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Received: 4 July 2020    Revised: 11 August 2020    Accepted: 21 August 2020

DOI: 10.1111/jfbc.13470

FULL ARTICLE

Protective effect of pristine C60 fullerene nanoparticle in

combination with curcumin against hyperglycemia-induced
kidney damage in diabetes caused by streptozotocin

Ersin Demir1  | Abdullah Aslan2

1
Department of Agricultural Biotechnology,
Faculty of Agriculture and Natural Sciences,
Duzce University, Duzce, Turkey
2
Department of Biology-Molecular Biology
and Genetics Program, Faculty of Science,
Firat University, Elazig, Turkey
Correspondence
Ersin Demir, Duzce University, Faculty
of Agriculture and Natural Sciences,
Department of Agricultural Biotechnology,
81620, Duzce, Turkey.
Email: ersncan.dmr@gmail.com
Funding information
This study was supported by the Scientific
Research Projects Unit of Düzce University
(Grant Number: BAP-2017.11.05.657).
Abstract
The present study aims to examine the protective effects of C60 fullerene (C60),
Curcumin (CUR; Curcuma longa), C60 + CUR combination against oxidative stress,
apoptosis, and changes in cellular level in kidney tissue of diabetic rats. Treatment
practices were administered separately to groups for 8 weeks following the approval
of diabetes induction. It was observed that the treatment groups had increased an-
tioxidant potential, decreased oxidative stress levels, decreased cholesterol, alpha
tocopherol, retinol levels along with improved important changes in fatty acid me-
tabolism compared with the diabetic group. C60, CUR, and C60 + CUR were also
determined to act in the direction of reducing kidney damage by activating apoptotic
pathways. It can be concluded based on these findings that C60, CUR, and especially
C60 + CUR combination has beneficial properties in maintaining kidney tissue and
function by effectively preventing oxidative stress, apoptotic changes, and changes
at the cellular level in kidney tissue under hyperglycemia conditions.
Practical applications
C60 and CUR have various biological activities which can be indicated as antioxidant,
anti-inflammatory, anticancer, neuroprotective, and hepatoprotective. It has been re-
ported that C60 and CUR protect the cells against oxidative injury brought about by
reactive oxygen species (ROS). Data acquired from the present study puts forth that
C60 and C60 + CUR may be promising agents to prevent damage induced by hyper-
glycemic conditions in kidney tissue.

KEYWORDS

apoptosis, C60 fullerene, curcumin, fat-soluble vitamins, lipotoxicity, oxidative stress

1 |  I NTRO D U C TI O N Diabetes develops due to deficiency of insulin secretion or de-

Today, diabetes has become a significant metabolic disease.
Sedentary lifestyle and obesity increase the incidence of this dis-
ease. The International Diabetes Federation states that approxi-
mately 425 million people are affected from diabetes worldwide as
of now, but by 2045, the number of patients is projected to exceed
600 million (Matoba et al., 2020).
creased tissue sensitivity to insulin. It leads to complications such
as uncontrolled hyperglycemia, diabetic nephropathy, cardiomy-
opathy, retinopathy in diabetic patients. Diabetic nephropathy is a
common complication linked to type-1 and type-2 diabetes, with
end-term kidney failure seen in almost 30% of individuals with di-
abetes (Chowdhury, Ghosh, Das, & Sil, 2019). There are numerous
studies reporting that chronic hyperglycemia causes an increase in

J Food Biochem. 2020;44:e13470. wileyonlinelibrary.com/journal/jfbc |

© 2020 Wiley Periodicals LLC.     1 of 12
https://doi.org/10.1111/jfbc.13470

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2 of 12       DEMIR and ASLAN
ROS to be produced while reducing antioxidant levels, thus, result-
ing in a redox imbalance which ultimately leads to oxidative stress
(Chowdhury et  al.,  2019; Sharavana, Joseph, & Baskaran,  2017;
Wagener, Dekker, Berden, Scharstuhl, & Van der Vlag, 2009).
ROS plays an important role in the formation and development
of diabetes and its complications. Increased ROS with hyperglyce-
mia has also been reported to be associated with the apoptosis of
kidney cells and the development of diabetic nephropathy (Wagener
et al., 2009). ROS leads to structural and functional changes in glo-
merular and renal tubular cells in diabetes causing kidney damage
due to oxidative stress (Kim et al., 2016). These cause structural
damages in DNA, RNA, lipids, and proteins resulting in glomerular
and tubular hypertrophy. Apoptosis or necrotic demise develops
due to the thickening of glomerular and tubular membranes which
finally results in glomerular dysfunction characterized by albumin-
uria, proteinuria, glomerulosclerosis, and tubule-interstitial fibrosis
(Asadi, Goodarzi, Karimi, Hashemnia, & Khodadadi,  2019). Various
studies propose that the suppression of oxidative stress may have a
dramatic impact in reducing the progression of diabetic nephropathy
significantly (Ahangarpour, Oroojan, Khorsandi, Kouchak, & Badavi,
2018; Kim et al., 2016).
C60 was discovered in 1985 by Harold Kroto et al. It is reported
that C60 and its derivatives are important carbon nanoparticle de-
rivatives. C60 is a spherical carbon molecule with a unique lattice
spheroid structure. It has been reported that this carbon molecule
has strong radical cleaning properties with higher antioxidant ac-
tivity than other antioxidant molecules thus making an important
contribution to the protection of redox homeostasis in the organ-
ism. Fullerene and its water soluble derivatives have been reported
to display neuron-protective, anti-proliferative, antitumor, and
anti-inflammatory properties (Gonchar et  al.,  2018). Biochemical
analyses revealed that C60 has not displayed acute or subacute tox-
icity under in vivo conditions (Chistyakov, Smirnova, Prazdnova, &
Soldatov, 2013; Gonchar et al., 2018). Due to its hydrophobic charac-
teristic, C60 can penetrate both the lipid bilayer and cell membranes.
Pristine C60 and its water-soluble derivative have been observed to
accumulate in mitochondria (Grebinyk et al., 2019). C60 has been
found to be present in the tail region of lipid molecules, preferably
in areas close to double bonds (prone to peroxidation) in accor-
dance with its antioxidant properties (Wong-Ekkabut et al., 2008).
Therefore, it has been reported that C60 fullerene should be consid-
ered as a promising therapeutic agent for the treatment of diseases
such as Parkinson's, Alzheimer's, diabetes, and cancer (Shershakova
et al., 2016).
Cur is one of the most important bioactive components derived
from the rhizomes of the turmeric (Curcuma longa) plant (Rhizoma
Curcumae), a yellow-flowered, large-leaved, perennial herbaceous
plant genus in the Zingiberaceae family (Toptaş & Alagöz, 2016). It
has been shown that CUR displays anti-oxidative, anticarcinogenic,
anti-inflammatory, anti-hyperlipidemic and hypoglycemic proper-
ties. Extensive studies have been carried out on CUR as a result of
which it has been observed to display protective effects against neu-
ropathy, nephropathy, retinopathy, vascular disease, and pancreatic
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HIGHLIGHTS
• C60 fullerene and curcumin induce the caspase-3 pro-
tein expression and it inhibits the bcl-2 protein expres-
sion in streptozotocin induced in diabetic rats.
• C60 fullerene and curcumin decreased the oxidative
stress levels in streptozotocin induced in diabetic rats.
• C60 fullerene and curcumin reduced the cholesterol lev-
els in streptozotocin induced in diabetic rats.
• The tissue fatty acid profile was improved with C60
fullerene and curcumin supplementation.
• The metabolism of fat-soluble vitamins were improved
with C60 fullerene and curcumin supplementation.
β-cell dysfunction in diabetes. In diabetes, CUR has been found to
reduce lipidemia, cholesterol, and lipid peroxidation products in
blood and urine (Kim et al., 2016; Selvi, Sridhar, Swaminathan, &
Sripradha, 2014). Various studies have reported that CUR plays a
critical role against oxidative stress-mediated diseases such as di-
abetes, obesity, cardiovascular disease due to its strong antioxidant
activity (Ghosh, Bhattacharyya, Rashid, & Sil, 2015; Kim et al., 2016;
Selvi et al., 2014).
We examined in the present study whether the combination of
CUR and C60, with strong antioxidant properties, in addition to tra-
ditional medications displayed therapeutic effects against oxidative
stress, apoptosis, and cellular changes in kidney tissue caused by
diabetes.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Chemicals
The primer antibodies (anti-caspase-3 sc-70497, anti bcl-2 sc-509,
anti-beta actin sc-47778) and the secondary antibodies (sc-516102,
ab-97023) were acquired from Santa Cruz Biotechnology and
Abcam. All chemicals were purchased from Sigma-Aldrich, Bio-Rad,
BioShop, and Merck.
2.2 | Animals
Wistar albino breed rats were acquired in the present study from
Duzce University Experimental Animal Practice and Research
Center (DUDAM; 8 to 10  weeks old, 229.46  ± 32.36 g). Ethical
permits for using animals in the present study were obtained
from the local Ethics Committee of Duzce University (meeting
date: 05/05/2020 decision no: 2020/5/2). Rats were subject to
a lighting period of 12  hour light/12  hour dark. An environment
with a controlled ventilation system at an ambient temperature of
22 ± 2ºC and relative humidity of 55% ± 5% was used for keeping

DEMIR and ASLAN
the animals. Significant attention was given to keeping the cages
clean during the study in which the rats were fed in their cages.
The procedures were carried out only after the 7 day period
ended during which the rats were allowed to adapt to the stand-
ard conditions. The Guide for the Care and Use of Laboratory
Animals, Public Health Service Policy on Human Care and Use of
Laboratory Animals, and Animal Welfare Act were used as guide-
lines for the experiments carried out during the study. All studies
on animals and the data obtained from this study were reported
in accordance with the ARRIVE directive (McGrath, Drummond,
McLachlan, Kilkenny, & Wainwright, 2010).
2.3 | Diabetes induction in experimental rats
All streptozotocin (STZ) groups were intraperitoneally adminis-
tered with a single dose of STZ (60 mg/kg b.wt) prepared by dis-
solving in citrate buffer (0.1  M, pH: 4.5) for inducing diabetes.
The dose of STZ in the present study was calculated based on the
live weight of each rat. This calculated dose was administered to
the rats with the help of a 1 ml injector. Only citrate buffer was
administered to the animals in the control group. A glucometer
(Glucometer, Accu-Check Active, Roche Diagnostics) was used for
measuring the blood glucose levels from a drop of blood drawn
from the tail of the rats 1 week after the injection of STZ fasted for
the night and rats with blood glucose levels of ≥250  mg/dl were
accepted as diabetic (Tabatabaei, Ghaderi, Bahrami-Tapehebur,
Farbood, & Rashno, 2017).
2.4 | Experimental Design (in vivo)
Nine groups of seven animals each were randomly prepared from a
total of 63 Wistar albino breed rats. Groups;
1. Control (n  = 7) group: rats fed on the standard diet and ad-
ministered physiological saline solution (1 ml/kg rat),
2. Olive oil (n = 7) group: rats fed on the standard diet and adminis-
tered olive oil (oral gavage-2.5 ml/kg body weight/week),
3. C60 (n = 7) group: rats fed on the standard diet and administered
C60 (oral gavage-1.0 mg/kg body weight/week, 2.5 ml with con-
centration C60 0.4 mg/ml),
4. CUR (n = 7) group: rats fed on the standard diet and administered
CUR (oral gavage-1.0 mg/kg body weight/ week, 2.5 ml with con-
centration CUR 10 mg/ml),
5. STZ (n = 7) group: rats fed on the standard diet and administered
STZ (60 mg/ kg i.p),
6. STZ + Olive oil (n = 7) group: rats fed on the standard diet, STZ
(60 mg/kg i.p), and olive oil (oral gavage-2.5 ml/kg body weight/
week),
7. STZ + C60 (n  = 7) group: rats fed on the standard diet, STZ
(60 mg/kg i.p), and C60 administered rats (oral gavage - 1.0 mg/kg
body weight/ week, 2.5 ml with concentration C60 0.4 mg/ml),
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8. STZ + CUR (n  = 7) group: rats fed on the standard diet, STZ
(60 mg/kg i.p), and CUR administered rats (oral gavage – 1.0 mg/
kg body weight/week, 2.5 ml with concentration CUR 10 mg/ml),
9. STZ 
+ C60+CUR (n = 7) group: rats fed on the standard diet, STZ
(60 mg/kg i.p), C60 (oral gavage – 1.0 mg/kg body weight/week,
2.5 ml with concentration C60 0.4 mg/ml), and CUR administered
rats (oral gavage – 1.0 mg/kg body weight/week, 2.5 ml with con-
centration CUR 10 mg/ml). Rats were first administered C60, and
then, CUR.
The procedure described by (Baati et al., 2012) was implemented
for dissolving C60 in olive oil after which it was stored in the dark
at a working concentration of 0.4 mg/ml at 4°C (Aly, Kotb, Haridy,
& Hammad, 2018). CUR was also dissolved in olive oil and stored in
the dark and working concentration of 10 mg/ml at 4°C (Abdel Aziz
et al., 2012).
A dose of 1.0 mg/ kg/ week of olive oil extract of pristine C60
were administered orally three times per week for a period of
8 weeks (Group 3, 7). A dose of 1.0 mg/ kg/ week of olive oil extract
of CUR were administered orally three times per week for 8 weeks
(Group 4, 8). Oral administration of olive oil extract of pristine C60
and CUR was made three times per week for 8 weeks at a dose of
1.0 mg/ kg/ week and 1.0 mg/ kg/ week for C60 and CUR, respec-
tively (Group 9).
Mild ether anesthesia was used for sacrificing the animals fasted
overnight following the experiment period of 8 weeks. Their kidney
tissues were removed without delay and placed into sterile plastic
locked bags to be stored at −80°C until further analyses can be car-
ried out on the tissue samples.
2.5 | Preparation of tissue homogenate
The kidney tissue samples were homogenized via a cold buffer so-
lution (0.1  M Tris-HCl buffer pH 7.4). The upper phase was then
separated from the tissue samples via centrifugation. Further
experimental procedures were carried out on the upper phase
obtained in this manner. Malondialdehyde (MDA), reduced glu-
tathione (GSH), antioxidant enzymes, and total protein analyses
were also carried out using the resulting upper phase. Whereas
the lower phase was used for fatty acid, fat-soluble vitamins, cho-
lesterol, and sterol analyses.
2.5.1 | Determination of the amount of MDA as a
lipid peroxidation marker in kidney tissue
The MDA amount that is characteristic for lipid peroxidation in
kidney tissue was measured using the method developed by
(Ohkawa, Ohishi, & Yagi, 1979). The absorbance values of all sam-
ples were read at a wavelength of 532 nm. 1.1′,3,3′-tetraethoxy-
propane solution was used as standard. Results were calculated as
nmol/g tissue.

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4 of 12       DEMIR and ASLAN
2.5.2 | Determination of GSH in kidney tissue
The amount of GSH in kidney tissue was measured using Ellman's
method (Ellman, 1959). The absorbance values of all samples were
read at a wavelength of 412 nm. Calibration curve was created using
pure GSH as standard.
2.5.3 | Lipid extraction in kidney tissue
The method described by Hara and Radin was implemented for ex-
tracting fatty acids, fat-soluble vitamins, cholesterol, and sterol in
kidney tissue samples (Hara & Radin, 1978).
2.5.4 | Determination of lipophilic vitamins,
cholesterol, and phytosterols in kidney tissue
A Shimadzu full VP series HPLC was used for analyzing the fat-soluble vi-
tamins and cholesterol, stigmasterol, and β-sitosterol levels based on the
method of (Lopez-Cervantes, Sanchez-Machado, & Rios-Vazquez, 2006;
Sánchez-Machado, López-Hernández, & Paseiro-Losada,  2002). The
carrier phase used for analysis was an acetonitrile/methanol (60 + 40,
v/v) solution. Mobile phase flux rate was obtained as 1.0 ml/minute.
Lipophilic vitamins were analyzed using a DAD-UV detector. Nucleodur
LC 18 (15 × 4.6 cm, 5 µm) column was utilized as the HPLC column.
2.5.5 | Preparation of fatty acid methyl esters from
kidney samples
Total lipid was used by acid catalyzed methylation at 55°C
for 15 hour based on the method of (Christie, 1990, 1992) for
preparing the fatty acid methyl esters. The required analy-
ses were carried out using a GC-17A Shimadzu gas chromato-
graph equipped with SPTM-2380 fused silica capillary column
30 m × 0.25 mm × 0.2 μm film thickness.
2.5.6 | Gas chromatographic analysis of fatty acid
methyl esters
Analyses for the standard fatty acid methyl ester mixture were car-
ried out prior to the analysis of the fatty acid methyl ester of kidney
samples. The retention times of each fatty acid were determined at
this stage (Tvrzicka, Vecka, Stankova, & Zak, 2002).
2.5.7 | Determination of Antioxidant
Enzyme Activities
Determination of Catalase (CAT) activity
The activity of CAT (EC 1.11.1.6) was measured by monitoring the re-
duction of hydrogen peroxide (H2O2) and 240 nm with an extinction
17454514, 2020, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13470 by Cochrane Colombia, Wiley Online Library on [09/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
coefficient of 0.04 mM−1cm−1 (Aebi, 1984). The quartz assay cuvette
contained 50 μl sample solution in a final volume of 250 μl containing
67 mM of phosphate buffer pH 7.0 and 20 mM of H2O2. The amount
of enzyme that decomposes 1.0 μmol of H2O2 per minute is repre-
sented by a single unit of CAT.
Determination of Glutathione S-transferase (GST) activity
GST (EC 2, 5, 1, 18) activity measurements were carried out at
340  nm with 1.0  mM of 1-chloro-2,4-dinitrobenzene (CDNB) and
1.0 mM of GSH in 100 mM of potassium phosphate buffer, pH 6.5.
The reaction was initiated by adding a 50 ml sample following the
preparation of the quartz assay cuvette containing 100 mM of po-
tassium phosphate buffer pH 6.5. 100 ml GSH and 100 ml CDNB.
An extinction coefficient of 9.6 mM−1cm−1 was used for determining
specific activities (Bell, Cowey, Adron, & Shanks, 1985).
2.5.8 | Kidney tissue homogenization for
Western blotting
A mechanical homogenizator was used for breaking down the kid-
ney tissue samples (0,5 M Tris; pH: 8, EDTA, β-mercaptoethanol,
phenylmethylsulfonyl fluoride [PMSF]) which were divided into
small parts. The tissue samples broken down in this manner were
subject to centrifugation at 15,000 rpm for 45 min. Supernatant
was removed and stored at −80°C until the time of its usage
(Laemmli, 1970).
2.5.9 | Analysis of proteins via SDS-PAGE and
western blotting method
The dilution ratio for the primary antibodies (Santa Cruz and
Abcam) was 1/500, whereas the secondary antibodies were di-
luted at a ratio of 1/1000. Protein density measurements were
carried out via a Lowry kit with 30 μg protein loaded to each
well. SDS-PAGE method was utilized and accordingly, the pro-
tein samples of this tissue were run on 12% of gel. These proteins
(caspase-3, bcl-2, and beta-actin) were then relocated to the ni-
trocellulose membrane in accordance with the western blotting
method after which their synthesis ratios were examined (Aslan,
Gok, Erman, & Kuloglu, 2018; Aslan et al., 2020; Laemmli, 1970).
Density measurement analysis system (Image J; National Institute
of Health, Bethesda, USA) was used afterward for determining the
protein levels.
2.6 | Statistical analysis
SPSS (version 16) package software was used for the statistical
analyses of the resulting data. One-way variance analysis (ANOVA)
was used for determining the differences between groups. Duncan
multiple comparison test (DMRT) and LSD (Least significant differ-
ence) were used as post hoc analysis in binary comparisons of groups
 17454514, 2020, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13470 by Cochrane Colombia, Wiley Online Library on [09/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DEMIR and ASLAN |
      5 of 12

for parameters. p  < .05 and p  < .001 were considered for statisti-

Note: Values are mean ± standard error for seven rats in each group. For groups bearing different letters on the same line, the difference is statistically significant (One-way analysis of variance (ANOVA)
36.29 ± 4.80 b,c,d
cal significance. The data were presented in the form of arithmetic

2,870.55 ± 141.78a 2,803.02 ± 43.64a 2,839.59 ± 83.36a 2,848.72 ± 112.99a 1674.01 ± 616.76c 1711.07 ± 103.25c 2,165.16 ± 178.70 b 2,211.46 ± 223.96b 2,357.35 ± 75.26b

18.95 ± 1.72a,b
STZ + C60+ CUR

137.98 ± 8.54b
mean ± standard error.

3 | R E S U LT S

3.1 | Changes in the level of oxidative stress in the

39.31 ± 2.34b,c

17.36 ± 1.92b
b
134.47 ± 9.78

kidney tissue of diabetic rats

STZ + CUR

MDA level was increased at a significant level (p < .001) in the STZ

group compared with the Control group. MDA levels were reduced at
a significant level in the STZ + C60, STZ + CUR, and STZ + C60+CUR
b
138.51 ± 20.07
41.19 ± 2.23b

16.97 ± 1.51b

groups in comparison with the STZ group (p < .001; Table 1).

STZ + C60

GSH level was reduced at a significant level (p < .001) in the STZ

group in comparison with the Control group. There was a significant
increase in the GSH levels of the STZ + C60 (p < .001), STZ + CUR
(p < .01), and STZ + C60+CUR (p < .001) groups compared with that
c
111.65 ± 12.34

of the STZ group (Table 1).

STZ + Olive oil

13.42 ± 1.66c
49.64 ± 3.52a

CAT and GST levels were reduced at a significantly level (p < .001)

in the STZ group in comparison with the Control groups. While the
CAT and GST levels increased at a significant level in the STZ + C60
(p < .01, p < .05), STZ + CUR (p < .01), and STZ + C60+CUR (p < .001)
groups compared to the STZ group (Table 1).
52.82 ± 6.80a
c

13.09 ± 4.92c
99.93 ± 6.39

3.2 | Changes in fatty acid composition in kidney

STZ

tissue of diabetic rats

34.72 ± 1.59c,d
TA B L E 1   Changes in the level of oxidative stress in the kidney tissue of diabetic rats

21.02 ± 0.67a

Palmitic acid (16:0) level was significantly reduced (p < .001) in the

166.19 ± 7.22

STZ group in comparison with the Control group. There was a signifi-
cant increase in the Palmitic acid levels in the STZ + C60 (p < .001),
CUR

STZ + CUR (p < .01), and STZ + C60+CUR (p < .001) groups in com-

parison with the STZ group (Table 2).
a
168.93 ± 16.46
34.41 ± 2.60 c,d

A significant increase was observed in the Stearic acid (18:0)

20.76 ± 1.01a

level (p  < .001) in the STZ group in comparison with the Control
group. There was a significant decrease in the Stearic acid levels in
C60

the STZ + C60, STZ + CUR, and STZ + C60+CUR groups in compar-

ison with the STZ group (p < .001).
34.59 ± 0.99c,d
a
166.66 ± 19.95

There was a significantly decrease in the Palmitoleic acid (16:1)

21.08 ± 1.22a

level (p  < .001) in the STZ group in comparison with the Control
Olive oil

group. Palmitoleic acid levels were significantly increased in the

Duncan's and LSD post hoc tests, p < .05)

STZ + C60 (p < .001), STZ + CUR (p < .01), and STZ + C60+CUR

(p < .01) groups in comparison with the STZ group (Table 2).
Oleic acid (18:1) level was significantly reduced in (p < .001) the
a
167.07 ± 17.98

21.27 ± 1.84a
32.44 ± 2.89d

STZ group in comparison with the Control group. Oleic acid levels
were significantly increased in the STZ + C60, STZ + CUR, and
Control

STZ + C60+CUR groups compared with the STZ group (p < .001).

STZ group Linoleic acid (18:2) level was significantly reduced
MDA nmol/g

(p < .001) in comparison with the Control group. Linoleic acid levels

μg/g/1 min
GSH μmol/g
CAT μg/g

were increased at a significant level in the STZ + C60, STZ + CUR,

and STZ + C60+CUR groups in comparison with the STZ group
GST

(p < .001; Table 2).
 17454514, 2020, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13470 by Cochrane Colombia, Wiley Online Library on [09/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
6 of 12       | DEMIR and ASLAN

Arachidonic acid (20:4) level was increased significantly in

Note: Values are mean ± standard error for seven rats in each group. For groups bearing different letters on the same line, the difference is statistically significant (One-way analysis of variance (ANOVA)
STZ+C60+CUR
(p  < .001) the STZ group in comparison with the Control group.

18.13±1.53b,c
1.45±0.33b

16.62±1.30 b

24.84±2.70 b
16.02±1.15b

18.72±1.16b

2.46±0.10
Arachidonic acid levels were reduced at a significant level in the
STZ + C60, STZ + CUR, and STZ + C60+CUR groups compared with
the STZ group (p < .001; Table 2).
Changes in the docosahexaenoic acid levels were not significant
(p > .05) among the groups (Table 2).

17.09±1.42b,c
b

b
b

16.46±1.29b
c

16.24±0.88
17.85±0.69

25.93±0.93
1.73±0.17

2.84±2.11
STZ+CUR

3.3 | Changes in the amount of cholesterol,

phytosterol, and fat-soluble vitamins in the kidney
tissue of diabetic rats
b
b
16.38±0.42b
b
c

16.18±2.12c
25.25±0.80
2.47±0.85
15.76±0.70
17.81±1.64
1.48±0.17
STZ+C60

The vitamin K2 amount was reduced at a significant level in the STZ

group in comparison with the Control group (p < .05). The amount
of vitamin K2 was increased at a significant level in the STZ + C60,
STZ  + CUR, and STZ + C60+CUR groups compared with the STZ
12.46±1.20 d
c

a
20.09±0.96a
d

c
1.02±0.21

29.06±0.18
13.54±1.33

2.95±0.74
14.07±2.75
STZ+Olive

group (p < .05; Table 3).

The α-tocopherol and retinol amounts were significantly in-
oil

creased in the STZ group in comparison with the Control group

(p < .001). The α-tocopherol and retinol levels were significantly re-
TA B L E 2   Changes in fatty acid composition in the kidney tissue of diabetic rats (Percentage of fatty acid/500 mg)

duced in the STZ + C60 (p < .001), STZ + CUR (p < .001, p < .01), and

d

11.28±2.06d
a
20.41±0.91a
c
c
13.05±4.04

2.95±0.22
30.61±0.46
12.14±1.38
0.86±0.11

STZ + C60+CUR (p < .001, p < .001) groups when compared with

the STZ group (Table 3).
STZ

A significant (p < .001) increase was observed in the cholesterol

level in the STZ group compared with the Control group. The cho-
lesterol levels were reduced at a significant level in the STZ + C60,
22.59±0.54a
a

13.84±0.22c
a

c
21.77±0.88
2.40±0.38

2.37±0.27
20.13±1.41

21.44±1.11

STZ  + CUR, and STZ + C60+CUR groups in comparison with the

STZ group (p < .001; Table 3).
CUR

A significant increase was observed in the stigmasterol level in

the STZ group in comparison with the Control group (p < .001). The
stigmasterol levels were reduced significantly in the STZ + C60,
a,b

13.71±0.64c
a

c
22.95±0.61a

2.11±0.30
2.36±0.20

20.62±0.86

21.90±0.85
21.48±1.26

STZ + CUR, and STZ + C60+CUR in comparison with the STZ group

(p < .001; Table 3).
C60

Changes in 25 hydroxy vitamin D, vitamin K1, Beta sitosterol, and

vitamin D2 levels were not significant among the groups (p  > .05;
Table 3).
a,b

23.06±0.26a
a

c
13.70±0.11c

1.90±0.60
2.30±0.33
21.58±0.90

20.84±0.75

21.03±1.08
Olive oil

3.4 | Expression of Caspase-3 and Bcl-2 proteins in

kidney tissue of diabetic rats
Duncan's and LSD post hoc tests, P<0.05).
a

13.49±1.03c
a
a

23.99±1.96a
c

1.60±0.06
2.44±0.27

Caspase-3 protein expression levels were reduced at a significant

22.61±1.27

21.15±1.52
20.98±1.90

level (p < .05) in the STZ group in comparison with the Control group.
Control

Caspase-3 protein expression levels were significantly increased in

the STZ + C60, STZ + CUR, and STZ + C60+CUR groups compared
C22:6 Docosahexaenoic

to the STZ group (p < .05; Figure 1).

C20:4 Arachidonic acid
C16:1 Palmitoleic acid

Bcl-2 protein expression levels were increased at a significant

C18: 2 Linoleic acid
C16:0 Palmitic acid

C18:0 Stearic acid

level (p  < .05) in the STZ group in comparison with the Control
C18:1 Oleic acid

group. A significant decrease was observed in the bcl-2 protein ex-

pression in the STZ + C60, STZ + CUR, and STZ + C60+CUR groups
acid

compared to the STZ group (p < .05; Figure 1).


DEMIR and ASLAN
4 | D I S CU S S I O N
This was the first study to address the effects of the combination of
C60 and CUR against oxidative stress, apoptosis, and changes at the
cellular level in kidney tissue in a diabetic rat model. Since the mech-
anisms involved in the development of diabetic nephropathy have
not been fully clarified, (Sifuentes-Franco, Padilla-Tejeda, Carrillo-
Ibarra, & Miranda-Díaz, 2018) the use of specific therapeutic targets
or antioxidant adjuvants in the control of oxidative stress in diabetic
nephropathy may offer alternative approaches.
While on the one hand, ROS production continues in chronic hy-
perglycemic conditions, the activity of the antioxidant defense system
decreases and these conditions exacerbate oxidative stress thus caus-
ing serious structural and functional deteriorations in various tissues
(Ighodaro, 2018; Kim et al., 2016; Selvi et al., 2014). The hyperglycemic
environment is known to induce apoptosis and contribute to gradual
loss of kidney function in diabetic nephropathy (Sifuentes-Franco
et al., 2018). Diabetic rats have been reported to have increased MDA
levels and decreased GSH levels in kidney tissue. Moreover, it has been
set forth that there is a decrease in antioxidant enzyme activity in the
kidney tissue of diabetic rats (Chowdhury et al., 2019). In accordance
with previous study data, the present study also reported increased
MDA levels in the renal tissue of diabetic rats, decreased activity of
antioxidant defense systems such as GSH, CAT, and GST. However,
C60, CUR, and the combination of C60 + CUR administered to dia-
betic rats resulted in a decrease in the MDA levels of kidney tissue
along with an increase in the activity of antioxidant defense systems
such as GSH, CAT, GST (Table  1). In previous studies, CUR adminis-
tered to diabetic rats has been reported to decrease the level of MDA
and increase the activity of GSH and antioxidant enzymes in kidney
tissue (Kim et al., 2016; Selvi et al., 2014). It appears that C60 fuller-
ene reduces oxidative stress in different tissues of diabetic rats (Bal
et al., 2011; Li et al., 2019), however, no information has been found on
its effects against oxidative stress in kidney tissue. Therefore, it can be
concluded as a result of the present study that the combination of C60,
C60 + CUR applied to diabetic rats is effective in reducing oxidative
stress in kidney tissue (Table 1). It can be put forth due to the strong
antioxidant and regulatory role of CUR and C60 that the activity of the
antioxidant defense system is restored by reducing oxidative stress in
the kidney tissue, contributing to the preservation of the tissue struc-
ture and function by supporting the protection of kidney tissue.
Diabetes-induced hyperglycemia has been reported to bring
about oxidative stress and apoptosis in the kidney (Wagener
et al., 2009). Caspase-3 protein levels have been reported to de-
crease in different tissues of diabetic rats (Dorfman et al., 2015;
Hamza, Fikry, Abdallah, & Amin, 2018; Yuan et al., 2016). Bcl-2 pro-
tein expression has been reported to increase in diabetic rats (He,
Sun, & Huang, 2018). Upregulation of bcl-2 and downregulation of
Bax induce apoptosis (Gai et  al.,  2019). Under normal conditions
caspase-3 is in the form of a zimogen; but once activated by stress,
caspase-3 hydrolyzes-specific substrates, resulting in apoptotic cell
death (Parrish, Freel, & Kornbluth, 2013). The bcl-2 family of pro-
teins functions as a critical regulator in the apoptosis pathway. Bcl-2
17454514, 2020, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13470 by Cochrane Colombia, Wiley Online Library on [09/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
|
      7 of 12
can inhibit and promote cell death. Bcl-2 inhibits apoptosis by sup-
pressing Bax (Hardwick & Soane,  2013). It was found in the pres-
ent study that caspase-3 protein level decreased and bcl-2 protein
level increased in the kidney tissue of diabetic rats compared with
the control group. However, when compared with the STZ group,
caspase-3 levels were increased and bcl-2 protein levels were de-
creased in the kidney tissue of diabetic rats where CUR, C60, and
their combinations were applied (Figure 1). It has been reported that
caspase-3 levels decreased in rats where toxic compounds were
administered and caspase-3 levels increased in tissue as a result
of therapeutic phytochemicals (Aslan et  al.,  2020). CUR has been
reported to weaken in vitro high glucose-induced podocyte apop-
tosis and in vivo diabetic nephropathy due to its strong antioxidant
properties (Sun, Liu, Chen, Guan, & Liu, 2016). It has been reported
in previous studies that CUR displays protective effects in diabe-
tes via regulating apoptosis and autophagy in kidney podocyte cells
under diabetes conditions (Ghosh et al., 2015; Zhang, Fang, Zhang,
Ding, & Gan,  2020). A previous study has been reported that C60
is able to restore increased apoptosis in conditions of diabetes (Bal
et  al.,  2011). Natural antioxidants have been recognized as show-
ing protective or regulatory properties against diabetes-induced in-
flammation, oxidative stress, and apoptosis in in vivo studies (Ghosh
et al., 2015; Nedzvetsky, Sukharenko, Baydas, & Andrievsky, 2019).
According to these findings, it can be concluded that C60, CUR, and
their combination (C60 + CUR) reduce kidney damage by activating
apoptotic pathways with upregulation of caspase-3 and downregu-
lation of bcl-2 in STZ-induced kidney damage.
Although lipids are essential for normal cellular functions; in-
creasing evidence suggests that abnormal lipid accumulation in
nonfat tissues contributes to organ damage and dysfunction. The
disorder in lipid metabolism and storage has been reported to be a
risk factor for the development and progression of diabetic kidney
disease (Gai et al., 2019; Stadler, Goldberg, & Susztak, 2015). In par-
ticular, excess free fatty acids have been shown to trigger apopto-
sis in renal tubular epithelial cells and podocytes through different
mechanisms (Gai et al., 2019). Therefore, improving the lipid profile
may be a good approach in terms of reducing the progression of dia-
betic nephropathy with formation. It was observed in this study that
the kidney tissue fatty acid profile of diabetic rats changed (Table 2).
Palmitic acid is synthesized as de novo, with the activity of important
enzymes such as fatty acid synthase (FAS) and acetyl CoA carbox-
ylase (ACC).
Both ACC and FAS activity can be regulated at the transcrip-
tion level with the sterol regulatory element-binding protein-1c
(SREBP-1c) in insulin control (Kwan et al., 2015). Diabetes reduces
the activities of the enzyme ACC (Wakil & Abu-Elheiga, 2009). It
is capable of activating insulin, ACC, and FAS (Abu-Elheiga, Wu,
Gu, Bressler, & Wakil, 2012; Griffin & Sul, 2004). It is well known
that insulin induces enzymes involved in the synthesis of fatty acid
and triacylglycerol (Griffin & Sul, 2004). The activity of SREBP-1c
is controlled by insulin (Griffin & Sul, 2004). In the present study,
palmitic acid levels decreased in the kidney tissue of diabetic rats
(Table 2). However, a combination of C60, CUR, and C60 + CUR
 17454514, 2020, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13470 by Cochrane Colombia, Wiley Online Library on [09/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 of 12       | DEMIR and ASLAN

was found to increase the amount of palmitic acid in the kidney

1725.79 ± 258.89b,c

Note: Values are mean ± standard error for seven rats in each group. For groups bearing different letters on the same line, the difference is statistically significant (One-way analysis of variance (ANOVA)
6.67 ± 0.56a,b,c
STZ + C60+CUR tissue of diabetic rats. The amount of palmitic acid may be de-

137.34 ± 15.89b
32.91 ± 2.89b,c

1.16 ± 0.62b
0.44 ± 0.30
0.58 ± 0.23

0.61 ± 0.42

7.91 ± 7.49
creased as a result of impaired insulin metabolism and reduced
activity of ACC and FAS enzymes in the kidney tissue of diabetic
rats. The amount of palmitic acid may have increased due to the
positive effect of the combination of C60, CUR, and C60 + CUR
on glucose metabolism. Both CUR and C60 have been reported
b
1827.65 ± 124.82
a,b,c

b
144.72 ± 18.22
35.38 ± 4.56b

1.47 ± 0.22b
to have regulatory roles on glucose metabolism (Bal et  al.,  2011;
0.83 ± 0.08

8.54 ± 3.28
6.74 ± 0.85

0.62 ± 0.21

0.63 ± 0.12
STZ + CUR

Halenova et  al.,  2018; Kim et  al.,  2016). The synthesis of other
fatty acids (Palmitoleic acid, Stearic acid, Oleic acid, Linoleic acid,
Arachidonic acid, and Docosahexaenoic acid) takes place through
a series of desaturation and elongation reactions of palmitic acid
b
2,388.51 ± 190.88 2,218.18 ± 237.02 1814.48 ± 101.53
b,c,d

(Nelson & Cox,  2005). The synthesis of these fatty acids varies
b
147.29 ± 23.27
35.87 ± 4.87b

1.76 ± 0.61b
0.64 ± 0.44
7.67 ± 5.50
0.51 ± 0.35
6.23 ± 0.37
0.79 ± 0.59

depending on the activity of desaturation enzymes. Stearoyl-CoA

STZ + C60

desaturase 1 ((SCD-1) 16: 1n-7/16:0; SCD-16 and 18: 1n 9/18:0;

SCD-18), Δ-5-desaturase (D5D; 20: 4n6/ 20: 3n-6) and Δ-6 de-
saturase (D6D; 18:3 nm 6/18: 2n-6), monounsaturated fatty acids
a

and polyunsaturated fatty acids are the main enzymes responsible

c,d

a
TA B L E 3   Changes in the amount of cholesterol phytosterol and vitamins (A, D, E, K) in the kidney tissue of diabetic rats (µg/g)

183.39 ± 18.75
46.86 ± 5.01a

3.67 ± 1.93a
1.06 ± 0.25

0.67 ± 0.58

1.06 ± 0.92
5.15 ± 1.10

9.33 ± 7.45
STZ + Olive oil

for the synthesis of endogenous. D5D and D6D are key enzymes
that catalyze rate-limiting stages in the conversion of linoleic acid
and α-linolenic acid (C18: 3n-3) into long-chain n-6 and n-3 PUFAs
(Dunder, Halin Lejonklou, Lind, Risérus, & Lind, 2018). Fatty acid
desaturases form double bonds in elongated fatty acid chains. Key
a
d

4.13 ± 2.36a
48.66 ± 6.75a

a
4.18 ± 0.86

0.82 ± 0.79

1.14 ± 0.98
206.27 ± 5.15
9.37 ± 9.30
0.92 ± 0.16

desaturases in mammals are D5D, D6D, and SCD-1. Fatty acid de-
saturases are thought to play a role in metabolic disorders such
as insulin resistance and dyslipidemia (Sjögren et al., 2008). Gene
expression of unsaturated fatty acid desaturases is basically reg-
STZ

ulated by insulin (Czumaj & Śledziński, 2020). Significant changes

a,b,c

have been observed in this study in the level of unsaturated fatty

c
1,540.01 ± 25.19

0.56 ± 0.31b
c
25.58 ± 0.31c

76.44 ± 3.22
7.29 ± 1.88

0.40 ± 0.20
0.43 ± 0.60

0.57 ± 0.06
7.11 ± 5.97

acids in the kidney tissue of diabetic rats. However, changes in

unsaturated fatty acid levels were found to improve the kidney
tissue of diabetic rats to which a combination of C60, CUR, and
CUR

C60 + CUR was administered (Table 2).

It has been reported that there is an accumulation of cholesterol
c
1517.54 ± 26.38
c
a,b

in kidney tissue under conditions of diabetes. β-hydroxy-β-methylgl-

76.04 ± 22.08

0.57 ± 0.19b
25.58 ± 7.56c
0.41 ± 0.06
0.57 ± 0.26
7.48 ± 1.24

6.68 ± 4.93

0.49 ± 0.13

utaryl coenzyme A (HMG CoA) reductase catalyzes the rate-limiting

step in cholesterol biosynthesis, and its activity is closely related
C60

to the rate of tissue cholesterol synthesis. As previously reported,

HMG-CoA reductase activity was found to be significantly increased
c
1533.15 ± 64.19
a,b

in diabetic rats due to insulin deficiency (Pari & Murugan, 2007). In

0.52 ± 0.20 b
c
25.88 ± 2.18c
0.40 ± 0.04

0.44 ± 0.38
8.18 ± 2.58

75.00 ± 2.28
0.31 ± 0.05

7.31 ± 1.05

this study, it was found that the level of cholesterol increased in the
Olive oil

kidney tissue of diabetic rats (Table 3). In previous studies, CUR has

Duncan's and LSD post hoc tests, p < .05).

been reported to reduce cholesterol levels in the tissues of diabetic

rats (Pari & Murugan, 2007; Sudirman, Lai, Yan, Yeh, & Kong, 2019).
c
1505.62 ± 46.75

Retinol and alpha tocopherol are fat-soluble vitamins with anti-

a

0.52 ± 0.17b
c
25.21 ± 4.51c
0.39 ± 0.04
8.82 ± 0.33
0.31 ± 0.23

5.82 ± 4.68

0.42 ± 0.36
74.43 ± 2.51

oxidant properties at the forefront. Retinol is carried in plasma via

Control

retinol-binding protein (RBP). It has been reported that retinol levels

have been decreased in serum of diabetic rats, as well as the level
of retinol-binding protein in kidney and liver and serum samples
Beta sitosterol
α-tocopherol

(Tuitoek, Ritter, Smith, & Basu,  1996). Changes in retinol metabo-

Stigmasterol
Cholesterol
25 hydroxy

Vitamin D2
vitamin D
Vitamin K 2

Vitamin K1

lism have been reported to be associated with insulin resistance and

Retinol

metabolic syndrome (Starkey et al., 2010). Plasma retinol levels in

diabetic rats were reduced significantly, but retinol levels increased

DEMIR and ASLAN
F I G U R E 1   Rat kidney tissue western blotting mean protein expression results; (A): Caspase-3, (B): Bcl-2, (C): western blotting protein
bands. a–h: different letters means that statistically significant difference between groups. Values are statistically significant at p < .05 one-
way analysis of variance (ANOVA) Post hoc Duncan Test
again with insulin therapy (Everts & Berdanier,  2002). It was also
found that the amount of retinol in liver samples of deceased dia-
betic patients increased. The level of retinol in tissue varies due both
to the increase in de novo fatty acid synthesis and the mobilization
of fatty acids (Everts & Berdanier, 2002). In this study, retinol levels
increased in the kidney tissue of diabetic rats (Table 3). There may
be increased levels of retinol in the kidney tissue due to the impaired
transport mechanism related with diabetes. Various studies have
been carried out which highlight this (Basu & Basualdo, 1997).
The alpha-tocopherol transfer protein (α-TTP) is responsible for
the traffic of α-tocopherol in plasma and peripheral tissues. Previous
diabetes studies have reported increased levels of α-tocopherol in
plasma and liver tissue. Oxidative stress is able to regulate α-TTP
transcription upward (Miranda et al., 2018; Miyazaki et al., 2013). It
has been announced that hyperglycemia, or insulin resistance, can
lead to increase a-tocopherol levels by regulating α-TTP gene ex-
pression (Arulselvan & Subramanian,  2007; Miyazaki et  al.,  2013).
There have been reports of the transport of alpha tocopherol from
the liver to the plasma in response to oxidative stress through α-TTP
upregulation (Traber, 2013). Alpha tocopherol levels were observed
to increase in the present study in the kidney tissue of diabetic rats
17454514, 2020, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13470 by Cochrane Colombia, Wiley Online Library on [09/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
|
      9 of 12
(Table 3). In accordance with previous studies, increased levels of
alpha tocopherol have been reported in the tissue of diabetic rats
(Miranda et al., 2018; Miyazaki et al., 2013). The accumulation of
retinol and alpha tocopherol in the kidney tissue of diabetic rats may
have been decreased due to the beneficial effects of CUR and C60
on glucose metabolism. Thus, possible toxicity is avoided.
As a result, important information has been obtained indicat-
ing that the combination of C60 and C60 + CUR reduces oxidative
stress in kidney tissue in addition to reducing kidney damage by
activating apoptotic pathways. Moreover, important information
has been reached regarding the preservation of kidney tissue and
function by reducing fatty acid and cholesterol accumulation in
kidney tissue of the combination of C60, CUR, and C60 + CUR.
It was also determined that the combination of C60, CUR, and
C60  + CUR showed beneficial effects in reducing the accumula-
tion of retinol and alpha tocopherol in the kidney tissue of diabetic
rats. The combination of C60 and C60 + CUR may be promising
agents to prevent damage induced by hyperglycemic conditions
in kidney tissue. Further detailed studies should be carried out for
exploring the exact mechanisms governing the renoprotective po-
tential of C60 (Figure 2).

|
10 of 12       DEMIR and ASLAN
F I G U R E 2   The effect of C60 and CUR on caspase-3, bcl-2, MDA, GSH, cholesterol, fatty acid profile, and antioxidant enzymes.
Caspase-3, GSH, CAT, and GST are increased; bcl-2, MDA, cholesterol, and fatty acid profile are reduced in the kidney
AC K N OW L E D G M E N T S
Authors thanks to Prof. Dr. Ökkeş Yılmaz and Dr. Can Ali Ağca for
contributions.
C O N FL I C T O F I N T E R E S T
The authors declare that they have no conflicts of interest.
AU T H O R C O N T R I B U T I O N
Ersin Demir: Investigation; Methodology; Resources; Validation;
Visualization; Writing-original draft; Writing-review & editing.
Abdullah Aslan: Investigation; Methodology; Writing-review &
editing.
ORCID
Ersin Demir  https://orcid.org/0000-0002-7676-5953
Abdullah Aslan  https://orcid.org/0000-0002-6243-4221
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How to cite this article: Demir E, Aslan A. Protective effect
of pristine C60 fullerene nanoparticle in combination with
curcumin against hyperglycemia-induced kidney damage in
diabetes caused by streptozotocin. J Food Biochem.
2020;44:e13470. https://doi.org/10.1111/jfbc.13470