Author’s Accepted Manuscript

Protection against oxidative stress-inducedapoptosis in kidney epithelium by Angelica andAstragalus

Muhammad Shahzad, David M Small, ChristudasMorais, Ken Wojcikowski, Arham Shabbir, GlendaC Gobe

PII: S0378-8741(15)30278-6DOI: http://dx.doi.org/10.1016/j.jep.2015.12.027Reference: JEP9877

To appear in: Journal of Ethnopharmacology

Received date: 2 June 2015Revised date: 6 December 2015Accepted date: 20 December 2015

Cite this article as: Muhammad Shahzad, David M Small, Christudas Morais,Ken Wojcikowski, Arham Shabbir and Glenda C Gobe, Protection againstoxidative stress-induced apoptosis in kidney epithelium by Angelica andA s t r a g a l u s , Journal of Ethnopharmacology,http://dx.doi.org/10.1016/j.jep.2015.12.027

This is a PDF file of an unedited manuscript that has been accepted forpublication. As a service to our customers we are providing this early version ofthe manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting galley proof before it is published in its final citable form.Please note that during the production process errors may be discovered whichcould affect the content, and all legal disclaimers that apply to the journal pertain.

www.elsevier.com/locate/jep

1

Protection against oxidative stress-induced apoptosis in

kidney epithelium by Angelica and Astragalus

Muhammad Shahzad a,b

, David M Small a, Christudas Morais

a, Ken Wojcikowski

c,

Arham Shabbir b, and Glenda C Gobe

a*

aCentre for Kidney Disease Research, School of Medicine, University of Queensland,

Translational Research Institute, Brisbane, Australia

bDepartment of Pharmacology, University of Health Sciences, Lahore, Pakistan

cDepartment of Natural and Complementary Medicine, Southern Cross University, Lismore,

Australia

* Corresponding author: Dr. Glenda C Gobe, Centre for Kidney Disease Research, University

of Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba, Brisbane,

Queensland, AUSTRALIA 4102. Tel: (+61) 7 344 38011, Email: g.gobe@uq.edu.au

2

ABSTRACT

Ethnopharmacological relevance: Astragalus membranaceus either alone or in combination with

Angelica sinensis has been used traditionally for kidney disease in East Asia and China for

thousands of years. Previous studies using in vivo animal models have shown the benefits of

these medicinal herbs in kidney diseases that involve oxidative stress. However, the mechanisms

by which these medicinal herbs protect kidney cells remain largely unknown.

Aim of the study: To investigate the mechanisms by which ethanol, methanol and aqueous crude

extracts of roots of A. membranaceus and A. sinensis afford protection to human kidney proximal

tubular epithelial cells, using an in vitro model of oxidative stress.

Materials and Methods: Ethanol, methanol and aqueous extracts of roots of A. membranaceus

and A. sinensis were prepared by a three-solvent sequential process. HK2 human kidney

proximal tubular epithelial cells were treated with H2O2 alone (0.5 mM) or in combination with

different concentrations of extracts. Cell mitosis and death (microscopy) and cell viability (MTT

assay) were compared. Western immunoblot was used to study expression of apoptosis-related

proteins (pro-apoptotic Bax andanti-apoptotic Bcl-XL), and cell survival (NFκB subunits p65 and

p50), pro-inflammatory (TNF-α) and protective (TGFβ1) proteins.

Results: H2O2-induced oxidative stress significantly increased apoptosis and reduced cell

survival; upregulated pro-apoptotic and down-regulated Bcl-XL; increased NFκB (p65, p50);

increased TNFα; and decreased TGFβ1. All changes indicated kidney damage and dysfunction.

All were modulated by all extracts of both plant species, except for NFκB which was only

modulated by extracts of A. membranaceus.

Conclusions: In conclusion, in a model of oxidative stress that might occur after nephrotoxicity,

the plant extracts were protective via anti-apoptotic and anti-inflammatory mechanisms.

3

Keywords: Apoptosis; Oxidative stress; Kidney epithelium; Inflammation; TNF-α

4

1. Introduction

Oxidative stress is a known contributor to the progression and development of acute and

chronic kidney diseases (Agarwal, 2003; Oberg et al., 2004; Calabrese et al., 2007; Gao et al.,

2012). Chronic kidney disease (CKD) is an increasing problem worldwide, with current

conventional medicine treatments having limited positive effects (Wojcikowski et al., 2004 a,b;

Small et al., 2012a). Alternative therapies that can replace or complement conventional

medicines may improve outcome for CKD patients. There is also a need to investigate the

mechanism of action of the alternative therapies, especially how these complementary and

alternative therapies protect intrinsic kidney cell populations that may be involved in tubular

atrophy, and modulate inflammation that may be damaging to the kidney.

Oxidative stress is a result of perturbations in normal oxidant signalling networks,

primarily regulated by reactive oxygen species (ROS) and endogenous antioxidants (Small et al.,

2012b). Kidney proximal tubular epithelial cells contain large numbers of mitochondria and are

the most reliant upon oxidative phosphorylation and most susceptible to ROS-induced injury of

cells of the kidney nephron (Agarwal, 2003). They therefore present an important site of

dysfunction and destruction when oxidant loads exceed antioxidant capabilities. Hydrogen

peroxide (H2O2) is an important and essential precursor to harmful ROS such as the hydroxyl

radical (OH-) and peroxynitrite (ONOO

-) and is considered a significant mediator in the

progression of kidney diseases (Singh et al., 2007), often via induction of apoptosis.

One of the most commonly studied of the pro-apoptotic members of the B-cell

lymphoma-2 (Bcl-2) family is Bax, and of the anti-apoptotic members, Bcl-XL appears to be

effective in kidney tissue protection (Cuttle et al., 2001). The ratio of Bax to Bcl-XL is a crucial

factor which regulates susceptibility of cells to apoptosis (Kroemer, 1997; Gobe et al., 2001).

5

The nuclear factor-kappa B (NF-κB) transcription factor gene family has many functions,

including those in apoptosis, cell survival, and inflammation (Meteoglu et al., 2008; Morais et

al., 2011). TNF-α acts as an inflammatory mediator and plays a significant role in the immune

response in chronic kidney disease (Frigo et al., 2005; Lee et al., 2015). Importantly, ROS are

potent activators of TNF-α-mediated apoptosis (Kim et al., 2010). Studies have demonstrated the

anti-apoptotic role of TGFβ1 in different cells, such as, hepatic stellate cells, microglia,

mammary epithelial cells, and osteoblasts (Sanchez-Capelo, 2005). However, in many cases of

oxidant-induced injury in the kidney, TGFβ1 may be pro-apoptotic, or pro-fibrotic (Cummins et

al., 2010; Zhao et al., 2014). Robust analysis of how these apoptotic or inflammatory pathways

are modulated by various alternative therapies would be beneficial.

Angelica sinensis (Oliv.) Diels (Apiaceae) and Astragalus membranaceus (Fisch.) Bunge

(Fabaceae) are folkloric Chinese herbs and are used traditionally for treatment of kidney

diseases. In the traditional system of medicine, extracts of the roots of A. membranaceus are used

alone or in combination with extracts of A. sinensis roots to treat patients with chronic kidney

disease (Li et al., 2011; Zhong et al., 2013). Previous studies by using in vivo animal models

have shown their protective benefits against ischaemia-reperfusion injury (Cai et al., 2001),

unilateral urinary obstruction and fibrosis in rat (Wojcikowski et al., 2010), chronic puromycin-

aminonuclease nephrosis (Wang et al., 2004), and improved renal microvascular insufficiency in

nephrectomized rats (Song et al., 2009). Nonetheless, the mechanisms by which these medicinal

herbs protect kidney cells remain largely unknown. This study aimed to investigate the

mechanisms by which extracts of roots of A. sinensis and A. membranaceus afford protection to

oxidative stress-injured human kidney proximal tubular epithelial cells, using an in vitro model.

6

2. Materials and Methods

2.1.Plant extracts

Roots of Angelica sinensis (Oliv.) Diels (Chinese names Danggui, Dong quai, Donggui;

Voucher number CP-04-0079) and Astragalus membranaceus (Fisch.) Bunge (Chinese name

Huang-Qi; Voucher number CP-04-206) were obtained from a reliable supplier and

authenticated by a pharmacognosist (Dr H. Wohlmuth, Southern Cross University Medicinal

Plant Herbarium) by chemical and morphological comparison with an authentic reference

specimen. The plant names were checked with www.theplantlist.org at 29-05-2015. Dried plant

root material was ground to a powder and extracted by a three-solvent sequential process. 20 g

ground material was sonicated (10 min) in 200 ml anhydrous solvent (sequentially, ethanol at 40

°C, methanol at 40 °C, and water at 90 °C) and filtered (Whatman No. 3, gravity filtration) and

then repeated for a second and third extraction (Wojcikowski et al., 2007). All the filtrates were

vacuum dried using a rotary vacuum centrifuge. The resultant products were weighed then

resuspended with dimethyl sulphoxide (DMSO) at a predetermined concentration, so that DMSO

content in culture medium, at concentrations selected for the plant extracts, never exceeded 1 %

in the growth medium. At this concentration DMSO had negligible effects on HK2 cells.

2.2.Cell culture

The human renal proximal tubular epithelial cell line, HK2, was obtained from the

American Type Culture Collection (ATCC) (Rockville, MA, USA). Cells were cultured in

7

Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 containing 10 % fetal bovine serum

(FBS; BioWhittaker Australia, Mt Waverley, Australia) with penicillin (1000 U/ml) and

streptomycin (1000 U/ml) (Life Technologies Pty Ltd, Mt. Waverley, Australia) in 5 % CO2

under a humidified atmosphere at 37 °C. For microscopy, cells were seeded onto sterile glass

coverslips in multi-well cell culture plates (1x104

cells/ml). For protein extractions, cells were

grown in 10 cm Petri dishes, also containing glass coverslips for concurrent microscopy. Cells

were grown in 96-well plates for cell viability studies. Cell number was determined using the

trypan blue exclusion method with an improved Neubauer haemocytometer.

2.3.Treatments

Dose response studies were carried out for an H2O2 concentration that induced apoptosis

but not necrosis in HK2 cells (range 0-1 mM H2O2). HK2 cells were treated at 80-90 %

confluence. From these pilot studies, a dose of 0.5 mM H2O2 for a treatment time of 24 h was

selected. Similar treatment concentration and time were used by Small et al. (2014). Cells were

pre-treated for 2 h or 24 h with ethanol, methanol and aqueous extracts of A. sinensis and A.

membranaceus at concentrations of 0.3, 0.6 and 1.2 mg/ml. These concentrations were selected

following initial dose-response experiments using concentrations of each extract ranging from

0.0045 – 5.0 mg/ml. DMSO vehicle control was used in the experiments. For Western blotting,

only one dose (1.2 mg/ml) for each extract type of A. sinensis and A. membranaceus was chosen,

because this showed best protection against H2O2-induced apoptosis in MTT assays and

microscopy studies. Initial MTT assays showed better protection of extracts with 2 h pre-

8

treatment compared with 24 h pre-treatment. Thus, all further experiments were carried out with

2 h pre-treatment. Experiments were carried out in triplicate (n = 3).

2.4.Cytology

Cells on glass coverslips were fixed overnight in 4 % buffered paraformaldehyde at 4 ºC,

washed with PBS and stored at 4 ºC. Cells were stained using haematoxylin and eosin (HE).

Microscopy (X 200) and morphology were used to determine the number of apoptotic and

mitotic cells. Aperio Image Scope digital histology software was used to assist in distinguishing

the morphological features. Data were obtained by counting ten frames of cells for each

treatment and calculating % apoptotic and mitotic cells per total number of cells in each frame.

Means ± standard deviation (SD) were then calculated and comparisons made amongst controls

and treatments using ANOVA. The morphological characteristics for apoptosis were: (i)

shrunken eosinophilic cells with condensed, marginated nuclear chromatin and intact cell

membrane (ii) discrete apoptotic bodies compromising large, dense, pyknotic nuclear fragments

surrounded by a narrow eosinophilic cytoplasm and (iii) clusters of small apoptotic bodies

(assessed as a single apoptotic occurrence) (Gobe, 2009). Morphological features for necrosis

were pale swollen cells with indistinct nuclear and cellular membranes, plus eosinophilia in the

cytoplasm. The morphological characteristics used to distinguish mitosis were: (i) formation of

mitotic spindles occurring during metaphase and remaining visible in anaphase, or (ii) cells in

the later stages of mitosis, telophase or undergoing cytokinesis. An additional analysis would

have been flow cytometry to identify apoptosis and mitosis, but we believe morphology remains

the gold standard for identifying these parameters.

9

2.5.Cell viability

Cell viability was measured by MTT assay using the tetrazolium dye 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich Pty Ltd, St. Louis, MO).

Briefly, the culture medium was removed and 100 μL of fresh culture medium containing MTT

(0.5 mg/ml) were added to the culture and incubated in a humidified atmosphere of 95 % air and

5 % CO2 at 37oC for 90 min. The medium was removed and 100 μl of DMSO were added to

each well to dissolve the purple formazan crystals. The absorbance was read at 570 nm with a

background correction of 690 nm in a Multiscan Go Microplate Reader (Thermo Scientific,

Waltham, MA, USA). The percentage of cell viability was calculated relative to the untreated

control wells, which were designated as 100 %. All appropriate controls were applied as per

manufacturer’s instructions and published in our previous papers, for example, Morais et al.

(2014).

2.6.Protein analysis by Western immunoblotting

Protein assay kits were from Bio-Rad Pty Ltd (Regents Park, Australia) or Pierce Pty Ltd

(Quantum Scientific, Murarrie, Australia) and polyvinylidene difluoride (PVDF) membrane was

from Perkin Elmer (Waltham Massachusetts, USA). Cells on ice were washed with PBS then

scraped into RIPA cell lysis buffer (0.15 M sodium chloride, 0.025 M sodium fluoride, 0.5 M

ethylenediaminetetraacetic acid, 0.1 % sodium dodecylsulphate/SDS, 1.0 % Igepal in 50 mM

Tris-Cl, pH 7.5) containing phosphatase and protease inhibitors (10 µg/ml aprotinin, 10 µg/ml

10

leupeptin, 1 mM sodium orthovanadate and 100 µg/ml phenylmethylsulfonyl chloride). After

cell lysis by sonification, cell debris was removed by centrifugation (13000 g, 20 min, 4 ºC).

Protein concentration was determined using a Pierce bicinchoninic acid (BCA) protein assay. 40

µg of each protein were separated by a 10 % SDS-polyacrylamide gel, and then

electrophoretically transferred onto PVDF membrane. Membranes were soaked in a blocking

solution of 5 % skim milk powder in TBS with Tween-20 pH 7.4 (TBST) (5 % blotto) for 1 h.

Membranes were incubated with primary antibodies diluted at 1:1000 in 1 % blotto solution for

18-24 h at 4 ºC. Primary antibodies (Santa Cruz Biotechnology, CA) were: Bax (Sc-526); Bcl-XL

(Sc-7195); TGF-β1 (Sc-146); NFκB p50 (Sc-7178); NFκB p65 (Sc-372); and TNFα (Sc-1350).

Even loading of proteins was verified using glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) (Sigma-G9545). Membranes were washed three times in TBST for 5 min. Anti-rabbit

HRP-conjugated secondary antibody diluted at 1:2000 in 1 % blotto was used. Protein bands

were visualised using enhanced chemiluminescence and X-ray film. Developed films were

scanned using a Canon Canoscan 8400F at 300 dpi. Scion Image software version Alpha 4.0.3.2

was used to quantify the density of the bands. GAPDH immunoblots were used to normalise

densitometry.

2.7.Statistical analysis

Values reported are mean ± SD. Data were analysed using one-way analysis of variance

(ANOVA) and Tukey’s post hoc analysis, two-way ANOVA and Bonferroni’s post hoc test, or

Student’s t test where appropriate, with statistical significance at P < 0.05.

11

3. Results

3.1. A. sinensis and A. membranaceus protect against H2O2 induced apoptosis

Morphological assessment revealed that 24 h treatment with 0.5 mM H2O2 alone

produced significantly increased apoptosis (the Angelica set 16.24 ± 1.49 % and the Astragalus

set 13.33 ± 0.96 %) and reduced mitosis (the Angelica set 1.23 ± 0.58 % and the Astragalus set

1.39 ± 0.36 %) in HK2 cells compared with untreated control cells (apoptosis Angelica set 1.55 ±

0.53 % and Astragalus set 1.54 ± 0.55 %; mitosis Angelica set 5.96 ± 0.87 % and Astragalus set

5.02 ± 0.58 %) (all P < 0.05). The 2 h pre-treatment with ethanol, methanol and aqueous extracts

of A. sinensis and A. membranaceus significantly reduced levels of apoptosis observed following

H2O2 alone (range 2.57 ± 0.48 to 4.01 ± 0.54 %; P < 0.05), yet did not significantly improve

levels of mitosis (Fig. 1). Non-significant difference was found when extract treated groups were

compared with each other. Fig. 2 demonstrates HE-stained cells: A is an untreated HK2 control

cell culture; B is an H2O2-treated HK2 culture: and C is an H2O2–treated culture co-treated with

1.2 mg/ml methanol extract of A. sinensis, showing restoration of HK2 cell viability, lack of

apoptosis, and some restored mitosis. The results for A. membranaceus were morphologically

similar to Fig. 2C.

3.2. A. sinensis and A. membranaceus promote cell survival and viability in response to H2O2

Fig. 3 demonstrates the results from the MTT assay, presented as % cell viability in

control cultures without Angelica and Astragalus pre-treatment for 2 h. Cell viability of the 0.5

12

mM H2O2-treated cultures markedly decreased by approximately 4 fold and 7 fold (P < 0.001)

following 24 h 0.5 mM H2O2 treatment compared with untreated control cells. Pre-treatment of

ethanol, methanol and aqueous extracts of A. sinensis or A. membranaceus for 2 h or 24 h

prevented H2O2-induced cytotoxicity. Results for 2 h pre-treatment are demonstrated in Fig. 3.

With 2 h pre-treatment, % cell viability was improved by approximately 2.5 to 6 fold (P < 0.01),

compared with the H2O2-treated cultures (Fig. 3). On the basis of these data, the remaining

experiments were conducted with 2 h pre-treatment for all extracts of both plants.

For A.sinensis, ethanolic and methanolic extracts showed maximum cell viability (non-toxic to

cells) at dose of 1.2mg/ml while among all of its aqueous extracts, the dose of 1.2mg/ml yielded

the highest cell viability. On comparison, ethanolic and methanolic extracts showed better results

as compared to the aqueous one.

In case of A. membranaceus, highest improvement in % cell viability was shown by all

three extracts i.e. ethanol, methanol, and aqueous extracts at the dose of 1.2 mg/ml. No

significant difference was observed in the effect of these extracts.

3.3. A. sinensis and A. membranaceus prevent oxidative stress-induced apoptosis through

modulation of Bcl-2 proteins

Fig. 4 demonstrates expression of anti-apoptotic Bcl-XL and pro-apoptotic Bax was

investigated using Western blots and densitometry. Densitometry (Fig. 4A) revealed a significant

decrease in this protein (P < 0.001) compared with untreated control cells following 24 h 0.5 mM

H2O2 treatment. 2 h pre-treatment with extracts of A. sinensis and A. membranaceus significantly

enhanced protein levels of Bcl-XL (P < 0.001). Non-significant difference was found when

13

extract treated groups were compared with each other. Expression of pro-apoptotic Bax (Fig. 4)

was significantly increased following H2O2 treatment (P < 0.001) compared with untreated

control cells. 2 h pre-treatment with extracts of A. membranaceus significantly lowered the

expression of Bax (P < 0.001) to levels seen in untreated control cells. A. sinensis also lowered

protein levels of Bax but to a lesser degree than A. membranaceus (P < 0.05). Representative

Western blots are found in Fig. 4B. Treatment with aqueous extract of A. sinensis displayed

significantly lower Bax expression levels as compared with ethanol extract. Methanolic extract

of A. membranaceus showed lowest Bax expression levels however, the results were non-

significant when compared with each other. Statistical comparison between aqueous extract of

Angelica and methanol extract of Astragalus exhibited non-significant results.

3.4. A. sinensis and A. membranaceus improve cell survival through modulation of NFκB

proteins

Fig. 4 demonstrates expression of NFκB (p50, p65) using Western blotsand densitometry.

Densitometry is demonstrated in Fig. 4A. This revealed a significant increase in NFκB (p50,

p65) (P < 0.05) compared with untreated control cells following 24 h 0.5 mM H2O2 treatment. 2

h pre-treatment with all extracts of A. membranaceus significantly lowered the expression of

NFκB p50 (P < 0.05) and p65 (P < 0.01) to that seen in untreated control cells. Non-significant

difference was found when extract treated groups were compared with each other. Only the

methanol extract of A. sinensis significantly decreased levels of NFκB p50 (P < 0.05) and p65 (P

< 0.01). Non-significant difference was found between methanolic extract of A. sinensis and A.

membranaceus treated groups. Representative western immunoblots can be found in Fig. 4B.

14

3.5.A. sinensis and A. membranaceus modulate H2O2-induced changes in TGFβ and TNFα

Expression of TGFβ1 and TNFα proteins was investigated using Western blots and is

demonstrated in Fig. 4. Densitometry (results in Fig. 4A) revealed a significant decrease in

TGFβ1 expression (P < 0.05) compared with untreated control cells following 24 h 0.5 mM H2O2

treatment. 2 h pre-treatment with extracts of A. sinensis and A. membranaceus significantly

enhanced protein levels of TGFβ1 (P < 0.05) to levels seen in untreated cells. Non-significant

difference was found when all Angelica extracts treated groups were compared with each other.

0.5 mM H2O2-induced a significant increase in TNFα (P < 0.05) compared with untreated

control cells. 2 h pre-treatment with all extracts of A. membranaceus significantly lowered the

expression of TNFα (P < 0.05) to levels seen in untreated control cells. A. sinensis did not

decrease or otherwise alter protein levels of TNFα after H2O2 treatment (Fig. 4). Representative

Western immunoblots can be found in Fig. 4B. Non-significant difference was found when all

Astragalus extracts treated groups were compared with each other.

4. Discussion

CKD is characterized by a continuous reduction in structural and functional decline in the

kidney function that involves increased inflammation and oxidative stress, and reduced

antioxidant capacity (Small et al., 2012b). The objective of this study was to assess the

mechanisms for any protective effects of extracts of A. sinensis and A. membranaceus on

oxidant-induced injury and inflammation in a cell culture model of oxidative stress-induced

kidney injury. The benefits of extracts such as the ones tested here are not well-characterized,

15

especially their protective mechanisms in the kidney tubular epithelial cell population. Loss of

this population contributes to tubular atrophy and loss of kidney function in vivo. The protective

mechanisms of ethanol, methanol and aqueous extracts of A. sinensis and A. membranaceus were

tested. The results provide new evidence that these extracts prevent oxidative stress-induced

apoptosis via modulation of the balance of pro- and anti-apoptotic Bcl-2 family proteins,

maintain cell viability possibly by normalization of NFκB isoforms, and also reduce the

expression of a known pro-inflammatory mediator, TNFα. TGFβ1 was decreased when cells

were injured with oxidative stress and then increased with use of the extracts, and we believe this

also helps mediate cell survival in this model (Sanchez-Capelo, 2005), but the pro-fibrotic role of

TGFβ1 cannot be discounted.

Apoptosis has a role in many processes, from immune modulation, to tissue atrophy, to

cancer development and regression (Pawlowski and Kraft, 2000). Oxidative stress induced-

apoptosis is well-documented and manifests in renal pathologies as kidney atrophy and reduced

kidney function (Small et al., 2012b). Oxidative stress induced a pattern of expression in Bcl-2

proteins favouring apoptosis (increased Bax, decreased Bcl-XL) that was prevented by ethanol,

methanol and aqueous extracts of A. sinensis and A. membranaceus. Bax is pro-apoptotic when

overexpressed and indicates activation of the intrinsic cell death pathway involving

mitochondrial pore formation and the release of cytochrome-c, thereby inducing caspase

activation causing apoptosis (Pawlowski and Kraft, 2000). Bcl-XL is anti-apoptotic and regulates

apoptosis by both homodimerizing with itself or heterodimerizing with Bax to protect the

mitochondrial membrane pores and stop release of cytochrome-c (Ding et al., 2014). Our results

are consistent with previous studies demonstrating that H2O2 induced Bax-dependent apoptosis

and inhibited Bcl-XL in kidney cells (Cuttle et al., 2001; Gao et al., 2012) and cancer (HeLa)

16

cells (Singh et al., 2007). Ethanol, methanol and aqueous extracts of both A. sinensis and A.

membranaceus prevented the increase in Bax expression, while maintaining Bcl-XL to prevent

cell death. Similar results have been shown elsewhere. In those cases, astragaloside-IV, an active

antioxidant constituent of A. membranaceus, was considered responsible for this protection in

glucose-induced podocyte apoptosis (Gui et al., 2012) as well as upregulating Bcl- XL in a rat

model of Alzheimer’s disease (Yin et al., 2010). A. sinensis reduced the expression of Bax-

dependent apoptosis in lipopolysaccharide-induced liver damage in mice (Ding et al., 2001).

Oxidative stress is also known to induce paracrine stimulation of TNFα and its receptor.

TNFα is a pro-inflammatory growth factor, acting in the inflammatory pathway of many kidney

diseases (Speeckaert et al., 2012). In addition, the ligation of TNFα to its receptor may trigger

apoptosis via activation of the caspase pathway (Sanchez-Nino et al., 2010). A. membranaceus

prevented the oxidative stress-induced increase in TNFα, perhaps thereby protecting kidney

proximal epithelial tubular cells against oxidative stress, but just as importantly perhaps acting in

an anti-inflammatory manner. This is consistent with previous studies that found injection of A.

membranaceus resulted in the reduction of serum levels of TNFα in patients with chronic heart

failure (Zhang et al., 2005). Important to note was the lack of TNFα suppression with A. sinensis

pre-treatment indicating that A. sinensis is protective against oxidative stress-induced apoptosis

by TNFα-independent mechanisms. In the kidney, Gao et al. (2012) reported that extracts of the

roots of Astragalus species lowered insulin-induced increases in oxidative stress, measured by

increased malondialdehyde and lowered superoxide dismutase, in diabetic rats. They also found

decreased TNFα and interleukin-6 (IL-6), another pro-inflammatory cytokine. Similar anti-

oxidant effects were reported for another Angelica species (A. dahurica) in a non-kidney model

17

using RAW264.7 macrophage cells (Lee et al., 2011). The ethanol extracts upregulated heme-

oxygenase-1, a marker of oxidative stress typically increased in cells surviving the injury.

NFκB is a dimeric complex that is activated by pro-inflammatory extracellular signals

and cellular stress, resulting in the transcriptional regulation of several hundreds of cellular genes

related to cell immunity, inflammation, cell survival and proliferation, differentiation, and

apoptosis (del Nogal et al., 2012; Shabbir et al., 2014). An NFκB-dependent pro-survival effect

occurs when this transcription factor regulates expression of various anti-apoptotic proteins, such

as Bcl-2, Bcl-XL and Mcl-1 (Karin, 2006). Specific stimuli were found to determine the pro-

inflammatory or pro-survival behaviours of NFκB using pervanadate- and H2O2-induced

apoptosis (Kaltschmidt et al., 2000). Another study demonstrated that hypoxia activated NFκB

and reduced Bcl-2, which caused apoptosis and aortic endothelial cell death (Matsushita et al.,

2000). Our study determined that oxidative stress increased NFκB levels, indicating that here,

too, NFκB overexpression was linked with reduced anti-apoptotic Bcl-XL and increased

apoptosis. All extracts of A. membranaceus were able to significantly reduce the raised levels of

NFκB (p50, p65) and protect against apoptosis. Only the methanol extract of A. sinensis was able

to produce a significant reduction in NFκB (p50, p65) levels. In a study using aqueous extracts of

roots of Astragalus species and A. sinensis, diabetic retinopathy was reduced by significantly

decreasing expression of pro-inflammatory factors such as NFκB, IL-1β and IL-6 (Gao et al.,

2013).

In the kidney, TGFβ1 is typically thought of as a profibrotic cytokine (Yang et al., 2007;

Zhou et al., 2014) but it has also been shown to possess both pro- and anti-apoptotic properties

(Sanchez-Capelo., 2005). It is thought that an immunosuppressive property of TGFβ1 acts in the

inhibition of apoptosis induced by TNFα and Fas/FasL binding in activated T cells, but the

18

mechanism is not fully understood (Wahl et al., 2000; Chen and Wahl, 2003). Our results may

demonstrate that TGFβ1 maintains cell viability and is anti-apoptotic against oxidative stress.

Extracts of A. sinensis and A. membranaceus prevented a decrease in TGFβ1 expression, thereby

inducing a protective effect against oxidative stress in human kidney proximal tubular cells. The

pro-survival nature of TGFβ1 is consistent with findings in hepatic stellate cells (Saile et al.,

2011), osteoblasts (Sowa et al., 2003), and mammary epithelial cells (Shin et al., 2001). The pro-

fibrotic nature of TGFβ1 needs further analysis for the effects of A. sinensis and A.

membranaceus in oxidative stress injury of human kidney proximal tubular cells.

In summary, the antioxidant properties of these two traditional Chinese medicinal herbs

have been well-documented and most likely reduce oxidative stress prior to activation of

apoptotic pathways and harmful downstream processes. There have been limited studies

investigating the specific antioxidant constituents of A. sinensis or A. membranaceus and more

importantly, the mechanism of action of cellular and molecular targets, other than antioxidant

potential, in kidney cell populations. These findings demonstrate the overall promotion of cell

survival against oxidative stress-induced damage in proximal tubular epithelial cells by A.

sinensis and A. membranaceus. We previously evaluated the antioxidant effects of 55 herbs

including A. sinensis and A. membranaceus (Wojcokowski et al, 2007). The combined data of 55

herbs provided useful comparison of the antioxidant activity for understanding the potency of

each extract. The clinical benefits, or toxicities, of the natural plant extracts in human kidney

disease need to be determined. However, despite these gaps in knowledge, the present study

favours the application of extracts of A. sinensis and A. membranaceus in use against oxidative

stress-induced kidney disease.

19

5. Conclusions

In conclusion, in a model of oxidative stress that might occur after nephrotoxicity, the plant

extracts were protective via anti-apoptotic and anti-inflammatory mechanisms.

Conflict of Interest

All authors declare no financial/commercial conflicts of interest.

Acknowledgement

Dr. Muhammad Shahzad was supported for this project by an Australian Endeavour Research

Fellowship.

References

Agarwal, R., 2003. Proinflammatory effects of oxidative stress in chronic kidney disease: role of

additional angiotensin II blockade. Am. J. Physiol. Renal Physiol. 284, F863-869.

Cai, Q., Li, X., Wang, H., 2001. Astragali and Angelica protect the kidney against ischemia and

reperfusion injury and accelerate recovery. Chin. Med. J. (Engl). 114, 119-123.

Calabrese, V., Mancuso, C., Sapienza, M., Puleo, E., Calafato, S., Cornelius, C., Finocchiaro,

M., Mangiameli, A., Di Mauro, M., Stella, A.M., Castellino, P., 2007. Oxidative stress

and cellular stress response in diabetic nephropathy. Cell Stress Chaperon. 12, 299-306.

Chen, W., Wahl, S.M., 2003. TGF-beta: the missing link in CD4+CD25+ regulatory T cell-

mediated immunosuppression. Cytokine Growth Factor Rev. 14, 85-89.

20

Cummins, T.D., Barati, M.T., Coventry, S.C., Salyer, S.A., Klein, J.B., Powell, D.W., 2010.

Quantitative mass spectrometry of diabetic kidney tubules identifies GRAP as a novel

regulator of TGF-beta signaling. Biochim. Biophys. Acta. 1804, 653-661.

Cuttle, L., Zhang, X.J., Endre, Z.H., Winterford, C., Gobe, G.C., 2001. Bcl-X(L) translocation in

renal tubular epithelial cells in vitro protects distal cells from oxidative stress. Kidney Int.

59, 1779-1788.

del Nogal, M., Luengo, A., Olmos, G., Lasa, M., Rodriguez-Puyol, D., Rodriguez-Puyo, M.,

Calleros, L., 2012. Balance between apoptosis or survival induced by changes in

extracellular-matrix composition in human mesangial cells: a key role for ILK-NFkappaB

pathway. Apoptosis. 17, 1261-1274

Ding, H., Peng, R., Yu, J., 2001. Modulation of angelica sinensis polysaccharides on the

expression of nitric oxide synthase and Bax, Bcl-2 in liver of immunological liver-injured

mice]. Zhonghua. Gan. Zang. Bing. Za. Zhi. 9 Suppl, 50-52.

Ding, J., Mooers, B.H., Zhang, Z., Kale, J., Falcone, D., McNichol, J., Huang, B., Zhang, X.C.,

Xing, C., Andrews, D.W., Lin, J., 2014. After embedding in membranes antiapoptotic

bcl-xl protein binds both Bcl-2 homology region 3 and helix 1 of proapoptotic Bax

protein to inhibit apoptotic mitochondrial permeabilization. J. Biol. Chem. 289, 11873-

11896..

Frigo, D.E., Vigh, K.A., Struckhoff, A.P., Elliot, S., Beckman, B.S., Burow, M.E., McLachlan,

J.A., 2005. Xenobiotic-induced TNF-alpha expression and apoptosis through the p38

MAPK signaling pathway. Toxicol. Lett. 155, 227-238.

21

Gao, Y., Zhang, R.R., Li, J.H., Ren, M., Ren, Z.X., Shi, J.H., Pan, Q.Z., Ren, S.P., 2012. Radix

Astragali lowers kidney oxidative stress in diabetic rats treated with insulin. Endocrine.

42, 592-598.

Gao, D., Guo, Y., Li, X., Li, Z., Xue, M., Ou, Z., Liu, M., Yang, M., Liu, S., Yang, S., 2013. An

aqueous extract of Radix astragali, Angelica sinensis, and Panax notoginseng is effective

in preventing diabetic retinopathy. Evid. Based Complement. Alternat. Med. 578165.

Gobe, G., Rubin, M., Williams, G., Sawczuk, I., Buttyan, R., 2002. Apoptosis and expression of

Bcl-2, Bcl-XL, and Bax in renal cell carcinomas. Cancer Invest. 20, 324-332.

Gobe, G., 2009. Identification of apoptosis in kidney tissue sections. Methods Mol. Biol. 466,

175-192.

Gui, D., Guo, Y., Wang, F., Liu, W., Chen, J., Chen, Y., Huang, J., Wang, N., Astragaloside,

I.V., 2012. a novel antioxidant, prevents glucose-induced podocyte apoptosis in vitro and

in vivo. PLoS One. 7, e39824.

Kaltschmidt, B., Kaltschmidt, C., Hofmann, T.G., Hehner, S.P., Droge, W., Schmitz, M.L., 2000.

The pro- or anti-apoptotic function of NF-kappaB is determined by the nature of the

apoptotic stimulus. Eur. J. Biochem. 267, 3828-3835.

Karin, M., 2006. Nuclear factor-kappaB in cancer development and progression. Nature. 441,

431-436.

Kim, J.J., Lee, S.B., Park, J.K., Yoo, Y.D., 2010. TNF-alpha-induced ROS production triggering

apoptosis is directly linked to Romo1 and Bcl-X(L). Cell Death Differ. 17, 1420-1434.

Kroemer, G., 1997. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. Med. 3,

614-620.Lee, B.T., Ahmed, F.A., Hamm, L.L., Teran, F.J., Chen, C.S., Liu, Y, Shah, K,

Rifai, N, Batuman, V., Simon, E.E., He, J., Chen, J. 2015. Association of C-reactive

22

protein, tumor necrosis factor-alpha, and interleukin-6 with chronic kidney disease. BMC

Nephrol. 16, 77. doi: 10.1186/s12882-015-0068-7.

Lee, M.Y., Lee, J.A., Seo, C.S., Ha, H., Lee, H., Son, J.K., Shin, H.K., 2011. Anti-inflammatory

activity of Angelica dahurica ethanolic extract on RAW264.7 cells via upregulation of

heme oxygenase-1. Food Chem. Toxicol. 49, 1047-1055.

Li, M., Wang, W., Xue, J., Gu, Y., Lin, S., 2011. Meta-analysis of the clinical value of

Astragalus membranaceus in diabetic nephropathy. J. Ethnopharmacol. 133(2), 412-419.

Matsushita, H., Morishita, R., Nata, T., Aoki, M., Nakagami, H., Taniyama, Y., Yamamoto, K.,

Higaki, J., Yasufumi, K., Ogihara, T., 2000. Hypoxia-induced endothelial apoptosis

through nuclear factor-kappaB (NF-kappaB)-mediated bcl-2 suppression: in vivo

evidence of the importance of NF-kappaB in endothelial cell regulation. Circ. Res. 86,

974-981.

Meteoglu, I., Erdogdu, I.H., Meydan, N., Erkus, M., Barutca, S., 2008. NF-KappaB expression

correlates with apoptosis and angiogenesis in clear cell renal cell carcinoma tissues. J.

Exp. Clin. Cancer Res. 27, 53. doi: 10.1186/1756-9966-27-53.

Morais, C., Gobe, G., Johnson, D.W., Healy, H., 2011. The emerging role of nuclear factor

kappa B in renal cell carcinoma. Int. J. Biochem. Cell. Biol. 43, 1537-1549.

Morais, C., Small, D.M., Vesey, D.A., Martin, J., Johnson, D.W., Gobe, G.C., 2014. Fibronectin

and transforming growth factor beta contribute to erythropoietin resistance and

maladaptive cardiac hypertrophy. Biochem. Biophys. Res. Commun. 444, 332-337.

Oberg, B.P., McMenamin, E., Lucas, F.L,, McMonagle, E., Morrow, J., Ikizler, T.A.,

Himmelfarb, J., 2004. Increased prevalence of oxidant stress and inflammation in patients

with moderate to severe chronic kidney disease. Kidney Int. 65, 1009-1016.

23

Pawlowski, J., Kraft, A.S., 2000. Bax-induced apoptotic cell death. Proc. Natl. Acad. Sci. USA.

97, 529-531.

Saile, B., Matthes, N., El, A.H., Neubauer, K., Ramadori, G., 2011. The Bcl, NFkappaB and

p53/p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic

effect of TGF-beta or TNF-alpha on activated hepatic stellate cells. Eur. J. Cell Biol. 80,

554-556.

Sanchez-Capelo, A., 2005. Dual role for TGF-beta1 in apoptosis. Cytokine Growth Factor Rev.

16, 15-34.

Sanchez-Nino, M.D., Benito-Martin, A., Goncalves, S., Sanz, A.B., Ucero, A.C., Izquierdo,

M.C., Ramos, A.M., Berzal, S., Selgas, R., Ruiz-Ortega, M., Egido, J., Ortiz, A., 2010.

TNF superfamily: a growing saga of kidney injury modulators. Mediators Inflamm. doi:

10.1155/2010/182958.

Shabbir, A., Shahzad, M., Ali, A., Zia-ur-rehman, M., 2014. Anti-arthritic activityof N0-[(2,4-

dihydroxyphenyl)methylidene]-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-

c][1,2]benzothiazin-1(4H)-yl)acetohydrazide. Eur. J. Pharmacol. 738, 263-272.

Shin, I., Bakin, A.V., Rodeck, U., Brunet, A., Arteaga, C.L., 2001. Transforming growth factor

beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1. Mol.

Biol. Cell. 12, 3328-3339

Singh, M., Sharma, H., Singh, N., 2007. Hydrogen peroxide induces apoptosis in HeLa cells

through mitochondrial pathway. Mitochondrion. 7, 367-373.

Small, D.M., Coombes, J.S., Bennett, N., Johnson, D.W., Gobe, G.C., 2012a. Oxidative stress,

anti-oxidant therapies and chronic kidney disease. Nephrology (Carlton). 17: 311-321.

24

Small, D.M,, Bennett, N.C., Roy, S., Gabrielli, B.G., Johnson, D.W., Gobe, G.C., 2012b.

Oxidative stress and cell senescence combine to cause maximal renal tubular epithelial

cell dysfunction and loss in an in vitro model of kidney disease. Nephron Exp. Nephrol.

122, 123-130.

Small, D.M., Morais, C., Coombes, J., Bennett, N.C., Johnson, D.W., Gobe, G.C., 2014.

Oxidative stress-induced alterations in PPAR-γ and associated mitochondrial

destabilization contribute to kidney cell apoptosis. Am. J. Physiol. Renal Physiol. 307,

F814-F822.

Song, J., Meng, L., Li, S., Qu, L., Li, X., 2009. A combination of Chinese herbs, Astragalus

membranaceus var. mongholicus and Angelica sinensis, improved renal microvascular

insufficiency in 5/6 nephrectomized rats. Vascul. Pharmacol. 50, 185-193.

Sowa, H., Kaji, H., Iu, M.F., Tsukamoto, T., Sugimoto, T., Chihara, K., 2003. Parathyroid

hormone-Smad3 axis exerts anti-apoptotic action and augments anabolic action of

transforming growth factor beta in osteoblasts. J. Biol. Chem. 278: 52240-52252.

Speeckaert, M.M., Speeckaert, R., Laute, M., Vanholder, R., Delanghe, J.R., 2012. Tumor

necrosis factor receptors: biology and therapeutic potential in kidney diseases. Am. J.

Nephrol. 36: 261-270.

Wahl, S.M., Orenstein, J.M., Chen, W., 2000. TGF-beta influences the life and death decisions

of T lymphocytes. Cytokine. Growth Factor Rev. 11, 71-79.

Wang, H., Li, J., Yu, L., Zhao, Y., Ding, W., 2004. Antifibrotic effect of the Chinese herbs,

Astragalus mongholicus and Angelica sinensis, in a rat model of chronic puromycin

aminonucleoside nephrosis. Life Sci. 74, 1645-1658.

25

Wojcikowski, K., Johnson, D.W., Gobe, G., 2004a. Medicinal herbal extracts--renal friend or

foe? Part two: herbal extracts with potential renal benefits. Nephrology (Carlton). 9, 400-

405.

Wojcikowski, K., Johnson, D.W., Gobe, G., 2004b. Medicinal herbal extracts -- renal friend or

foe? Part one: the toxicities of medicinal herbs. Nephrology (Carlton). 9, 313-318.

Wojcikowski, K., Stevenson, L., Leach, D., Wohlmuth, H., Gobe, G., 2007. Antioxidant capacity

of 55 medicinal herbs traditionally used to treat the urinary system: a comparison using a

sequential three-solvent extraction process. J. Altern. Complement. Med. 13, 103-109.

Wojcikowski, K., Wohlmuth, H., Johnson, D.W., Gobe, G., 2010. Effect of Astragalus

membranaceus and Angelica sinensis combined with Enalapril in rats with obstructive

uropathy. Phytother. Res. 24, 875-884.

Yang, T., Vesey, D.A., Johnson, D.W., Wei, M.Q., Gobe, G.C., 2007. Apoptosis of

tubulointerstitial chronic inflammatory cells in progressive renal fibrosis after cancer

therapies. Transl. Res. 150, 40-50.

Yin, Y., Liu, Y., Huang, L., Huang, S., Zhuang, J.H., Chen, X., Zhang, L., Wu, H.S., 2010. Anti-

apoptosis effect of astragalaside Iv on alzheimer's disease rat model via enhancing the

expression of bcl-2 and bcl-xl. Scand. J. Lab. Anim. Sci. 37: 75-82.

Zhang, J.G., Yang, N., He, H., 2005. Effect of astraglus injection on serum apoptosis relevant

factors in patients with chronic heart failure. Zhongguo. Zhong. Xi. Yi. Jie. He. Za. Zhi.

25, 400-403.

Zhao, L.X., Jiang, B,C., Wu, X.B., Cao, D.L., Gao, Y.J., 2014. Ligustilide attenuates

inflammatory pain via inhibition of NFkappaB-mediated chemokines production in spinal

astrocytes. Eur. J. Neurosci. 39, 1391-1402.

26

Zhong, Y., Deng, Y., Chen, Y., Chuang, YP., and He, J.C., 2013. Therapeutic use of traditional

Chinese herbal medications for chronic kidney diseases. Kidney Int. 84(6),

10.1038/ki.2013.276.

Zhou, T.B., Qin, Y.H., Lei, F.Y., Huang, W.F., Drummen, G.P., 2014. Association of prohibitin-

1 and 2 with oxidative stress in rats with renal interstitial fibrosis. Mol. Biol. Rep. 41,

3033-3043.

27

Figure Legends

Fig. 1: Apoptosis and mitosis with hydrogen peroxide (H2O2) without and with A. sinensis and A.

membranaceus are demonstrated. % apoptosis and mitosis were compared in untreated control

cultures, DMSO vehicle-treated cultures, and cultures treated with H2O2 (0.5 mM) for 24 h

without and with A. sinensis and A. membranaceus as a 2 h pre-treatment. There was no

significant difference between untreated and DMSO-vehicle treated cultures. Compared with

control untreated cultures, H2O2 significantly increased apoptosis and decreased mitosis (#). All

extracts of A. sinensis and A. membranaceus significantly reduced apoptosis induced by H2O2 (*,

P < 0.001). Effects on mitosis were not significant, although there was a trend for improved

mitosis with the plant extracts. All data are expressed as the means ± SD.

Fig. 2: Examples of cell cultures stained with hematoxylin and eosin are demonstrated. Eosin

gives a pale-pink stain to the cytoplasm (pale grey in black and white photos) and hematoxylin

stains nuclei as an intense blue (dark grey in black and white photos) that is enhanced in

apoptotic nuclei and apoptotic bodies. A is an untreated HK2 culture, B is treated with 0.5 mM

H2O2 for 24 h, and C is an H2O2–treated culture co-treated with 1.2 mg/ml methanol extract of A.

sinensis. A. sinensis. Apoptosis (examples arrowed in B and C, seen as condensed cells with

dense nuclei, or as groups of apoptotic bodies) increased in B with H2O2 treatment compared

with the untreated cultures. A. sinensis protected the cells from oxidative stress (seen in C).

Mitosis (M with arrow) is demonstrated by a metaphase plate in A and a cell in cytokinesis in C.

Mitosis was rarely seen in H2O2-treated cultures (not seen in B) but was partly restored by

treatment with A. sinensis. Magnification x 200.

28

Fig. 3: Cell viability determined by the MTT assay is demonstrated as a % of untreated control

cells that had no pre-treatment with Angelica or Astragalus. There was no significant difference

between control cells and DMSO vehicle-treated cells. H2O2 (0.5 mM) treatment for 24 h

significantly decreased cell viability (###; P < 0.001) in cultured HK2 cells compared with

control cells. 2 h pre-treatment with ethanol, methanol and aqueous extracts of A. sinensis and A.

membranaceus at all concentrations significantly increased cell viability against H2O2 (***; P <

0.001). All data are expressed as means ± SD.

Fig. 4: Examples of Western immunoblots and graphs for densitometry for TGFβ1, Bcl-XL, Bax,

NFκB (p50 and p65), TNFα and GAPDH. H2O2 (0.5 mM) treatment for 24 h significantly

increased the expression level of Bax (P < 0.001), NFκB (p50 and p65) (P < 0.05), TNFα (P <

0.05) and reduced the expression level of Bcl-XL (P < 0.001) and TGFβ1 (P < 0.05) compared

with untreated controls. 2 h pre-treatment with all extracts of A. sinensis (1.2 mg/ml) and A.

membranaceus (1.2 mg/ml) significantly increased the expression of Bcl-XL (P < 0.001) and

TGFβ1 (P < 0.05) and decreased levels of Bax (P < 0.05) compared with H2O2 treated cells.

Expression of NFκB (p50) and NFκB (p65) was significantly decreased (P < 0.05; P < 0.01,

respectively) compared to H2O2 treated cells. A. membranaceus pre-treatment (2 h) significantly

decreased (P < 0.05) TNFα protein levels compared with H2O2 treated cells. Densitometry was

normalised to GAPDH, calculated as a fold change of controls. All data are expressed as the

means ± SD. # denotes a significant difference compared with untreated control cells, while

*

represents a significant difference compared with H2O2 treated cells.

Fig

ure

Fig

ure

Fig

ure

Figure

Ba

x

B

cl-

XL

Ke

y:

Up

-re

gu

late

:

Do

wn

-re

gu

late

:

e:

NF

-κB

(p6

5)

NF

-κB

,

(p5

0)

TNFα

TGFβ1

T

N

Eff

ects

of

A. si

nen

sis

and A

.

mem

bra

nace

us

on m

arker

s of

apopto

sis,

cell

su

rviv

al, an

d i

nfl

amm

atio

n

*Gra

ph

ical A

bstr

act

(fo

r re

vie

w)