Suppression of Nitric Oxide Implicated in the Protective
Effect of Echinacoside on H2O2-Induced PC12 Cell Injury
Rong Kuanga,b, Yiguo Suna and Xiaoxiang Zhenga,*
aDepartment of Biomedical Engineering, Key Laboratory of Biomedical Engineering, Ministry of
Education, Zhejiang University (Yuquan Campus), Zheda Road 38, 310027 Hang Zhou, PR China
bZhejiang Food and Drug Institute for Control, Jichang Road 86, 310004, Hang Zhou, PR China
Received: April 12th, 2009; Accepted: February 6th, 2010
We investigated whether suppression of nitric oxide (NO) implicated in the protective effect of echinacoside (ECH), a
phenylethanoid glycoside, on H2O2-induced injury to the rat pheochromocytoma cell line (PC12 cells). Data show that
application of ECH to H2O2-injured PC12 cells (HIPCs) increased cell viability and decreased the necrotic ratio. Laser
scanning confocal microscopic (LSCM) analysis suggested that ECH exerted an inhibitory effect on the formation of NO. In
addition, RT-PCR analysis revealed that ECH down-regulated p65 and iNOS mRNA expressions in HIPCs. In summary,
suppression of NO is related to the protective effect of ECH on HIPCs.
Keywords: Echinacoside, ECH, apoptosis, NO, iNOS,NF-κB.
Alzheimer’s and Parkinson’s diseases are
neurodegenerative disorders characterized by apoptosis
and neuron loss where the effects of oxidative stress are
thought to play critical roles . Because H2O2 has been
used extensively to induce cell apoptosis, and PC12
cells show many properties just like primary cultured
neuronal cells , the HIPCs model has been used
widely to develop drugs for neurodegenerative diseases
in vitro [3,4]. Studies have demonstrated that NO
synthesized by iNOS promotes neurotoxicity and plays
an important role in H2O2–mediated cell apoptosis .
NF-κB is an essential transcription factor mediating
iNOS induction and can be inversely activated by NO
. Although the complicated interaction among NF-κB,
iNOS and NO in the process of cell apoptosis remains
unknown, it is a fact that inhibition of NO makes a
major contribution to cytoprotection, and a compound
suppressing the generation of NO could be a
potential candidate for use in the intervention of
ECH (Figure 1) was extracted from Echinacea
angustifolia in 1950 and defined as a phenylethanoid
glycoside. It exists also in several other plants such
as Cistanche tubulosa (Schrenk) Wight. In China,
glycosides isolated from the stems of C. tubulosa have
been approved as a treatment for vascular dementia.
This is a government- approved drug containing
Figure 1: Chemical structure of ECH.
antioxidant properties and a neuroprotective function
with ECH as a quality control compound (content more
than 25%) [7,8]. It has recently been demonstrated that
ECH protects against hepatotoxicity and is a potent
promoter of neuronal survival that is induced by TNF-α
and MPTP [9,10]. However, these studies provide little
information about the relationship between NO and the
protective effect of ECH on oxidative stress-induced
injury in vitro. In order to explore this subject, the cell
survival, apoptosis and necrosis, and the change of NO,
iNOS and p65 mRNA expressions were determined.
Cell viability determined by MTT reduction was
markedly decreased and the necrosis ratio detected
using flow cytometry was increased after PC12 cells
were exposed to H2O2. However, when the cells were
pre- and co-incubated with ECH, H2O2-induced cell
glycosides which have shown
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2 Natural Product Communications Vol. 5 (0) 2010 Kuang et al.
Table 1: Attenuation of H2O2-induced PC12 cell injury by ECH.
Control 100.0 ± 5.1
H2O2 27.9 ± 8.0 ###
H2O2+ECH 5μg/mL 35.5 ± 10.8
H2O2+ECH 10μg/mL 51.0 ± 13.4 **
H2O2+VE 10μg/mL 56.6±12.4***
Data are expressed as percentages of cell viability relative to the control, with
mean ± SD (n = 5). ###P < 0.001 compared with control; **P < 0.01, ***P <
0.001 compared with H2O2.
1.67 ± 0.20
4.83 ± 0.78###
3.54 ± 0.50*
2.59 ± 0.68**
3.22 ± 0.73*
Figure 2: H2O2 induced cell apoptosis stained by Hoechst 33324 and the effect
of ECH. “→” shows the apoptotic cells. A: Control; B: H2O2; C: ECH 5μg/
mL; D: ECH 10μg/ mL; E: VE 10μg/ mL.
toxicity was significantly attenuated (Table 1). The
pictures of PC12 cells stained by Hoechst 33324 are
shown in Figure 2. The apoptotic cells can be
distinguished by their typical morphological appearance:
chromatin condensation and nuclear fragmentation with
nuclei being stained much lighter than normal cells.
To determine whether NO was involved in the
protection of ECH on H2O2-induced PC12 cell injury,
LSCM was utilized to monitor the change of NO level.
After PC12 cells were treated with 200 μM of H2O2 for
30 min, NO increased rapidly during the first 28 min
and then attained a peak. The treatment with ECH (5, 10
μg/mL) for 30 min prior to H2O2 treatment significantly
suppressed the time course curve (Figure 3A).
Moreover, ECH application caused a remarkable
decrease in the peak value of NO by 33.1% (P < 0.05)
and 43.3% (P < 0.01), (Figure 3B). The results suggest
that ECH reduces the NO level elevated by H2O2.
We also measured the effects of ECH on H2O2-induced
up-regulation of p65 and iNOS mRNA by RT-PCR. As
shown in Figure 4, p65 and iNOS mRNA expressions in
PC12 cells increased (p < 0.01) when cells were treated
with 400 μM of H2O2. However, ECH significantly
suppressed the up-regulated p65 and iNOS mRNA.
Figure 3: Effect of ECH on the time course of NO and peak value of Ft/F0 (n =
20-25).Data are expressed as Ft/F0 and mean ± SD. ###P < 0.001 compared
with control; **P < 0.01 compared with H2O2.
Figure 4: Effect of ECH on the expressions of p65 and iNOS mRNA in H2O2-
induced PC12 cells. (A): Photographs of agarose gel electrophoresis of the p65
and iNOS and β-actin mRNA RT-PCR product. The level of mRNA was
quantified by densitometric analysis and expressed as targeted mRNA / β-actin
and mean ± SD. ###P < 0.001 compared with control; *P < 0.05, **P < 0.01,
***P < 0.001 compared with H2O2.
NO, a free radical, is synthesized by three different
types of NO synthase including the constitutive
Ca2+/calmodulin-dependent neuronal and endothelial
isoforms (nNOS and eNOS) and the inducible
Ca2+/calmodulin-independent isoform (iNOS). NO is
synthesized by different types of NO synthase, which
shows divergent effects on neuronal cells. Unlike the
protective effect of eNOS, NO produced by nNOS and
iNOS has a neurotoxic action which derives in part
from the reaction of NO with superoxide anions to form
peroxynitrite to alter cell functions . iNOS was
assumed to have an important role during CNS injuries
. For example, it has been reported that cerebral
Protective effect of ECH on H2O2-induced injury
Natural Product Communications Vol. 5 (0) 2010 3
ischemia-reperfusion injury in rats induced NF-κB and
iNOS activation and taxifolin ameliorated the injury by
modulating iNOS and NF-κB expressions . The
relationships among the NF-κB, iNOS and NO remain
unclear in HIPCs. We propose that H2O2 can elicit a
rapid rise in intracellular Ca2+, which could activate
Ca2+-dependent nNOS, and NO produced by nNOS
could stimulate NF-κB activity, leading to increased
synthesis of iNOS, which would then promote greater
NO release and further activation of NF-κB, resulting in
a vicious cycle in which NO seems to have a critical
The results indicate that ECH protects PC12 cells
against H2O2 -induced apoptosis, necrosis and increases
cell viability through a route of suppressing NO
formation. Several mechanisms are proposed to explain
this suppression. First, experiments showed that ECH
decreased the level of NO by down-regulating the
expressions of NF-κB and iNOS. Secondly, ECH
reduced the concentration of Ca2+ in HIPCs with a
lower Ca2+-dependent nNOS activity. Thirdly, a
possible mechanism is
polyphenolic structure, since polyphenolic compounds
are known to be potent antioxidants and free radical
scavengers. Therefore, suppression of NO by ECH
protects H2O2-induced PC12 cells injury.
Materials and cell culture: ECH (purity >98%, HPLC)
and vitamin E (VE, purity >98.8%, GC) were supplied
by the National Institute for the Control of the
Pharmaceutical and Biological Products (Nicpbp,
China). PC12 cells were purchased from Shanghai
Institute of Biochemistry and Cell Biology (Sibcb,
China). Cells were maintained in DMEM (Gibco, USA)
supplemented with 5% heat-inactivated horse serum,
10% fetal bovine serum (Gibco, USA), 100 U/mL
penicillin, and 100 U/mL streptomycin. Culture flasks
were kept in humidified 5% CO2/95% air at 37ºC. The
medium was changed every 3 days.
Experimental protocols: PC12 cells were grown to 80-
90% confluence and then replanted at an appropriate
density (according to the particular experiment) on
either culture plates or dishes. To induce oxidative
stress, fresh H2O2 was prepared from a 30% stock
solution prior to each experiment. In all experiments,
two doses (5, 10 μg/mL) of either ECH or 10 μg/mL of
VE were pre-incubated with PC12 cells for 30 min, after
which 400 μM of H2O2 was added (except in NO
evaluation). Assays were performed 4 h after H2O2 was
added. VE, a strong antioxidant that has anti-apoptosis
properties , was used as a positive control.
suggested by ECH’s
Measurement of cell viability: PC12 cells were seeded
in a 96-well plate at a density of 2 × 104 cells per well.
After treatment with ECH and H2O2, as described
above, cells were incubated with 0.5 mg/mL MTT
solution (Sigma, USA) for 4 h at 37ºC. Formazan
crystals that formed in intact cells were dissolved with
200 μL of dimethylsulfoxide (DMSO), and the
absorbance was measured at 570 nm using a microplate
reader (Molecular Devices Co., Spectra MAX 340,
USA). Cell viability measurements were calculated as a
percentage of the absorbance of the control sample.
Determination of apoptotic cells with Hoechst 33342
staining and necrotic cells with PI combined flow
cytometry: To further evaluate the effect of ECH on the
H2O2-induced apoptosis of PC 12 cells ， apoptotic
nuclei staining in H2O2-administered PC12 cells was
examined. After treatment with H2O2 for 4 h with or
without ECH，the cells were rinsed with PBS and fixed
with 4% paraformaldehyde for 5 mins. The fixed cells
were washed twice with PBS and incubated with the
DNA-binding dye Hoechst 33342 at 10 µg/mL. Nuclear
morphology was examined by Olympus IX 70
fluorescence microscopy (Tokyo, Japan). Individual
nuclei were visualized to distinguish the apoptotic cells
by their typical morphological appearance: chromatin
condensation, nuclear fragmentation with nuclei being
stained more lightly or smaller than normal cells .
In addition, separate groups of cells were also harvested
and stained with PI (propidium iodide, Caltag, USA) to
evaluate the percentage of necrotic cells using flow
cytometry (Becton-Dickinson, USA). Data were
analyzed using CellQuest software (Becton-Dickinson)
Measurement of NO change: A LSCM (Zeiss 510,
Germany) was used to evaluate the relative change of
NO by detecting DAF-2 fluorescence (10 μΜ,
Molecular probes, USA). Fluorescence was measured at
an excitation of 488 nm and emission of 515 nm - 565
nm. Following the addition of 200 μM of H2O2 to the
dish, laser scanning was used to obtain a series of
images. Image frames were acquired every 30 secs and
image acquisition was completed within 30 mins
following H2O2 addition. Images were analyzed
quantitatively using Zeiss LSM software to determine
the change in fluorescence intensity within a cell (20-25
cells were analyzed in each group). The fluorescence at
each time point (Ft) after H2O2 addition was divided by
the fluorescence before H2O2 addition (F0) to obtain the
ratio Ft/F0 . ECH and VE were incubated with PC12
cells for 30 mins prior to H2O2 irritation.
RT-PCR analysis of NF-κB p65 and iNOS mRNA:
After treatment, as mentioned in “Experimental
protocols”, 5× 107 cells were rinsed twice with cold
4 Natural Product Communications Vol. 5 (0) 2010 Kuang et al.
PBS and total RNA was extracted according to the Kit
supplier’s instructions. For PCR amplification, the
specific primers used were as follows: (1) iNOS sense
primer: 5′-GCCTCGCTCTGGAAAGA-3′, antisense
primer: 5′-TCCATGCAGACAACCTT-3′; (2) p65:
sense primer: 5′-AAGATCAATGGCTACACAGG-3′;
anti-sense primer: 5′- CCTCAATGTCTTCTTTCTGC-
3′; (3) β-actin:
GAGGGAAATCGTGCGTGAC-3′, anti-sense primer:
5′-TTGGCATAGTAGGTCTTTACGG-3′. The primer
sets yield PCR products of 477, 493 and 272 bp for
iNOS , p65 and β-actin, respectively. The PCR
procedure was performed at 42ºC for 30 min, and 94ºC
for 3 min, followed by 35 cycles at 94ºC for 20 s, 60ºC
for 1 min, and extension at 72ºC for 5 min. PCR
products were examined by electrophoresis on 2%
agarose ethidium bromide gel and visualized by an
ultraviolet gel documentation system. The intensity of
the bands was quantified by scanning densitometry
 Butterfield DA, Howard B, Yatin S, Koppal T, Drake J, Hensley K, Aksenov M, Aksenova M, Subramaniam R, Varadarajan S,
Harris-White ME, Pedigo NW Jr, Carney JM.(1999) Elevated oxidative stress in models of normal brain aging and Alzheimer's
disease. Life Sciences, 65, 1883-1892.
 Greene LA, Tischler AS. (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which
respond to nerve growth factor. Proceedings of National Academy of Sciences U S A, 73, 2424-2428.
 Li RC, Morris MW, Lee SK, Pouranfar F, Wang Y, Gozal D. (2008) Neuroglobin protects PC12 cells against oxidative stress. Brain
Research, 23, 159-166.
 Kim JW, Li MH, Jang JH, Na HK, Song NY, Lee C, Johnson JA, Surh YJ.(2008) 15-Deoxy-delta (12,14)-prostaglandin J (2)
rescues PC12 cells from H2O2-induced apoptosis through Nrf2-mediated upregulation of heme oxygenase-1: potential roles of Akt
and ERK1/2. Biochemical Pharmacology, 76, 1577-1589.
 Xu HB, Li L, Liu GQ. (2008) Protection against hydrogen peroxide-induced cytotoxicity in PC12 cells by guggulsterone. Yao Xue
Xue Bao, 43, 1190-1197.
 Carbone DL, Moreno JA, Tjalkens RB. (2008) Nuclear factor kappa-B mediates selective induction of neuronal nitric oxide
synthase in astrocytes during low-level inflammatory stimulation with MPTP. Brain Research, 27, 1-9.
 Liu FX, Wang XW, Luo L, Xin H, Wang XF. (2006) The effects of glycosides of Cistanche on learning and memory in β-amyloid
peptide induced Alzheimer’s disease in mice and its possible mechanism. Chinese Pharmacological Bulletin, 22, 595-599.
 Mourboul A, Mao XM , Rena K, Ma SY, Leininger M, Gérard S. (2008) Antioxidant property of general cistanosides in human
HL-60 cell line. Chinese Pharmacological Bulletin, 24, 362-364.
 Deng M, Zhao JY, Tu PF, Jiang Y, Li ZB, Wang YH. (2004) Echinacoside rescues the SHSY5Y neuronal cells from TNFalpha-
induced apoptosis. European Journal of Pharmacology, 505, 11-18.
 Geng X, Tian X, Tu P, Pu X. (2007) Neuroprotective effects of echinacoside in the mouse MPTP model of Parkinson's disease.
European Journal of Pharmacology, 564, 66-74.
 Jhaveri KA, Toth LA, Sekino Y, Ramkumar V. (2006) Nitric oxide serves as an endogenous regulator of neuronal adenosine A1
receptor expression. Journal of Neurochemistry, 99, 42-53.
 Koh PO. (2008) Melatonin regulates nitric oxide synthase expression in ischemic brain injury. Journal of Veterinary Medical
Science, 70, 747-750.
 Wang YH, Wang WY, Chang CC, Liou KT, Sung YJ, Liao JF, Chen CF, Chang S, Hou YC, Chou YC, Shen YC. (2006) Taxifolin
ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation.
Journal of Biomedical Science, 13, 127-141.
 Hong H, Liu GQ. (2004) Protection against hydrogen peroxide-induced cytotoxicity in PC12 cells by scutellarin. Life Sciences, 74,
 Mosmann T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
Journal of Immunological Methods, 65, 55-63.
 Zeng XH, Zhang SM, Zhang L, Zhang KP, Zheng XX. (2006) A study of the neuroprotective effect of the phenolic glucoside
gastrodin during cerebral ischemia in vivo and in vitro. Planta Medica, 72, 1359-1365.
sense primer: 5′-
using NIH Image 1.61 software. The level of gene
mRNA expression was expressed as the ratio of the
intensity of the gene PCR products to the corresponding
β-actin PCR product.
Statistical analysis: Data are presented as mean ± SD.
Differences between group mean values were calculated
using analysis of variance, and comparisons between
means were performed by the two-tailed Student’s t-
test. Statistical significance was defined as P < 0.05.
Acknowledgments - This work was supported by the
Zhejiang Provincial Key Laboratory of Chinese
Medicine Screening, Exploitation
Effectiveness Appraisal for Cardio-cerebral Vascular &
Nervous System, and by the Key Laboratory for
Biomedical Engineering of the Ministry of Education of