Introduction
Diabetic nephropathy (DN) is one of the most serious
microvascular complications of diabetes and a relatively common
disease, which is the leading cause of end-stage renal disease
(1–4). According to reports, the prevalence
rate of DN is 33–40% in type 1 diabetes (5). The prevalence rate of DN is 20–25% in
type 2 diabetes (6). Increasing
research has focused on the genetics of lesion formation in the
coronary artery of patients with type 2 diabetes (7). Actin, α2, smooth muscle, aorta (ACTA2)
is located on chromosome 10q(23.3) and is the most abundant α actin
content of protein in vascular smooth muscle cells (8). ACTA2 accounts for 40% of the total
protein in vascular smooth muscle cells, and 70% of total actin
protein (9). Thus, ACTA2 is an
important protein required for the contraction of vascular smooth
muscle cells. p38 is a key component of mitogen-activated protein
kinase-mediated signaling pathways, and is involved in a variety of
physiological processes, including inflammation, proliferation and
apoptosis (10). Rutin is a drug
that has been used clinically for many years for the regulation of
cardiovascular disease and for diabetes (11,12).
Therefore, the present study aimed to investigate whether rutin is
able to inhibit the expression of p38 and ACTA2 and improve the
severity of diabetic nephropathy. The protective effects of rutin
on diabetic kidney disease were investigated by examining mesangial
cell viability, energy metabolism, cell cycle and protein
expression with the aim of providing valuable data for the clinical
prevention and treatment of DN.
Materials and methods
Materials
ACTA2 (BM1693) and p38 (MK2101) antibodies were
purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan,
China). G secondary antibodies (AP130P) were obtained from
Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). An MCO-5AC CO2
thermostat incubator was obtained from Sanyo Electric Biomedical
Co., Ltd. (Tokyo, Japan).
Cell culture
Glomerular mesangial cells were provided by Jilin
University School of Pharmaceutical Sciences (Changchun, China).
Glomerular mesangial cells were divided into a control group (PBS
washing), high glucose-induced group (incubation with DMEM high
glucose to mimic an environment that would induce DN; Invitrogen;
Thermo Fisher, Scientific, Inc., Waltham, MA, USA), high glucose +
captopril group (0.4 µmol/l, 24 h), and high glucose + low, middle
and high rutin groups (0.2, 0.4 and 0.8 µmol/l rutin,
respectively). Cells were cultured in DMEM high glucose at 37°C in
a humidified atmosphere containing 5% CO2 for 24 h. Cells in the
high glucose + rutin groups were treated with rutin for 24 h.
Subsequently, mesangial cells were prepared for assays of cell
viability, adenosine 5′-triphosphate (ATP) content, cell cycle, and
ACTA2 and p38 protein expression levels.
Cell viability assay
A cell viability assay was carried out with an MTT
staining kit (Sigma-Aldrich; Merck KGaA), according to the
manufacturer's protocol. Results were analyzed using a
spectrophotometer (UV2550; Shimadzu Corporation, Kyoto, Japan) at
450 nm.
ATP content
Intracellular ATP was quantified using an ATP Assay
Kit (S0026; Biyun Tian Biotechnology Research Institute). Cell
suspension (100 µl) was transferred to the wells of a 96-well
microplate and ATP Assay reagent (100 µl) was added. The plate was
shaken using a plate shaker for 10 min and luminescence was
measured using a spectrophotometer (UV2550, 450 nm).
Cell cycle analysis
Glomerular mesangial cells were cultured in
Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher
Scientific, Inc.) in 50-cm2 plates (1×106
cells/well) for 24 h at 37°C then treated with 0.2, 0.4 and 0.8
µmol/l (low, medium and high) rutin. Following incubation for 24 h,
the cells were digested with 0.25% trypsin. DNA profiles were
evaluated using a flow cytometry station (MACSQuant Analyzer,
Miltenyi Biotec, Inc., Cambridge, MA, USA). Histograms or density
plots were generated using the MACSQuant digital software.
Immunofluorescence and DAPI
staining
Cells were fixed with 4% paraformaldehyde
(Sigma-Aldrich; Merck KGaA) for 15 min at room temperature, then
washed three times with PBS for 10 min. Cells were incubated with
ACTA2 (BM1693) and p38 (MK2101) antibodies (1:200) at 4°C
overnight, washed three times for 5 min and subsequently incubated
with anti-mouse immunoglobulin G secondary antibodies (1:500;
AP130P; Sigma-Aldrich; Merck KGaA) at 4°C for 2 h. DAPI was then
used for nuclear staining. Cells were mounted and images were taken
using a Nikon eclipse 80i fluorescence microscope (Nikon
Corporation, Tokyo, Japan). The protein expression levels of ACTA2
and p38 and apoptotic cell count (DAPI) were analyzed using Image
Pro Plus 6.0 software (Media Cybernetics, Inc., Rockbille, MD,
USA).
Statistical analysis
All data were obtained from at least three
independent experiments and presented as the mean ± standard error
of the mean. Statistical comparisons were made using Student's
t-tests. Statistical analysis of the data was performed using SPSS
v.11.0 for Windows (SPSS, Inc., Chicago, IL, USA). P<0.05 was
considered to indicate a statistically significant difference.
Results
Mesangial cell viability
Mesangial cells were treated with different doses of
rutin. Results demonstrated that cell viability was significantly
increased in the high glucose-induced group at 24 h compared with
the control group (P<0.01; Fig.
1). However, cell viability was significantly reduced after
treatment with rutin (low, middle and high doses) and captopril
(P<0.05) compared with high glucose-induced cells. These results
indicated that rutin was able to inhibit the viability of mesangial
cells.
ATP content analysis
The present results demonstrated that ATP content
was significantly increased in all high glucose-induced mesangial
cells compared with the control cells (P<0.05; Fig. 2).
Cell cycle analysis
Flow cytometry was performed following treatment of
mesangial cells with 0.2, 0.4 and 0.8 µmol/l rutin for 24 h.
Results demonstrated that the cell cycle of mesangial cells changed
markedly in the high glucose-induced mesangial cells group.
However, the cell cycle progression of mesangial cells improved
markedly in cells treated with rutin (Fig. 3; Table
I). These results suggested that rutin may affect the
regulation of the mesothelial cell cycle.
 | Table I.Cell cycle distribution in each group
of mesangial cells. |
Table I.
Cell cycle distribution in each group
of mesangial cells.
| Group | G1, % | G2, % | S, % |
|---|
| Control | 83.60 | 3.13 | 13.30 |
| High
glucose-induced | 80.40 | 8.21 | 11.40 |
| Captopril | 81.70 | 6.84 | 11.50 |
| Rutin (0.2
µmol/l) | 85.00 | 1.08 | 13.90 |
| Rutin (0.4
µmol/l) | 82.70 | 7.94 | 9.29 |
| Rutin (0.8
µmol/l) | 80.80 | 7.79 | 11.50 |
Immunofluorescence staining
As demonstrated in Fig.
4 and Table II, the
experimental results indicated apoptotic morphological changes of
mesangial cells after 24 h rutin treatment was observed by
fluorescence microscopy. Relative to the control group, cell groups
treated with rutin exhibited higher numbers of detached cells with
round and shrunken morphologies. DAPI nuclear staining also
identified signs of mesangial cell apoptosis in the rutin treatment
groups.
| Table II.ACTA2 and p38 protein expression in
each mesangial cell group (%). |
Table II.
ACTA2 and p38 protein expression in
each mesangial cell group (%).
| Group | Nucleus | ACTA2 | p38 |
|---|
| Control | 10.27±1.19 | 18.047±0.81 | 12.543±0.24 |
| High
glucose-induced |
25.22±1.75a |
28.41±1.92a |
26.85±0.11a |
| Captopril |
12.26±0.97b |
22.57±1.36b |
17.05±0.60b |
| Rutin (0.2
µmol/l) |
17.92±1.79b |
25.61±1.66b |
22.63±0.72b |
| Rutin (0.4
µmol/l) |
17.84±2.35b | 35.60±2.53 |
19.26±0.65b |
| Rutin (0.8
µmol/l) |
14.63±1.09b |
20.77±1.40b |
19.93±0.73b |
ACTA2 protein expression
To validate the expression of ACTA2 in mesangial
cells, ACTA2 specific fluorescent staining was performed on
mesangial cells. The results demonstrated that cells in the control
group had uniform chromatin, with a large nucleus and that they
maintained integrity of the nuclear envelope. Expression of ACTA2
protein increased significantly in the high glucose group compared
with the control (P<0.05; Fig. 5;
Table II). The expression of ACTA2
protein decreased significantly in the rutin groups at 24 h
compared with the high glucose group (P<0.05; Fig. 5; Table
II), with the exception of the middle dose of rutin group.
p38 protein expression
To validate the expression of p38 in mesangial
cells, p38 specific fluorescent staining was performed on mesangial
cells. The results demonstrated that cells in the control group had
uniform chromatin, a large nucleus and maintained integrity of the
nuclear envelope. The expression of p38 protein increased
significantly in high glucose group compared with the control
(P<0.05; Fig. 6; Table II). The expression of p38 protein
decreased significantly in the rutin groups at 24 h compared with
the high glucose group (P<0.05; Fig.
6; Table II).
Discussion
DN is one of the most serious microvascular
complications of diabetes (13).
Glucose metabolism and renal hemodynamics have an important role in
the pathogenesis of DN (14). In the
present experiment, high glucose induced the viability of mesangial
cells. Rutin inhibited the viability of high glucose-induced
mesangial cells. These results indicated that rutin had
hypoglycemic effects. ATP is a versatile nucleotide and has an
important role in cell biology as a coenzyme that is the ‘molecular
unit of currency’ of intracellular energy transfer (15). The present experiment demonstrated
that ATP content increased in high glucose-induced mesangial cells,
indicating that high glucose may induce a change in ATP content.
The content of ATP decreased after intervention with rutin.
Therefore, rutin may have a hypoglycemic effect on cells and
regulate energy metabolism. ACTA2 exists in birds and mammals.
ACTA2 is predominantly distributed in vascular myofibroblastic
cells, myoepithelial cells and smooth muscle cells under normal
circumstances (16).
In the present experiment, the expression of ACTA2
increased in high glucose-induced mesangial cells. Expression of
ACTA2 reduced after treatment with rutin. These results indicated
that rutin may inhibit the expression of ACTA2 protein and protect
against DN. p38 protein kinase is able to promote leukocyte
aggregation and activation, regulate the activity of transcription
factors and the synthesis of cell factors, and it has an important
role in the regulation of inflammatory reactions (17). In the present experiment, the
expression of p38 increased in high glucose-induced mesangial
cells. p38 expression decreased after treatment with rutin. These
results indicated that rutin may inhibit the expression of p38
protein, with protective effects against DN.
In conclusion, a diabetic model was successfully
induced by high glucose, which promoted glomerular cell viability,
increased ATP content, induced changes in the cell cycle, and
increased the expression of ACTA2 and p38 proteins. Results
suggested that rutin has hypoglycemic effects. Rutin may inhibit
the expression of ACTA2 and p38 and may confer protective effects
against DN. Rutin may also regulate the expression of p38.
Acknowledgments
The present study was supported by the Science and
Technology Department of Jilin Province, China (grant nos.
20140312002ZG and YYZX201259).
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