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Effects of Shuang Dan Ming Mu Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and the VEGF receptor, Flk-1, in Diabetic Retinopathy Rats
228
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Digital Chinese Medicine journal homepage: http://dcmhi.com.cn
Effects of Shuang Dan Ming Mu Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and the VEGF receptor, Flk-1, in Diabetic Retinopathy Rats Jun PENGa, Kun PANa, Zheng-Rong LIUa, Yu-Hui QINa* , Qing-Hua PENGa* a. Department of Traditional Chinese Medicine Ophthalmology, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China
ARTICLE INFO
ABSTRACT
Article history
Objective The effects of Shuang Dan Ming Mu capsule on expression of VEGF-a, VEGF-b, VEGF-c and the VEGF receptor, Flk-1, were examined in a diabetic retinopathy rats model.
Received 25 Apr. 2018 Accepted 1 Jun. 2018 Available online 26 Sep. 2018 Keywords Shuang Dan Ming Mu capsule Diabetic rats model VEGF-a VEGF-b VEGF-c Flk-1 *Corresponding author: Yu-Hui QIN, Professor, Research Scientist Grade II. Research direction: TCM diagnosis and treatment of fundus cornea disease. E-mail: [email protected]. Qing-Hua PENG, Professor Grade II. Research direction: TCM treatment and prevention of ocular fundus diseases, glaucoma and ocular surface diseases and the normalization of ophthalmic symptoms. E-mail: [email protected]. Peer review under the responsibility of Hunan University of Chinese Medicine.
Methods Forty Sprague-Dawley (SD) rats were randomized into Groups A (blank), B (model), C (Shuang Dan Ming Mu) and D (positive control) group, with each group containing 10 rats and 20 eyes. Rats from groups B, C and D were administered one dose of 50 mg/kg streptozotocin (STZ) by tail vein injection to establish a diabetic rat model. One week after model preparation, medication was continuously administered by gavage. After gavage for 8 weeks, the animals were sacrificed and retinal expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 was quantified by immunohistochemical analysis. Results At week 8 of drug administration after model preparation, the average protein expression grayscale values for VEGF-a, VEGF-b, VEGF-c and Flk-1 in the rats model, Shuang Dan Ming Mu and positive control groups were all lower than those in the normal group, while the mean optical density values were higher than those in the normal group. When the model group was compared to the normal group, the difference was extremely significant (P < 0.01). The mean grayscale values of VEGF-a, VEGF-b, VEGF-c and Flk-1 in the Shuang Dan Ming Mu and positive control groups were all higher than those in the model group, while the mean optical density values were lower than those in the model group (P < 0.05 or 0.01). Conclusion Shuang Dan Ming Mu capsule can significantly decrease the expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in the retinas of diabetic model rats and exhibit some protective effects in their retinas.
Copyright © 2018 Hunan University of Chinese Medicine Publishing services provided by Elseiver B.V. On behalf of KeAi This is an open access article under the CC BY-NC-ND License (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 229
2.1.2 Experimental drugs
1 Introduction Diabetic retinopathy (DR) is the most common and severe microvascular complication in diabetes and the main cause of vision loss in patients with diabetes mellitus (DM). If DR is not treated in the early stages, it may lead to irreversible visual impairment. Qin suggested that the main pathogenesis of DR is liver and kidney deficiencies and pulse stagnation. Based on long-term clinical practice, he created China’s first new traditional Chinese medicine (TCM), Shuang Dan Ming Mu capsule, to specifically treat DR. Previous studies showed that the mechanisms of Shuang Dan Ming Mu capsule may involve effective reduction of blood glucose and glycated hemoglobin levels in DR rats, improvements in the retina and pancreas structures in rats, reduce platelet adhesion rate and blood rheology and promotion of blood circulation
[1-3]
. In this study, we evaluated the
changes in the vascular endothelial growth factor (VEGF) signaling pathway and its receptor and related molecules to determine the effects of Shuang Dan Ming Mu capsule on retinal VEGF-a, VEGF-b,
Shuang Dan Ming Mu capsule were composed of Ligustri Lucidi Fructus (Nv Zhen Zi, 女 贞 子 ), Ecliptae Herba (Mo Han Lian, 墨 旱 莲 ), Corni Fructus (Shan Zhu Yu, 山茱萸), Dioscoreae Rhizoma (Shan Yao, 山药), Poria (Fu Ling, 茯苓)， Alismatis Rhizoma (Ze Xie, 泽泻), Moutan Cortex (Mu Dan Pi, 牡丹皮), Salviae Miltiorrhizae Radix Et Rhizoma (Dan Shen, 丹参), Notoginseng Radix Et Rhizoma (San Qi, 三七), Smilacis Glabrae Rhizoma (Tu Fu Ling, 土 茯 苓) and Achyranthis Bidentatae Radix (Niu Xi, 牛膝). Shuang Dan Ming Mu capsule were provided by the TCM Research Institute, Hunan Academy of Chinese Medicine (Hunan, China; Batch number: Guoyaozhun No. Z20080062). Calcium dobesilate (250 mg/tablet) was obtained from Ningxia Kangya Pharmaceutical Co. Ltd. (Ningxia, China; Batch
number:
Guoyaozhun
No.
H20030809).
Recombinant human endostatin injection (Endostar, Warsaw, Poland; 15 mg/2 mL/vial) was obtained from Yantai Medgenn Co., Ltd. (Nanjing, China; Batch number: Guoyaozhun No.S20050088).
VEGF-c and Flk-1 expression in diabetic rats.
2.1.3 Experiment reagents
2 Materials and Methods
VEGF-a, VEGF-b, VEGF-c and Flk-1 were obtained from Boster Biological Technology Co. Ltd. (Wuhan,
2.1 Experimental materials
China). Streptozocin (STZ) was produced by Sigma (St.
2.1.1 Experimental animals
Louis, MO, USA). Sodium pentobarbital was obtained from
Shanghai
Chemical
Dispensing
Factory
Forty one-month old, healthy, specific pathogen-free-grade, outbred Sprague-Dawley (SD)
(Shanghai, China). Hematoxylin-eosin was obtained
rats with no ocular fundus disease and weighing
Ltd. (Beijing, China). The 4% paraformaldehyde, 1%
200–250 g were obtained from Beijing Vital River
osmic acid, acetone. Epon812 epoxy resin, uranium
Laboratory Animal Technology Co., Ltd. (Beijing, China). Before the experiment, the fundus of both
acetate, lead citrate and isoamyl acetate were obtained from Hunan Fuzitang Biotechnology Co., Ltd. (Hunan,
eyes was examined for any abnormalities and rats
China). The 4% paraformaldehyde was obtained from
with systemic lesions were excluded. The animals
the pathology laboratory of Hunan University of
were housed at 18–20℃ with ventilation in dry
Chinese Medicine. Harris hematoxylin, eosin and 3%
specific pathogen-free animal laboratories with a relative humidity of 60%–70% and fed laboratory
peracetic acid were obtained from Beijing Zhongshang
rat growth pellets that had been stringently disinfected and Grade III drinking water. The
from Beijing Dingguo Changsheng Biotechnology Co.,
Jinqiao Biotechnology Co., Ltd. (Beijing, China). 2.2 Experimental methods
cages and bottles were cleaned and disinfected
2.2.1 Grouping
every 2 days.
After feeding the animals routinely for 1 week,
230
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
random numbers were used to divide the 40 rats into 4
solution was used for gavage combined with
groups (A, B, C and D). Each group contained 10 rats
intravitreal administration of recombinant human
and 20 eyes. These groups were A: blank group
endostatin
(normal group), B: model group, C: Shuang Dan
administered once per day and Endostar intravitreal
Ming Mu group and D: positive control group.
injections were started on day 10 after model
2.2.2 Model construction
(Endostar).
Calcium
dobesilate
was
construction. Before intravitreal injection, the anterior chamber was punctured to release 20 μL of aqueous
Except for group A, the 30 rats in the other groups
humor. A 30G needle microinjector was used to pierce
were used for model construction. Before model
the sclera at 0.5 mm behind the corneal limbus of the
construction, rats were fasted for 10 h but given adlibitum access to water. One dose of 50 mg/kg STZ
superior
administration of 20 μL (5 g/L) Endostar once every 10
was injected through the tail vein into the rats. Rats in
days [5].
group A were given tail vein injections with an equal volume of physiological saline. Blood was drawn
temporal
quadrant
for
intravitreal
2.2.4 Material collection
from the tail vein 72 h later and blood glucose was
After rats were anesthetized with an overdose of 2%
measured using a Johnson & Johnson Blood Glucose
sodium pentobarbital, they were sacrificed by
Meter (New Brunswick, NJ, USA). Urine test strips
decapitation. The eyeballs were immediately removed
were used to measure urine glucose levels. Rats with
and fixed with 10% formalin for 24 h. The anterior
fasting blood glucose concentration >16.7 mM and
segment was removed and retinal tissues were
urine glucose of +++ and above were considered s diabetic. After model construction, the rats were
isolated and washed with running water for 8 h. After
observed for 1 week and rats with stable glucose
impregnation, embedding and sectioning (thickness of
concentrations were considered as successful models.
4 μm) were carried out.
The disease course was then calculated. No rats died after model construction. However, 2 rats showed low
dehydration
using
an
gradient,
wax
2.2.5 Marker detection
blood glucose values, which met the standards after a
Immunohistochemistry
second tail vein injection.
VEGFa,
2.2.3 Drug administration method
alcohol
VEGFb,
quantification
VEGFc
and
of
retinal
Flk-1
protein
expression: The SP method was used to examine the protein expression of VEGFa, VEGFb, VEGFc and
groups, continuous gavage was used for drug
Flk-1 under high magnification. After the immunohistochemistry reaction, the cytoplasm of
administration for 8 weeks before the animals were
positively labeled cells was colored brown or deep
sacrificed. All rats were given ad libitum access to the
brown. Ten retina fields were randomly chosen under
diet and the bedding was changed once per day.
high magnification and MOTIC 6.0 image analysis
Regular ventilation was carried out and a quiet
software (Motic Co. Ltd) was used for image analysis
environment was maintained.
and processing and quantitative analysis of the mean
One week after model construction of the various
(1) Blank group (Group A), Model group (Group B): These two groups were gavaged with 10 mL/kg of physiological saline. (2) Shuang Dan Ming Mu capsule group (Group C): Shuang Dan Ming Mu capsule solution was used for gavage at a dose of 12.5 mL/kg. (3) Positive control group (Group D): Relevant studies
[4]
were referenced and calcium dobesilate
optical density of the image (OD value). 2.3 Statistical analysis SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA) was to analyze the experimental data. A difference of P < 0.05 was considered statistically significant and a difference of P < 0.01 was considered extremely significant. All data was expressed as the mean ±
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 231
standard deviation (x̄ ± s). 3 Results 3.1 Retinal VEGF-a protein expression status After 8 weeks of drug treatment after model construction, the mean optical density of VEGF-a protein expression was compared. As shown in Table 2, the mean optical density for VEGF-a protein expression in the model, Shuang Dan Ming Mu and positive control groups were all higher than that in the normal group. When the model group was compared to the normal group, the difference was extremely significant (t = 4.905, P < 0.01). The results showed widespread expression of VEGF-a in the retinas in the
difference was extremely significant (t = 4.445 and 4.608, both P < 0.01). After treatment with Shuang Dan Ming Mu capsule or positive control drugs, retinal VEGF-a expression in the rats was significantly downregulated. When the positive control group was compared to the Shuang Dan Ming Mu group, the difference was not significant (t = 0.124，P > 0.05) (Table 1 and Figure 1). Table 1 VEGF-a mean optical density comparison at week 8 of drug treatment after model construction Group
n
Mean optical density
A
10
0.356 ± 0.097
B
10
0.571 ± 0.099**
C
10
0.382 ± 0.091△△
model group. The mean optical densities of VEGF-a protein expression in the Shuang Dan Ming Mu and
Notes: Comparison of Groups B, C and D with Group A: *P <
positive control groups were all lower than that in the
0.05, **P < 0.01; Comparison of Groups C and D with Group
model group. Statistical analysis showed that the
B: △P < 0.05,
△△
P < 0.01.
Fig. 1 Immunohistochemical observation of VEGF-a protein expression at week 8 after model construction (×400) a. Normal group: In normal retinas, there were small amounts of positive VEGF- b protein expression in the nerve fiber layer, ganglion cell layer and inner plexiform layer (VEGF- b ×400). b. Model group: Positive retinal VEGF- b protein expression was significantly increased and gradually shifted towards the inner plexiform layer (VEGF-b ×400); c. Shuang Dan Ming Mu group: Positive retinal VEGF- b protein expression showed a slight increase but was lower than the model group (VEGF-b ×400); d. Positive control group: Positive retinal VEGF- b protein expression showed a slight increase but was lower than the model group (VEGF-b ×400)
232
3.2 Retinal VEGF-b protein expression status After 8 weeks of drug treatment after model construction, the mean optical density of VEGF-b protein expression was compared. As shown in Table 2, the mean optical density of VEGF-b protein expression in the model, Shuang Dan Ming Mu and positive control groups were all higher than that in the normal group. When the model group was compared to the normal group, the difference was extremely significant (t = 5.252, P < 0.01). The results showed widespread expression of VEGF-b in the retinas in the model group. The mean optical densities of VEGF-b protein expression in the Shuang Dan Ming Mu and positive control groups were all lower than that in the model group. Statistical analysis showed that the difference was extremely significant (t = 4.649 and 4.784, both P < 0.01). After treatment with Shuang
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Dan Ming Mu capsule or positive control drugs, retinal VEGF-b expression in the rats was significantly downregulated. When the positive control group was compared to the Shuang Dan Ming Mu group, the difference was not statistically significant (t = 0.071, P > 0.05) (Table 2 and Figure 2). Table 2 VEGF-b mean optical density comparison at week 8 of drug treatment after model construction Group
n
Mean optical density
A
10
0.326 ± 0.097
B
10
0.561 ± 0.103**
C
10
0.352 ± 0.098△△
D
10
0.349 ± 0.095
△△
Notes: Comparison of Groups B, C and D with Group A: *P < 0.05, **P < 0.01; Comparison of Groups C and D with Group △ B: P < 0.05,
△△
P < 0.01.
Fig. 2 Immunohistochemical observation of VEGF-b protein expression at week 8 after model construction (×400) a. Normal group: In normal retinas, there were small amounts of positive VEGF- b protein expression in the nerve fiber layer, ganglion cell layer and inner plexiform layer (VEGF- b ×400); b. Model group: Positive retinal VEGF- b protein expression was significantly increased and gradually shifted towards the inner plexiform layer (VEGF-b ×400); c. Shuang Dan Ming Mu group: Positive retinal VEGF- b protein expression showed a slight increase but was lower than the model group (VEGF-b ×400); d. Positive control group: Positive retinal VEGF- b protein expression showed a slight increase but was lower than the model group (VEGF-b ×400)
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 233
3.3 Retinal VEGF-c protein expression status After 8 weeks of drug treatment after model construction, the mean optical density of VEGF-c protein expression was compared. As shown in Table 6, the mean optical density of VEGF-c protein expression in the model, Shuang Dan Ming Mu and positive control groups were all higher than that in the normal group. When the model group was compared with the normal group, the difference was extremely significant (t = 4.871, P < 0.01). The results showed widespread expression of VEGF-c in the retinas in the model group. The mean optical densities of VEGF-c protein expression in the Shuang Dan Ming Mu and positive control groups were lower than that in the model group. Statistical analysis showed that the difference was extremely significant (t = 4.376 and 4.323, both P < 0.01). After treatment with
Shuang Dan Ming Mu capsule or positive control drugs, retinal VEGF-c expression in the rats was significantly downregulated. When the positive control group was compared to the Shuang Dan Ming Mu group, the difference was not significant (t = 0.124, P > 0.05) (Table 3 and Figure 3). Table 3 VEGF-c mean optical density comparison at week 8 of drug treatment after model construction Group
n
Mean optical density
A
10
0.369 ± 0.083
B
10
0.568 ± 0.099**
C
10
0.381 ± 0.092
D
10
0.386 ± 0.089△△
△△
Notes: Comparison of Groups B, C and D with Group A: *P < 0.05, **P < 0.01; Comparison of Groups C and D with Group △
B: P < 0.05,
△△
P < 0.01.
Fig. 3 Immunohistochemical observation of VEGF-c protein expression at week 8 after model construction (×400) a. Normal group: In normal retinas, there were small amounts of positive VEGF-c protein expression in the nerve fiber layer, ganglion cell layer and inner plexiform layer (VEGF-c ×400); b. Model group: Positive retinal VEGF- c protein expression was significantly increased and gradually shifted towards the inner plexiform layer (VEGF-c ×400); c. Shuang Dan Ming Mu group: Positive retinal VEGF- c protein expression showed slight increase but lower than the model group (VEGF-c ×400); d. Positive control group: Positive retinal VEGF- c protein expression showed slight increase but lower than the model group (VEGF-c ×400)
234
3.4 Retinal Flk-1 protein expression status After 8 weeks of drug treatment after model construction, the mean optical density of Flk-1 protein expression was compared. As shown in Table 8, the mean optical density of Flk-1 protein expression in the model, Shuang Dan Ming Mu and positive control groups were all higher than that in the normal group. When the model group was compared to the normal group, the difference was extremely significant (t = 5.109, P < 0.01). The results showed widespread expression of Flk-1 in retinas in the model group. The mean optical densities of Flk-1 protein expression in the Shuang Dan Ming Mu and positive control groups were lower than that the model group. Statistical analysis showed that the difference was extremely significant (t = 4.789 and 4.953, both P < 0.01). After
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
treatment with Shuang Dan Ming Mu capsule or positive control drugs, retinal Flk-1 expression in rats was significantly downregulated. When the positive control group was compared to the Shuang Dan Ming Mu group, the difference was not significant (t = 0.125, P > 0.05) (Table 4 and Figure 4). Table 4 Flk-1 mean optical density comparison at week 8 of drug treatment after model construction Group
n
Mean optical density
A
10
0.419 ± 0.093
B
10
0.642 ± 0.102**
C
10
0.436 ± 0.090
D
10
0.431 ± 0.088△△
△△
Notes: Comparison of Groups B, C and D with Group A: *P < 0.05, **P < 0.01; Comparison of Groups C and D with Group △
B: P < 0.05,
△△
P < 0.01.
Fig. 4 Immunohistochemical observation of Flk-1 protein expression at week 8 after model construction (×400) a. Normal group: In normal retinas, there were small amounts of positive Flk-1 protein expression in the nerve fiber layer, ganglion cell layer and inner plexiform layer (Flk-1 ×400); b. Model group: Positive retinal Flk-1 protein expression was significantly increased and gradually shifted towards the inner plexiform layer (Flk-1 ×400); c. Shuang Dan Ming Mu group: Positive retinal Flk-1 protein expression showed a slight increase but was lower than in the model group (Flk-1 ×400); d. Positive control group: Positive retinal VEGF- c protein expression showed a slight increase but was lower than in the model group (Flk-1 ×400)
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 235
Modern physicians have employed ocular fundus
4 Discussion
examinations and fluorescein angiography to study 4.1 Traditional Chinese medicine understanding of diabetic retinopathy
this disease based on blood rheology, microcirculation and blood biochemistry; they found that “blood
In TCM, diabetic retinopathy is known as “diabetic
stasis” plays an important role in the pathogenesis of
. According to vision changes and
this disease. Therefore, professor Yu-Hui QIN found
reduced visual acuity in the patient, DR is involved in
that the etiology of this disease is that kidney vacuity
different diseases, such as “indistinct vision,” “clouds
is the root, yin vacuity is the cause and blood stasis is
and mist moving over the eye,” “blood passing
the symptom and “kidney vacuity and blood stasis” is
cataracts”
[6]
. The
the main pathogenesis of this disease. Therefore, DR
Jin dynasty physician Zi-He ZHANG pointed out in
treatment should follow the basic principle of
“Confucians’ Duties to Parents” that diabetes is due to
“enriching the kidney and quickening the blood.”
[7]
through the pupil,” “sudden blindness,” etc.
excessive consumption of the essence-spirit and
Based on this principle, Professor Yu-Hui QIN
over-exuberant dryness-heat..., and can evolve into
developed Shuang Dan Ming Mu capsule, which is
sparrow vision or cataracts.” He suggested that
the first new traditional Chinese medicine for
dryness-heat
main
specifically treating DR in China. This drug nourishes
dynasty
the kidneys and liver, increases blood glow and clears
physician Xian ZHAO suggested that “diabetes
the eyes and is mainly used to treat DR caused by
treatment cannot be classified as low, moderate and
liver-kidney yin vacuity and static blood blocking the
high and it is imperative to treat the kidneys by
network
six-ingredient pill or eight-ingredient pill to decrease
demonstrated that the drug has good efficacy
heart fire nourishment of kidney water, which will
improving fundus lesions in patients, promoting the
self-stop diabetes.” The Ming dynasty physician
recovery of visual acuity and treating DR. Our
Ken-Tang WANG’s book “Standards of diagnosis and
previous pharmacological studies
treatment”
three
Shuang Dan Ming Mu capsule (originally known as
dissipation-thirst results in depletion of essential Qi,
Jiang Tang Ming Mu capsule) can be combined with
causing vision loss. He believed that DR is caused by
glucose-lowering drugs to greatly promote the latter’s
the depletion of essential Qi and vision loss.
glucose-lowering effect. Shuang Dan Ming Mu
Subsequently, generations of physicians examined DR
capsule monotherapy can also decrease platelet
based on these foundations. Most researchers
adhesion, improve blood rheology and promote blood
suggested that long periods of diabetes result in
circulation.
reduced essential qi, which cannot be transmitted to
4.2 Examination of effector mechanisms of Shuang
pathogenesis
that
damages
mechanism.
also
described
yin The
as
the
Ming
that
long
the eyes, resulting in undernourishment of the eyes or
vessels.
Multi-center
trials
[1-3]
have [8-10]
in
also found that
Dan Ming Mu capsule on diabetic rats
liver-kidney yin vacuity causes yin vacuity and yang hyperactivity, upflaming vacuity fire that burns the eyes, resulting in blurred vision or even blindness.
Neovascularization
(NV)
is
a
characteristic
pathological change of DR that occurs from non-proliferative diabetic retinopathy to proliferative
236
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
diabetic retinopathy. NV occurrence is regulated by
developing
multiple growth factors, among which VEGF is a key
differentiation and growth of blood vessels. In
factor in angiogenesis. VEGF is a family that includes
endothelial cells, VEGF-c acts on the VEGFR-2 and
VEGF-a, VEGF-b, VEGF-c, VEGF-d, VEGF-e and
VEGFR-3 receptors to induce endothelial cell
placental growth factor, among others
[11]
. VEGF-a is
embryo,
VEGF-c
promotes
the
proliferation, particularly capillary endothelial cells
a highly conserved, disulfide linked homodimer.
[15]
Under reducing conditions, the homodimers separate
showed that infusion of VEGF-c plasmids through a
into monomers and all biological activity is lost.
catheter promoted the growth of blood vessels in
VEGF-a can promote the proliferation of vascular
ischemic regions and significantly improved the blood
endothelial cells. As a specific mitogen for vascular
supply.
endothelial cells, VEGF can stimulate in vitro
potential for treating ischemic diseases
cultured vascular endothelial cells to undergo mitosis
promote endothelial cell proliferation. Flk-1/KDR
and migration. VEGF also acts on in vivo vascular
plays a dominant role in VEgF signal transduction
endothelial cells to cause endothelial cell division and
and formation of the vascular endothelium. Flk-1 acts
proliferation, induce blood vessel formation and is
as a negative regulator of Flk-1/KDR and antagonizes
currently the most potent pro-angiogenic factor.
the effects of Flk-1/KDR in promoting vascular
VEGF can increase vascular permeability, particularly
endothelial cell proliferation [17].
. A study using a rabbit ischemic hindlimb model
Therefore,
VEGF-c
shows
application
[16]
. Flk-1 can
that of the capillaries, causing plasma proteins to leak
In this study, we constructed diabetic rat models
into the extracellular matrix and be deposited in the
and observed the effects of Shuang Dan Ming Mu
extracellular matrix to provide a conditional matrix
capsule on retinal expression of VEGF-a, VEGF-b,
for the migration of fibroblasts and vascular
VEGF-c and the VEGF receptor, Flk-1, in a diabetic
endothelial cells. This increases nutrient levels
rat model. At week 8 of drug administration after
available for the growth of tumor cells and
model construction, we compared the mean optical
[12-13]
.
density of retinal VEGF-a, VEGF-b, VEGF-c and
VEGF-b is mainly distributed in the heart, skeletal
Flk-1 expression. The results showed that the mean
muscles, brain and kidneys in rats and the heart,
optical density of VEGF-a protein expression in the
skeletal muscles, pancreas and prostate gland in
model, Shuang Dan Ming Mu and positive control
humans. Researchers have analyzed [3H] labeled
groups were higher than that in the normal group.
thymidine in human umbilical vein endothelial cells
When the model groups were compared to the normal
and bovine capillary endothelial cells and found that
group, the difference was extremely significant (P <
VEGF-b can promote endothelial cell proliferation.
0.01). The mean optical density of VEGF-a, VEGF-b,
VEGF-b is also intimately associated with tumor
VEGF-c and Flk-1 protein expression in the Shuang
growth: VEGF-b and VEGF can promote local blood
Dan Ming Mu and positive groups were lower than that
establishment of new capillary networks
[14]
.
in the model group. Statistical analysis showed that
VEGF-c has potent angiogenic effects in the body and
these differences were significant (P < 0.05 or P <
has more persistent effects than VEGF. In the
0.01). This demonstrates that after treatment with the
vessel growth, thereby promoting tumor growth
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 237
Shuangdanmingmu
Shuang Dan Ming Mu capsule or positive control drug,
capsule
on
retinal
vascular
morphology and VEGF expression in rats with diabetic
the expression of VEGF-a, VEGF-b, VEGF-c and
retinopathy. Int Eye Sci, 2015, 15(1): 29-33.
Flk-1
in
the
rat
retinas
was
significantly
[2] QIN Y H， LI W J， ZHANG X, et al. Effects of
downregulated. Additionally, when the positive
Shuangdanmingmu
control group was compared to the Shuang Dan Ming
retinopathy in rats with diabetic retinopathy. Int Eye Sci,
capsule
on
blood
glucose
and
2014, 14(11): 1943-1945.
Mu group, the difference was not significant (P >
[3] QIN Y H， LI W J， ZHANG X, et al. Effects of
0.05). Our study showed that Shuang Dan Ming Mu
Shuangdan Mingmu Capsule on the Expression of Retinal
capsule can significantly decrease the mean optical
VEGF and VEGFR Protein Activity in Rats with Diabetic
density of VEGF-a, VEGF-b, VEGF-c and Flk-1
Retinopathy. Journal of Hunan University of Chinese Medicine, 2015, 35(6): 1-3.
expression in the retinas of rat models, i.e. Shuang [4]
Dan Ming Mu capsule can significantly decrease the
NAGAOKA
T,
SATO
E,
TAKAHASHI
A,
et
al． Impaired retinal circulation in patients with type 2
expression of VEGF-a, VEGF-b, VEGF-c and Flk-1
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in the retinas, which can protect retinal capillaries in
Invest Ophthalmol Vis Sci, 2010, 51(12): 6729. [5] CHEN L J, MIAO L. Inhibitive effects of recombinant
diabetic rats.
human endostatin on choroidal neovascularization. Rec Adv Ophthalmol, 2012, 32(10): 922-925.
Acknowledgements
[6] PENG Q H. Ophthalmology of Chinese Medicine [M].
We thank for the funding support from the National Natural Science Foundation General Program (No. 814737370); Hunan Province Graduate Student
Chinese Press of Traditional Chinese Medicine, 2012:187. [7] CHEN X D, PENG Q H, YAN J C, et al. Clinical observation of Fuming Table on diabetic retinopathy after retinal
Research Innovation Program (No. CX2016B377 and No. CX2017B432); Traditional Chinese Medicine Prevention and Treatment of Facial Feature Disease Hunan
Province
Key
Laboratory
Construction
laser
photocoagulation.
Journal
of
Hunan
University of Chinese Medicine, 2016, 36(1): 63-65. [8] TU L Y, WANG W C, WANG Y L. Efficacy and safety of Shuangdan
Mingmu
capsule
in
treating
diabetic
retinopathy. Chinese Journal of New Drugs, 2009, 18(24): 2331-2336.
Program (No. 2017TP1018); Changsha City Science
[9] PANG Y H. Clinical observation on treatment of diabetic
and Technology Program (No. kc1704005); Central
retinopathy with Shuang Dan Ming Mu capsule. Diabetes
Finance Supported Local High School Construction
New World, 2015,(3): 39.
Project; State Administration of Traditional Chinese
[10] QIN Y H, LI F, TU L Y, et al. Multicentric clinical study of Shuangdan Mingmu capsule on diabetic retinopathy.
Medicine
Ophthalmology
Key
Discipline
Construction Project; Hunan Province Traditional Chinese Medicine Facial Feature Key Discipline Construction Project.
Journal of Hunan University of Chinese Medicine, 2010, 30(1): 46-51. [11] LU C, SHI C H. Relationship between VEGF family/VEGF receptors and pathogenesis of diabetic retinopathy. Int Eye Sci, 2007, 7(5): 1400-1402.
Competing Interests
[12] NAGY J A, BENJAMIN L, ZENG H, et al. Vascular permeability,
The authors declare no conflict of interest.
vascular
hyperpermeability
and
angiogenesis. Angiogenesis, 2008(11): 109-119. [13] ZHANG D M, QING C. Vascular Endothelial Growth
References [1] QIN Y H， LI W J， ZHANG X, et al. Effects of
Factor Family and Tumor Angiogenesis. Medical Recapitulate, 2017, 23(3): 417-420. [14] SALVEN P, LYMBOUSSAKI A, HEIKKILA P, et al.
238
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Vascular endothelial growth factors VEGF-B and
et
VEGF-C are expressed in human tumors. Am J Pathol,
(VEGF-C/VEGF-2) promotes angiogenesis in the setting
1998, 153(1): 103-108.
of tissue ischemia. Am J Pathol, 1998, 153(2): 381-394.
al.
Vascular
endothelial
growth
factor-C
[15] CAO Y, LINDEN P, FAMEBO J, et al. Vascular
[17] ANTONERTTI D A, KLEIN R, GARDNER T W.
endothelial growth factor C induces angiogenesis in vivo.
Diabetic retionpathy. N Engl J Med, 2012, 366(13):
Proc Natl Acad Sci USA, 1998, 95(24): 14389-14394.
11227-11239.
[16] WITZENBICHLER B, ASAHARA T, MUROHARA T,
双丹明目胶囊对糖尿病大鼠模型视网膜 VEGF-a、VEGF-b、VEGF-c 及 其受体 Flk-1 表达的影响 彭俊 a，潘坤 a，刘峥嵘 a，秦裕辉 a*，彭清华 a*
a.湖南中医药大学中医眼科学重点学科，湖南 长沙 410208，中国 【摘要】目的 观察双丹明目胶囊对糖尿病大鼠模型视网膜 VEGF-a、VEGF-b、VEGF-c 及其受体 Flk-1 表 达的影响。方法 将 40 只 SD 大鼠，采用随机数字法分为空白组 (A)、模型组 (B)、双丹明目组 (C)、阳性 对照组 (D) 4 组，每组 10 只 20 眼。将 B、C、D 三组实验大鼠采用 STZ 50mg/kg 的剂量一次性大鼠尾静脉 注射法建立糖尿病视网膜病变大鼠模型，造模后 1 周开始连续灌胃用药，灌胃后 8 周，处死动物，采用免 疫组化法检测视网膜组织中 VEGF-a、VEGF-b、VEGF-c 及其受体 Flk-1 的表达。结果 成模后用药第 8 周， 模型组、双丹明目组、阳性对照组视网膜中 VEGF-a、VEGF-b、VEGF-c 及其受体 Flk-1 蛋白表达平均灰度 值均低于正常组，平均光密度均高于正常组，其中：模型组与正常组比较，差异均具有统计学意义（P＜ 0.01） ；双丹明目组、阳性对照组 VEGF-a、VEGF-b、VEGF-c 及其受体 Flk-1 表达的平均灰度值均高于模型 。结论 双丹明目胶囊能明显降低 VEGF-a、VEGF-b、 组、平均光密度值均低于模型组（P＜0.05 或 P＜0.01） VEGF-c 及其受体 Flk-1 在糖尿病模型大鼠视网膜中的表达，对其视网膜有一定的保护作用。 【关键词】 双丹明目胶囊；糖尿病大鼠模型；VEGF-a；VEGF-b；VEGF-c；Flk-1
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Digital Chinese Medicine journal homepage: http://dcmhi.com.cn
Effects of Shuang Dan Ming Mu Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and the VEGF receptor, Flk-1, in Diabetic Retinopathy Rats Jun PENGa, Kun PANa, Zheng-Rong LIUa, Yu-Hui QINa* , Qing-Hua PENGa* a. Department of Traditional Chinese Medicine Ophthalmology, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China
ARTICLE INFO
ABSTRACT
Article history
Objective The effects of Shuang Dan Ming Mu capsule on expression of VEGF-a, VEGF-b, VEGF-c and the VEGF receptor, Flk-1, were examined in a diabetic retinopathy rats model.
Received 25 Apr. 2018 Accepted 1 Jun. 2018 Available online 26 Sep. 2018 Keywords Shuang Dan Ming Mu capsule Diabetic rats model VEGF-a VEGF-b VEGF-c Flk-1 *Corresponding author: Yu-Hui QIN, Professor, Research Scientist Grade II. Research direction: TCM diagnosis and treatment of fundus cornea disease. E-mail: [email protected]. Qing-Hua PENG, Professor Grade II. Research direction: TCM treatment and prevention of ocular fundus diseases, glaucoma and ocular surface diseases and the normalization of ophthalmic symptoms. E-mail: [email protected]. Peer review under the responsibility of Hunan University of Chinese Medicine.
Methods Forty Sprague-Dawley (SD) rats were randomized into Groups A (blank), B (model), C (Shuang Dan Ming Mu) and D (positive control) group, with each group containing 10 rats and 20 eyes. Rats from groups B, C and D were administered one dose of 50 mg/kg streptozotocin (STZ) by tail vein injection to establish a diabetic rat model. One week after model preparation, medication was continuously administered by gavage. After gavage for 8 weeks, the animals were sacrificed and retinal expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 was quantified by immunohistochemical analysis. Results At week 8 of drug administration after model preparation, the average protein expression grayscale values for VEGF-a, VEGF-b, VEGF-c and Flk-1 in the rats model, Shuang Dan Ming Mu and positive control groups were all lower than those in the normal group, while the mean optical density values were higher than those in the normal group. When the model group was compared to the normal group, the difference was extremely significant (P < 0.01). The mean grayscale values of VEGF-a, VEGF-b, VEGF-c and Flk-1 in the Shuang Dan Ming Mu and positive control groups were all higher than those in the model group, while the mean optical density values were lower than those in the model group (P < 0.05 or 0.01). Conclusion Shuang Dan Ming Mu capsule can significantly decrease the expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in the retinas of diabetic model rats and exhibit some protective effects in their retinas.
Copyright © 2018 Hunan University of Chinese Medicine Publishing services provided by Elseiver B.V. On behalf of KeAi This is an open access article under the CC BY-NC-ND License (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 229
2.1.2 Experimental drugs
1 Introduction Diabetic retinopathy (DR) is the most common and severe microvascular complication in diabetes and the main cause of vision loss in patients with diabetes mellitus (DM). If DR is not treated in the early stages, it may lead to irreversible visual impairment. Qin suggested that the main pathogenesis of DR is liver and kidney deficiencies and pulse stagnation. Based on long-term clinical practice, he created China’s first new traditional Chinese medicine (TCM), Shuang Dan Ming Mu capsule, to specifically treat DR. Previous studies showed that the mechanisms of Shuang Dan Ming Mu capsule may involve effective reduction of blood glucose and glycated hemoglobin levels in DR rats, improvements in the retina and pancreas structures in rats, reduce platelet adhesion rate and blood rheology and promotion of blood circulation
[1-3]
. In this study, we evaluated the
changes in the vascular endothelial growth factor (VEGF) signaling pathway and its receptor and related molecules to determine the effects of Shuang Dan Ming Mu capsule on retinal VEGF-a, VEGF-b,
Shuang Dan Ming Mu capsule were composed of Ligustri Lucidi Fructus (Nv Zhen Zi, 女 贞 子 ), Ecliptae Herba (Mo Han Lian, 墨 旱 莲 ), Corni Fructus (Shan Zhu Yu, 山茱萸), Dioscoreae Rhizoma (Shan Yao, 山药), Poria (Fu Ling, 茯苓)， Alismatis Rhizoma (Ze Xie, 泽泻), Moutan Cortex (Mu Dan Pi, 牡丹皮), Salviae Miltiorrhizae Radix Et Rhizoma (Dan Shen, 丹参), Notoginseng Radix Et Rhizoma (San Qi, 三七), Smilacis Glabrae Rhizoma (Tu Fu Ling, 土 茯 苓) and Achyranthis Bidentatae Radix (Niu Xi, 牛膝). Shuang Dan Ming Mu capsule were provided by the TCM Research Institute, Hunan Academy of Chinese Medicine (Hunan, China; Batch number: Guoyaozhun No. Z20080062). Calcium dobesilate (250 mg/tablet) was obtained from Ningxia Kangya Pharmaceutical Co. Ltd. (Ningxia, China; Batch
number:
Guoyaozhun
No.
H20030809).
Recombinant human endostatin injection (Endostar, Warsaw, Poland; 15 mg/2 mL/vial) was obtained from Yantai Medgenn Co., Ltd. (Nanjing, China; Batch number: Guoyaozhun No.S20050088).
VEGF-c and Flk-1 expression in diabetic rats.
2.1.3 Experiment reagents
2 Materials and Methods
VEGF-a, VEGF-b, VEGF-c and Flk-1 were obtained from Boster Biological Technology Co. Ltd. (Wuhan,
2.1 Experimental materials
China). Streptozocin (STZ) was produced by Sigma (St.
2.1.1 Experimental animals
Louis, MO, USA). Sodium pentobarbital was obtained from
Shanghai
Chemical
Dispensing
Factory
Forty one-month old, healthy, specific pathogen-free-grade, outbred Sprague-Dawley (SD)
(Shanghai, China). Hematoxylin-eosin was obtained
rats with no ocular fundus disease and weighing
Ltd. (Beijing, China). The 4% paraformaldehyde, 1%
200–250 g were obtained from Beijing Vital River
osmic acid, acetone. Epon812 epoxy resin, uranium
Laboratory Animal Technology Co., Ltd. (Beijing, China). Before the experiment, the fundus of both
acetate, lead citrate and isoamyl acetate were obtained from Hunan Fuzitang Biotechnology Co., Ltd. (Hunan,
eyes was examined for any abnormalities and rats
China). The 4% paraformaldehyde was obtained from
with systemic lesions were excluded. The animals
the pathology laboratory of Hunan University of
were housed at 18–20℃ with ventilation in dry
Chinese Medicine. Harris hematoxylin, eosin and 3%
specific pathogen-free animal laboratories with a relative humidity of 60%–70% and fed laboratory
peracetic acid were obtained from Beijing Zhongshang
rat growth pellets that had been stringently disinfected and Grade III drinking water. The
from Beijing Dingguo Changsheng Biotechnology Co.,
Jinqiao Biotechnology Co., Ltd. (Beijing, China). 2.2 Experimental methods
cages and bottles were cleaned and disinfected
2.2.1 Grouping
every 2 days.
After feeding the animals routinely for 1 week,
230
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
random numbers were used to divide the 40 rats into 4
solution was used for gavage combined with
groups (A, B, C and D). Each group contained 10 rats
intravitreal administration of recombinant human
and 20 eyes. These groups were A: blank group
endostatin
(normal group), B: model group, C: Shuang Dan
administered once per day and Endostar intravitreal
Ming Mu group and D: positive control group.
injections were started on day 10 after model
2.2.2 Model construction
(Endostar).
Calcium
dobesilate
was
construction. Before intravitreal injection, the anterior chamber was punctured to release 20 μL of aqueous
Except for group A, the 30 rats in the other groups
humor. A 30G needle microinjector was used to pierce
were used for model construction. Before model
the sclera at 0.5 mm behind the corneal limbus of the
construction, rats were fasted for 10 h but given adlibitum access to water. One dose of 50 mg/kg STZ
superior
administration of 20 μL (5 g/L) Endostar once every 10
was injected through the tail vein into the rats. Rats in
days [5].
group A were given tail vein injections with an equal volume of physiological saline. Blood was drawn
temporal
quadrant
for
intravitreal
2.2.4 Material collection
from the tail vein 72 h later and blood glucose was
After rats were anesthetized with an overdose of 2%
measured using a Johnson & Johnson Blood Glucose
sodium pentobarbital, they were sacrificed by
Meter (New Brunswick, NJ, USA). Urine test strips
decapitation. The eyeballs were immediately removed
were used to measure urine glucose levels. Rats with
and fixed with 10% formalin for 24 h. The anterior
fasting blood glucose concentration >16.7 mM and
segment was removed and retinal tissues were
urine glucose of +++ and above were considered s diabetic. After model construction, the rats were
isolated and washed with running water for 8 h. After
observed for 1 week and rats with stable glucose
impregnation, embedding and sectioning (thickness of
concentrations were considered as successful models.
4 μm) were carried out.
The disease course was then calculated. No rats died after model construction. However, 2 rats showed low
dehydration
using
an
gradient,
wax
2.2.5 Marker detection
blood glucose values, which met the standards after a
Immunohistochemistry
second tail vein injection.
VEGFa,
2.2.3 Drug administration method
alcohol
VEGFb,
quantification
VEGFc
and
of
retinal
Flk-1
protein
expression: The SP method was used to examine the protein expression of VEGFa, VEGFb, VEGFc and
groups, continuous gavage was used for drug
Flk-1 under high magnification. After the immunohistochemistry reaction, the cytoplasm of
administration for 8 weeks before the animals were
positively labeled cells was colored brown or deep
sacrificed. All rats were given ad libitum access to the
brown. Ten retina fields were randomly chosen under
diet and the bedding was changed once per day.
high magnification and MOTIC 6.0 image analysis
Regular ventilation was carried out and a quiet
software (Motic Co. Ltd) was used for image analysis
environment was maintained.
and processing and quantitative analysis of the mean
One week after model construction of the various
(1) Blank group (Group A), Model group (Group B): These two groups were gavaged with 10 mL/kg of physiological saline. (2) Shuang Dan Ming Mu capsule group (Group C): Shuang Dan Ming Mu capsule solution was used for gavage at a dose of 12.5 mL/kg. (3) Positive control group (Group D): Relevant studies
[4]
were referenced and calcium dobesilate
optical density of the image (OD value). 2.3 Statistical analysis SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA) was to analyze the experimental data. A difference of P < 0.05 was considered statistically significant and a difference of P < 0.01 was considered extremely significant. All data was expressed as the mean ±
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 231
standard deviation (x̄ ± s). 3 Results 3.1 Retinal VEGF-a protein expression status After 8 weeks of drug treatment after model construction, the mean optical density of VEGF-a protein expression was compared. As shown in Table 2, the mean optical density for VEGF-a protein expression in the model, Shuang Dan Ming Mu and positive control groups were all higher than that in the normal group. When the model group was compared to the normal group, the difference was extremely significant (t = 4.905, P < 0.01). The results showed widespread expression of VEGF-a in the retinas in the
difference was extremely significant (t = 4.445 and 4.608, both P < 0.01). After treatment with Shuang Dan Ming Mu capsule or positive control drugs, retinal VEGF-a expression in the rats was significantly downregulated. When the positive control group was compared to the Shuang Dan Ming Mu group, the difference was not significant (t = 0.124，P > 0.05) (Table 1 and Figure 1). Table 1 VEGF-a mean optical density comparison at week 8 of drug treatment after model construction Group
n
Mean optical density
A
10
0.356 ± 0.097
B
10
0.571 ± 0.099**
C
10
0.382 ± 0.091△△
model group. The mean optical densities of VEGF-a protein expression in the Shuang Dan Ming Mu and
Notes: Comparison of Groups B, C and D with Group A: *P <
positive control groups were all lower than that in the
0.05, **P < 0.01; Comparison of Groups C and D with Group
model group. Statistical analysis showed that the
B: △P < 0.05,
△△
P < 0.01.
Fig. 1 Immunohistochemical observation of VEGF-a protein expression at week 8 after model construction (×400) a. Normal group: In normal retinas, there were small amounts of positive VEGF- b protein expression in the nerve fiber layer, ganglion cell layer and inner plexiform layer (VEGF- b ×400). b. Model group: Positive retinal VEGF- b protein expression was significantly increased and gradually shifted towards the inner plexiform layer (VEGF-b ×400); c. Shuang Dan Ming Mu group: Positive retinal VEGF- b protein expression showed a slight increase but was lower than the model group (VEGF-b ×400); d. Positive control group: Positive retinal VEGF- b protein expression showed a slight increase but was lower than the model group (VEGF-b ×400)
232
3.2 Retinal VEGF-b protein expression status After 8 weeks of drug treatment after model construction, the mean optical density of VEGF-b protein expression was compared. As shown in Table 2, the mean optical density of VEGF-b protein expression in the model, Shuang Dan Ming Mu and positive control groups were all higher than that in the normal group. When the model group was compared to the normal group, the difference was extremely significant (t = 5.252, P < 0.01). The results showed widespread expression of VEGF-b in the retinas in the model group. The mean optical densities of VEGF-b protein expression in the Shuang Dan Ming Mu and positive control groups were all lower than that in the model group. Statistical analysis showed that the difference was extremely significant (t = 4.649 and 4.784, both P < 0.01). After treatment with Shuang
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Dan Ming Mu capsule or positive control drugs, retinal VEGF-b expression in the rats was significantly downregulated. When the positive control group was compared to the Shuang Dan Ming Mu group, the difference was not statistically significant (t = 0.071, P > 0.05) (Table 2 and Figure 2). Table 2 VEGF-b mean optical density comparison at week 8 of drug treatment after model construction Group
n
Mean optical density
A
10
0.326 ± 0.097
B
10
0.561 ± 0.103**
C
10
0.352 ± 0.098△△
D
10
0.349 ± 0.095
△△
Notes: Comparison of Groups B, C and D with Group A: *P < 0.05, **P < 0.01; Comparison of Groups C and D with Group △ B: P < 0.05,
△△
P < 0.01.
Fig. 2 Immunohistochemical observation of VEGF-b protein expression at week 8 after model construction (×400) a. Normal group: In normal retinas, there were small amounts of positive VEGF- b protein expression in the nerve fiber layer, ganglion cell layer and inner plexiform layer (VEGF- b ×400); b. Model group: Positive retinal VEGF- b protein expression was significantly increased and gradually shifted towards the inner plexiform layer (VEGF-b ×400); c. Shuang Dan Ming Mu group: Positive retinal VEGF- b protein expression showed a slight increase but was lower than the model group (VEGF-b ×400); d. Positive control group: Positive retinal VEGF- b protein expression showed a slight increase but was lower than the model group (VEGF-b ×400)
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 233
3.3 Retinal VEGF-c protein expression status After 8 weeks of drug treatment after model construction, the mean optical density of VEGF-c protein expression was compared. As shown in Table 6, the mean optical density of VEGF-c protein expression in the model, Shuang Dan Ming Mu and positive control groups were all higher than that in the normal group. When the model group was compared with the normal group, the difference was extremely significant (t = 4.871, P < 0.01). The results showed widespread expression of VEGF-c in the retinas in the model group. The mean optical densities of VEGF-c protein expression in the Shuang Dan Ming Mu and positive control groups were lower than that in the model group. Statistical analysis showed that the difference was extremely significant (t = 4.376 and 4.323, both P < 0.01). After treatment with
Shuang Dan Ming Mu capsule or positive control drugs, retinal VEGF-c expression in the rats was significantly downregulated. When the positive control group was compared to the Shuang Dan Ming Mu group, the difference was not significant (t = 0.124, P > 0.05) (Table 3 and Figure 3). Table 3 VEGF-c mean optical density comparison at week 8 of drug treatment after model construction Group
n
Mean optical density
A
10
0.369 ± 0.083
B
10
0.568 ± 0.099**
C
10
0.381 ± 0.092
D
10
0.386 ± 0.089△△
△△
Notes: Comparison of Groups B, C and D with Group A: *P < 0.05, **P < 0.01; Comparison of Groups C and D with Group △
B: P < 0.05,
△△
P < 0.01.
Fig. 3 Immunohistochemical observation of VEGF-c protein expression at week 8 after model construction (×400) a. Normal group: In normal retinas, there were small amounts of positive VEGF-c protein expression in the nerve fiber layer, ganglion cell layer and inner plexiform layer (VEGF-c ×400); b. Model group: Positive retinal VEGF- c protein expression was significantly increased and gradually shifted towards the inner plexiform layer (VEGF-c ×400); c. Shuang Dan Ming Mu group: Positive retinal VEGF- c protein expression showed slight increase but lower than the model group (VEGF-c ×400); d. Positive control group: Positive retinal VEGF- c protein expression showed slight increase but lower than the model group (VEGF-c ×400)
234
3.4 Retinal Flk-1 protein expression status After 8 weeks of drug treatment after model construction, the mean optical density of Flk-1 protein expression was compared. As shown in Table 8, the mean optical density of Flk-1 protein expression in the model, Shuang Dan Ming Mu and positive control groups were all higher than that in the normal group. When the model group was compared to the normal group, the difference was extremely significant (t = 5.109, P < 0.01). The results showed widespread expression of Flk-1 in retinas in the model group. The mean optical densities of Flk-1 protein expression in the Shuang Dan Ming Mu and positive control groups were lower than that the model group. Statistical analysis showed that the difference was extremely significant (t = 4.789 and 4.953, both P < 0.01). After
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
treatment with Shuang Dan Ming Mu capsule or positive control drugs, retinal Flk-1 expression in rats was significantly downregulated. When the positive control group was compared to the Shuang Dan Ming Mu group, the difference was not significant (t = 0.125, P > 0.05) (Table 4 and Figure 4). Table 4 Flk-1 mean optical density comparison at week 8 of drug treatment after model construction Group
n
Mean optical density
A
10
0.419 ± 0.093
B
10
0.642 ± 0.102**
C
10
0.436 ± 0.090
D
10
0.431 ± 0.088△△
△△
Notes: Comparison of Groups B, C and D with Group A: *P < 0.05, **P < 0.01; Comparison of Groups C and D with Group △
B: P < 0.05,
△△
P < 0.01.
Fig. 4 Immunohistochemical observation of Flk-1 protein expression at week 8 after model construction (×400) a. Normal group: In normal retinas, there were small amounts of positive Flk-1 protein expression in the nerve fiber layer, ganglion cell layer and inner plexiform layer (Flk-1 ×400); b. Model group: Positive retinal Flk-1 protein expression was significantly increased and gradually shifted towards the inner plexiform layer (Flk-1 ×400); c. Shuang Dan Ming Mu group: Positive retinal Flk-1 protein expression showed a slight increase but was lower than in the model group (Flk-1 ×400); d. Positive control group: Positive retinal VEGF- c protein expression showed a slight increase but was lower than in the model group (Flk-1 ×400)
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 235
Modern physicians have employed ocular fundus
4 Discussion
examinations and fluorescein angiography to study 4.1 Traditional Chinese medicine understanding of diabetic retinopathy
this disease based on blood rheology, microcirculation and blood biochemistry; they found that “blood
In TCM, diabetic retinopathy is known as “diabetic
stasis” plays an important role in the pathogenesis of
. According to vision changes and
this disease. Therefore, professor Yu-Hui QIN found
reduced visual acuity in the patient, DR is involved in
that the etiology of this disease is that kidney vacuity
different diseases, such as “indistinct vision,” “clouds
is the root, yin vacuity is the cause and blood stasis is
and mist moving over the eye,” “blood passing
the symptom and “kidney vacuity and blood stasis” is
cataracts”
[6]
. The
the main pathogenesis of this disease. Therefore, DR
Jin dynasty physician Zi-He ZHANG pointed out in
treatment should follow the basic principle of
“Confucians’ Duties to Parents” that diabetes is due to
“enriching the kidney and quickening the blood.”
[7]
through the pupil,” “sudden blindness,” etc.
excessive consumption of the essence-spirit and
Based on this principle, Professor Yu-Hui QIN
over-exuberant dryness-heat..., and can evolve into
developed Shuang Dan Ming Mu capsule, which is
sparrow vision or cataracts.” He suggested that
the first new traditional Chinese medicine for
dryness-heat
main
specifically treating DR in China. This drug nourishes
dynasty
the kidneys and liver, increases blood glow and clears
physician Xian ZHAO suggested that “diabetes
the eyes and is mainly used to treat DR caused by
treatment cannot be classified as low, moderate and
liver-kidney yin vacuity and static blood blocking the
high and it is imperative to treat the kidneys by
network
six-ingredient pill or eight-ingredient pill to decrease
demonstrated that the drug has good efficacy
heart fire nourishment of kidney water, which will
improving fundus lesions in patients, promoting the
self-stop diabetes.” The Ming dynasty physician
recovery of visual acuity and treating DR. Our
Ken-Tang WANG’s book “Standards of diagnosis and
previous pharmacological studies
treatment”
three
Shuang Dan Ming Mu capsule (originally known as
dissipation-thirst results in depletion of essential Qi,
Jiang Tang Ming Mu capsule) can be combined with
causing vision loss. He believed that DR is caused by
glucose-lowering drugs to greatly promote the latter’s
the depletion of essential Qi and vision loss.
glucose-lowering effect. Shuang Dan Ming Mu
Subsequently, generations of physicians examined DR
capsule monotherapy can also decrease platelet
based on these foundations. Most researchers
adhesion, improve blood rheology and promote blood
suggested that long periods of diabetes result in
circulation.
reduced essential qi, which cannot be transmitted to
4.2 Examination of effector mechanisms of Shuang
pathogenesis
that
damages
mechanism.
also
described
yin The
as
the
Ming
that
long
the eyes, resulting in undernourishment of the eyes or
vessels.
Multi-center
trials
[1-3]
have [8-10]
in
also found that
Dan Ming Mu capsule on diabetic rats
liver-kidney yin vacuity causes yin vacuity and yang hyperactivity, upflaming vacuity fire that burns the eyes, resulting in blurred vision or even blindness.
Neovascularization
(NV)
is
a
characteristic
pathological change of DR that occurs from non-proliferative diabetic retinopathy to proliferative
236
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
diabetic retinopathy. NV occurrence is regulated by
developing
multiple growth factors, among which VEGF is a key
differentiation and growth of blood vessels. In
factor in angiogenesis. VEGF is a family that includes
endothelial cells, VEGF-c acts on the VEGFR-2 and
VEGF-a, VEGF-b, VEGF-c, VEGF-d, VEGF-e and
VEGFR-3 receptors to induce endothelial cell
placental growth factor, among others
[11]
. VEGF-a is
embryo,
VEGF-c
promotes
the
proliferation, particularly capillary endothelial cells
a highly conserved, disulfide linked homodimer.
[15]
Under reducing conditions, the homodimers separate
showed that infusion of VEGF-c plasmids through a
into monomers and all biological activity is lost.
catheter promoted the growth of blood vessels in
VEGF-a can promote the proliferation of vascular
ischemic regions and significantly improved the blood
endothelial cells. As a specific mitogen for vascular
supply.
endothelial cells, VEGF can stimulate in vitro
potential for treating ischemic diseases
cultured vascular endothelial cells to undergo mitosis
promote endothelial cell proliferation. Flk-1/KDR
and migration. VEGF also acts on in vivo vascular
plays a dominant role in VEgF signal transduction
endothelial cells to cause endothelial cell division and
and formation of the vascular endothelium. Flk-1 acts
proliferation, induce blood vessel formation and is
as a negative regulator of Flk-1/KDR and antagonizes
currently the most potent pro-angiogenic factor.
the effects of Flk-1/KDR in promoting vascular
VEGF can increase vascular permeability, particularly
endothelial cell proliferation [17].
. A study using a rabbit ischemic hindlimb model
Therefore,
VEGF-c
shows
application
[16]
. Flk-1 can
that of the capillaries, causing plasma proteins to leak
In this study, we constructed diabetic rat models
into the extracellular matrix and be deposited in the
and observed the effects of Shuang Dan Ming Mu
extracellular matrix to provide a conditional matrix
capsule on retinal expression of VEGF-a, VEGF-b,
for the migration of fibroblasts and vascular
VEGF-c and the VEGF receptor, Flk-1, in a diabetic
endothelial cells. This increases nutrient levels
rat model. At week 8 of drug administration after
available for the growth of tumor cells and
model construction, we compared the mean optical
[12-13]
.
density of retinal VEGF-a, VEGF-b, VEGF-c and
VEGF-b is mainly distributed in the heart, skeletal
Flk-1 expression. The results showed that the mean
muscles, brain and kidneys in rats and the heart,
optical density of VEGF-a protein expression in the
skeletal muscles, pancreas and prostate gland in
model, Shuang Dan Ming Mu and positive control
humans. Researchers have analyzed [3H] labeled
groups were higher than that in the normal group.
thymidine in human umbilical vein endothelial cells
When the model groups were compared to the normal
and bovine capillary endothelial cells and found that
group, the difference was extremely significant (P <
VEGF-b can promote endothelial cell proliferation.
0.01). The mean optical density of VEGF-a, VEGF-b,
VEGF-b is also intimately associated with tumor
VEGF-c and Flk-1 protein expression in the Shuang
growth: VEGF-b and VEGF can promote local blood
Dan Ming Mu and positive groups were lower than that
establishment of new capillary networks
[14]
.
in the model group. Statistical analysis showed that
VEGF-c has potent angiogenic effects in the body and
these differences were significant (P < 0.05 or P <
has more persistent effects than VEGF. In the
0.01). This demonstrates that after treatment with the
vessel growth, thereby promoting tumor growth
Jun PENG, et al./Digital Chinese Medicine 1 (2018) 228-238
Effects of SDMM Capsule on Expression of VEGF-a, VEGF-b, VEGF-c and Flk-1 in Rats 237
Shuangdanmingmu
Shuang Dan Ming Mu capsule or positive control drug,
capsule
on
retinal
vascular
morphology and VEGF expression in rats with diabetic
the expression of VEGF-a, VEGF-b, VEGF-c and
retinopathy. Int Eye Sci, 2015, 15(1): 29-33.
Flk-1
in
the
rat
retinas
was
significantly
[2] QIN Y H， LI W J， ZHANG X, et al. Effects of
downregulated. Additionally, when the positive
Shuangdanmingmu
control group was compared to the Shuang Dan Ming
retinopathy in rats with diabetic retinopathy. Int Eye Sci,
capsule
on
blood
glucose
and
2014, 14(11): 1943-1945.
Mu group, the difference was not significant (P >
[3] QIN Y H， LI W J， ZHANG X, et al. Effects of
0.05). Our study showed that Shuang Dan Ming Mu
Shuangdan Mingmu Capsule on the Expression of Retinal
capsule can significantly decrease the mean optical
VEGF and VEGFR Protein Activity in Rats with Diabetic
density of VEGF-a, VEGF-b, VEGF-c and Flk-1
Retinopathy. Journal of Hunan University of Chinese Medicine, 2015, 35(6): 1-3.
expression in the retinas of rat models, i.e. Shuang [4]
Dan Ming Mu capsule can significantly decrease the
NAGAOKA
T,
SATO
E,
TAKAHASHI
A,
et
al． Impaired retinal circulation in patients with type 2
expression of VEGF-a, VEGF-b, VEGF-c and Flk-1
diabetes mellitus: retinal laser doppler velocimetry study.
in the retinas, which can protect retinal capillaries in
Invest Ophthalmol Vis Sci, 2010, 51(12): 6729. [5] CHEN L J, MIAO L. Inhibitive effects of recombinant
diabetic rats.
human endostatin on choroidal neovascularization. Rec Adv Ophthalmol, 2012, 32(10): 922-925.
Acknowledgements
[6] PENG Q H. Ophthalmology of Chinese Medicine [M].
We thank for the funding support from the National Natural Science Foundation General Program (No. 814737370); Hunan Province Graduate Student
Chinese Press of Traditional Chinese Medicine, 2012:187. [7] CHEN X D, PENG Q H, YAN J C, et al. Clinical observation of Fuming Table on diabetic retinopathy after retinal
Research Innovation Program (No. CX2016B377 and No. CX2017B432); Traditional Chinese Medicine Prevention and Treatment of Facial Feature Disease Hunan
Province
Key
Laboratory
Construction
laser
photocoagulation.
Journal
of
Hunan
University of Chinese Medicine, 2016, 36(1): 63-65. [8] TU L Y, WANG W C, WANG Y L. Efficacy and safety of Shuangdan
Mingmu
capsule
in
treating
diabetic
retinopathy. Chinese Journal of New Drugs, 2009, 18(24): 2331-2336.
Program (No. 2017TP1018); Changsha City Science
[9] PANG Y H. Clinical observation on treatment of diabetic
and Technology Program (No. kc1704005); Central
retinopathy with Shuang Dan Ming Mu capsule. Diabetes
Finance Supported Local High School Construction
New World, 2015,(3): 39.
Project; State Administration of Traditional Chinese
[10] QIN Y H, LI F, TU L Y, et al. Multicentric clinical study of Shuangdan Mingmu capsule on diabetic retinopathy.
Medicine
Ophthalmology
Key
Discipline
Construction Project; Hunan Province Traditional Chinese Medicine Facial Feature Key Discipline Construction Project.
Journal of Hunan University of Chinese Medicine, 2010, 30(1): 46-51. [11] LU C, SHI C H. Relationship between VEGF family/VEGF receptors and pathogenesis of diabetic retinopathy. Int Eye Sci, 2007, 7(5): 1400-1402.
Competing Interests
[12] NAGY J A, BENJAMIN L, ZENG H, et al. Vascular permeability,
The authors declare no conflict of interest.
vascular
hyperpermeability
and
angiogenesis. Angiogenesis, 2008(11): 109-119. [13] ZHANG D M, QING C. Vascular Endothelial Growth
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[16] WITZENBICHLER B, ASAHARA T, MUROHARA T,
双丹明目胶囊对糖尿病大鼠模型视网膜 VEGF-a、VEGF-b、VEGF-c 及 其受体 Flk-1 表达的影响 彭俊 a，潘坤 a，刘峥嵘 a，秦裕辉 a*，彭清华 a*
a.湖南中医药大学中医眼科学重点学科，湖南 长沙 410208，中国 【摘要】目的 观察双丹明目胶囊对糖尿病大鼠模型视网膜 VEGF-a、VEGF-b、VEGF-c 及其受体 Flk-1 表 达的影响。方法 将 40 只 SD 大鼠，采用随机数字法分为空白组 (A)、模型组 (B)、双丹明目组 (C)、阳性 对照组 (D) 4 组，每组 10 只 20 眼。将 B、C、D 三组实验大鼠采用 STZ 50mg/kg 的剂量一次性大鼠尾静脉 注射法建立糖尿病视网膜病变大鼠模型，造模后 1 周开始连续灌胃用药，灌胃后 8 周，处死动物，采用免 疫组化法检测视网膜组织中 VEGF-a、VEGF-b、VEGF-c 及其受体 Flk-1 的表达。结果 成模后用药第 8 周， 模型组、双丹明目组、阳性对照组视网膜中 VEGF-a、VEGF-b、VEGF-c 及其受体 Flk-1 蛋白表达平均灰度 值均低于正常组，平均光密度均高于正常组，其中：模型组与正常组比较，差异均具有统计学意义（P＜ 0.01） ；双丹明目组、阳性对照组 VEGF-a、VEGF-b、VEGF-c 及其受体 Flk-1 表达的平均灰度值均高于模型 。结论 双丹明目胶囊能明显降低 VEGF-a、VEGF-b、 组、平均光密度值均低于模型组（P＜0.05 或 P＜0.01） VEGF-c 及其受体 Flk-1 在糖尿病模型大鼠视网膜中的表达，对其视网膜有一定的保护作用。 【关键词】 双丹明目胶囊；糖尿病大鼠模型；VEGF-a；VEGF-b；VEGF-c；Flk-1