Sugar composition analysis of Fuzi polysaccharides by HPLC-MSn and their protective effects on Schwann cells exposed to high glucose

Article abstract of DOI:10.3390/molecules21111496

Fuzi has been used to treat diabetic complications for many years in china. In a previous study, we have shown that Fuzi aqueous extract can attenuate Diabetic peripheral neuropathy (DPN) in rats and protect Schwann cells from injury. Thus, the protective

Full text of DOI:10.3390/molecules21111496

molecules
Article
Sugar Composition Analysis of Fuzi Polysaccharides
by HPLC-MSn and Their Protective Effects on
Schwann Cells Exposed to High Glucose
Bei-Bei Wang 1,†, Jia-Li Wang 1,†, Jiang Yuan 1, Qing-Hua Quan 1, Rui-Fang Ji 1, Peng Tan 1,
Jing Han 2,* and Yong-Gang Liu 1,
*
1
School of Chinese Materia Medica, Beijing University of Chinese Medicine,
Wangjing Zhonghuan Road No. 6 School Range, Chaoyang District, Beijing 100102, China;
wbb20131681@163.com (B.-B.W.); wangjl120@163.com (J.-L.W.); Bucmyj2015@163.com (J.Y.);
18810689952@163.com (Q.-H.Q.); 17801164351@163.com (R.-F.J.); tanpengtcm@163.com (P.T.)
Beijing Chinese Medicine Research Institute, Beijing University of Chinese Medicine,
North Third Ring Road No. 11 School Range, Chaoyang District, Beijing 100029, China
Correspondence: hanjing8585@163.com (J.H.); liuyg0228@163.com (Y.-G.L.);
Tel.: +86-108-4738658 (Y.-G.L.); Fax: +86-108-4738611 (Y.-G.L.)
2
*
†
These authors contributed equally to this work.
Academic Editor: Quan-Bin Han
Received: 26 September 2016; Accepted: 2 November 2016; Published: 9 November 2016
Abstract: Fuzi has been used to treat diabetic complications for many years in china. In a previous
study, we have shown that Fuzi aqueous extract can attenuate Diabetic peripheral neuropathy (DPN)
in rats and protect Schwann cells from injury. Thus, the protective effect of Fuzi polysaccharides
(FPS) on high glucose-induced SCs and the preliminary mechanism were investigated. Firstly, the
FPS were obtained and their monose composition was analyzed by the combination of pre-column
derivatization and high performance liquid chromatography coupled with electrospray ionization
multi-tandem mass spectrometry (HPLC/ESI-MSn). The results witnessed the efficiency of this
method and seven monosaccharides were tentatively identified, among which fucose was first
reported. Simultaneously, m/z 215 can be considered as diagnostic ions to confirm the number
of monosaccharides. Next, high glucose-induced SC model was applied and divided into model
group, treated group of FPS, normal and osmotic control group. After treatment for 48 h, the data
showed FPS could significantly decrease the intracellular ROS and apoptosis, which were determined
by the corresponding fluorescent probes. Then, the expression of oxidative stress-related proteins
in SCs were measured by Western blot. Furthermore, the protein tests found that FPS markedly
up-regulated superoxide dismutase (SOD), catalase (CAT) and peroxisome proliferator-activated
receptor gamma coactivator 1-alpha (PGC-1
(Nox1) protein level. Moreover, FPS could also increase AMP-activated protein kinase (AMPK)
activation significantly. Hence, we preliminary deduced that AMPK-PGC-1 pathway may play an
α) protein level, but down-regulated NADPH oxidase-1
α
important role in the protective effect of FPS against high glucose-induced cell damage.
Keywords: Fuzi polysaccharides; HPLC-ESI-MSn; monosaccharide composition; Schwann cells;
antioxidant; AMPK-PGC-1α pathway
1. Introduction
Diabetic peripheral neuropathy (DPN), the commonest diabetes complication, may affect 30%–90%
diabetics and forms a leading cause of foot ulceration and lower limb amputation in patients with
DM and consequently burdens their quality of life [
1]. Numerous mechanisms can result in DPN,
including the activation of polyol pathway flux and protein kinase C, the stack of advanced glycation
Molecules 2016, 21, 1496; doi:10.3390/molecules21111496
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end products and the overproduction of oxygen free radicals, etc. Thus, it is difficult to find drugs to
ameliorate the multiple symptoms of DPN due to the complex mechanism. However, in 2001, a uniform
pathological mechanism about diabetic complications was proposed that the increased oxidative stress
might intimately associate with nerve dysfunction and regenerative capacity diminishment [
Subsequently, antioxidants became a hot spot in the management of DPN and drug target [4–6].
2,3].
Schwann cells (SCs), a constituent of myelin cells, play a crucial role in the peripheral nervous
system by providing myelin sheath; maintaining, nurturing, and restoring axons and neurons;
and producing neurotrophic factors such as nerve growth factor (NGF), and fibroblast growth factor
(FGF) [7]. Therefore, the injury of SCs can often cause depressed nerve conduction velocity, atrophic
axon, and the impaired regeneration ability of the axon. It has been reported that when SCs are
exposed to high glucose concentration, the cell damage could be triggered via induced oxidative stress,
activated mitochondrial pathway and promoted apoptosis [8,9]. Hence, high glucose stimulated SCs
have been developed as a valuable model for exploring novel therapeutic approaches and further
mechanisms of DPN [10–14].
Fuzi, the lateral root of Aconitum carmichaeli Debx, was listed in materia medica classics,
Shen Nong Ben Cao Jing (Shennong’s Herbal) from 2000 years ago and its medicinal effect
against diabetes can be traced back to Shang Han Lun (Treatise on Cold-Induced Diseases) [15].
Besides, modern clinical studies also proved that prescriptions with Fuzi have unexpected effect on
treatment of DPN [16,17]. Due to the complex component of the TCM (traditional Chinese medicine)
combination formulas, it is not sufficient to validate the effectivity of Fuzi for DPN. In a previous
study [18], we have demonstrated that the aqueous extract of Fuzi could ameliorate the symptom of
rats with DPN, reduce hydrogen peroxide and superoxide anion induced by high glucose in Schwann
cells. In this paper, further exploration of the main functional components of Fuzi for improving the
oxidation damages in SCs and the underlying mechanisms were given.
In accordance with our screen, polysaccharides of Fuzi were found to be the active ingredient for
cytoprotective effect on SCs. FPS were extracted and purified and its monosaccharide composition was
identified using HPLC-MSn for the first time. Then protective effects of FPS on oxidative injury were
evaluated with high glucose-induced SCs. Additionally, oxidative stress-related signaling pathways
were analyzed by Western blot method for validating the potential molecular mechanism.
2. Results
2.1. The MS Analysis of FPS
Monosaccharide composition analysis of polysaccharides is of fundamental importance for the
research on polysaccharide structure and its characteristics. Pre- or post-column derivatization methods
are commonly used in chromatography separation and detection in that monosaccharides have strong
polar, similar structure, a lack of optical activity. The reagent PMP (1-Phenyl-3-methyl-5-pyrazolone),
one of the common labels that can react with reducing carbohydrate under mild condition, started to be
applied in analysis saccharides in 1989 by the Honda [19]. Then pre-column derivatization with PMP
method started to be applied in analysis saccharides by HPLC-MS in 1998 [20]. However, with salt
formation in traditional derivative reaction, the desalinization treatment has to be done before mass
spectrometry. Over the past decade, an improved PMP derivatization method had been developed,
which could be applied directly to mass spectrometry [21]. In this study, the improved method was
applied to analyze FPS for the first time by HPLC-MSn.
2.1.1. The Fragmentation Regulations of PMP-Labeled Monosaccharides
Firstly, through the comparison between the blank and PMP-labeled group, it can be sure
that derivative reaction was completed successfully (see Figure 1). Compared with positive-ion,
negative-ion mode has a weaker interference of background, although both ion modes have high
response (see Figure 2). The [M
−
H]−, [M
−
PMP
−
H]− were obtained easily in MS. Then, in MS3,

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a characteristic fragment ions m/z 215 were detected, which could be used to confirm the number
of monosaccharide with negative ion mode (see Figure 3). The fragmentation pathway is shown in
Figure 4.
Figure 1. Chromatogram of three double PMP-labeled monosaccharides: (
) PMP-labeled mannose; ( ) PMP-labeled rhamnose; ( ) PMP-labeled glucose; and (
PMP-labeled monosaccharides.
A
) Blank control;
(
B
C
D
E
) three mixed

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Figure 2.
Positive and negative total ion current chromatogram (TIC) of three double
PMP-labeled monosaccharides.
Figure 3. MS (left); MS2 (middle); and MS3 (right) spectra of PMP-labeled: mannose (
and Glucose (C).
A); rhamnose (B);

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Figure 4. Fragmentation pathway of double PMP-labeled monosaccharides in ESI-MSn.
2.1.2. Monosaccharide Composition Analysis of the Hydrolyzed FPS
Total ion chromatograms (TIC) of the separated PMP-labeled monosaccharides were detected
simultaneously (Figure 5). In our study, nine PMP-labeled monosaccharides were separated, in which
seven were identified according to fragmentation characterization of PMP-labeled monosaccharides
and references [22–24] (Table 1).
Peaks 2, 3, and 5 could be undoubtedly identified as PMP-labeled mannose, rhamnose, and glucose
according to the retention times and MS data of standard sample. The number of monosaccharide
was determined through the characteristic fragment ions m/z 215. Further, the molecular weight was
obtained by the fragments [M
columns, the monosaccharide composition was tentatively identified.
−
H]−, [M
−
PMP
−
H]−. Based on the retention times on the C18
Figure 5. The total ion chromatogram and chromatogram of PMP-labeled monosaccharide composition
of polysaccharides.

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Table 1. Identification of monosaccharide composition of FPS.
No.
tR (min)
MS
MS2
MS3
Mw
Saccharides
1
2
3
4
5
6
7
8
9
9
508.2
509
334.1
335.1
319
335
335
335.1
305
305
213.8
214.9
214.8
214.7
214.8
214.7
214.7
214.8
214.7
180
180
164
180
180
180
150
150
164
Unknown (C6H12O6)
Mannose (C6H12O6)
Rhamnose (C6H12O5)
Glucose (C6H12O6)
Galactose (C6H12O6)
Unknown (C6H12O6)
Xylose (C5H10O5)
10.8
15.8
24.9
27.4
29.4
31
493.2
509.2
509.3
509.3
479.2
479.2
493.3
31.9
38.5
Arabinose (C5H10O5)
Fucose (C6H12O5)
319.1
To recap, FPS was composed of mannose, glucose, rhamnose, galactose, xylose, fucose and
arabinose, which mainly were glucose.
2.2. Evaluation of the Effects of FPS on Reactive Oxygen Species in High Glucose-Stimulated RSC96 Cells
The antioxidative effects of FPS on high glucose-stimulated RSC96 cells cultivated in different
treatment for 48 h are shown in Figure 6. The levels of peroxide and superoxide anions in NG + M group
increased slightly compared to the NG group. However, the HG group increased ROS significantly,
which revealed that the sugar concentration was the main causes of oxidative injury in RSC96 cells.
Moreover, the result displayed that all doses of FPS could dramatically down-regulate peroxide and
superoxide anion levels in a dose-dependent manner (p < 0.01). Therefore, FPS has an inhibitory effect
on hyperglycemia-induced ROS in RSC96 Cells.
Figure 6. The effects of FPS on antioxidant in injured RSC96 Cells: (
with DCFH2DA and DHE; and ( ) the quantized data of peroxide and superoxide anion levels were
expressed by mean S.D. (n = 3). ## or ** p < 0.01; * p < 0.05; “##”, HG vs. NG; “*” or “**”, HG vs.
HG + FPS. The ordinate is a percentage relative to NG group.
A) RSC96 cells were stained
B
±

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2.3. Evaluation of the Effects of FPS on Apoptosis in High Glucose-Stimulated RSC96 Cells
The antiapoptotic effect of FPS on high glucose-stimulated RSC96 cells was measured by flow
cytometry using annexin V-PE and 7-AAD staining. As can be seen in Figure 7, the apoptotic
ratio had no obvious change between normal control and osmotic control group. Unsurprisingly,
significant increases were observed in the apoptotic ratio of RSC96 cells stimulated by high glucose.
Compared with HG group, the apoptotic ratio was restored (p < 0.01) in medicated group of FPS,
whereas the curative effects did not show significant changes as the concentration of FPS rose.
Figure 7. The effects of FPS on antiapoptotic in injured RSC96 Cells: (
staining was represented by flow cytometry. Q1: necrotic; Q2: late apoptotic cells; Q3: normal viable
cells; Q4: early stage apoptotic cells; ( ) Apoptosis rate = early apoptosis + late apoptosis, the date
A) Annexin V-PE and 7-AAD
B
were expressed by mean ± S.D. (n = 3). ## or ** p < 0.01; “##”, HG vs. NG; “**”, HG vs. HG + FPS.
2.4. Evaluation of the Effects of on the Expression of Antioxidant Enzymes and Nox1
To clarify the mechanism underlying the inhibitory effect of FPS on ROS production, we detected
the expression of enzymes including SOD, CAT, and Nox1 which were directly related to the formation
and removal of ROS [25
reduced in HG group (p < 0.05). Moreover, the Nox1 protein level was increased in HG group (p < 0.05).
As expected, FPS could suppress the expression of Nox1 at different concentrations (p < 0.05 or p < 0.01
and, simultaneously, FPS upregulated CAT and SOD protein expression (p < 0.05 or p < 0.01) (Figure 8).
,26]. Compared with NG, the protein expression of SOD and CAT were
)
Figure 8. Cont.

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Figure 8. The effects of FPS on the expression of antioxidant enzymes and Nox1: (
expression levels were assayed by Western blotting; (B) The statistical data of protein were expressed
by mean S.D. (n = 3). ** p < 0.01; # or * p < 0.05; “#”, HG vs. NG; “*” or “**”, HG vs. HG + FPS.
A) The protein
±
The ordinate is the percentage relative to NG group.
2.5. The Effects of FPS on AMPK-PGC-1α Pathway
Then, we further investigated PGC-1
of many ROS-detoxifying enzymes and a master regulator of mitochondrial biogenesis and AMPK,
an essential upstream mediator of PGC-1 27 29]. The results showed that HG attenuated both the
ratio of p-AMPK/AMPK) and the expression of PGC-1 (p < 0.05 or p < 0.01). Moreover, FPS increased
α, a transcriptional coactivator required for the induction
α
[ –
α
levels of p-AMPK and PGC-1α in a dose-dependent manner (p < 0.01).
Combining with the results above, we deduced that FPS could decrease ROS production by
regulating SOD, CAT and Nox1, which was mediated by AMPK-PGC-1α pathway (Figure 9).
In this study, we used mannitol as an osmotic control. Data showed that compared to mannitol,
high glucose had more significant effect on the production of intracellular ROS and protein expression
levels of antioxidant enzymes. However, there was no remarkable difference in the level of activated
AMPK (p-AMPK) between the high glucose group and mannitol group. This is probably because
AMPK could be regulated by a variety of stimulation such as energy depletion, hypoxia, and
hypertonicity [30,31].

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Figure 9. The effects of FPS on expression of AMPK and PGC-1
α
: (
A
) The protein expression was
S.D. (n = 3).
assayed by Western blotting; ( ) The quantized data of protein were expressed by mean
B
±
## or ** p < 0.01; #, p < 0.05; “#”or “##”, HG vs. NG; “**”, HG vs. HG + FPS. The ordinate is a percentage
relative to NG group.
3. Experimental Section
3.1. Materials and Reagents
Aconitum carmichaeli Debx was collected in July 2011 from Sichuan Province of China. The plant
was identified by Dr. Peng Tan (School of Chinese Medicine, Beijing University of Chinese Medicine,
Beijing, China) and voucher specimens (voucher number 20110728) were kept at materia medica
Specimen Museum of this school.
D-Mannose, L-rhamnose, D-glucose were obtained from the Shanghai Yuanye Bio-Technology
Co., Ltd. (Shanghai, China). Trifluoroacetic acid (TFA) and 1-Phenyl-3-methyl-5-pyrazolone (PMP)
were from Shanghai Macklin Chemical Co., Ltd. (Shanghai, China). Acetonitrile (HPLC-grade)
was purchased from Merck (E. Merck, Darmstadt, Germany). Other chemistry reagents were all
analytical grade.
RIPA Lysis Buffer, 29% acrylamide: 1% bisacrylamide, Tris-base, glycine, TBST, ammonium
persulfate (APS), tetramethylethylenediamine (TEMED), Resolving gel buffer, Stacking gel Buffer and
prestained protein marker were purchased from APPLYGEN (Beijing, China). Polyvinylidene fluoride
(PVDF) membrane was provided from Millipore ((Billerica, MA, USA) and all main antibodies used in
this paper were obtained from Abcam (Cambridge, UK).
3.2. Chemistry
3.2.1. Extraction of the Polysaccharides from Fuzi
The coarse powder (200 g) of Fuzi was extracted by 10 times of water under refluxing at 100 ◦C
for 3 h. The resultant extracting solution was concentrated to 500 mL under reduced pressure.
◦
Then precipitated the polysaccharides with 2000 mL anhydrous alcohol and kept at 4 C overnight [32].
The crude polysaccharides precipitations were obtained by centrifugation at 4000 rpm for 20 min and
then the precipitate was dissolved in water again and repeated 80% alcohol-precipitation operation.
After 24 h, the precipitate was re-suspended with pure water and protein was dislodged by Sevag
method. The layer of water was freeze-dried after pressure reduction condensation and the Fuzi

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polysaccharides were obtained. The total sugar content of polysaccharides was 70.24% with method of
Phenol-sulfuric acid.
3.2.2. The Monosaccharide Composition Analysis of FPS
Acid Hydrolysis of the Polysaccharides
The polysaccharide sample (10 mg) was hydrolyzed in boiling water bath for 8 h with 2 M TFA
(2 mL) [23,24]. After restoring at ambient temperature, the sample was centrifuged at 4000 rpm for
15 min. Then, the supernatant was gathered and dried under a reduced pressure.
Derivatization Treatment
The monosaccharides were dissolved in ammonia to prepare a solution with 1 mmol/L
concentration. Then, the 450 µL standard solution was mixed with a same volume of 0.5 M methanol
solution of PMP in a clean tube with stopper. The reaction occurs at 70 ◦C water-bath for 30 min and
then cool the mixture to normal temperature and neutralize with the amount of glacial acetic acid.
Then chloroform (2.0 mL) was mixed. They were shaken vigorously, and the extraction process was
repeated three times. The chloroform layer was abandoned and the supernatant was centrifuged at
8000 rpm for 15 min. After filtering through 0.45
µm nylon membrane, the sample was obtained for
later HPLC-MSn analysis. The hydrolytic polysaccharide was dissolved in 1 mL ammonia and derived
with the same processes used in monosaccharide [21
any monosaccharide was used as blank control.
,23]. In this process, the ammonia solvent without
Chromatography and Mass Spectrometry Conditions
Firstly, the analysis was performed on a Shimadzu HPLC system (Shimadzu LC-6A, Shimadzu
Corporation, Kyoto, Japan). Samples were isolated with a Zorbax Extend-C18 column (250 4.6 mm,
m, Agilent Corporation, Palo Alto, CA, USA) at room temperature. The injection volume was
L with 1.0 mL/min flow rate and peaks were detected at 327 nm. The mobile phase consisted
×
5
µ
20
µ
of 20 mM ammonium acetate (added 1 mL glacial acetic acid per 100 mL)–acetonitrile (80:20, v/v).
Then Agilent 1100 series LC/MSD XCT plus ion-trap mass spectrometer was used for HPLC-MSn
analysis (Agilent, Waldbronn, Germany). Chromatographic conditions: see above. Mass spectrometer
conditions were as follows: capillary voltage 3.5 kV at 350 ◦C; the drying gas flow rate was 11 L/min
and a nebulizer pressure was 35 psi; The drying gas temperature was set at 350 ◦C. The MS spectrum
was recorded in scanning range of m/z 100–1000.
3.3. Biological Effectiveness
3.3.1. Cell Cultivation and Treatment Allocation
RSC96 cells, a Schwann cell line, were purchased from Cells Resource Center of Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and incubated with Dulbecco’s
modified Eagle Medium (DMEM) (Hyclone, Logan, UT, USA) containing 10% (v/v) fetal bovine serum
(Gibco, UK) in the incubator with a 5% CO2 humidified atmosphere at 37 ◦C.
Treatment allocation of FPS was investigated as the following groups: 5.5 mM glucose as
the normal control group (NG, normal glucose), 69.5 mM of mannitol plus 5.5 mM glucose as
osmotic controls (NG + M, normal glucose + mannitol), 75 mM high glucose (HG) as model group,
75 mM glucose plus FPS with different concentrations (1, 10, and 100
Furthermore, the treatment time was 48 h.
µg/mL) as medicated group.
3.3.2. Detection of the Level of Active Oxygen Species (ROS)
When RSC 96 cells were cultured in high glucose condition, they often display over-production
of ROS [33]. For affirming the effects of FPS on the ROS, the environment with 75 mM glucose was

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used to induce oxidative injury in RSC96. Then the contents of intracellular peroxide were detected by
hydrogen peroxides-sensitive fluorescent probe DCFH2DA and superoxide anions were determined
with superoxide anions-sensitive fluorescent probe DHE. Briefly, Cells (1
in 12-well plates under different culture conditions for 48 h, were collected, treated with 10
DCFH2DA or 10
M of DHE under 37 ◦C for 30 min and dark condition and then rinsed by PBS for
×
105 cells/well), cultivated
M of
µ
µ
2 times. Images were taken by a laser scanning confocal microscope (FV1000, excitation 488 nm for
DCFH2DA and 633 nm for DHE). The fluorescent intensity was quantified with Image J software.
3.3.3. Measurement of Cell Apoptosis Rate
The equal numbers of RSC96 cells (1
plates and incubated overnight for cell adherence. Next, cells were handled with different
concentrations of glucose or FPS. After 48h of treatment, the cells attached to the wall and suspended
×
106 cells/well) were inoculated into six-well flat-bottomed
in medium were collected and washed twice with PBS. Afterwards, 1
×
binding buffer was added to
obtain a density of 1 L were incubated by using the Annexin V-PE/7-AAD
×
106 cells/mL and 100
µ
(Apoptosis Detection Kit from BD Biosciences, San Diego, CA, USA) double staining for 15 min under
shade environment. Stained cells were analyzed by flow cytometry (DIVA) (BD FACS Canto II).
3.4. Mechanism
Determination of the Protein Expression Levels Related Oxidative Stress
About 8
cell attachment. After the same treatment with Section 3.3.3, the each group’s cells were trypsinized,
rinsed three times with PBS and collected. Then the cells were added 100 L of ice cold RIPA lysis
×
105 RSC96 cells were seeded in a 25 cm2 cell culture bottle and incubated overnight for
µ
buffer containing a protease inhibitor mixture, lysed in an ice bath for 1 h and centrifuged 10 min at
◦
12,000 g at 4 C. The protein dissolved in supernatant was acquired and concentration was determined
by using the pierce BCA protein assay reagent kit (Thermo Scientific, Rockford, IL, USA).
SDS-PAGE gels with 12% Acrylamide were prepared and used to separate the mixtures of
proteins sample (50 µg). The protein bands were transferred onto PVDF membrane and blocked for
◦
◦
1 h with 5% non-fat dry milk at 3 C and then incubated at 4 C over night in primary antibodies
which were rabbit polyclonal anti-AMPK alpha 1 + AMPK alpha 2 antibody (ab131512, dilutions:
1:800), anti-superoxide dismutase 1 antibody (ab13498, dilutions: 1:1000), anti-NOX1 antibody
(ab131088, dilutions: 1:1000), anti-catalase antibody (ab15834, dilutions: 1:1000), anti-PGC1 alpha
antibody (ab54481, dilutions: 1:800), phospho-AMPKα (Thr172) (#2535, dilutions: 1:600; Cell Signaling)
respectively. Subsequently, the blots were rinsed and incubated for 1.5 h at ambient temperatures with
secondary antibody to Rabbit IgG-H&L (HRP) (ab16284, dilution: 1:2000). The proteins were visualized
using an enhanced chemiluminescence (ECL) kit and quantified using image analysis software. In all
instances, the membranes were blotted again with antibody against β-actin (dilutions: 1:1000).
3.5. Statistical Analysis
The quantified data were analyzed by one factor analysis of variance (ANOVA) using
SPSS19.0 software (IBM Corporation, Armonk, NY, USA.). A P-value less than 0.05 was considered
statistically significant.
4. Discussion
In traditional Chinese medicine, Fuzi, a famous herb, has been widely and successfully used for
many years. In our previous study, the aqueous extract of Fuzi has been shown to ameliorate DPN in
rats and improve the oxidative stress and apoptosis in Schwann cells [18]. However, the material base
of Fuzi aqueous extract is unclear. Thus, in this study, we attempted to clarify the effective component
of Fuzi aqueous extract. It is generally accepted that the diverse bioactivities of Fuzi predominantly
originated from the diterpenoid alkaloids or polysaccharides, especially alkaloids [34]. Thus, firstly,

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we evaluated the antioxidant activity of the five main alkaloids of Fuzi using high glucose-stimulated
Schwann cell line RSC96. However, the results showed that neoline could reduce the level of ROS
without dose-dependent relationship and 14-benzoylaconine, only high concentrations, presented the
antioxidant effect (data not shown). This indicated that these alkaloids may be not the main antioxidant
components. Then, we extracted the polysaccharides of Fuzi (FPS) and assessed its protective potential
against ROS and apoptosis. The findings indicated that FPS diminished the peroxide and superoxide
anion levels in dose-dependent manner to some extent and lowered the apoptotic ratio. Meanwhile, the
MS analysis results demonstrated that FPS was composed of mannose, glucose, rhamnose, galactose
and so on. The previous and present studies indicate altogether that Fuzi aqueous extract may be used
to improve DPN and polysaccharides are probably the effective component of Fuzi aqueous extract.
In addition, we investigated the underlying molecular mechanisms of FPS against the oxidative
stress. SOD and CAT play important roles in protecting the cell from oxidative damage by ROS [26].
SOD catalyzes the dismutation of the superoxide radical into oxygen and hydrogen peroxide.
CAT catalyzes the decomposition of hydrogen peroxide to water and oxygen [26]. In contrast, Nox1 is
a member of Nox family, which transfers electrons from to oxygen to produce superoxide anion [35].
AMPK is a metabolic master switch in regulating cellular energy homeostasis and AMPK activation is
currently considered an important component of cellular responses to stresses that threaten cell viability,
where the common one is oxidative stress [30
survival, such as metformin and dithiolethiones [38
mechanism of diabetic complications, oxidative stress, AMPK signaling pathways has been as a novel
therapeutic approach for antidiabetic treatment [40]. PGC-1 is involved not only mitochondrial
,31
,36
,37]. However, AMPK activators could increase cell
,39]. Moreover, owing to the unified pathological
α
biogenesis, but also ROS metabolism by regulating the transcription of a group of genes involved in
ROS detoxification, such as superoxide dismutase and uncoupling protein [41]. Recently, studies have
shown that AMPK can promote PGC-1α transcription [42]. Thus, in preliminarily exploring the FPS
effects, the AMPK-PGC-1-alpha pathway was more particularly considered. Some other downstream
targets could also be activated and function protectively in SCs, which need further exploration.
5. Conclusions
In summary, combining the phytochemical and pharmacological studies, our study was a
comprehensive evaluation of FPS. FPS, which includes at least nine different monosaccharides and
consists of vast glucose, could ameliorate high glucose-induced oxidative injury and these benefits
might work through AMPK-PGC-1α pathway.
Acknowledgments: We are very grateful for the financial support from the National Natural Science Foundation
of China (No. 30901959).
Author Contributions: B.-B.W. and J.-L.W. performed the experiments and wrote the paper; Y.-G.L. and J.H.
designed and guided the study; J.Y. analyzed the data; Q.-H.Q. and R.-F.J. participated in the discussion;
P.T. revised the paper and answered the reviewers’ comments.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds FPS are available from the authors.
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