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Protective effects of degraded soybean polysaccharides on renal epithelial cells exposed to oxidative damage Xin-Yuan Sun, Jian-Ming Ouyang, Poonam Bhadja, Qin Gui, Hua Peng, and Jie Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03323 • Publication Date (Web): 05 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016
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Protective effects of degraded soybean polysaccharides on oxidatively damaged epithelial cells 119x44mm (300 x 300 DPI)
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Protective effects of degraded soybean polysaccharides on renal epithelial cells exposed to oxidative damage
Xin-Yuan Sun,†,‡ Jian-Ming Ouyang,*,†, ‡ Poonam Bhadja,‡ Qin Gui,‡ Hua Peng,‡ Jie Liu*,† † ‡
Department of Chemistry, Jinan University, Guangzhou 510632, China Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632, China
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ABSTRACT: This study aimed to investigate the protective effects of degraded soybean
3
polysaccharides (DSP) on oxidatively damaged African green monkey kidney epithelial (Vero) cells.
4
Low DSP concentration (10 μg/mL) elicited an evident protective effect on H2O2-induced cell injury
5
(0.3 mmol/L). The cell viabilities of the H2O2-treated group and the DSP-protected group were
6
57.3% and 93.1%, respectively. The cell viability decreased to 88.3% when the dosage was increased
7
to 100 μg/mL. DSP protected Vero cells from H2O2-mediated oxidative damage by enhancing
8
cellular superoxide dismutase activity and total antioxidant capacity and by decreasing
9
malonaldehyde content and lactate dehydrogenase release. The H2O2-treated cells stimulated the
10
aggregation of calcium oxalate monohydrate crystals. DSP could also reduce the crystal size,
11
decrease the attached crystal content, and prevent the cell aggregation by alleviating oxidative injury
12
and lipid peroxidation, enhancing antioxidant capacity, and decreasing hyaluronan expression on
13
cellular surfaces. The internalization ability of the injured cells was improved after these cells were
14
exposed to DSP solution. The regulation ability of DSP-repaired cells on calcium oxalate dihydrate
15
formation, crystal attachment, aggregation, and internalization was lower than that of normal cells
16
but was higher than that of the injured cells. DSP may be a potential green drug to prevent CaOx
17
stone formation because DSP could protect cells from oxidative damage and inhibit CaOx crystal
18
formation.
19 20
KEYWORDS:
soybean
polysaccharide,
cell
protection,
oxidative
damage,
CaOx
crystallization, crystal attachment and internalization
21 22 23 24
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INTRODUCTION
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Kidney stones have emerged as a frequently occurring health problem worldwide. Calcium
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oxalate (CaOx) is the predominant inorganic component of most stones. Its main constituent is
28
calcium oxalate monohydrate (COM), followed by calcium oxalate dihydrate (COD). Although
29
kidney stones have been extensively investigated, their formation mechanism has yet to be fully
30
elucidated, and effective clinical preventive methods have yet to be developed 1,2. Renal epithelial cell injury is a major risk factor for crystallization and crystal retention in
31
3,4
32
kidneys and thus cause kidney stone formation
33
anionic molecules on the cell membrane of renal tubular cells after cell injury affect the presence of
34
peripheral crystals because this environment influences crystal adhesion and cell regulation of
35
crystal growth, aggregation, or transformation
36
the mechanism of kidney stone formation. Crystal adhesion is essential for physiological crystal
37
retention and in the early stages of CaOx renal stone formation
38
epithelial cell surfaces are subsequently internalized by cells, where these crystals can stimulate cell
39
proliferation and prevent stone formation 10. Nevertheless, the regulatory effect of renal tubular cell
40
membranes on CaOx crystals should be further investigated.
5-7
. Negatively charged environments provided by
. Crystal-cell interaction is the basis for studies on
8,9
. Crystals attached to renal
41
Normal renal tissues possess effective antioxidant defense systems, such as superoxide
42
dismutase (SOD) and other enzymes, which hinder the generation of free radicals and metabolites
43
and protect cells from oxidative damage. In a pathological state, excessive free radical generation
44
decreases the scavenging activities of antioxidant enzymes, which directly damage renal tissues and
45
trigger kidney stone formation and other diseases
46
polysaccharide molecules that function as antioxidants can significantly alleviate oxidative damage
47
13,14
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plays significant protective roles in renal-tissue injury
11,12
. Cells pre-protected with exogenous
. For example, fucoidan, laminaran, and alginic acid are potent antioxidants, 17
15,16
and Sargassum
. In clinical practice, the protection and
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repair for injured epithelial cells are markedly necessary to prevent stone recurrence. Therefore,
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plant polysaccharides may be used to repair damaged renal tubular epithelial cells and inhibit stone
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formation. Soybean polysaccharide (SPS) is a dietary fiber extracted and refined from okara, a byproduct
52
18
53
of soy manufacturing
. It is a common and inexpensive molecule that exhibits potent antioxidant
54
and biological activities
55
enzymatic degradation. SPS is an acidic polysaccharide containing 18% of galacturonic acid and
56
possessing a pectin-like structure
57
composed of –4)-α-D-GalA-(1–, and rhamnogalacturonan (RG), which is composed of the
58
diglycosyl repeating unit –4)-α-D-GalA-(1→2)-α-L-Rha-(1–. The radius of the gyration of globular
59
SPS is approximately 23.5 ±2.8 nm, as indicated by static and dynamic light scattering 22.
19,20
. The detailed structure of SPS has been determined through stepwise
21
. Its main backbone consists of galacturonan (GN), which is
60
We previously reported that degraded soybean polysaccharides (DSP) can effectively inhibit
61
the formation and aggregation of COM crystals and induce COD formation in the urine environment
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23,24
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oxidative damage, to examine the responses of controlled, injured, and DSP-pre-protected Vero cells
64
to CaOx crystallization, attachment, aggregation, and internalization, and to provide a basis for
65
further studies on new drugs that can prevent and treat urolithiasis.
. This study aimed to investigate the protective effects of DSP on renal epithelial cells exposed to
66 67
MATERIALS AND METHODS
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Materials and Equipments. African green monkey renal epithelial (Vero) cells were purchased
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from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). DMEM culture medium
70
was purchased from HyClone Biochemical Products Co., Ltd. (UT, USA). Fetal bovine serum was
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purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China).
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Penicillin and streptomycin were purchased from Beijing Pubo Biotechnology Co., Ltd. (Beijing,
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China). Propidium Iodide (PI), and anti-quencher were purchased from Haibi sky Biotechnology Co.,
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Ltd. Biotinylated hyaluronan binding protein (bHABP) was purchased from MERCK Inc.
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(Germany). Fluorescein isothiocyanate-avidin (FITC-Avidin) was purchased from Wuhan Boster
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Biological Engineering Co., Ltd. Cell proliferation assay kit (CCK-8) was purchased from Dojindo
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Molecular Technologies Inc. (Japan), Superoxide dismutase (SOD) kit, total antioxidant capacity (T-
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AOC) kit, lactate dehydrogenase (LDH) kit and malondialdehyde (MDA) kit were purchased from
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Nanjing Jiancheng Bioengineering Institute, and cell culture plates were purchased from Japan Iwaki
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Company. Other conventional reagents were analytically pure and purchased from Guangzhou
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Chemical Reagent Factory.
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Soybean polysaccharide was provided by Professor Fu Liang, Jinan University. DSP was
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obtained by the hydrogen peroxide (H2O2) degradation method, which was reported in our previous
84
study
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group was about 11.8% and the solubility was 40 g/L. DSP did not show any characteristic
86
absorption peak at 260–280 nm; therefore, DSP did not contain proteins 23.
24
. The average molecular weight of DSP was found to be 10,200, the content of carboxyl
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The following experimental apparatuses were used: an X-L type environmental scanning
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electron microscope (ESEM, Philips, Eindhoven, Netherlands), an inverted fluorescence microscope
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(IX51, Olympus, Japan), a microplate reader (SafireZ, Tecan, Switzerland), a laser scanning confocal
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microscope (LSM510 META DUO SCAN, ZEISS, Germany), an inductively coupled plasma atomic
91
emission spectrometer (ICP-AES, Optima 2000DV, Perkin Elmer, CT, USA), and X-L type
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environmental scanning electron microscope (SEM, Philips, Eindhoven, Netherlands).
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Cell Culture and Grouping. Cells were incubated in a 5% CO2 humidified atmosphere at
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37 °C, when the cells were trypsinized up to 80%, dispersed into single cell suspensions. The cell
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suspension (100 μL) with a density of 1×105 cells/mL was seeded in the appropriate size plates and
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cultured in DMEM containing 10% fetal bovine serum for 24 h. The medium was then aspirated, and
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the cells were kept in the serum-free medium for 12 h to achieve synchronization. Afterward, the
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cells were divided into three groups: 1) Control group: only serum-free DMEM culture medium was
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added. 2) Injured group: cells were exposed to a serum-free medium containing 0.3 and 1.0 mmol/L
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H2O2 for 1 h. 3) Protection group: serum-free medium containing DSP with the concentration of 1,
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10, 100 μg/mL was added, and the culture medium was aspirated after 12 h. The cells were treated
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with 0.3 and 1.0 mmol/L H2O2 solution for 1 h.
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Measurement of Biochemical Indicators. The cell suspension with a density of 1×105
104
cells/mL was plated per well in 96-well plates and incubated in DMEM containing 10% fetal bovine
105
for 24 h. The cells were divided into three groups, and a cell viability assay was carried out by using
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Cell Counting Kit-8 (CCK-8). Simultaneously, extracellular SOD activity, T-AOC, LDH release, and
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MDA content were determined by using the SOD, T-AOC, LDH, and MDA kits, respectively,
108
according to the instructions provided with kits.
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Propidium Iodide (PI) Assay. PI staining assay was performed on the cells of each
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experimental group. After the treatment time was reached, the supernatant was removed by suction,
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and the cells were washed thrice with PBS. Afterward, 5 μL of PI staining solution was added and
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incubated at 4 °C for 20–30 min. The cells were washed again with PBS thrice, and the dead cells
113
were observed under a fluorescence microscope.
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In PI quantitative analysis, the cells were inoculated in 96-well plates with the concentration of
115
1×105 cells/mL or 100 μL per well, and the PI fluorescence intensity was determined directly in
116
accordance with the method by using a microplate reader.
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Hyaluronan (HA) Detection. The cell suspension (1 mL) with a cell concentration of 1×105
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cells/mL was inoculated per well in 12-well plates. The cells were grouped after synchronization was
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completed. The supernatant was then aspirated. The cells were washed twice with PBS, fixed with
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the fixation fluid composed of 5% glacial acetic acid, 10% formalin, and 70% ethyl alcohol, and
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washed thrice. bHABP solution (100 μL of 5 mg/mL) was then added to the cells and incubated at
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4 °C overnight. The cells were washed thrice with PBS again, and 100 μL of FITC-avidin was added
123
to the cells and incubated for 1 h. The cells were washed with PBS three times (every time for 5 min).
124
Afterward, DAPI staining solution was added to the cells and incubated for 4 min. The cells were
125
again washed thrice with PBS (for 5 min). Finally, the prepared samples were mounted with anti-
126
fade fluorescence mounting medium and observed using a confocal microscope. HA expression and
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cell nuclei were stained green and blue, respectively.
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Quantitative analysis: HA fluorescence intensity was analyzed by Axiovision software (ZEISS, Jena, Germany). HA expressions in 100 cells were quantitatively detected for each group.
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Observation of CaOx Crystallization, Attachment and Internalization. After the
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predetermined time was reached, the culture medium was removed by suction, and the cells were
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washed twice with PBS. The serum-free culture medium with the supersaturated solution of CaOx
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was added to each orifice plate, and the final concentrations of both Ca2+ and C2O42− ions were 0.5
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mmol/L because the formation of the crystalline particles evidently began from supersaturation,
135
which was undoubtedly essential for the stone formation
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humidified atmosphere at 37 °C to induce the growth of CaOx crystals. Subsequently, the cells were
137
prepared for SEM images and ICP analysis after 6 h.
25
. The cells were incubated in a 5% CO2
138
Electron Microscopic Observation. After the cells were incubated with CaOx supersaturated
139
solution for 6 h, the supernatant was removed by suction, the cells were washed thrice with PBS,
140
fixed in 2.5% glutaraldehyde at 4 °C for 24 h, and then fixed with 1% OsO4, washed three times with
141
PBS again, dehydrated in gradient ethanol (30%, 50%, 70%, 90% and 100%, respectively), dried
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under the critical point of CO2, and treated with gold sputtering. Finally, the Vero cells response to
143
crystals were observed by SEM.
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ICP Quantitative Determination. Cell suspension (1 mL) with cell concentration of 1×105
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cells/mL was inoculated per well in 12-well plates and incubated for 12 h. After incubating with
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CaOx supersaturated solution for 6 h, the supernatant was aspirated and the cells were washed three
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times with PBS to remove unbound crystals. The samples were then transferred to 25 mL beaker and
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mixed with 4.0 mL concentrated HNO3 and 1.0 mL HClO4 solution for digestion. A separate HClO4
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solution was heated until smoke appeared; the heat was then used to dry the solution. After cooling,
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3 mL of 2% dilute HNO3 was added. An ICP-AES was used to measure the concentration of Ca2+
151
ions, which was then converted to determine the amount of formed crystals. The control group was
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treated using the same method to determine the interference of intracellular Ca2+ in Vero cells.
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Statistical Method. The experimental results were analyzed statistically in SPSS 13.0 (SPSS
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Inc., Chicago, IL, USA) and expressed as mean ± SD from three independent experiments. The
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differences of means between the experimental groups and the injured group were analyzed by
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Tukey. If p < 0.05, there was significant difference; if p < 0.01, the difference was extremely
157
significant; if p > 0.05, there was no significant difference.
158 159
RESULTS
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Effect of DSP on Vero Cell Viability. The results of the cell viability examination are shown in
161
Fig. 1. When the cells were separately treated with 0.3 and 1.0 mmol/L H2O2 for 1 h, cell viability
162
rapidly decreased; the 1 mmol/L H2O2, in particular, seriously damaged the cells. However, Vero
163
cells pre-protected with different concentrations of DSP for 8 h and later treated with 0.3 mmol/L
164
H2O2 achieved significantly (p < 0.01) higher cell viability than the injury group, indicating that DSP
165
can improve the ability of Vero cells to resist oxidative damage induced by 0.3 mmol/L H 2O2. 10
166
μg/mL DSP provided the highest resistance (Fig. 1), the cell viability significantly (p < 0.01)
167
increased to 93.1%, which was higher than that of the injured group (57.3%). Cells pre-protected
168
with different concentrations of DSP and further damaged by 1.0 mmol/L H2O2 showed less cell
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viability because the damage caused by 1 mmol/L H2O2 was too severe for the cells to resist H2O2
170
damage.
171 172
Effect of DSP on Cell Antioxidant Capacity. Superoxide dismutase (SOD) activity and total
173
antioxidant capacity (T-AOC) can reflect the function of the antioxidant system. Fig. 2 shows that,
174
after Vero cells were injured with 0.3 and 1.0 mmol/L H2O2, the SOD activity and T-AOC decreased,
175
suggesting that antioxidant capacity of the cells decreased. The extracellular SOD and T-AOC
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activity of the protection group (injured by 0.3 mmol/L H2O2) were significantly (p < 0.05) higher
177
than that of the injury group, indicating that DSP can aid cells in resisting oxidative damage. The
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SOD and T-AOC activity of 1.0 mmol/L H2O2-treated DSP pre-protected cells were slightly higher
179
than that of the injury group. DSP with the concentration of 10 μg/mL showed superlative
180
antioxidant capacity on injured Vero cells.
181 182
Effect of DSP on Malonaldehyde (MDA) and Lactate Dehydrogenase (LDH) Release.
183
Changes in MDA content can reflect the degree of lipid peroxidation in the biomembrane, and
184
LDH% is considered a marker of cell membrane integrity. Fig. 3 shows the MDA content (Fig. 3a)
185
and LDH% (Fig. 3b) in controlled, H2O2-injured, and DSP-protected group cells. The released MDA
186
and LDH% of injured Vero cells increased with increasing H2O2 concentration, indicating that
187
different concentrations of H2O2 produce different degrees of cell damage. However, the amount of
188
MDA and LDH% released by the protection group were lower than that of the injury group,
189
indicating the oxidative damage level of cells decreased due to DSP protection. In particular, 10
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μg/mL DSP showed superlative protection ability, the amount of MDA was significantly (p < 0.01)
191
reduced to 1.98 nmol/mL compared with that of the injured group (3.57 nmol/mL), and LDH%
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significantly (p < 0.05) decreased to 23.73% compared with that of the injured group (32.21%).
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DSP Reduces H2O2-mediated Cell Death Rate. Propidium iodide (PI) is a fluorescent dye
195
commonly used to stain the nucleus. The number of red-stained nuclei was positively related to cell
196
mortality. Fig. 4A shows one or two nuclei stained red in control cells, indicating normal cells. PI
197
stained nuclei of cells treated with H2O2 gradually increased as H2O2 concentration increased. When
198
H2O2 concentration was 1.0 mmol/L, most of the nuclei were stained red, indicating that a large
199
number of cells were in late apoptosis or necrosis. When DSP-protected cells were treated with 0.3
200
mmol/L H2O2, cell death rate decreased, with lowest deaths achieved at 10 μg/mL DSP (p < 0.01;
201
Fig. 4B). However, the red nuclei in the DSP protection group only decreased slightly compared
202
with the injury group when c(H2O2) = 1.0 mmol/L.
203 204
DSP Decreases Hyaluronan (HA) Expression on Cell Surface. HA was identified as a major
205
crystal adhesion molecule following cell injury 26. Fig. 5A shows the HA expression of the control,
206
injured, and DSP pre-protection groups of Vero cells. The green fluorescence of the control group
207
cells was weak, indicating less expression of HA. After the cells were damaged with 0.3, 1.0 mmol/L
208
H2O2, intensity of the green fluorescence on cell surfaces gradually increased. The fluorescence
209
intensity of the protecting group was lower than that of the injury groups, but higher than that of the
210
control cells. Accordingly, the fluorescence intensity in the 10 μg/mL DSP protection group was
211
significantly (p < 0.01) lower than that in the injured group (Fig. 5B), and the former expressed the
212
least HA among DSP-treated groups.
213 214
SEM Observation of Differences of CaOx Crystallization Induced by Various Group Cells.
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Fig. 6 shows SEM images of CaOx crystallization induced by control, injury, and DSP pre-protective
216
groups of Vero cells. The crystals induced by the control group cells were mostly tetragonal
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bipyramid calcium oxalate dihydrate (COD; Fig. 6a). The 0.3 mmol/L H2O2-injured cells (Fig. 6b)
218
induced crystals that were considerably larger than those of the control group. The edges and corners
219
of the crystals were sharp, and the internalization of the crystals notably decreased. Furthermore, 1.0
220
mmol/L H2O2-injured cells increased the crystals, which were mostly calcium oxalate monohydrate
221
(COM) crystals on cell surfaces. Crystal aggregation also markedly increased, and no crystal
222
internalization was observed (Fig. 6f).
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DSP pre-protected cells treated with 0.3 mmol/L H2O2 induced rounder crystals (Figs. 6c–e),
224
which were considerably smaller than those induced by un-treated cells (Fig. 6b). Furthermore,
225
internalization notably increased. Crystals induced by the 10 μg/mL DSP-protected cells were almost
226
COD (Fig. 6d), closing to the control group (Fig. 6a), and cells exhibited strong internalization
227
capacity. DSP-protected cells treated with 1.0 mmol/L H2O2 lost their internalization ability (Figs.
228
6g–i) and stimulated the formation of COM crystals with a different degree of aggregation.
229 230
ICP Quantitative Determination of the Formed Crystals Induced by Various Groups Cells.
231
The amounts of crystals which formed to the control, injury, and DSP pre-protection groups of cells
232
were measured by ICP (Fig. 7). When the control Vero cells were injured with 0.3 and 1.0 mmol/L
233
H2O2, the formed CaOx crystals on cell surface increased from 15.0 μg/cm2 (control group) to 29.1
234
μg/cm2 and 82.3 μg/cm2, respectively.
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When the cells were pre-protected with 1, 10, and 100 μg/mL DSP and then injured by 0.3
236
mmol/L H2O2, the corresponding amounts of formed CaOx crystals were reduced to 25.7 (p > 0.05),
237
18.1 (p < 0.01), and 20.5 μg/cm2 (p < 0.05), respectively, compared with that in the injured group
238
(29.1 μg/cm2). The amounts of formed CaOx crystals on DSP-protected cells (treated with 1.0
239
mmol/L H2O2) were 44.1 μg/cm2 for 1.0 μg/mL DSP (p < 0.01), 32.1 μg/cm2 for 10 μg/mL DSP (p
0.05), which were all lower than the amounts for the
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injury group (82.3 μg/cm2). The 10 μg/mL DSP-protected cells induced the lowest crystal formation.
242 243
DISCUSSION
244
H2O2 Oxidative Damage Pathway. Renal epithelial cell injury associated with oxidative stress
245
is one of the key factors in stone formation. H2O2 is a reactive oxygen species (ROS), which may
246
damage cells through direct oxidation of lipids, proteins, or DNA, or act as a signaling molecule to
247
trigger intracellular pathways leading to cell death
248
generating free radicals. Excessive ROS formation leads to lipid peroxidation, destruction of the
249
structure and functions of the cell membrane, decrease of the antioxidant capacity of cells, damage to
250
intracellular organelles, and finally, cell death
251
lipid peroxidation by increasing the amount of MDA in Vero cells (Fig. 3a). Increased extracellular
252
LDH level induced by H2O2 reflected damage in cell membrane integrity (Fig. 3b). Excessive ROS
253
generation resulted in depletion of SOD enzyme activity and T-AOC of cells (Figs. 2a and b),
254
indicating the H2O2 had reduced the antioxidant capacity of cells. This study also demonstrated that
255
H2O2-treated Vero cell surfaces expressed a large degree of HA (Fig. 5), which indirectly reflected
256
cell damage.
28
27
. H2O2 produced oxidative damage in cells by
. We found that exogenously adding H2O2 induced
257 258
Protective Effect of DSP on Oxidative Damaged Vero Cells. H2O2 oxidation-damaged
259
pathways showed that H2O2 deteriorated the functions of cells, while DSP could protect and repair
260
cells by blocking or inhibiting injury pathways. The results showed that DSP effectively protected
261
cells against lower oxidative stress (0.3 mmol/L H2O2 treatment). However, higher oxidative stress,
262
such as 1.0 mmol/L H2O2 treatment, could damage even though DSP was applied to pre-protect the
263
cells. The cell viability (Fig. 1), extracellular SOD activity (Fig. 2a), and T-AOC (Fig. 2b) were
264
significantly increased (p < 0.01) when the normal cells were pre-protected with 10 μg/mL DSP and
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treated with 0.3 mmol/L H2O2, and the MDA content (Fig. 3a, p < 0.01) and LDH leakage (Fig. 3b, p
266
< 0.05) were significantly reduced compared with the injured cells. The results of PI staining after
267
this method yielded the fewest dead cells (p < 0.01; Fig. 4) and the lowest HA expression (p < 0.01;
268
Fig. 5) on the cell surface compared with the injured cells. These results are in accordance with
269
previous observation, in which Li et al.29 confirmed that the pretreatment of Cordyceps sinensis
270
polysaccharide on cultured rat pheochromocytoma PC12 cells provides a potent protective effect
271
against H2O2-induced insult.
272
The protective effect of overdosage DSP (100 μg/mL) to Vero cells was reduced compared with
273
that of 10 μg/mL. With very high DSP concentration, some DSP molecules likely cover the cell
274
surface, and this condition likely affects cellular normal respiration, increases extracellular osmotic
275
pressure
276
(100 μg/mL) was lower than that of 10 μg/mL. Similar results were reported in many studies
277
Fang et al.
278
concentration because of high osmotic pressure. Zha et al.
279
polysaccharide stimulated the proliferation of human lens epithelial (HLE) cells when the DHPD1
280
concentration ranged from 10 μg/mL to 200 μg/mL, but the cell viability was decreased rapidly
281
when the dosage was increased to 200–1600 μg/mL. Xue et al. 33 examined the effects of Astragalus
282
polysaccharide (APS) at various concentrations on porcine kidney (PK-15) cell proliferation. The
283
viability of PK-15 cells was not significantly affected by APS up to a concentration of 40 μg/mL, but
284
APS significantly reduced the cell viability of PK-15 cells at concentrations of 60 and 80 μg/mL.
285
These results revealed that excessive polysaccharide exposure is not beneficial as an antioxidant.
30,31
, and decreases cellular activity. Thus, the protective effect of excessive DSP dosage
31
31-33
.
revealed that the cell growth was inhibited by a relatively higher initial sugar 32
found that Dendrobium huoshanense
286 287 288
Comparison of Regulation Ability of Control, Injured, and DSP Pre-protected Cells on CaOx Crystallization.
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Crystal Phase Difference. COM exhibits a stronger affinity to renal tubule cell membranes than 34
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COD does
291
amount of COM calculi detected in supersaturated urine is twice higher than that of COD 35. Hence,
292
stabilization of COD crystals is more beneficial than stabilization of COM crystals for lowering the
293
possibility of stone formation. It has been found that the intact cells transform COM into COD
294
crystals
295
normal renal tubular cell could transform crystal habit from COM to COD. In the current experiment,
296
normal Vero cells (Fig. 6a) and 10 μg/mL DSP pre-protected cells (Fig. 6d) promoted the formation
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of COD, rather than COM crystals, which was in agreement with the findings of Zhang et al. 38, who
298
found that Sargassum polysaccharides (SGP) inhibited the growth of COM and induced the
299
formation of COD crystals.
36
. As such, the former is more difficult to be excreted with urine than COD, and the
. This result also confirmed by Lieske et al.
37
, who determined that the apical surface of
300
Crystals induced by pre-protected cells after treatment with 1.0 mmol/L H2O2 did not
301
significantly differ from those of the injury group, and mostly COM aggregates were found, likely
302
due to the stronger damage capacity of 1.0 mmol/L H2O2. Cells were damaged despite pre-protection
303
with DSP. Exposure to 1.0 mmol/L H2O2 caused severe damage to the structure and functions of Vero
304
cell surfaces, possibly due to the conversion of COM crystals to COD being abolished, leaving
305
aggregated COM crystals (Figs. 6f, g and i).
306 307
Crystal Size Difference. Small crystals were stimulated by control Vero cells (Fig. 6a), as the
308
structure and functions of normal cells were intact, and the charge density on normal cell surfaces
309
was lower. Therefore, normal cells can undergo anti-crystal growth and anti-crystal adhesion.
310
H2O2-treated cells expressed HA molecules on the apical cell surface, increasing the negative
311
charge density, which increased the adsorption sites to Ca2+ ions and the attachment to positively-
312
charged COM crystals. These attached COM crystals promoted further injury in Vero cells, leading
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to further CaOx crystal nucleation and growth
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group cells were larger (Figs. 6b and f) than those induced by control group cells (Fig. 6a).
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, hence, the sizes of crystals induced by injured
315
Cells pre-protected by DSP had considerably improved resistance to oxidative damage, and
316
reduced HA molecule expression; hence, crystal sizes were lower than those of injured group cells.
317
Crystals induced by 10 μg/mL DSP protection cells (Fig. 6d) were of similar size to those of the
318
control cells (Fig. 6a), which was in accordance with previous in vivo studies where plant extracts
319
inhibited kidney stone formation by reducing the size and growth of the crystals 40,41. DSP contained
320
negatively charged groups, which can break free-radical chains by donating electrons or hydrogen
321
atoms. Therefore, the cells are protected against injury. These negatively charged groups eventually
322
inhibited crystal growth. Farmanesh et al.42 reported that chondroitin sulfate, a polysaccharide with
323
several functional moieties including anionic groups (i.e. carboxylic acid and sulfate) and hydrogen-
324
bonding groups, is an effective inhibitor of CaOx crystal growth. However, DSP-protected cells
325
could not resist injury caused by 1.0 mmol/L H2O2, in which case, induced CaOx crystals were of
326
similar size to those in the injury group.
327 328
Crystal Adhesion Difference. SEM observation (Fig. 6) and ICP quantitative determination (Fig.
329
7) revealed that crystal adhesion increased as degree of injury increased. When cells were injured by
330
H2O2, a large number of negatively charged HA molecules were expressed on the cell surface (Fig.
331
5), facilitating more crystal attachment. Although the cells were pre-treated by DSP, their antioxidant
332
capacity increased, and the HA expressed on the cell surface decreased to lower than those of the
333
injured group, which decreased crystal binding to the cell surface. 1,2,3,4,6-penta-O-galloyl-beta-D-
334
glucose (PGG) can also inhibit crystal binding on cell surfaces because this molecule could coat
335
crystals that arise from tubular fluid and prevent their retention in the kidney. PGG can also decrease
336
the cell surface expression of the crystal binding molecule hyaluronan 43. Moreover, medicinal plant
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extracts, including Heruniaria hirusta and Quercus salicina, likely play similar roles in inhibiting
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the attachment of crystals to renal tubular cells by coating the crystal surface 44,45.
339 340
Crystal Aggregation Difference. Normal cells inhibited crystal aggregation due to their low
341
surface charge density, as well as their anti-crystal growth and anti-crystal adhesion. Cells of the pre-
342
protection group (injured by 0.3 mmol/L H2O2) exhibited less crystal aggregation (Figs. 6c-e).
343
Previous study confirmed that acidic polymers can be applied as effective therapies for kidney stones
344
by preventing CaOx crystal aggregation
345
mmol/L H2O2 were mainly stimulated crystals in aggregation state, which was attributed to the
346
expression of more negatively-charged molecules on the cell surface, due to cell injury. Following
347
treatment with 1.0 mmol/L H2O2, the cell membrane was damaged by free radicals, gradually
348
forming a large amount of cell debris. The two factors promoted heterogeneous nucleation and
349
provided favorable conditions for secondary nucleation, leading to formation of more and larger
350
crystals, as well as promoting crystal aggregation. The crystals adhering to the apical cell surface
351
damaged the cell membrane again, which aggravated crystal growth and crystal aggregation.
46
. However, DSP pre-protected cells treated with 1.0
352 353
Comparison of Internalization Ability of Cells: Protected Group Cells Enhanced the
354
Ability of Vero to Internalize Crystals. The control Vero cells had intact structure and functions,
355
showing strong internalization ability (Fig. 6a). However, the internalization ability of injured group
356
cells declined (Fig. 6b). The resistance of DSP pre-protected cells against 0.3 mmol/L H2O2-
357
mediated oxidative damage was improved and cell damage was reduced. The ability of these cells to
358
internalize crystals was also considerably increased, particularly at DSP concentrations of 10 μg/mL
359
(Fig. 6d).
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Fig. 8 shows the crystal internalization by Vero cells. Tetragonal bipyramid COD crystals
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formed on the cell surface when Vero cells were incubated with CaOx supersaturated solution (Fig.
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8a). Fig. 8b shows the cell membrane was partially deformed, with an unclear boundary between a
363
cell and a crystal, and this finding indicated that the cells internalized the COD crystal. Over time,
364
the remaining crystal further interacted with cells and slightly absorbed into the cells (Fig. 8c). The
365
cell membrane was further depressed or formed pseudopodia to enclose the entire crystal and ingest
366
it within the cell (Fig. 8d). Subsequently, the internalized crystals appeared to dissolve in lysosomes
367
under the continuous action of acid hydrolases (Fig. 8e) 47.
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The increased internalization of crystals can decrease the rates of crystal aggregation 48 possibly
369
because of the elimination of potential crystal growth sites on the cell membrane after the crystal
370
underwent internalization, which is one of the reasons for the weak crystal aggregation of DSP-
371
protected cells (0.3 mmol/L H2O2-damage). However, DSP protection was insufficient to allow the
372
cells to internalize the crystals when 1.0 mmol/L H2O2-treated cells were exposed to CaOx
373
supersaturated solution because of the severe damage. The crystals adhering to the cell surface
374
damage cells, and this phenomenon was a key factor in the formation of kidney stones. Cells
375
internalized the adhering crystals, which could reduce damage to renal epithelial cells. In addition,
376
the occurrence of crystal internalization could reduce the supersaturation of urine. Following
377
internalization, crystals dissolved within intracellular lysosomes over 5–7 weeks
378
formation of kidney stones.
48
, slowing the
379
Although our results showed that soybean polysaccharide could prevent CaOx crystal
380
deposition and protect renal epithelial cells from oxidative damage in vitro, the in vivo environment
381
is much more complicated than the cell lines. The development of experimental urolithiasis requires
382
more complex physiopathological mechanisms than the simple contact of calcium oxalate crystals
383
with renal epithelial cells. Animal experiments are underway in our laboratory to further investigate
384
the possible significance of DSP in the treatment of CaOx renal stone disease.
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In conclusion, DSP can effectively protect Vero cells from H2O2-mediated oxidative damage by
386
maintaining stable cell viability, decreasing lipid peroxidation, LDH levels and HA expression, and
387
enhancing cellular SOD activity and total antioxidant capacity. In addition, DSP can reduce crystal
388
size and aggregation, weaken cell–crystal attachment, and improve the internalization of injured
389
Vero cells. DSP can protect cells from oxidative damage and inhibit CaOx crystal formation.
390
Therefore, DSP may be a potential green drug that can be used to prevent CaOx stone formation.
391 392
AUTHOR INFORMATION
393
Corresponding Author
394
(J.-M. OY.) *E-mail address:
[email protected]. Tel.: +86-20-85223353.
395
(J. L.) *E-mail address:
[email protected]. Tel: +86-20-85220223.
396
Funding
397
This work was supported by the National Natural Science Foundation of China (No. 81670644).
398
Notes
399
The authors declare no competing financial interest.
400
ACKNOWLEDGMENTS
401
We thank Jinan university analytical and testing center for technical assistance.
402 403
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Captions
555 556
Fig. 1. Effect of DSP on H2O2-induced Vero cell viability. Cells were treated with different
557
concentration of DSP (1, 10, 100 μg/mL) for 12 h, afterwards cells were treated with 0.3, 1.0
558
mmol/L H2O2 solution respectively for 1 h. Data were expressed as mean±SD from three
559
independent experiments. Compared with injured group, * indicates p < 0.05. # indicates p < 0.01.
560
Fig. 2. SOD activity (a) and T-AOC (b) assay of Vero cells in the control group, H2O2-treated
561
groups, and DSP-pretreated groups (1, 10, and 100 μg/mL). Compared with injured group, *
562
indicates p < 0.05. # indicates p < 0.01.
563
Fig. 3. MDA content (a) and released LDH% (b) of Vero cells in the control group, H2O2-
564
treated groups, and DSP-pretreated groups (1, 10, 100 μg/mL) prior to 0.3, 1.0 mmol/L H2O2
565
incubation. Compared with injured group, * indicates p < 0.05. # indicates p < 0.01.
566
Fig. 4. Protective effect of DSP on 0.3, 1.0 mmol/L H2O2-treated cell death rate, detected using
567
propidium iodide staining assay. (A) images by fluorescence microscope; (B) quantitative histogram
568
of relative fluorescence intensity by microplate reader. Compared with injured group, * indicates p