Capsaicin Alleviates Vascular Endothelial Dysfunction and Cardiomyopathy via TRPV1/eNOS Pathway in Diabetic Rats
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Oxidative Medicine and Cellular Longevity
Volume 2022, Article ID 6482363, 11 pages
https://doi.org/10.1155/2022/6482363
Research Article
Capsaicin Alleviates Vascular Endothelial Dysfunction and
Cardiomyopathy via TRPV1/eNOS Pathway in Diabetic Rats
Qiuyue Wang,1 Caihui Zhang,1 Chen Yang ,1 Yue Sun,1 Keyang Chen,2 and Yao Lu 1,3
1
Department of Anesthesiology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
2
Department of Health Inspection and Quarantine, School of Public Health, Anhui Medical University, Hefei 230032, China
3
Ambulatory Surgery Center, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
Correspondence should be addressed to Chen Yang; yangchen199806@163.com and Yao Lu; luyao-mz@163.com
Received 2 February 2022; Accepted 26 April 2022; Published 12 May 2022
Academic Editor: Jianlei Cao
Copyright © 2022 Qiuyue Wang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Endothelial dysfunction and cardiomyopathy are considered to be important vascular complications associated with
diabetes. This study was designed to investigate whether capsaicin (CAP), a selective TRPV1 agonist, could prevent diabetes-
induced endothelial dysfunction and cardiomyopathy. Methods. Male Sprague Dawley rats aged 8 weeks were injected
intraperitoneally with streptozotocin (STZ, 50 mg/kg) to establish the diabetes model. The diabetic rats were randomly divided
into the untreated diabetes group (DM, 10/group) and diabetes plus CAP treatment group (DM+CAP, 10/group); meanwhile,
the nondiabetic healthy rats were used as normal controls (10/group). DM+CAP group were treated with CAP by gavage for 8
weeks. The cultured mouse vascular endothelial cells were exposed to different concentrations of glucose in the presence or
absence of CAP treatment. The TRPV1 inhibitor capsazepine (CPZ) and eNOS inhibitor L-NAME were used in vivo and
in vitro experiment. Results. CAP treatment significantly decreased the serum total cholesterol (TC) and total triglyceride (TG)
and ameliorated the pathogenesis and fibrosis in the heart, while did not significantly improve plasma glucose level and the
body weights of diabetic rats. In addition, CAP enhanced the expression of TRPV1 and eNOS in the heart and normalized the
vascular permeability under diabetic state. Similarly, CAP treatment also increased nitric oxide and reduced reactive oxygen
species. The same results were observed in cultured mouse vascular endothelial cells by CAP treatment. These beneficial effects
of CAP were abolished by either CPZ or L-NAME. Conclusions. CAP might protect against hyperglycemia-induced endothelial
dysfunction and diabetic cardiomyopathy through TRPV1/eNOS pathway.
1. Introduction eNOS is one of the critical signal molecules, functioning as a
crux vasoactive factor related to endothelium-dependent relax-
Diabetic cardiovascular disease is a major cause of mortality ation [3]. Physiologically, NO curbs inflammation and vascular
and morbidity in diabetic patients. In fact, diabetes-associated
hyperplasia. Besides, high-glucose level leads to an increase in
cardiac pathophysiological condition is considered a distinct
reactive oxygen species (ROS) in endothelial cells, which accel-
process referred to as diabetic cardiomyopathy, also leads to
poor prognosis [1]. Vascular endothelial dysfunction is thought erates NO inactivation and decreases NO bioavailability [4, 5].
to play a crucial role in the situation. High blood glucose sabo- Oxidative stress is widely believed to play a role in the pathogen-
tages the integrity of blood vessels and induces endothelial esis of diabetes and its complications [6]. Research has proved
dysfunction. Reduction in the bioavailability of the nitric oxide that appropriate treatments aimed at nitric oxide and oxidative
(NO) subsequent to the reduction of the endothelial nitric stress have the potential to ameliorate hyperglycemia-induced
oxide synthase (eNOS) is a characteristic of vascular endothe- vascular lesions [7].
lial dysfunction [2]. eNOS is involved in the regulation of Transient receptor potential channels are a diverse group of
vascular function. In the circulatory system, NO derived from proteins conserved in many species of mammals and are divided
2 Oxidative Medicine and Cellular Longevity
into 7 subgroups, one of which is the transient receptor potential 2.2. Detection of Total Cholesterol and Triglycerides. The con-
vanilloid (TRPV) subgroup [8]. TRPV1 exists in many tissues tents of TC and TG in serum were, respectively, determined
including vascular endothelial cells [9]. Activation of TRPV1 by assay kits (Nanjing Jiancheng Bioengineering Research Insti-
by capsaicin-mediated Ca2+ influx in endothelial cells can tute, China).
increase eNOS activity, then stimulates NO production [10].
Therefore, capsaicin is considered to have potential cardiovascu- 2.3. In Vivo Permeability Assay. Endothelial permeability in
lar protective effects [11]. Obviously, our previous studies have the aortic arch was detected using Evans blue (1%, 30 min).
also confirmed this conclusion [12, 13]. Similarly, there is evi- The anesthetized rats were injected with Evans blue dye
dence suggesting that TRPV1 can significantly increase NO pro- through the caudal vein, and the aorta was taken out after
duction and improve endothelial function through specific circulating for half an hour. After the stained aorta was
targeting of PKA/eNOS phosphorylation [14]. Impairment in photographed, the dye was extracted with deionized form-
capsaicin-mediated vasodilation is associated with downregula- amide, and the absorbance at 620 nm was measured by
tion of TRPV1, but whether activated TRPV1 is able to alleviate microplate reader [21, 22].
endothelial cell dysfunction is unclear [15]. Interestingly, in a
large Mediterranean population, regular consumption of chili 2.4. Immunohistochemistry. Rats were anesthetized and then
pepper is associated with a lower risk of total and lower death sacrificed by exsanguination; the aortic arch and heart were
of cardiovascular disease independent of cardiovascular risk fac- isolated, dissected, and fixed in 4% paraformaldehyde and
tors or adherence to a Mediterranean diet [16]. Capsaicin, as a embedded in paraffin. After paraffin section, 4 mm sections
major component of chili pepper, is a TRPV1 agonist, can were stained with hematoxylin and eosin. At the same time,
improve endothelial function by diet administration in rat the heart’s paraffin sections were stained with Aniline blue
model of renovascular hypertension [17]. Consequently, activa- and Ponceau S to observe tissue fibrosis. Prepared heart
tion of TRPV1 is quite possible to protect noninsulin-dependent slides were stained with periodic acid Schiff’s reagent
diabetes patients. However, in animal experiments, whether oral
(PAS) for glycogen and evaluated by light microscopy. ′
administration of capsaicin can increase NO and improve vas-
cular endothelial function in T1DM rats is still not clear. There-
fore, this study aims at investigating the intervention effects of 2.5. Cell Culture. MVECs (catlog# C166 ATCC, Allendale,
the high selective agonist capsaicin in T1DM rats and related NJ, USA) were grown in DMEM supplemented with 10%
mechanism. FBS and 1% antibiotics. Cultured cells were maintained at
37°C in a humidified atmosphere of 95% O2, 5% CO2. The
cells were seeded with suitable density and divided into dif-
2. Materials and Methods ferent groups, which included normal-glucose concentration
group (NG: 5.5 mmol/l), high-glucose concentration group
The animal study protocol was approved by the Animal Care (HG: 25 mmol/l), and high-glucose concentration plus
and Use Committee of Anhui Medical University and the proce- 1 μmol/l capsaicin group (HG + CAP) [23].
dures were conducted in accordance with the National Institutes
of Health Animal Research Advisory Committee guidelines. 2.6. Western Blot Analysis. Total proteins were separated on a
10% Bis–Tris CriterionTM XT Precast Gel (Bio-Rad, Mar-
neLa-Coquette, France) and transferred to an Immobilon poly-
2.1. Animals. Male Sprague–Dawley rats (200-250 g) at 8 weeks vinylidene difuoride (PVDF) membrane (Millipore, Molsheim,
old were bought from Anhui Medical Laboratory Animal France) as described (REF: PMID: 30797815). Antibodies
Center and acclimated for 1 week then were injected intraper- against -TRPV1 (1 : 1000, rabbit) from Abcam (Oakville, Can-
itoneally with streptozotocin (STZ, 50 mg/kg body weight) ada) and -eNOS (1/1000, rabbit) were incubated with mem-
dissolved in 0.1 mol/l citrate buffer (pH 4.5) [18] to establish branes overnight at 4°C. Membranes were incubated for 1 h at
diabetes model, which was confirmed by fasting plasma glucose room temperature with a corresponding horseradish peroxi-
level more than 16.7 mmol/l. The diabetic rats were randomly dase- (HRP-) conjugated secondary antibody (1/2000, Sigma-
divided into untreated diabetic group (DM) and diabetes plus Aldrich) and developed using the Luminata™ Forte Western
capsaicin treatment group (DM+CAP); meanwhile, the nondi- HRP substrate with BeyoECL Plus (Beyotime, Wuhan, China).
abetic healthy rats were used as normal controls (NC). The rats The relative quantity of the protein of interest compared with
in DM+CAP group received oral gavage of CAP at the dose of the reference protein β-actin (1/5000, mouse, Santa Cruz) was
0.5 mg/kg/per rat for a duration of 8 weeks, and the rats in the measured with Image J software (NIH, USA).
untreated DM group received the vehicle solution, using
disposable feeding needle. Diabetic rats were, respectively, 2.7. Immunofluorescence. The expression of TRPV1 and eNOS
gavageded with the combination of capsaicin and the TRPV1 in the heart and aorta of rats was detected using immunofluo-
inhibitor capsazepine (CPZ, 5 mg/kg) or the eNOS inhibitor rescence as described (Ref. PMID: 20950828; PMID:
Nnitro-L-arginine methyl ester (L-NAME, 100 mg/kg) in the 27733157). Concisely, frozen slides were incubated with anti-
DM+CAP+CPZ group or DM+CAP+L-NAME group [19]. TRPV1 (1 : 100, NeuroMab, USA) and anti-eNOS (1 : 100, Cell
After the completion of the 8 weeks’ treatment, all rats were Signaling Technology, USA), followed by corresponding
anesthetized and then sacrificed [20]. Then, the heart and aor- Alexa Fluor-conjugated secondary antibody (1 : 200) before
tic arch were removed and deep frozen until analyzed. imaging using a fluorescence microscope.
Oxidative Medicine and Cellular Longevity 3
2.8. Nitric Oxide Detection. The rat heart tissue and mouse vas- in the lower compartment (20 min) to those in the upper com-
cular endothelial cells were used to detect the content of nitric partment (0 min). The data were expressed as a percentage
oxide with Griess reagents (Beyotime, Wuhan, China) by the compared with control [27].
manufacturer’s instruction as described (Ref: PMID:
16390827). Also, nitric oxide concentrations of cell culture 2.12. Annexin V/PI Double Staining. The cell culture medium
medium and of rats’ serum were tested by the same way [24]. in wells of the plate was removed and washed it once by PBS.
5 μl annexin V-FITC and 10 μl propidium iodide staining
2.9. Hydrogen Peroxide Detection. Preparation of serum solution were, respectively, added into the wells and gently
samples: 50 mM phosphate buffer was prepared at pH 6.0. mixed. The plate was incubated at room temperature (20-
The sample was diluted 50 times with 50 mM phosphate 25°C) in dark for 20 min and then placed in ice bath. The plate
buffer. 96 well UV microplate method was used to determine was observed under the fluorescence microscope, annexin V-
the marker curve. Determination of hydrogen peroxide con- FITC showed green fluorescence and propidium iodide (PI)
centration: the hydrogen peroxide detection reagent was dis- showed red fluorescence [28].
solved and kept on ice. 50 μl sample and 100 μl hydrogen
peroxide detection reagent were, respectively, added into 2.13. Fluo-4 AM Staining. The cell culture medium in wells
each well that was shaken gently and mixed thoroughly. of the plate was removed and washed it once by PBS. The
The plate was leaved at room temperature (15-30°C) for 30 5 μM fluo-4 am probe solution diluted with PBS was added
minutes and then detected at A560 or A540-570 nm imme- into the wells and gently mixed. After incubated in the dark
diately. The concentration of hydrogen peroxide in the sam- at 37°C for 30 minutes, the six-well plate was observed under
ple was calculated according to the standard curve [25]. the fluorescence microscope. Fluo-4 showed green fluores-
cence [29].
2.10. Detection of Reactive Oxygen Species. After the rats were 2.14. Statistical Analysis. The results are expressed as the
euthanized and killed, the hearts were removed rapidly. The means ± SD, and n indicated sample size in each group. Sta-
heart tissues were soaked in paraformaldehyde, dehydrated, tistical analysis was performed using one-way analysis of
embedded, and made into frozen sections, stained with ROS variance (ANOVA), followed by Tukey’s post-hoc analysis
probe. In addition, DCFH-DA ROS fluorescent probe was to compare the differences among the groups. p < 0:05 was
used to detect the treated cell samples [26]. The results were considered significant.
observed by fluorescence microscope. The relative fluores-
cence intensities of the samples were measured with Image J
software (NIH, USA).
3. Results
3.1. Capsaicin Intervention Improved Body Weight, Blood Lipid,
2.11. Permeability Assay. The mixed base glue (Matrigel) was and Cardiac Fibrosis of Diabetic Rats. After 2 months’ interven-
dissolved with coating buffer (0.01 M Tris, pH 8.0 + 0.7% tion, it is obvious that capsaicin moderately increased the body
NaCl) to 200-300 μg/ml, 2 ml in total. The solution was placed weights of diabetic rats in the DM+CAP group but did not
on ice. The mixed coating solution was injected into 24 cham- improve the hyperglycemia caused by diabetes (Figures 1(a)
bers (Transwell insert, including filter membrane, pore size and 1(b)). In addition, diabetes was often accompanied by
0.4-8 μm) with a sterile syringe at 100 μl/well. The 24-well hyperlipidemia. Similarly, our current data suggested that capsa-
plate containing 24 chambers was incubated at 37°C for 2 h, icin treatment significantly lowered the serum levels of TC and
then the coating solution in each chamber was removed and TG (Figures 1(c) and 1(d)). To explore the role of capsaicin in
100 μl of mouse vascular endothelial cells at 2-5 × 105 was the correction of diabetic cardiomyopathy, myocardial tissue
injected into each chamber, which was incubated at 37°C for by HE staining was detected. Our current data showed that
1 h to make the cells adhere to the bottom, then 200 μl/well the overt pathological changes in myocardium were observed
complete medium was added into each chamber. At the same in diabetic rats, which was ameliorated by capsaicin treatment
time, 1 ml complete medium was added to each well of the (Figure 1(e)). Simultaneously, compared with the NC group,
supporting plate. For the treatments, normal-glucose (NC, Masson’s trichrome, and PAS staining of rat heart sections dem-
5.5 mM) and high-glucose (HG, 25 mM) medium and HG + onstrated more severe fibrosis in myocardium in DM group,
capsaicin were added to the corresponding chambers and while capsaicin treatment also ameliorated the fibrosis in DM
incubated at 37°C for 24 h. All chambers were transferred into + CAP group (Figure 1(e)). The protective effects of capsaicin
a new sterile 24-well supporting plate (each well contained on myocardial overt pathological changes and fibrosis in dia-
1 ml fresh complete medium), the old medium in the cham- betic rats were both abolished by either the inhibitor of TRPV1
bers was removed, and 150 μl FITC dextran solution was (CPZ) or the inhibitor of eNOS (L-NAME) (Figure 1(e)).
gently added into each chamber (final medium concentration,
10 μg/ml), which was placed in dark at room temperature and 3.2. Capsaicin by TRPV1/eNOS Activation Attenuates Heart
gently shaken for 20 minutes. 100 μl medium containing FITC Oxidative Stress and Increases the Level of NO in Diabetic
from each well of the supporting plate was taken and injected Rats. To investigate the mechanism underlying the capsaicin
into the corresponding well of the black 96-well plate, which function, the immunofluorescence was carried out to detect
was then placed in the fluorescence reader and measured at the signaling-related proteins. Our current results demon-
the absorption values of 485 and 535 nm, respectively. FITC- strated that TRPV1 and eNOS expression was sharply
dextran flux was valued as a ratio of fluorescence intensities decreased in diabetic rats, compared with that in healthy
4 Oxidative Medicine and Cellular Longevity
40 ⁎⁎⁎⁎ ⁎⁎⁎⁎
⁎⁎⁎⁎ ⁎⁎⁎⁎
600
Blood glucose (mmol/l)
⁎⁎⁎⁎ ⁎⁎⁎⁎⁎⁎⁎⁎
⁎⁎⁎⁎ ⁎⁎⁎⁎
⁎⁎⁎⁎ ⁎⁎⁎⁎ ⁎⁎⁎⁎
Body weight (g)
30 ## ⁎⁎⁎⁎⁎⁎⁎⁎
⁎⁎⁎⁎
400 # ⁎⁎⁎⁎
⁎⁎⁎⁎
⁎⁎
20 #### #
###⁎⁎⁎⁎
200 ⁎⁎
10
0 0
0 4 8 12 0 4 8 12
Ages (weeks) Ages (weeks)
NC NC
DM DM
DM+CAP DM+CAP
(a) (b)
⁎⁎ 2.0 ⁎⁎
3 ⁎⁎⁎ ⁎
TG (mmol/l)
1.5
TC (mmol/l)
2 1.0
1 0.5
0.0
0
NC
DM
DM+CAP
NC
DM
DM+CAP
(c) (d)
CPZ+DM L-NAME+
NC DM DM+CAP +CAP DM+CAP
HE
Masson
PAS
(e)
Figure 1: Blood glucose, weight, total cholesterol and triglycerides, myocardial pathological injury in rats. (a–b) Body weight is different in
the NC, DM, and CAP group. Blood glucose in the DM and CAP group are higher than NC group. (c–d) Total cholesterol and triglycerides.
(e) Representative images of H&E-stained, Masson-stained, and PAS-stained heart sections. The scale bar represents 50 μm. Data are
presented as mean ± SD (n =10 per group). ∗∗∗ p < 0:001, ∗∗p < 0:01, and ∗p < 0:05.
controls (Figure 2(a)). Nevertheless, this decrease caused by 3.3. Capsaicin by TRPV1/eNOS Activation Attenuates
diabetes was reversed by capsaicin treatment in DM+CAP Vascular Oxidative Stress and Increases the Level of NO in
group (Figure 2(a)). Consistent with the immunofluorescence Diabetic Rats. Vascular HE staining showed that the blood
data, protein expression in rat heart tissues was also assayed by vessel wall of diabetic rat was thicker than that of normal
immunoblots, showing that diabetes markedly lowered the rat, while CAP treatment significantly improved the patho-
expression of TRPV1 and eNOS, but capsaicin treatment dra- genic thickness (Figure 3(a)). Immunohistochemical assay
matically recovered the expression (Figures 2(b) and 2(c)). indicated that vascular intercellular adhesion molecules
Nitric oxide synthase (NOS) is an isoenzyme, which exists in (vcam-1) expression was significantly elevated in endothelial
endothelial cells, macrophages, nerve phagocytes, and nerve and smooth muscle cells from diabetic rats, and that capsa-
cells, respectively. In heart tissue, nitric oxide in vascular endo- icin treatment overtly mitigated the elevation of vcam-1 in
thelial cells mainly comes from eNOS. In the present study, we DM+CAP group (Figure 3(b)). For in vivo permeability
found that the change of nitric oxide content was consistent assay, Miles method was taken to look at the leakage of
with eNOS expression in different groups in rat heart tissue blood vessels in normal and diabetic rats. Our current data
(Figure 2(d)). In addition, fluorescent probe for detecting reac- showed the permeability was significantly increased in dia-
tive oxygen species indicated that capsaicin treatment reduced betes compared with normal rats, while capsaicin treatment
ROS production in diabetic rat hearts (Figure 2(e)). normalized the vascular permeability under diabetic state
Oxidative Medicine and Cellular Longevity 5
DAPI TRPV1 eNOS Merged
NC
DM
DM+CAP
(a)
1.5 ⁎⁎⁎⁎
⁎
NC DM DM+CAP
eNOS/actin
1.0
eNOS 140 KD
Actin 42 KD 0.5
0.0
NC
DM
DM+CAP
(b)
⁎⁎⁎
2.5
⁎⁎
(mmol/mg/protein)
2.0 ⁎ NO of heart tissue 2.0
NC DM DM+CAP ⁎
TRPV1/actin
1.5 1.5
TRPV1 75 KD
1.0 1.0
Actin 42 KD
0.5 0.5
0.0 0.0
NC
DM
DM+CAP
NC
DM
DM+CAP
(c) (d)
DAPI ROS Merged
NC
DM
DM+CAP
(e)
Figure 2: TRPV1/eNOS activation by capsaicin attenuates heart oxidative stress and increases the level of NO in diabetic rats. (a)
Representative immunofluorescence images showing the coexpression of TRPV1 and eNOS in the heart from the NC, DM, and CAP
group (bar denotes 50 μm). (b–c) The protein expression of TRPV1 and eNOS in the hearts of rat was determined by Western blotting.
Quantitative analysis of TRPV1 and eNOS for Western blotting (n =4 per group). (d) The content of nitric oxide in heart tissue. (e)
Representative oxidative stress images detected by DAF-2 DA in heart tissue. Data are presented as mean ± SD. ∗∗∗ ∗p < 0:001, ∗∗∗ p <
0:001, ∗∗p < 0:01, and ∗p < 0:05 represent significant differences in the NC, DM, and CAP group.
6 Oxidative Medicine and Cellular Longevity
NC DM DM+CAP
HE
(a)
NC DM DM+CAP
VCAM-1
(b)
NC DM DM+CAP 250
⁎⁎⁎⁎ ⁎
200
(% of control)
Permeability
150
100
50
0
NC
DM
DM+CAP
(c)
DAPI TRPV1 eNOS Merged
NC
5 ⁎⁎⁎
NO contens in serum
4 ⁎
DM
(umol/l)
3
2
DM+CAP 1
0
DM+CAP
DM
NC
(d) (e)
⁎
⁎⁎⁎
H2O2 contents in serum
80
60
(umol/l)
40
20
0
DM+CAP
DM
NC
(f)
Figure 3: TRPV1/eNOS activation by capsaicin attenuates vascular oxidative stress and increases the level of NO in diabetic rats. (a–b)
Vascular structure assessed by eosin/hematoxylin coloration, expression of vcam-1 by immunohistochemistry. The scale bar represents
500 μm and 50 μm. (c) Vascular permeability assay by Evans blue and the analysis of statistical. (d) Representative immunofluorescence
images showing the expression of TRPV1 and eNOS in the blood vessel. (e–f) The content of nitric oxide and hydrogen peroxide in
serum. Data are presented as mean ± SD. ∗∗∗ ∗p < 0:001, ∗∗∗ p < 0:001, and ∗p < 0:05 represent significant differences in the NC, DM,
and CAP group.
Oxidative Medicine and Cellular Longevity 7
DAPI TRPV1 eNOS Merged
NC
HG
HG+CAP
(a)
⁎⁎ ⁎ ⁎⁎
1.5 ⁎⁎⁎ ⁎⁎ 100 ⁎
1.5
(mmol/mg protein)
TRPV1/Actin
eNOS/Actin
NC HG HG+CAP 1.0
NO in cells
140 KD 1.0
eNOS
TRPV1 75 KD 50
0.5 0.5
Actin 42 KD
0.0 0.0
0
NG
HG
HG+CAP
NG
HG
HG+CAP
NG
HG
HG+CAP
(b) (c)
3 ⁎⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎⁎ NG HG HG+CAP 50
DCF-fluorescene
⁎⁎⁎⁎
NO in medium
40
2
(umol/l)
30
20
1
10
0
0
NG
HG
HG+CAP
NG
HG
HG+CAP
(d) (e)
⁎⁎⁎
Permeability (% of control)
150
⁎⁎ ⁎ ⁎⁎
100
50
0
NG
HG
HG+CAP
HG+CAP+CPZ
HG+CAP+L-NAME
(f)
Figure 4: Capsaicin by activation TRPV1/eNOS alleviates oxidative stress and permeability and increases the level of NO in mouse vascular
endothelial cells in high-glucose. (a) Representative immunofluorescence staining of TRPV1 and eNOS. The scale bar represents 20 μm. (b)
The protein expression of TRPV1 and eNOS was determined by western blotting and quantitative analysis. (c–d) The content of nitric oxide
in cell and cell culture medium. (e) Representative images detected by DAF-2 DA and quantitative analysis. (f) Permeability of mouse
vascular endothelial cells to FITC- dextran after stimulation with normal-glucose (NG, glucose 5.5 mmol/L), high-glucose (HG, glucose
25 mmol/L), HG+capsaicin (HG+CAP, Cap 1 μmol/L), high-glucose pretreated with CPZ 10 μmol/L 2 hr, then capsaicin 1 μmol/L 24 hr
(HG+CAP+CPZ) or high-glucose pretreated with 400 μmol/L NG-nitro-L-arginine methyl ester (L-NAME, an eNOS inhibitor) for 2 hr,
then capsaicin for 24 hr (HG+CAP+L-NAME). Data are presented as mean ± SD. ∗∗∗∗ p < 0:001, ∗∗∗ p < 0:001, ∗∗ p < 0:01, and ∗ p < 0:05.
8 Oxidative Medicine and Cellular Longevity
Phase contrast Annexin V-FITC PI Merged
NG
HG
HG+CAP
HG+CAP+CPZ
HG+CAP+L-NAME
(a)
NG HG HG+CAP HG+CAP+CPZ
HG+CAP+L-NAME
100 ⁎⁎
Fluo-4 fluorescence
80 ⁎⁎ ⁎⁎⁎
60
40
20
0
NG
HG
HG+CAP
HG+CAP+CPZ
HG+CAP+L-NAME
(b)
Figure 5: Capsaicin by activation TRPV1/eNOS prevents endothelial cell apoptosis induced by high glucose. Apoptosis (a) and calcium
concentration. (b) of endothelial cells after stimulation with normal-glucose (NG, glucose 5.5mmol/L), high-glucose (HG, glucose 25
mmol/L), HG+capsaicin (HG+CAP, Cap 1 μmol/L), high-glucose pretreated with CPZ 10 μmol/L 2 hr, then capsaicin 1 μmol/L 24 hr
(HG+CAP+CPZ) or high-glucose pretreated with 400 μmol/L NG-nitro-L-arginine methyl ester (L-NAME, an eNOS inhibitor) for 2 hr,
then capsaicin for 24 hr (HG+CAP+L-NAME).
(Figure 3(c)). The results of vascular immunofluorescence cellular permeability, the murine vascular endothelial cells were,
showed the drastic decrease of TRPV1 and eNOS expression respectively, cultured in normal-glucose (NG) and high-glucose
in rat blood vessel in untreated diabetic group, which was sub- (HG) medium, and capsaicin plus HG. The results of these cell
stantially ameliorated by capsaicin treatment (Figure 3(d)). immunofluorescence experiments demonstrated that the pro-
Concomitantly, capsaicin also greatly reversed the NO pro- tein expression levels of TRPV1 and eNOS were significantly
duction and sharply suppressed the ROS increase in diabetic downregulated in HG compared with those in NG, however,
rats (Figures 3(e) and 3(f)). the capsaicin treatment markedly reversed the decrease in HG
+CAP group (Figure 4(a)). Likewise, the WB assay indicated
3.4. Capsaicin by Activation TRPV1/eNOS Alleviates Oxidative the similar results in the cultured cells by HG or HG + capsaicin
Stress, Permeability, and Cell Apoptosis and Increases the Level (Figure 4(b)). The concentrations of NO produced by eNOS in
of NO in Mouse Vascular Endothelial Cells in High Glucose. cultured cells and medium were verified under the different
To further elucidate the capsaicin function in regulating inter- conditions, showing that HG significantly suppressed NO
Oxidative Medicine and Cellular Longevity 9
barrier composed of endothelial cells is essential for vascular
Diabetes
permeability, and vital to maintain vascular function. However,
overproduction of AGEs in diabetes increases endothelial cell
Heart fibrosis
Glucose Blood vessel permeability, inhibits eNOS activity, and activates both
high permeability NADPH oxidase (NOX) and NF-кB [32]. Another hallmark
of endothelial dysfunction is a deficiency in the bioavailability
TRPV1 of NO, and diabetes-induced inhibition of nitric oxide is
eNOS
attributable to the inhibition of eNOS, but it is not clear
whether it is related to TRPV1. Our current study is aimed
NO VCAM-1 Endothelial to explore the relationship between endothelial dysfunction
ROS dysfunction and TRPV1 in diabetes, particularly under capsaicin
treatment.
Our present findings reveal that in streptozotocin-induced
Capsaicin diabetic rats, the morphological fibrosis caused by diabetes is
improved by capsaicin treatment, not only in the heart but also
in the blood vessels. As an important factor mediating the
adhesion between leukocytes and vascular endothelial cells,
Figure 6: Upregulation of eNOS by TRPV1 activation attenuates vascular intercellular adhesion molecules (vcam-1) play an
hyperglycemia-induced ROS production in endothelial dysfunction. important role in vascular injury. In normal arteries, it is inter-
Schematic showing that hyperglycemia triggers endothelial mittently expressed in endothelial and smooth muscle cells,
dysfunction by downregulated expression of TRPV1/eNOS,
and the density is higher where plaques are easy to form [33].
exacerbates oxidative stress, and decreases the level of NO. Capsaicin
by activation TRPV1/eNOS alleviates oxidative stress and increases By vascular immunohistochemistry, we found that the upregu-
the level of NO in endothelial cells and reduces vascular permeability lation of vcam-1 in diabetes was downregulated after capsaicin
and ameliorates apoptosis and fibrosis in heart tissue. treatment. Thereafter, endothelial permeability in the aortic
arch was studied using Evans blue to better understand the
production, while capsaicin treatment normalized NO levels changes of vascular function. Our current results indicated that
compared with the NC (Figures 4(c) and 4(d)). Additionally, capsaicin may truly improve diabetic vascular disorders. In
oxidative stress is a key factor in the damage of endothelial cells order to reveal the role of TRPV1 in cardiovascular complica-
caused by HG. Our current data suggested that ROS levels were tions of diabetes, we performed immunofluorescence localiza-
sharply elevated by HG, implying the oxidative stress induced tion and protein quantitative analysis in cardiac tissue and
by HG. However, capsaicin substantially reduced the oxidative blood vessel. Immunofluorescence allowed us to observe that
stress (Figure 4(e)). Endothelial cell permeability assay also TRPV1 in blood vessels and heart tissues was downregulated
verified that endothelial cell barrier was destroyed by high- under hyperglycemia and recovered after capsaicin treatment.
glucose environment and capsaicin treatment improved its The double label staining of eNOS and TRPV1 highlighted that
function and reduced material leakage, which was invalidated eNOS was significantly activated. Quantitative analysis for tar-
after TRPV1 or eNOS inhibitor treatment (Figure 4(f)). In situ get protein assayed by Western blotting also verified our con-
fluorescence detection of annexin V-FITC/PI cell apoptosis, clusions. eNOS relaxes blood vessels through the synthesis of
the protective effect of capsaicin on apoptosis impelled by high nitric oxide, so we detected the changes of nitric oxide content
glucose disappeared after the addition of inhibitor of TRPV1 in the serum and heart to verify that TRPV1 activates eNOS.
or eNOS (Figure 5(a)). In situ fluorescence probe was applied Excessive release of free radicals damaged vascular function,
for the detection of endothelial cell intracellular calcium; the and eNOS can also reduce the production of reactive oxygen
result showed that the intracellular calcium ion concentration species while releasing nitric oxide. For the changes of reactive
decreased in high-glucose (HG) medium. Then, capsaicin oxygen species, we selected fluorescent probes to label heart tis-
treatment restored intracellular calcium ion concentration in sue and endothelial cells in vivo. Our findings revealed that the
HG medium, which was abolished by the addition of CPZ upregulation of eNOS by capsaicin treatment really suppressed
(TRPV1 inhibitor), but not by the L-NAME (eNOS inhibitor) the ROS production. Our in vitro cell experiments confirmed
(Figure 5(b)). the findings obtained in vivo. Specifically, high glucose reduced
the expression of TRPV1/eNOS and NO production, while
4. Discussion activation of TRPV1 by capsaicin-mediated Ca2+ influx in
endothelial cells can increase eNOS activity then stimulate
At present, ointments and patches containing capsaicin are NO production, which implies a novel mechanism underlying
often used for pain treatment [30, 31]. However, as an agonist capsaicin-mediated amelioration of cardiomyopathy and endo-
of TRPV1, the mechanism of capsaicin-mediated improvement thelial dysfunction under diabetes condition.
in the metabolic diseases is still not completely elucidated. Vas-
cular endothelial dysfunction, as a major hallmark in diabetic 5. Conclusions
cardiomyopathy, may potentially play a role in the development
of other diabetic cardiovascular complications like atherosclero- Taken together, our current study suggested that capsaicin
sis [32]. When the endothelial cell metabolism is normal, the intervention may improve the diabetic cardiomyopathy and
cells are quiescent and remain in vascular homeostasis. Vascular blood vessel endothelial dysfunction via upregulating TRPV1/
10 Oxidative Medicine and Cellular Longevity
eNOS pathway as illustrated in Figure 6. Our findings in the Natural Science Foundation of Anhui Province for Outstand-
current study may facilitate clinical trials to extend the applica- ing Youth (2008085J34 to YL).
tion of capsaicin in patients with diabetes, and also might shed
a new light into clinical treatment for diabetic complications.
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