Antioxidative and Antiinflammatory Activities of Asiatic Acid

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Antioxidative and antiinflammatory activities of asiatic acid, glycyrrhizic acid and oleanolic acid in human bronchial epithelial cells Shih-Ming Tsao, and Mei-Chin Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00102 • Publication Date (Web): 17 Mar 2015 Downloaded from http://pubs.acs.org on March 20, 2015

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Journal of Agricultural and Food Chemistry

Antioxidative and antiinflammatory activities of asiatic acid, glycyrrhizic acid and oleanolic acid in human bronchial epithelial cells

Shih-ming Tsaoø,§, Mei-chin Yin†,⊥,*

ø

Institute of Medicine, Chung Shan Medical University, Taichung City, Taiwan

§

Sections of Infectious Diseases and Chest Medicine, Department of Internal Medicine,

Chung Shan Medical University Hospital, Taichung City, Taiwan †

Department of Health and Nutrition Biotechnology, Asia University, Taichung City,

Taiwan ⊥

Department of Nutrition, China Medical University, Taichung City, Taiwan

Running title: protective activities of triterpenic acids *To whom correspondence should be addressed: Dr. Mei-chin Yin, Professor, Department of Nutrition, China Medical University, 91, Hsueh-shih Rd., Taichung City, Taiwan TEL: 886-4-22053366 ext. 7510, FAX: 886-4-22062891 Email: [email protected]

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ABSTRACT

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Protective effects of triterpenic acids, asiatic acid (AA), glycyrrhizic acid (GA) or

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oleanolic acid (OA), for two human bronchial epithelial cells, 16HBE and BEAS-2B cells,

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against hydrogen peroxide (H2O2) induced injury were examined.

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by triterpenic acid at 4 or 8 µmol/L, and followed by H2O2 treatment. Results showed

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that H2O2 significantly upregulated both Bax and cleaved caspase-3 expression; also

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downregulated Bcl-2 expression in test cells.

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but GA and OA only at 8 µmol/L reserved Bcl-2 expression.

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lowered cleaved caspase-3 expression dose-dependently.

Cells were pre-treated

AA at these doses retained Bcl-2 expression; Test triterpenic acids

H2O2 treatment lowered

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Na+-K+-ATPase activity and mitochondrial membrane potential in cells. Triterpenic acids

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pretreatments

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Na+-K+-ATPase activity.

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necrosis factor-α and prostaglandin E2 levels in test cells.

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treatments dose-dependently reversed these changes.

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expression of p47phox, gp91phox, cyclooxygenase-2 (COX-2), mitogen-activated protein

16

kinase and nuclear factor-κB (NF-κB).

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downregulated the expression of p47phox, COX-2, NF-κB p65 and p-p38; but only at 8

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µmol/L decreased gp91phox expression.

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could protect bronchial epithelial cells to attenuate apoptotic, oxidative and inflammatory

20

stress.

significantly

maintained

mitochondrial

membrane

potential

H2O2 enhanced reactive oxygen species, interleukin-6, tumor Three triterpenic acids

H2O2 promoted the protein

AA, GA or OA pretreatments dose-dependently

These results support that these triterpenic acids

21 22

and

KEYWORDS: triterpenic acids; bronchial epithelial cells; ROS; NF-κB; MAPK

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INTRODUCTION Environmental contamination including air pollution impairs the human respiratory

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system.

Bronchial epithelium acts as a barrier to prevent the invasion of foreign materials.

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Apoptosis of bronchial epithelial cells could be induced by toxins such as cigarette,

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particulate matter or heavy metals.1,2

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pathway, mainly mediated by BCL family, is involved in bronchial epithelial cell death.3

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This BCL family includes proapoptotic factors like Bax; and antiapoptotic factors like Bcl-2.

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Thus, agent(s) with antiapoptotic activity may potentially protect bronchial epithelium, and

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prevent the development of respiratory disorders.

It is reported that mitochondrial intrinsic apoptotic

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Oxidative injury resulted from the massive generation of reactive oxygen species

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(ROS) is a crucial pathophysiological factor for the progression of chronic airway

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disorders including asthma and lung cancer.4,5

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enzyme complex with several subunits such as p47phox and gp91phox, is the major ROS

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source in airway epithelial cells and responsible for oxidative cell death.6,7

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enhanced cyclooxygenase-2 (COX-2) protein expression and/or prostaglandin E2 (PGE2)

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formation in respiratory epithelium promote airway inflammatory stress.8,9

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documented that ROS could activate signaling pathways including mitogen-activated

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protein kinase (MAPK) and/or nuclear factor-κB (NF-κB) in airway epithelium, which in

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turn elicits the production of oxidative and/or inflammatory associated molecules, and

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facilitates the progression of respiratory diseases.10,11

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to reduce ROS and PGE2 generation; and/or suppress NADPH oxidase, COX-2, NF-κB

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and MAPK expression may potentially protect airway epithelial cells to attenuate oxidative

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and inflammatory injury.

The activation of NADPH oxidase, an

In addition,

It has been

Thus, any agent with the capability

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Asiatic acid (AA), glycyrrhizic acid (GA), and oleanolic acid (OA) are pentacyclic

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triterpenic acids occurring in some edible plants including hawthorn fruit (Crataegi

3



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Pinnatifidae Fructus), licorice (Glycyrrhiza glabra), brown mustard (Brassica juncea) and

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basil (Ocimum basilicum).12-14

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increased their bioavailability in tissues such as liver or heart.

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and OA possess antioxidative and antiinflammatory activities.15,16

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reported that some pentacyclic triterpenoids such as OA could protect respiratory system

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through antitubercular activity.

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at 3.2, 6.3, 12.5 and 25 µmol/L, protected human normal bronchial epithelial cells to

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alleviate cigarette smoke extract caused injury, and cell survival rates were in the range of

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73-86%.

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and GA might benefit survival and function of human bronchial epithelial cells.

Yin et al.12 indicated that dietary intake of AA and OA It is reported that AA, GA Wächter et al.17

Liu et al.18 indicated that ursolic acid, an isomer of OA,

Those previous studies suggest that OA and other triterpenic acids such as AA

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Both 16HBE and BEAS-2B cells are normal human bronchial epithelial cell lines, and

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have been widely used in bronchial diseases related researches.3,18 Hydrogen peroxide,

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an oxidant in cigarette smoke extract, could cause apoptotic and oxidative injury in those

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bronchial epithelial cells.19,20

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study to investigate the protective activities of AA, GA and OA at two doses, 4 and 8

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µmol/L, against hydrogen peroxide induced apoptotic, oxidative and inflammatory stress.

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The impact of these triterpenic acids upon the activity and protein expression of associated

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factors including COX-2, NADPH oxidase, MAPK and NF-κB was evaluated.

Thus, 16HBE and BEAS-2B cells were used in this present

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MATERIALS AND METHODS Materials. AA, GA, OA (95%) and Rhodamine123 (Rh123) were bought

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commercially (Sigma-Aldrich Chemical Co., Milwaukee, WI, USA).

16HBE and

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BEAS-2B cells were purchased from American Type Culture Collection (Rockville, MD,

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USA).

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from Difco Laboratory (Detroit, MI, USA).

Medium, antibiotics, chemicals and plates used for cell culture were obtained

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Experimental Design and Cell Culture. Our preliminary data indicated that 12, 24, 36

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and 48 hr incubation resulted in approximately 21.7, 48.6, 72.9 and 96.3% incorporation of

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test compound into test cells.

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addition, the improvement upon cell viability of test compounds at 0.5, 1, 2, 4 and 8

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µmol/L against H2O2 was also examined in preliminary test, and results showed that these

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compounds at 0.5, 1 or 2 µmol/L did not significantly improve cell viability.

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compounds at 4 and 8 µmol/L were further used.

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to prepare stock solution for three triterpenic acids; and stock solution was further diluted

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by bronchial epithelial growth medium (BEGM).

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culture was lower than 0.5%, which did not impact any measurements.

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BEAS-2B cells were routinely cultured in BEGM containing 2.5 mM L-glutamine, 10%

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fetal calf serum, 100 units/mL streptomycin and 100 units/mL penicillin (pH 7.4) under

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5% CO2 and 95% air at 37°C.

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sub-cultured every 7 days.

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saline (PBS) with pH at 7.2 for experiments and measurements.

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were pre-treated by test compound at 4 or 8 µmol/L at 37°C for 48 hr, and followed by

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washing twice with PBS.

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using a HPLC method described in Yin et al.12

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considered as incorporated one.

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97.1±1.3% and 95.8±0.9% incorporation into 16HBE and BEAS-2B cells, respectively.

95

Cells were then treated with 100 µM H2O2 for 4 hr at 37°C.

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compound and without H2O2 treatment.

97 98

Cell

Viability

Thus, 48 hr incubation was used for present study.

In

Thus, these

Dimethyl sulfoxide (DMSO) was used

Final concentration of DMSO in cell 16HBE and

Culture medium was renewed every 72 hr, and cells were

Cell number (105/mL) was adjusted by a phosphate buffer After aspiration, cells

The concentration of test compound in PBS was measured

Assay.

The compound left in cells was

Results showed that 48 hr incubation resulted in

Cell

viabiliaty

Control groups had no test

was

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).21

5


examined

by

Cells (105/mL)


99

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were treated by test compound for 48 hr, then H2O2 treatment for 4 hr. MTT at 0.25 mg

100

MTT/mL was added into medium, and followed by incubating at 37°C for 3 hr.

MTT

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formazan product was quantified by a Bio-Rad microplate reader (Hercules, CA, USA) to

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measure the absorbance at 570 nm, with a reference wavelength at 630 nm.

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was shown as a percent of control groups.

Cell viability

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Determination of DNA Fragmentation. DNA fragmentation was determined by

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using a cell death detection kit purchased from Roche Molecular Biochemicals (Mannheim,

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Germany).

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KCl (10 mM), MgCl2 (1.5 mM), EDTA (0.1 mM), PMSF (1 mM), DTT (1 mM) and

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Nonidet P-40 (0.6%) for 30 min, and followed by centrifugation for 10 min at 200 xg.

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Supernatant at 20 µL was added on plate pre-coated with streptavidin, and followed by

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adding 80 µL immunoreagent and incubating at 25°C for 2 hr.

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twice.

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The absorbance at 405 nm, with 490 nm as a reference wavelength, was measured by a

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microplate reader.

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equation: absorbance of sample (cells with test compound or/and H2O2) ÷ absorbance of

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control (cells without H2O2 and test compound).

Cells were mixed with 5 mL lysis buffer containing HEPES (10 mM, pH 7.9),

PBS was used to wash

After adding substrate solution, the mixture was incubated for a further 15 min.

DNA fragmentation was determined as enrichment factor by using an

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Mitochondrial Membrane Potential (MMP) Assay. MMP was assayed by a

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Beckman-FC500 flow cytometry (Beckman Coulter, Fullerton, CA, USA) and Rh123, a

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fluorescent dye.22

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BEGM medium, and further mixed with Rh123 at 100 µg/L.

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45 min, collected cells were washed twice by PBS.

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was then analyzed by a flow cytometry.

After centrifugation for 5 min at 1200 xg, cells were re-suspended in After incubating at 37°C for

Mean fluorescence intensity (MFI)

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Preparation of Mitochondrial Fractions. Cells were lysed first, and followed by

123

centrifugation at 4°C and 200 xg for 10 min. The supernatant was collected for further 6


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centrifugation at 4°C and 10,000 xg for 20 min to get mitochondrial pellet.

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to re-suspend this mitochondrial pellet, and protein concentration of this fraction was

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measured by a kit purchased from Pierce Biotechnology Inc. (Rockford, IL, USA).

127

serum albumin was used as a standard.

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PBS was used

Bovine

Assay of Na+-K+-ATPase Activity. Na+-K+-ATPase activity was assayed by

129

monitoring the released inorganic phosphate (Pi) from ATP.23

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fraction was mixed with 30 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl and 20

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mM KCl.

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37°C, assay was stopped by adding 15% trichloroacetic acid.

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was measured to determine Pi level.

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of control groups.

ATP at 2 mM was added to initiate this assay.

Cell mitochondrial

After 15 min incubation at The absorbance at 640 nm

The value of sample was shown as a percent of that

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Measurement of Caspase Activity. Fluorometric assay kits (Upstate, Lake Placid,

136

NY, USA) were obtained to meaure caspase-3 or -8 activity according to the instruction of

137

manufacturer.

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Cells were first lysed in 25 mL lysis buffer, and followed by incubating on ice for 10 min.

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Cell lysates at 50 µL was then added into 25 mL reaction buffer, and further mixed with 5

140

mL fluorogenic substrates which were specific for either caspase-3 or -8.

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incubating at 37°C for 60 min, a Hitachi F-4500 fluorophotometer (Tokyo, Japan) was

142

applied to determine fluorescence, in which excitation and emission were set at 400 nm

143

and 505 nm, respectively.

144

The inter-assay CV was 4.5-6.1%, and the intra-assay CV was 4.2-4.9%.

After

Value was expressed as a percent of control groups.

Analyses for ROS, 8-Hydroxydeoxyguanosine (8-OHdG) and Glutathione (GSH)

145

Levels. ROS level was measured by DCFH2-DA, a dye.

After washed and re-suspended

146

in medium, cells were mixed with DCFH2-DA at 50 µmol/L for 30 min. PBS was used to

147

wash and suspend cells, and followed by centrifugation at 4°C and 412 xg for 10 min.

148

Collected cells were dissolved by adding 1% Triton X-100.

7


A fluorescence microplate


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149

reader was used to measure the fluorescence change, in which excitation and emission

150

were set at 485 nm and 530 nm, respectively. Result was shown as relative fluorescence

151

unit (RFU)/mg protein, in which RFU was the change in fluorescence between 5 min and

152

time 0.

153

(Tokyo, Japan) was used to extract DNA fraction.

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dissolved in water.

155

Inc. (Portland, OR, USA) was used to determine the oxidative injury of DNA.

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detection limit of 8-OHdG was 10 pg/mg protein.

157

from OxisResearch (Portland, OR, USA) was used to measure GSH concentration (ng/mg

158

protein), and its detection limit was 10 ng/mg protein.

A DNA Extractor WB kit purchased from Wako Pure Chemical Industries Ltd. The obtained DNA at 1 mg/mL was

An ELISA assay kit for 8-OHdG bought from OXIS Health Products The

A colorimetric GSH kit purchased

159

Measurements for Tumor Necrosis Factor (TNF)-α α, Interleukin (IL)-6 and PGE2.

160

After cells were homogenized and centrifuged, TNF-α or IL-6 level (pg/mg protein) in

161

supernatant was assayed by cytoscreen immuno-assay kits obtained from BioSource

162

International (Camarillo, CA, USA).

163

pg/mg protein and 5 pg/mg protein, respectively.

164

measured by a PGE2 EIA kit purchased from Cayman Chemical Co. (Ann Arbor, MI,

165

USA), and its detection limit was 10 pg/mg protein.

The detection limit of TNF-α and IL-6 was 10 PGE2 level (pg/mg protein) was

166

Cytosolic or Nuclear Fraction Preparation. Cells were treated with cold buffer

167

containing 1.5 mM MgCl2, 10 mM KCl, 10 mM HEPES (pH 7.9), 0.5 mM DTT, 0.5 mM

168

PMSF, 1 mg/mL aprotinin and 1 mg/mL leupeptin for 15 min.

After centrifugation for 1

169

min at 4°C and 14,000 xg, cytosolic fraction was obtained.

Nuclear pellet was then

170

re-suspended in buffer containing 1.5 mM MgCl2, 450 mM NaCl, 20 mM HEPES (pH 7.9),

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25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.2 mM EDTA, 1 mg/mL aprotinin and

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1 mg/mL leupeptin for 30 min.

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nuclear fraction was obtained.

After centrifugation for 15 min at 4°C and 14,000 xg,

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Western Blot Analyses. PBS was used to wash plate twice.

Cells were then scraped

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and suspended in lysis buffer.

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(Hercules, CA, USA) were used to assay protein content.

177

used for gel electrophoresis containing 10% SDS-polyacrylamide, and followed by

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applying to a Millipore nitrocellulose membrane (Bedford, MA, USA) for 60 min.

179

treated by a solution of 5% non-fat milk for 60 min to block binding from non-specific

180

antibody, membrane was then reacted with monoclonal antibodies against Bcl-2, cleaved

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caspase-3 or Bax (1:2000); p47phox, gp91phox or COX-2 (1:1000); NF-κB p65, NF-κB p50

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or MAPK (1:500) at 4ºC overnight.

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Boehringer-Mannheim (Indianapolis, IN, USA).

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horseradish

185

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was a control for loading, and the

186

obtained bands were processed by an ATTO image analyzer (Tokyo, Japan).

187

Densitometric analysis was processed to quantify the blot, and data were normalized

188

against GAPDH.

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peroxidase

Reagents purchased from Bio-Rad Laboratories Inc.

conjugated

Forty µg protein sample was

After

These antibodies were bought from

antibody

Sample was further treated with at

25ºC

for

3.5

Statistical Analysis. Shapiro-Wilk test was used to assess normality of data.

190

preparations were used to examine the effects of treatments (n=9).

191

was undertaken by one-way ANOVA.

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Dunnett's t-test.

hr.

Nine

Statistical analysis

Post-hoc comparisons were processed by

Difference was P0.05).

197

H2O2 treatment decreased survival and raised DNA fragmentation of test cells (Figure 2,

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P

Antioxidative and Antiinflammatory Activities of Asiatic Acid

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