Protective Effects of Degraded Soybean Polysaccharides on Renal

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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.

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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

27

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

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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

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the formation and aggregation of COM crystals and induce COD formation in the urine environment

62

23,24

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oxidative damage, to examine the responses of controlled, injured, and DSP-pre-protected Vero cells

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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

69

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

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study

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group was about 11.8% and the solubility was 40 g/L. DSP did not show any characteristic

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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

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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

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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

118

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).

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Afterward, DAPI staining solution was added to the cells and incubated for 4 min. The cells were

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again washed thrice with PBS (for 5 min). Finally, the prepared samples were mounted with anti-

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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

134

mmol/L because the formation of the crystalline particles evidently began from supersaturation,

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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

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prepared for SEM images and ICP analysis after 6 h.

25

. The cells were incubated in a 5% CO2

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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,

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fixed in 2.5% glutaraldehyde at 4 °C for 24 h, and then fixed with 1% OsO4, washed three times with

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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

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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+

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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.

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RESULTS

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Effect of DSP on Vero Cell Viability. The results of the cell viability examination are shown in

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Fig. 1. When the cells were separately treated with 0.3 and 1.0 mmol/L H2O2 for 1 h, cell viability

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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

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H2O2 achieved significantly (p < 0.01) higher cell viability than the injury group, indicating that DSP

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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)

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increased to 93.1%, which was higher than that of the injured group (57.3%). Cells pre-protected

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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

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damage.

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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,

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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

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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

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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.

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Changes in MDA content can reflect the degree of lipid peroxidation in the biomembrane, and

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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)

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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

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H2O2 concentration was 1.0 mmol/L, most of the nuclei were stained red, indicating that a large

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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

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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

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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

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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

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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

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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

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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),

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which were considerably smaller than those induced by un-treated cells (Fig. 6b). Furthermore,

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internalization notably increased. Crystals induced by the 10 μg/mL DSP-protected cells were almost

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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.

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The amounts of crystals which formed to the control, injury, and DSP pre-protection groups of cells

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were measured by ICP (Fig. 7). When the control Vero cells were injured with 0.3 and 1.0 mmol/L

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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

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mmol/L H2O2, the corresponding amounts of formed CaOx crystals were reduced to 25.7 (p > 0.05),

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18.1 (p < 0.01), and 20.5 μg/cm2 (p < 0.05), respectively, compared with that in the injured group

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(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.

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DISCUSSION

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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.

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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

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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

297

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

338

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).

360

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.

368

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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1301.

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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