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Introduction
Diabetic nephropathy (DN) is one of the most common microangiopathies and ultimately leads to chronic renal failure.
Hypertrophy of the mesangial cell and accumulation of the extracellular matrix (ECM) in the mesangial region is mainly
composed of glomerulosclerosis. High glucose is presumed an initiating agent and increased transforming growth
factor-b1 (TGF-b1) is thought the key cytokine involved in the progression of
DN[1]. It has been found that the kidneys of DN rats
exhibit oxidative stress[2]. Studies have found that high glucose can induce oxidative stress or reactive oxygen species (ROS)
expressions and TGF-b1 synthesis in cultured mesangial
cells[1,3]. The addition of antioxidants to high glucose caused a
significant reversal of fibronectin and collagen IV gene
expressions[3,4]. TGF-b1 plays an important role in cell hypertrophy
and glomerular ECM accumulation by autocrine and paracrine methods. Interestingly, it has been found that ACE-inhibitors
or Ang II receptor blockers can lower enhanced
TGF-b1 and mesangial matrix
accumulation[5]. This effect may be via the
reduction of Ang II-stimulated TGF-b1 production[6]. With diabetes mellitus, high glucose and many factors including
TGF-b1, Ang II and ROS may also activate various kinds of signals that arrest cells in the
G1 phase inducing cell hypertrophy. It
has also been found that hypertrophic cells could secrete more
ECM[7]. Both create an infernal circle that leads to the
aggravation of DN.
A central component of
TGF-b1-stimulated ECM accumulation is the
TGF-b1 family-specific Smad signal transduction
pathway. TGF-b1 activates Smad2/3 by activating
TGF-b1 receptors, after which it
partners with Smad4 and translocates to the nucleus, where they act as transcriptional regulators of target genes including
TGF-b1, tissue inhibitors of metalloproteinase
(TIMP), collagen IV and laminin[8]. Smad6 and Smad7, the inhibitory Smads, appear to specifically inhibit Smad2/3 activation
by blocking its access to TGF-b1 receptors.
Ginkgo biloba extract (EGb) is taken from the leaves of ginkgo biloba by modern extraction techniques. It is a mixture
containing flavonoid glycosides (>24%) including quercetin, kaempferol, isorhamnethin, and terpene lactones (>8%)
including bilobalide, ginkgolide. It has been found that EGb ameliorates hemodynamics, suppresses platelet-activating factor
(PAF), scavenges ROS, relaxes vascular smooth muscles, and so
on[9,10]. It has also been found that flavonoid glycosides
have the effect of suppressing ACE activity and suppressing glycation end-products (AGE)
expression[11,12]. All of these offer us a pharmacological foundation of EGb for DN therapy. Many scholars have explored the effects of EGb and
ascertained its protective effects on DN in
vivo. Most of EGb's protective qualities were thought to be closely related to its
hemodynamic action. On the cellular level, however, the protective mechanisms of EGb on glomerulosclerosis of DN have
not been identified.
In our present work, using captopril as an antifibrotic control drug, we studied the possible influence of
EGb in mesangial cells on the level of cell cycle,
TGF-b1, Smad2/3, Smad7, collagen IV, laminin and antioxidases, which were all closely related
to the glomerulosclerosis of DN.
Materials and methods
Materials Rat mesangial cells were provided by the China Center for Type Culture Collection (CCTCC) in Wuhan
University (No HBZT-1); EGb (Lot No 040029) was provided by the Pizhou Fuwei Biochemical Company (Xuzhou, China);
Captopril (Lot No 050050) was provided by Changzhou Pharmaceutical Factory (Changzhou, China). Rabbit polyclonal
anti-Smad2/3 (Lot No 200506) was purchased from Boster Company (Wuhan, China). Goat anti-Rabbit IgG-horseradish
peroxidase (Lot No 015090) was provided by Zhongshan Golden Bridge Biotechnology Company (Beijing, China).
Considering that EGb can not dissolve in water completely, EGb and DMEM with D-glucose at 25 mmol/L were mixed with 1.0 %
ethanol in different concentrations.
Mesangial cell culture Taking the 5th_8th generation of mesangial cells, after incubating 24 h under normal conditions
(containing 5.56 mmol/L glucose, 10% FBS, 100 IU/mL penicillin and 100 mg/mL streptomycin, 37 ºC, 5%
CO2), we divided the cells into 7 groups, of which every group contained 6 bottles: normal glucose group (NS; DMEM), solvent control group (ET;
1.0% ethanol-DMEM), high glucose group (HG; 25 mmol/L glucose-DMEM), low dose of EGb group (GL; 10
µg/mL EGb-25 mmol/L glucose -DMEM), moderate dose of EGb group (GM; 20 µg/mL EGb-25 mmol/L glucose -DMEM), high dose of EGb
group (GH; 40 µg/mL EGb-25 mmol/L glucose -DMEM), and captopril group (1 µmol/mL captopril-25 mmol/L glucose
-DMEM). Cells were harvested after 48 h incubation. Adjusting the cell population to
1.5×106/mL, we took 1 mL of cell suspension from every group. The suspensions were then centrifuged three times at
100×g at 4 ºC for 8 min. The cell pellet
was then added into 2 mL of cell lysis solution. After being shaken severely, the mixture was centrifuged at
650×g at 4 ºC for 8 min. The supernatants were stored at -20 ºC for analysis at a later stage.
Cell cycle analysis by flow
cytometry[13] After incubation for 72 h, cells were harvested with 0.25% trypsin and washed
three times with PBS buffer for 100×g at 4 ºC for 8 min. The cell pellets were fixed with 70% ethanol at 4 ºC for 24 h. And the
ethanol was washed off with PBS buffer. The cell pellets were added into 0.5 mL propidium iodide-DNA fluorescein staining
solution for 30 min at 4 ºC. Then the mixture was put in the sample chamber to be examined at 488 nm of excitation wave. 10
000 cells were detected in every sample and the cell cycle was analyzed by Mod Fit 2.0 package (Becton Dickinson, USA).
Determining the quantity of collagen IV and
laminin[14] The stored supernatant at -20 ºC was tested by
radioimmunoassay kits from Shanghai High Biotech Center (Lot
No 200506, Shanghai, China).
Immunocytochemistry measurements of Smad2/3 and
Smad7[15] The glass slides were pretreated with 10% polylysine,
then placed on 24-well culture plates. The cells were transferred to the glass slides to incubate for 72 h. The supernatants
were displaced and the cells were fixed by 4% paraformaldehyde (PFA) for 30 min. After permeation with
0.1% Triton-X-100 for 15 min, the cells were incubated with rabbit polyclonal anti-Smad2/3 at a dilution of 1:100 at 37 ºC for 2
h. After being washed, goat anti-rabbit IgG-horseradish peroxidase was added. To visualize Smad2/3, cells were stained with
3.3-diaminobenzidine (DAB) for 30 min and then examined by light microscopy (×400). All steps were performed at room
temperature. Smad7 measurement was identical to Smad2/3. The stained Smad2/3 and Smad7 were quantified by gray scale
analysis (Leica Qwin Standard V2.6; Leica Microsystems, Welzlar, Germany).
RT-PCR for the relative quantities of
TGF-b1 mRNA of mesangial cells[16] A reverse transcription polymerase chain reaction
(RT-PCR) procedure was performed to determine the relative quantities of
TGF-b1 mRNA in mesangial cells, while b-actin mRNA, the house-keeping gene, was used as an internal control. Total RNA was
extracted from mesangial cells with Promega Totle RNA Isolation system (Lot
No 182207, Promega Corperation, Madison,WI,USA).
The upstream and downstream primers for rat
TGF-b1 mRNA were 5'-CCCGCA-TCCCAGGACCTCTCT-3' and 5'-CGGGGGACTGGCG AGCC-TTAG-3',
yielding a 519-bp product, whereas those for b-actin were 5'-GCTGCGTGTGGCCCCTGAG-3'and
5'-ACGCA-GGATGGCATGAGGGA-3', yielding a 252-bp product. Equal amounts (3 µL) of each total RNA sample were added in a 50 µL
reaction mixture exerting one-step amplification with Promega RT-PCR system (Lot
No 199676, Promega). The reaction mixture was incubated at 48 ºC for 45 min to reverse transcript, then went into cycles. The cycle conditions were set to: initial
denaturation for 5 min at 94 ºC, 40 cycles at 94 ºC
for 1 min, 57 ºC for 50 s, 72 ºC for 1 min, final elongation at 72
ºC for 7 min. The RT-PCR products were separated by 1% agarose electrophoresis, and the band densities were analyzed using laser densitometry.
The relative quantities of TGF-b1 mRNA in mesangial cells were represented
by the ratio of band density of TGF-b1 versus
that of b-actin.
Measurement of oxidative stress in mesangial
cells[14] Total antioxidative capability (T-AOC), catalase (CAT), total
superoxidase dismutase (T-SOD) and glutathione-peroxidase (GSH-Px) activities of the stored supernatants were measured
by spectrophotometry, using kits from Jiancheng Bioengineering Institute (Lot
No 20050522, Nanjing, China).
Statistical analysis Statistical analysis was performed to compare the effects of EGb on mesangial cells using one-way
analysis of variance (ANOVA) and Dunnett's t-test (2-side) for different groups using SPSS 10.0. Data were expressed as the
mean±SD. P<0.05 was considered statistically significant.
Results
Effect of EGb on cell cycle of mesangial cells The cell cycle of ET group using 1.0% ethanol as solvent was of no
significant difference to that of the NS group using normal sodium as solvent
(P>0.05), which suggested that 1.0% ethanol
had no significant effect on cell cycle and 1.0% ethanol could be used to incubate mesangial cells. The
G0/G1 phase percentage of the HG group was significantly higher and the S phase percentage was lower than that of the ET group
(P<0.05 or P<0.01). When compared with those of the HG group, the
G0/G1 phase percentages of GL, GM, and GH were
decreased and S phase percentages were increased in a concentration-dependent manner. The difference was significant
(P<0.05 or P<0.01). The
G0/G1 phase and S phase percentages of GM had no great difference to those of the ET group,
indicating that the moderate dose of EGb could reverse the cell cycle changes in high glucose. The
G0/G1 phase percentage of captopril was lowered and the S phase percentage was raised slightly, but they were not statistically significant
(P>0.05) (Table 1).
Effects of EGb on collagen IV and laminin of mesangial cells The levels of collagen IV and laminin of the ET group were
of no significant difference to those of the NS group
(P>0.05), suggesting that 1.0% ethanol had no significant effect on the
cell expressions of collagen IV and laminin. The levels of collagen IV and laminin of the HG group were significant increased,
when compared with those of the ET group (P<0.05 or P<0.01). Collagen IV levels of the GM, GH, and captopril group were
strikingly lower than those of the HG group
(P<0.05 or P<0.01). The level of the GH group was similar to that of the captopril
group (P>0.05). Laminin levels of the GL, GM, GH, and captopril groups were all decreased
(P<0.05 or P<0.01). These results
suggested that EGb could decrease the expressions of collagen IV and laminin in mesangial cells and captopril's capability of
decreasing expressions of collagen IV and laminin was between that of EGb's moderate and high dose
(P>0.05) (Figure 1).
Immunocytochemistry analysis of Smad2/3 and Smad7 of mesangial cells Mesangial cells looked like an irregular star or a fusiform. The color of the stained Smad2/3 or Smad7 protein was brown. Smad2/3 or Smad7 was expressed in cytoplasm,
but Smad2/3 could bind to Smad4 translocating into the nucleus. So Smad7 could be found only in the cytoplasm, whereas
Smad2/3 could be seen in both the cytoplasm and nucleus (Figures 2 and 3). The staining intensity of Smad2/3 of the HG
group was highly increased and Smad7 was markedly decreased, when compared with those of the ET group. After the
exposure of mesangial cells to EGb or captopril, the intensity of Smad2/3 became scarce and Smad7 became intense.
Using gray scale analysis to quantify Smad2/3 and Smad7 proteins, we found that the levels of Smad2/3 and Smad7 of the
ET group were of no significant difference to those of the NS group
(P>0.05), suggesting that 1.0% ethanol had no significant
effect on the cell expressions of Smad2/3 and Smad7 (Figure 4).
The level of Smad2/3 in the HG group was strikingly higher than that of the ET group and Smad7 was markedly lowered.
They had significant difference when compared to those of the ET group
(P<0.01). With the increasing concentration of EGb,
the expressions of Smad2/3 in GL, GM, and GH were significantly decreased
(P<0.01). The expressions in the captopril group
were also decreased (P<0.01), and the level of Smad2/3 in the captopril group was between the GL group and GM group. The
captopril group and the high dose of EGb could significantly increase the expression of Smad7
(P<0.01). The moderate dose of EGb also increased the expression of Smad7, but it had no significant difference when compared to that of the HG group.
All of these results suggested that EGb had a potent influence on cell expressions of Smad2/3 and Smad7 and EGb could
reverse the changes of Smad2/3 and Smad7 when mesangial cells were exposed to high
glucose.
Effect of EGb on the relative quantity of
TGF-b1 mRNA of mesangial cells The RT-PCR products of
TGF-b1 were separated by 1% agarose electrophoresis, after which we could see distinct bands (Figure 5A). The relative quantity of
TGF-b1 mRNA of the ET group was of no significant difference to that of the NS group
(P>0.05), suggesting that
1.0% ethanol had no significant effect on the cell expression of
TGF-b1 mRNA.
The relative quantity of
TGF-b1 mRNA of the HG group was greatly higher than that of the ET group
(P<0.01). The TGF-b1 mRNA level of the GH group and captopril group was strikingly decreased when compared with that of the HG group
(P<0.05). The levels of the GL and GM group were also decreased, but the differences were not significant
(P>0.05). The level of the GH group was similar to that of the captopril group. The results suggested that the high dose of EGb had a similar
capability to captopril of decreasing the expression of
TGF-b1 mRNA (Figure 5B).
Effects of EGb on oxidative stress The CAT, GSH-Px, T-AOC, and T-SOD activities of the ET group were of no significant
difference to those of the NS group (P>0.05), suggesting that 1.0% ethanol had no significant effect on the cell antioxidative
indexes activities. The activities of the four antioxidases of HG were all lower than those of the ET group
(P<0.05 or P<0.01), suggesting that mesangial cells in high glucose displayed oxidative stress. We also found that moderate and/or high doses
of EGb increased the four antioxidases activities
(P<0.05 or P<0.01). The activities of the GL group also increased but the
differences were not significant. These results indicated that EGb could ameliorate the oxidative stress state of mesangial
cells in high glucose. Captopril also significantly increased CAT and GSH-Px activities
(P<0.05), but it had no evident effect on
T-AOC and T-SOD (P>0.05) (Figure 6).
Discussion
Mesangial cells are a special kind of cell that can synthesize and secrete many protein factors regulating the structure and
function of glomerulus. Alteration in mesangial cell function is central to the progression of glomerular disease in numerous
models of chronic renal failure. It has been found that high glucose can stimulate the expression of Ang
II, ROS, and TGF-b1[17]. Ang II may directly induce the irregularity of hemodynamics in the
kidneys, and may also stimulate the expression of
TGF-b1, ROS, and ECM.
TGF-b1 itself can also induce accumulation of ECM mediated by signals such as MAPK, Smads, PKC, PKA,
Ca2+, and so on, and the signals interact with each other constituting a complicated network which leads DN to
aggravation[8]. The Smads protein is thought to be one of the most important factors in the process of ECM accumulation. Researchers
have found that mesangial cell hypertrophy and the accumulation of ECM in the mesangial region consists mainly of
glomerular sclerosis, while the autocrining cytokines of mesangial cells is a non-ignored element. The manifestations of
diabetic nephropathy may be a consequence of the actions of certain cytokines and growth factors. Therefore, the research
of mesangial cells has been a warm spot in DN research. In addition to blocking RAS and suppressing oxidative stress and
TGF-b1 expression, the interference in Smads signals following
TGF-b1 would be a new pathway to delay the progression of
DN.
Cell cycle is an elementary process in vital movements of cells, and it has a close connection with the cell proliferation,
differentiation and apoptosis. In the
G1 phase cells synthesize RNA and protein. If the main protein or RNA is modified in the
G1 phase cells can not step into the S phase. So
G1/S transition is a restriction point in the cell cycle. If cells can progress to
G1/S transition, cells will show proliferation; if cells can not progress to transition, cells will undergo cellular hypertrophy
through stimulated protein synthesis. High glucose-mediated expression of
TGF-b1 is pivotal for G1-phase arrest because
neutralizing anti-TGF-b1 antibodies convert the
G1-phase arrest into a proliferative
phenotype[18]. This neutralization
experiment clearly demonstrates that
TGF-b1 is a necessary prerequisite for the development of cell hyper-trophy.
G1 growth arrest is regulated by many elements of cell cycle machinery. Among regulators of
G1 progression, the increased cyclin inhibitors
p27 and p21 have been recognized as main
regulators of TGF-b1-induced cell-cycle arrest in
DN[19,20]. Flow cytometry is a technique for analyzing unicells rapidly. The cell cycle is analyzed acting on DNA contents. In our present study, we found
that the percentage of G0/G1 in the HG group was increased and S phase percentage was decreased
accompanied by increased
TGF-b1, which is consistent with former
reports[7,19]. When cells were cocultured with EGb,
G0/G1 percentage was decreased,
while the S percentage was increased correspondingly in a concentration-dependent manner. We also found that a middle
dose of EGb could reverse the cell cycle changes in high glucose, which indicates that EGb has a potent effect on cytoprotective
action, and that captopril do not significantly affect the cell
cycle. All of these results demonstrate that high
glucose/TGF-b1 induce the G1-phase arrest stimulating cell hypertrophy and that EGb could strikingly raise S phase percentage, therefore
leading cells to surpass G1/S restriction. In this respect, EGb might have much more protection on mesangial cells than
captopril. It has been found quercetin could suppress cell hypertrophy by degrading the expression of p27 in
glomerulus[21]. But little is known about EGb's cell protection, so it would be worthwhile to further investigate its potential mechanisms of
manifold ingredients on cell cycle.
The accumulation of ECM is the result of imbalance between synthesis system such as plasminogen activator
inhibitor-1 and TIMP and resolution system including matrix metalloproteinase.
TGF-b1 is closely associated with the accumulation of
mesangial ECM. The Smads protein following
TGF-b1 is thought to be one of the most important factors in the process of
ECM accumulation. In our experiment, the expressions of collagen IV and laminin of the HG group were strikingly increased.
To trace back to their upstream signals, we found the expressions of Smad2/3 and
the relative TGF-b1 level were also increased, while the inhibitory signal Smad7 was decreased. This indicates that it is high glucose that initially
induces the increased expression of
TGF-b1, ultimately resulting in the accumulation of collagen IV and laminin. In other words, it is
the activated TGF-b1/Smads signal pathway that
induces the accumulation of ECM.
TGF-b1/Smads/ECM is obviously a linked response. Our results are in correspondence with other
colleagues[22]. After cells were incubated with EGb, the
expressions of TGF-b1 and Smad2/3 were lowered significantly, and Smad7 was raised greatly, suggesting that EGb
suppress the TGF-b1/Smads/ECM response when mesangial cells are composed to high glucose. Recently Ang II blockade is rapidly becoming a
standard antifibrotic therapy in renal diseases, and its mechanism has been a theme of
research[23]. It is largely because
ACE-inhibitors block up TGF-b1 induced by Ang II. Colleagues
have proved that it is by suppressing
TGF-b1/Smads signal pathway that ACE-inhibitors
(N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP) decrease the accumulation of ECM in mesangial
cells[24]. In our study we also found that an ACE-inhibitor (captopril) had a potent effect on suppressing ECM. The effect of
EGb on ECM is similar to that of captopril. We know that the hypertrophic cells could express more ECM, so the potent effect
of EGb suppressing ECM expression might be inseparable from its action of powerfully regulating cell cycle.
Oxidative stress has been known to play an important role in the development and progression of DN, and ROS is a direct
consequence of hyperglycemia. It has been found that
TGF-b1 and AGE could also activate
ROS[25]. ROS activates other signaling molecules, such as PKC and MAPK and transcription factors including NF-kappa B, activator protein-1 and
specificity protein 1 leading to a transcription of genes encoding cytokines, growth factors, and ECM proteins, all of which
are closely relative to DN[26]. Various antioxidants inhibit mesangial cell activation by high glucose and ameliorate features
of DN. It has been reported that the glucose-induced collagen IV expression can be partially reversed by the addition of two
structurally unrelated antioxidants, trolox and a-lipoic acid, in porcine mesangial
cells[3]. So antioxidant treatment is a
potential antifibrotic therapy. N-acetylcysteine, a classic antioxidant, was used as an effective antifibrotic drug by some
scholars[27]. In our study, after the cells were incubated with EGb, we found that CAT, T-AOC, T-SOD, and GSH-Px activities
(common indicators for changes in the antioxidation system) were all increased significantly accompanied by decreased
collagen IV and laminin expressions, strongly suggesting that EGb has a potent antioxidative capability and antifibrotic
capability in vitro. This result is consistent with a former report, in which EGb suppressed oxidized LDL-stimulated fibronectin
production through an antioxidant action in rat mesangial
cells[28]. In another report EGb was used as a free radical
scavenger[10]. The control drug captopril also had significant effects on enhancing CAT and GSH-Px activities, but had no significant
effect on T-AOC and T-SOD. These data suggest that EGb may have a more profound effect than captopril on ameliorating
the oxidative stress state of mesangial cells and antifibrotic action.
More recently, scholars have paid more attention to the
interaction between ROS and TGF-b1/Smads in DN. It has been
found that AGE-RAGE-mediated ROS generation activates
TGF-b1-Smads signals and subsequently induces mesangial cell
hypertrophy and fibronectin synthesis by autocrine production of Ang
II[25]. It has also been found that
H2O2 mediated the activation of ERK with the Smads pathway in
TGF-b1 induction of p21[29].
Therefore, we could conclude that ROS and
TGF-b1/Smads signals interact mutually and upregulate each other in the pathogenesis of DN. In our present study, we could see
that EGb could act as an antioxidant suppressing
TGF-b1-Smads signals and could also suppress
TGF-b1 expression ameliorating the oxidative stress state. Both observations could suppress ECM
accumulation and cell hypertrophy in mesangial cells. Therefore, the non-hemodynamic effects of EGb on DN are also
significant.
In conclusion, EGb can regulate the mesangial cell cycle, ameliorate oxidative stress state, suppress the
accumulation of collagen IV and laminin, decrease
TGF-b1 and Smad2/3, and increase Smad7 expressions in cultured cells. That is to say, EGb
can suppress cell hypertrophy and the accumulation of ECM mediated
by TGF-b1/Smads and ROS signals on cell levels,
which means it could vitally postpone glomerulosclerosis of DN.
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