
3.4. The effect of CeO2 nanoparticles on of DRG neurons observations As shown in Fig. 4, the DRG neurons are smaller, more basophilic and have prominent vacuoles in DN rats compared to control group. Changes in DRG neurons were reversed by CeO2 nanoparticles administration. 7
3.5. The effect of CeO2 nanoparticles on morphometric parameters of A and B neurons In DN rat the, A cells count was significantly decreased while B cell count had increased compared to non diabetic rats (p< 0.05). Diameter and area of A cells and B cells were markedly decreased in DN compared to control group. The effect of diabetes was inhibited by CeO2 nanoparticles, but we did not observe any significance difference in diameter of B cell in CeO2 nanoparticles (85 mg/kg) in comparison with DN rats (Table 1).
3.6. Effect of CeO2 nanoparticles on the plasma LPO, TAP and TTM level: In control diabetic rat the MDA levels were increased in compare to control group (p< 0.001), whereas the level of TTP and FRAP decreased in these conditions (p< 0.001). Treatment of rats with CeO2 nanoparticles, markedly reduced lipid peroxidation, and reversed high glucose-induced increase in TTM and TAP compared to control diabetic rat (Table 2).
3.7. Effect of CeO2 nanoparticles on intracellular ADP/ATP ratio: A marked decrease in the ADP / ATP ratio was observed in diabetic rat as compared with non diabetic rat (p< 0.001). Administration of CeO2 nanoparticles for 8 weeks resulted in a significant recovery in the ADP/ATP level in comparison to the control diabetic rats (Fig. 5).
4. Discussion DN as the most common complication of diabetes is a type of nerve injury caused by diabetes. Several causative factors involved in pathogenesis of DN such as hyperglycemia and oxidative stress (Vinik, 2008). Glucose metabolism pathway is responsible for the development of microvascular complications. These include: 1) nonenzymatic glycation of protein 2) increased the activity of polyol pathway 3) activation of PKC signaling pathway 4) increased hexosamin pathway (Feldman, 2003) . Thus increasing of these pathways linked to hyperglycemia- mediated ROS over production through the mitochondrial electron transport chain which have delicious effects on the nerve (Greene et al., 2000). It seems that 8
sensory neuron is a primary target for neural complication of diabetes. Oxidative stress exerts more severe effect at the DRG neuron that as the nerve (Schmeichel et al., 2003). Previous studies indicated that the STZ- injected diabetes rat model used in our study revealed morphological and functional features of the DN (Hosseini et al., 2011). In this research, we examined the DRG morphometry, sensory nerve function and antioxidant status in diabetic rats. Our results showed a toxic effect of high glucose leading to neurotoxicity. Note after the administration of CeO2 nanoparticles these factors were considerably improved. Indeed, this study indicated for the first time that CeO2 nanoparticles can be used as an effective therapeutic regenerative agent which inhibits the nerve damage caused by diabetes. CeO2 nanoparticles have unique advantages compare to other antioxidants including: the ability of regeneration of free radicals scavenging function through spontaneously moving between reduction and oxidation status, having a large number of active sites that used for scavenging of free radical and the ability to pass cross the blood - brain barrier due to their small size thereby make CeO2 nanoparticles an appropriate option for treatment of neural injury (Korsvik et al., 2007; Rzigalinski et al., 2006). Protective effects of CeO2 nanoparticles have been investigated in several studies in vitro and invivo. The results have confirmed the idea that CeO2 nanoparticles play an effective role in the attenuation of oxidative damage in biological tissue (Amin et al., 2011; Dowding et al., 2014; Estevez et al., 2011; Ghaznavi et al., 2015; Hosseini et al., 2013; Hosseini et al., 2014). In the present study, the nocieptive threshold was markedly higher than the control group. Ceo2 treatment restores pain threshold. DN is associated with elevated thermal perception thresholds that eventually lead to the sensory loss and degeneration of fibers in the peripheral nerve. Several pathogenesis mechanisms involved in this phenomenon such as increased aldose reductase, PKC, PARP, oxidative stress and downstream effectors of ROS and oxidative stress (Calcutt et al., 2004; Obrosova, 2009). All these pathways results in TNF-α and NF-KB activation and COX2 induction. COX2 expression leads to change in prostaglandin profile with enhanced production of vasoconstricting PGH2, PGF2α and thromboxane and increased production of vasodilatory prostacyclin. COX2 also increases the ROS generation that 9
exacerbated oxidative stress. On the other hand, hyperglycemia-increased TNF-α increased the permeability of microvascular and nerve damage (Kuhad et al., 2008; Pop‐Busui et al., 2006). Sensory loss in diabetic rat can be prevented not only by several antioxidants such as alpha lipoic acid, but also by agents that able to counteracting downstream effectors such as PARP (Cameron et al., 2001; Li et al., 2005). Therefore free radical scavengers such as CeO2 nanoparticles can be act as a powerful analgesic agent for neuropathy pain. The other finding of this study is an increase of B cell number and reduction of A cell count and volume of the DRG neuron. Myelinated fibers connect mostly to type A (The large neurons) and unmyelinated fibers connect mostly to B type (the small neurons) of DRG neuron. conduction velocity has a direct relation to the size of neurons (Kishi et al., 2002). Modification of protein by ROS decrease protein function, slowed axonal transport, decrease intermediate growth factor delivery from the synapse to the cell body, leading to induction of apoptosis (Metodiewa and Kośka, 1999). In addition, oxidative stress leads to activation of poly ADP –ribos polymers pathway which control the expression of genes involved in neuronal dysfunction (Kuhad and Chopra, 2009). In the current study, we evaluated the biochemical parameters of oxidative stress. STZ- injected rat had a significantly higher MDA level compare to non diabetic rat. Administration of CeO2 nanoparticles caused a considerable reduction in the MDA concentration. Polyunsaturated fatty acids located in the plasma membrane as well as proteins and DNA, are major targets for peroxidation by ROS. MDA as a major end product of lipid peroxidation uses to assess the tissue injury by free radicals (Pizzimenti et al., 2010). Previous studies showed that the increased MDA concentration in diabetic rat reduced by CeO2 nanoparticles (Pourkhalili et al., 2011) . Moreover, we observed that CeO2 nanoparticles at varying doses increase the level of TTM and TAP. It seems that the ROS scavenging effect of CeO2 nanoparticles is responsible for the improvement of TTM. Thiol molecule contains a sulphydryl group. Thiols are the major compound of the total body antioxidants include glutathione, cystein, taurine and methionine that play an essential role in defense against free radicals (Mungli et al., 2009). 10
In this study, we observed that ADP/ATP ratio increased in diabetic rat, whereas CeO2 nanoparticles administration restored this change. Previous studies reveal the ability of CeO2 nanoparticles in increasing the level of ATP (Hosseini et al., 2014; Pourkhalili et al., 2012). The ADP/ATP ratio is a authentic index for the determination of cell death and cell survival (Schütt et al., 2012). hyperglycemia increased mitochondrial ROS generation and subsequent decreased mitochondrial membrane potential ultimately leading to reduced ATP synthesis (Herrera et al., 2001). It seems that CeO2 nanoparticles increase ATP levels by limiting the mitochondrial ROS generation and oxidative stress. Based on the present study, we conclude that CeO2 nanoparticles as an antioxidant agent can be used for treatment of diabetic neuropathy. However, further research is required to explore.
Acknowledgment This study was supported by a grant from Iran University of Medical Sciences
Conflict of interest: None.
11
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Whalen, K., Miller, S., Onge, E.S., 2015. The Role of Sodium-Glucose Co-Transporter 2 Inhibitors in the Treatment of Type 2 Diabetes. Clinical therapeutics.
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(a)
(b)
Fig.1. CeO2 nanoparticles characterization. (a) Histogram of CeO2nanoparticles size distribution. (b) SEM image of CeO2nanoparticles.
17
Weight (g)
400
†
**
300 200 100
)
rt ic le
(8 5
m
g/ kg
) g/ kg m pa no na
eO 2 C
C
eO 2
na
no
pa
D
rt ic le
(6 5
ia be tic
C
co
on tr ol
nt ro l
0
Fig. 2. Protective effects of CeO2 nanoparticles on weight of DN rats after two months. Values are given by mean ± S.E.M of seven animals. **P < 0.01 vs control group; †P < 0.05 vs diabetic group.
18
25
***
Latency (S)
20
†
††
15 10 5
) g/ kg m
rt ic le s
(8 5
m pa no na 2 eO C
C
eO
2
na
no
pa
D
rt ic le s
(6 5
ia be tic
C
co
g/ kg
)
nt ro l
on tr ol
0
Fig. 3. Effects of CeO2 nanoparticles on latency in diabetic rats after two months Values are given by .
mean ± S.E.M of six animals. ***P < 0.001 vs control group; †P < 0.05, ††P < 0.01 vs diabetic group.
19
(A)
(B)
20 µm
20 µm
(C)
(D)
20 µm
20 µm
Fig. 4. H&E-stained DRG neurons. (A) Control group, (B) diabetic neuropathy group, (C) Nanocerium oxide (65 mg/kg) + STZ and (D) Nanocerium oxide (85 mg/kg) + STZ groups. (A cell= red arrow, B cell= yellow arrow).
20
ADP/ATP ratio
10
*** ††
8 6
†††
4 2
) g/ kg
)
m
g/ kg
(8 5
m rt ic le s
2 eO C
C
eO
2
na
na
no
no
pa
pa
D
rt ic le (6 5
ia be tic
co
co nt ro l
nt ro l
0
Fig. 5. Effects of CeO2 nanoparticles on ADP/ATP level in diabetic rats after two months Values are .
given by mean ± S.E.M of six animals. ***P < 0.001 vs control group; †††P < 0.001, ††P < 0.01 vs diabetic group.
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Table 1. The effect of CeO2 nanoparticles on morphometric parameters of A and B neurons
control Number of A cells
Diabetic control ***
22.71 ± 0.82
11.71 ± 0.55
Number of B cells
5.786 ± 0.4824
14.93 ± 0.5393
Diameter of A cells (µm)
181.4 ± 2.251
117.5 ± 3.582
Diameter of B cells (µm)
84.69 ± 2.4
61.57 ± 2.54
Area of A cells (µm2) Area of B cells (µm2)
20290 ± 7552 1150 ± 28.65
***
***
***
13160 ± 477.8 829.5 ± 25.38
***
***
CeO2 nanoparticles (65 mg/kg) †††
19.57 ± 0.7
†††
8.500 ± 0.3743 165.0 ± 4.028
†††
72.98 ± 1.62
††
18010 ± 476.7
†††
†††
1079 ± 42.38
CeO2 nanoparticles (85 mg/kg) ††
15.21 ± 0.52
††
12.14 ± 0.6535 134.9 ± 2.184
64.81 ± 1.3 15700 ± 466.4
†
†
956.9 ± 24.54
Data are represented as mean ± SEM (n = 4). ***P < 0.05 vs control group; †P< 0.05, ††P< 0.01, 0.001 vs diabetic neuropathy group.
22
††
†††
P<
Table 2. Effect of CeO2 nanoparticles on the plasma LPO, TAP and TTM level
Groups
LPO (µM)
TAP (mM)
TTM (nM)
control
2.594±0.25
725.0±10.41
0.7960± 0.04
Diabetic control
5.445±0.13
550.0±12.91***
0.3928± 0.02***
Nanocerium oxide (65 mg/kg)
4.001±0.1
672.3±8.27†
0.6845± 0.02††
Nanocerium oxide (85 mg/kg)
4.344±0.21
669.0±13.91†
0.6283± 0.07†
***
†††
††
Data are represented as mean ± SEM (n = 4). ***P < 0.001 vs control group; †P< 0.05; ††P < 0.01 , †††P< 0.001 vs group was treated only glucose
23