SUPPLEMENTARY MATERIAL
Title: Luteolin attenuate the D-galactose induced renal damage by attenuation of oxidative
stress and inflammation
Author names and affiliations:
Yuqing Xu*1, Jingzheng Zhang*1 , Jing Liu*1, Sai Li*1, Cheng Li*1, Wen Wang*1, Rong Ma**2 and
Yi Liu**1
1 School of Pharmacy, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou, Jiangsu, 221002,
China
2 School of Environment, Nanjing University, Nanjing, 210000 Jiangsu Province, P. R. China
E-mail: rongmnju@163.com
Abstract
Luteolin is reported to have antioxidant and anti-inflammatory properties. In the present study, we
investigated the protective effect of luteolin against the renal damage induced by D-galactose
(D-gal). The levels of creatinine (Cr) and urea nitrogen (BUN) were evaluated in plasma, kidney
sections were stained with hematoxylin-eosin, followed by assessment of the antioxidant and
anti-inflammatory activity. Furthermore, we also investigate the expression of the p38
mitogen-activated protein kinase (p38 MAPK) and its phosphorylated active. The results of
luteolin treatment showed that the renal damages were attenuated. Luteolin could significantly
ameliorate D-gal induced oxidative damage and suppress the inflammatory response. Moreover,
the result also shows that luteolin could significantly inhibit the p38 MAPK phosphorylation in the
kidneys from the model of D-gal-treated mice. Therefore, our research suggests that luteolin might
be involved in the attenuated oxidative stress and inflammatory responses, hence the protective
effects against D-gal-induced renal damage.
Keywords: Luteolin; D-galactose; Oxidative stress; Inflammation; kidney
1. Experimental section
1.1 Materials
Luteolin was purchased from Shanxi Sciphar Biotechnology Co., Ltd.(Shanxi, China).
Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), catalase (CAT)
enzyme, blood urea nitrogen (BUN) and creatinine (Cr) assay kit were purchased from Nanjing
Jiancheng Biotechnology Co., Ltd (Nanjing, China), β-actin (Sigma-Aldrich, St. Louis, MO,
USA). Phospho and non-Phospho-p38 MAPK (Cell Signal Technology Inc. Danvers, MA, USA).
Enzyme-linked immunosorbent assay (ELISA) kit (Immuno-Biological Laboratories Co., Ltd.,
Japan). All other reagents used in the experiments were of analytical grade and purity.
1.2 Animal and tissue preparation
Adult male Kunming mice (6 weeks old, weighing 18-23g) were randomized into 4 groups (n = 12
each): Group 1 (control group): mice received daily intraperitoneally injected of saline (0.9%) and
received 0.5% sodium carboxymethylcellulose (CMC-Na) solution; Group 2 (D-gal group): mice
received daily intraperitoneally injected of D-gal (100 mg/kg) and received 0.5% (CMC-Na)
solution; Group 3 (D-gal + luteolin group): mice received daily intraperitoneally injected of D-gal
(100 mg/kg) and received daily treatment of luteolin (100mg/kg, p.o.) Which was suspended in
0.5% (w/v) CMC-Na solution; Groups 4 (luteolin group): mice received daily intraperitoneally
injected with saline (0.9%) and being administered luteolin (100mg/kg, p.o.). All groups of animal
were kept at a regulated temperature (22 ± 2◦C) and humidity (50 ± 10%) on a 12-hour light/dark
cycle with ad libitum access to standard commercial food and pure water. The mice received
humane care throughout the experiment according to the Chinese Council on Animal Care and
Institutional Care Committee of Xuzhou Medical College. At the end of treatment for 8 weeks, the
mice were sacrificed, followed by the blood sampling without the addition of anticoagulants for
centrifugation at 3000 × g for 10 min for subsequent determination of clear plasma, and the
isolation of kidneys on ice. The blood samples and kidney tissues were rapidly frozen and stored
at -80℃ until biochemical determinations.
1.3 Assessment of the Renal Function
Biochemical analyses of blood urea nitrogen (BUN) and creatinine (Cr) levels were made using
the standard diagnostic kits (Nanjing Jiancheng Bioengineering Institute, China).
1.4 Histomorphological evaluations
For histomorphological examinations, all samples were fixed in 4% formaldehyde buffer,
followed by embedment in paraffin and longitudinal slicing, with 5-μm-thick sections obtained for
haematoxylin-eosin staining for the histomorphological analysis.
1.5 Lipid peroxidation assay
The quantitative analysis of lipid peroxidation in the kidneys was performed as Wills described
(Wills 1966). The MDA content was measured by reaction with thiobarbituric acid at the
wavelength of 532 nm. The results were expressed as nmol/mg protein.
1.6 Superoxide dismutase activity
SOD activity was assayed according to the method of Kono (Kono 1978) wherein the reduction of
nitroblue tetrazolium chloride was inhibited by the superoxide which was measured at 560 nm
spectrophotometrically. Briefly, the reaction was initiated by the addition of hydroxylamine
hydrochloride to the reaction mixture containing NBT and post nuclear fraction of kidney
homogenate. The results were expressed as units/mg protein, with one unit of enzyme defined as
the amount of SOD required to inhibit the rate of reaction by 50%.
1.7 Estimation of GSH content
GSH concentrations were determined by the procedures of Ellman (Ellman 1959). Briefly, 0.5 ml
homogenate was mixed with 1.5 ml 0.15 M KCl and 3 ml deproteinization solution. Each sample
was centrifuged at 3000 rpm for 10 min and the supernatant was removed, followed by the
addition of 2 ml phosphate solution and 0.5 ml DTNB into the 0.5 ml supernatant, with the
absorbance read at 412 nm and compared with glutathione standards. The concentration of
reduced glutathione was expressed as milligram per gram tissue.
1.8 Estimation of CAT
CAT activity was measured according to the method of Aebi (Aebi 1984). Briefly, 0.1 ml of
supernatant was added to a cuvette containing 1.9 ml of 50 mmol/L phosphate buffer (pH 7.0).
The reaction was initiated by the addition of 1 ml freshly prepared 30 mmol/L H2O2. The rate of
decomposition of H2O2 was measured spectrophotometrically by changes in absorbance at 240 nm.
The activity of catalase was expressed as units per milligram protein.
1.9 Western blot
To determine the phosphorylation and protein expression levels of p38 MAPK, whole kidney
tissues were homogenized at 4°C in homogenizing buffer (50 mM Tris-Cl, pH 7.5, containing 2
mM dithiothreitol (DTT), 2 mM ethylene diamine tetraacetic acid (EDTA), 2 mM ethylene glycol
tetraacetate (EGTA), 5 mg/ml each of leupeptin, aprotinin, pepstatin A, and chymostatin, 50 mM
potassium fluoride (KF), 50 nM okadaic acid, 5 mM sodium pyrophosphate, and 2% sodium
dodecyl sulfate (SDS)). The homogenate was then sonicated to dissolve the tissue completely, and
the protein quantities of each sample were determined by a BCA Protein Assay kit ((ibid.) Equal
amounts of protein (100 μg) were isolated by 10% SDS-polyacrylamide gel electrophoresis and
were electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked
with 5% skim milk powder in a washing buffer [Tris-buffered saline containing 0.05% (v/v)
Tween 20] for 2 h at 25°C, and was incubated with primary rabbit polyclonal antibodies against
Thr180/Tyr182-phosphorylated p38MAPK (Phospho-p38 MAPK, Sigma-Aldrich, St. Louis, MO,
USA) at a 1:500 dilution at 4°C overnight. Each membrane was thrice rinsed for 15 min and
incubated with the secondary horseradish peroxidase-linked antibodies. β-actin was used as an
internal control for the cytosolic extracts. Comparison between different treatment groups was
performed by determination of the T-P38/β-actin protein ratio of the immunoreactive area by
densitometry and the ratio of Phospho-p38 MAPK to T-p38 MAPK was recognized at the protein
activation level.
1.10 Measurement of TNF-α and IL-6 levels
TNF-α levels in the kidneys were determined with a commercial enzyme-linked immunosorbent
assay (ELISA) kits (Roche Eurofin) for mice according to the manufacturer’s instructions.
Similarly, the IL-6 concentrations in the kidney samples were also respectively analyzed with
ELISA kits (Roche Eurofin).
Statistical analyses
Data were expressed as the mean ± SEM. The data were analyzed with SPSS 16.0 (SPSS, Inc.,
Chicago, Ill., USA). The statistical significance of differences in the dependent measures between
experimental groups was determined by one-way ANOVA followed by the Tukey’s tests.
Probability values less than 0.05 were considered significant.
References
Wills E. 1966. Mechanisms of lipid peroxide formation in animal tissues. Biochem J.
99:667-676.
Kono Y. 1978. Generation of superoxide radical during autoxidation of hydroxylamine and
an assay for superoxide dismutase. Arch Biochem Biophys. 186:189-195.
Ellman GL. 1959. Tissue sulfhydryl groups. Arch Biochem Biophys. 82:70-77.
Aebi H. 1984. Catalase in vitro. Methods Enzymol. 105:121-126.
Figure S1.（A, B）Effects of luteolin on creatinine (Cr) and urea nitrogen (BUN) in plasma of
D-gal -treated mice. Values are expressed as mean ± SEM. *p < 0.01 vs. Control group. #p < 0.05
vs. D-gal group (n = 6).
Figure S2. Haematoxylin and eosin stained sections of mice kidneys. (A) Control kidney section.
(B) Kidney section of D-gal-treated mice showing severe tubular necrosis and cast formation. (C)
Kidney section of luteolin treated mice showing moderate necrosis and cast formation. (D)
Kidney section of single luteolin treated mice showing moderate necrosis and cast formation.
Figure S3. Effect of luteolin on Malondialdehyde (MDA) level(A), reduced glutathione (GSH)
level(B), superoxide dismutase (SOD) activity(C), catalase (CAT) activity(D) in the kidneys of
D-gal-treated mice. Values are expressed as mean ± SEM. **p < 0.01 vs. Control group. #p < 0.05
vs. D-gal group (n = 6)
.
Fig S4. Increased phosphorylation of p38 MAPK in kidney homogenates from D-gal group
compared with D-gal + luteolin group (A) Representative results of Western blots show that the
levels of phosphorylated p38 MAPK (phospho-p38 MAPK ), but not total-p38 MAPK (T-p38
MAPK), are significantly increased in D-gal group to D-gal + luteolin group. (B) Quantitative
analysis indicated a significant increase of phospho-p38 MAPK in the kidney homogenates from
D-gal group compared to D-gal + luteolin group. (Values are presented as mean ± SEM. n = 3
for each group. **p < 0.01 vs. Control group #p < 0.05 vs. D-gal group) phospho-p38 MAPK: Thr
180/Tyr 182-phosphorylated p38 MAPK; T-p38 MAPK: total protein of p38 MAPK including
both phospho- and nonphospho-p38 MAPKs.
Figure S5. (A, B) Effect of luteolin on tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)
level in the kidneys of D-gal-treated mice .Values are expressed as mean ±SEM. **p < 0.01 vs.
Control group. # #p < 0.01 vs. D-gal group (n = 6).