Radix puerariae extracts ameliorate paraquat-induced pulmonary fibrosis by attenuating follistatin-like 1 and nuclear factor erythroid 2p45-related factor-2 signalling pathways through downregulation of miRNA-21 expression

Preparation of RPE

Radix puerariae, the root of Pueraria lobata (Willd) Ohwi (Shanghai Leiyunshang Pharmaceutical
Co. Ltd., Shanghai, China) was ground, extracted three times with 70 % alcohol for
2 h, concentrated with a vacuum rotary evaporator and freeze-dried. The dried powder
was then dissolved in distilled water and filtered. It was chromatographed on a macroporous
resin D101 column (11.5 cm?×?85.5 cm) eluted with distilled water followed by 70 %
ethanol. The 70 % ethanol eluate was dried with a rotary evaporator and stored in
the dark at 4 °C. A yield of 3.89 g RPE was obtained from 100 g of dried Radix puerariae.

Extraction and analysis of Radix puerariae

The dried powder sample (200 mg) of RPE was extracted with 5 mL methanol for 12 h
on a test tube rotator and centrifuged at 2000?×?g for 10 min and the supernatant
collected and evaporated in a Speed-Vac sample concentrator (model SPD 111 V, Thermo
Savant, LaJolla, CA, USA). For HPLC analysis, extracts were redissolved in HPLC grade
methanol and filtered through a nylon filter (0.45 ?m, National, Salt Lake City, UT,
USA). Sample injection volume for HPLC was 20 ?L. The HPLC system was equipped with
a pump (L2130; Hitachi, Tokyo, Japan), auto sampler (L-2200; Hitachi) and a UV detector
(L-2400; Hitachi) controlled with “Lachrome Elite” software (Waters 2487 instrument
software).

HPLC separation was performed on a 250 mm?×?4 mm C18 reversed-phase column (5 ?m,
LichroCART; Merck KGaA, Darmstadt, Germany) protected by a guard column of the same
material. The HPLC method was as described by Goyal and Ramawat 8] with small modifications. The solvent system was composed of solvent A (0.0025 %
trifluoroacetic acid in water) and solvent B (80 % acetonitrile (E. Merck, Mumbai,
India) in solvent A). The column was eluted with successive gradients of solvent A
and solvent B, with the percent solvent A programmed as follows: 0–2 min, 85 %; 2–5 min,
85 %???80 %; 5–15 min, 80 %???50 %; 15–20 min, 50 %???40 %; 20–30 min, 40 %???30 %;
30–35 min, 30 %???20 %; 35–45 min, 20 %???0 %; 45–48 min, 0 %; 48–50 min, 0 %???85 %;
50–55 min, 85 %. Separation was performed at a flow rate of 1.0 mL/min and chromatographic
peaks at 254 nm were monitored. The compounds were quantified from a calibration curve
constructed with standard solutions. To analyse components of RPE, the concentrations
of the chemical reference substance standards used for plotting the calibration curve
ranged from 1.0 to 5.0 ?g. Each HPLC run was repeated three times.

The HPLC analysis indicated that 100 mg RPE contained 49.4 mg puerarin, 1.3 mg daidzin,
and 0.16 mg daidzein, indicating that puerarin was its major component (Fig. 1).

Fig. 1. Chromatogram of the chemical reference substances and sample. a Chromatogram of the chemical reference substances. b Chromatogram of various samples: 1, hydroxypuerarin; 2, puerarin; 3, daidzin; 4,
genistin; 5, daidzein; 6, genistein; 7, formomonetin; 8, biochanin A

Animals

C57BL/6J (B6) mice were purchased from Kunming Medical University Laboratory Animal
Center (Kunming, China). All mice were housed in the Animal Care Facility of Kunming
Medical University and maintained in a pathogen-free environment. The mice (8–9 week
old and weighing 20–30 g) used in the experiment were housed in a vivarium maintained
at 23 °C with a 12:12 h light/dark cycle (lights off at 7.00 p.m.). They received
a standard laboratory diet and water ad libitum. All experiments were approved by the Ethics Committee of Kunming Medical University
(Yunnan, China) (Approval number: TCM-2011-041-E17) and performed according to The
Guidelines of the Animal Care Committee of Kunming Medical University.

In vivo miR-21 knockdown using locked nucleic acid-modified anti-miR-21

Locked nucleic acid (LNA)-modified scrambled or anti-miR-21 oligonucleotides (Exiqon,
Woburn, MA, USA) were diluted in saline (5 mg/mL) for administration through intraperitoneal
(i.p.) injection (10 mg/kg) at least 30 min before PQ exposure 18].

Reagents

PQ aqueous solution (active ingredient content of 200 g/L, product license number:
XK13-003-00058) was from Chuandong Agrochemical Co., Ltd, (Guangdong, China). An enhanced
chemiluminescence (ECL) kit was from Perkin Elmer Life Sciences, Inc. (Boston, MA,
USA). SYBR fluorescence quantitative reverse transcription polymerase chain reaction
(PCR) kit was from Takara Company (Tokyo, Japan). TRIzol reagent was from Invitrogen
Corporation (Carlsbad, CA, USA), Mouse TGF-?1 and MMP-9 ELISA kits were from Bender
Medsystem (Vienna, Austria).

Animal treatments

In the first experiment, mice were weighed and randomly divided into five groups (five
mice per group) to assess protective effects of puerarin on pulmonary fibrosis. For
induction of pulmonary fibrosis, PQ (10 mg/kg) or saline, as a control, was injected
i.p. into mice 3]. Group 1 was untreated (or, where indicated, received only saline) and served as
the control group; Group 2 received PQ (10 mg/kg) to induce pulmonary fibrosis and
served as the “model group”; Group 3 received PQ to induce pulmonary fibrosis and
was also treated with RPE at a dose of 30 mg/kg i.p. once per day. On day 14 after
PQ injection, mice were killed and lungs harvested. A small portion of each lung was
fixed with 10 % formalin and embedded in paraffin for haematoxylin-eosin (HE) and
Masson’s trichrome staining.

Real-time PCR analysis

For RNA isolation, lung tissues were frozen in liquid nitrogen and stored in a ?80 °C
freezer until use. Total RNA was extracted from frozen lung tissue (left lung) with
TRIzol reagent and amplified with a PCR single-step kit (Promega, Madison, WI, USA)
according to the manufacturer’s instructions. Real-time-PCR was performed with a PTC-200
DNA Engine PCR cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers
(Table 1) were designed based on published sequences of these genes and synthesised by Invitrogen
19], 20]. PCR was performed in a total volume of 20 ?L containing 20 mM Tris–HCl, 50 mM KCl,
1.25 mM MgCl2, 0.2 mM dNTP, 0.5 mMprimer, 1 U TaqDNA polymerase and 0.5 ?L cDNA. Cycle
parameters were as follows: 95 °C for 3 min, 25 cycles (98 °C for 30 s, 60 °C for
40 s, and 72 °C for 60 s) and 72 °C for 5 min. Ordinary PCR products were separated
on a 2 % agarose gel. ?2-Actin was used as an endogenous controland values for each
sample were normalised according to its ?2-actin content. The mRNA expression levels
of target genes were calculated using the 2
^-??Ct
method 21], 22].

Table 1. Primer sequences for the genes to validate microarray analysis by real-time-PCR

Western blotting

Lung homogenates were prepared in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 % NP-40,
0.5 % sodium deoxycholate, 2 mM NaF, 2 mM EDTA, 0.1 % SDS and a protease inhibitor
cocktail tablet (Roche Applied Science, Indianapolis, IN, USA). Protein concentrations
were quantified by the BCA method (Pierce Biotechnology, Inc., Rockford, IL, USA).
An equal amount of protein (30 mg) from each sample was loaded on respective gel lanes.
Samples and pre-stained molecular weight markers (Bio-Rad) were electrophoresed on
12 % Tris-glycine polyacrylamide gels, then protein bands were electrophoretically
transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Marlborough,
MA, USA). Membranes were blocked for 1 h at room temperature with 5 % bovine serum
albumin (BSA) and then incubated overnight at 4 °C with the following primary antibodies:
anti-MMP-9 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-p-p38MAPK (Bio-Rad Laboratories
Inc.), anti-NF-kB65 (DAKOCorp, Carpinteria, CA, USA), anti-HO-1 (Cell Signaling, Beverly,
MA, USA), anti-Nrf2 (Cell Signaling), anti-FSTL1 (RD systems, Minneapolis, MN, USA),
anti-p-Smad2/3 (Serotec Ltd, Oxford, United Kingdom), anti-TGF-?1 (Sigma, St. Louis,
MO, USA), anti-CTGF (Santa Cruz Biotechnology), anti-collagen III (Santa Cruz Biotechnology),
anti-collagen-1 (Invitrogen), and anti-?-actin (Sigma-Aldrich), each diluted 1:1000
in Tris-buffered saline with Tween-20 (TBST). ?-actin blotting served as the control
to confirm protein loading. After washing with TBST, membranes were incubated with
the corresponding horseradish peroxidase-linked anti-rabbit antibody (Pierce) diluted
in TBST (1:20000) for 1 h at room temperature. After further washing with TBST, immunoreactive
bands were visualised by enhanced chemiluminescence (ECL) and quantified by densitometry
using Bio-Rad Universal Hood and Quantity One software (Bio-Rad). Results were normalised
to ?-actin levels in the same lanes.

Enzyme-linked immunosorbent assay (ELISA)

At 24 h after the last challenge, bronchoalveolar lavage (BAL) fluid was obtained
from the anaesthetised mice with 1 mL sterile isotonic saline. Lavage was performed
four times and the total volumes pooled for each mouse. Lavage fluid samples were
immediately centrifuged at 2000?×?g for 10 min at room temperature and stored at ?80 °C
until use. TGF-?1 and MMP-9 levels were then assayed with TGF-?1 and MMP-9 ELISA kits
according to the manufacturers’ instructions.

Immunohistochemistry

Immunostaining was performed on lung sections after antigen retrieval using Retrievagen
A (Zymed, South San Francisco, CA, USA) at 100 °C for 20 min and then quenching endogenous
peroxidases with 3 % H
₂
O
₂
. Sections were blocked with 2 % BSA in PBS followed by staining with primary anti-FSTL1
and ?-SMA at room temperature for 1 h. Sections were then washed. After application
of the secondary antibody (Sigma-Aldrich), tissue staining was developed with Vectastain
ABC (Vector Labs, Burlingame, CA, USA) and 3,3?-diaminobenzidine (Vector Labs). With
Image Pro Plus image analysis software (Media Cybernetics, Bethesda, MD, USA), FSTL1
and ?-SMA positive staining in lung tissue were determined and each staining intensity
was expressed as positive units.

Total lung collagen

Total lung collagen was determined by measuring total soluble collagen (Sircol Collagen
Assay, Biocolor, Belfast, Northern Ireland). The left lung was homogenised in 5 mL
0.5 M acetic acid containing pepsin (1 mg/10 mg tissue; Sigma-Aldrich) and incubated
(24 h, 24 °C, with mixing at 240 rpm). Sircol dye was added (1 mL/100 mL, with mixing
for 30 min) and then the sample was centrifuged (12,000?×?g for 12 min). The pellet
was resuspended in 1 mL 0.5 M NaOH. The optical density at 540 nm was measured with
a spectrophotometer.

Measurement of intracellular ROS

ROS were measured as previously described 23]. BAL cells were washed with PBS and incubated for 10 min at room temperature with
PBS containing 3.3 ?M 2?,7?-dichlorofluorescein (DCF) diacetate (Molecular Probes,
Eugene, OR, USA) to label intracellular ROS. DCF stained cells were analysed by fluorescence-activated
cell sorting (1?×?10
⁴
cells).

SOD activity assay

SOD activity was estimated as described by Kakar et al. 24]. The reaction mixture contained 0.1 mL phenazine methosulphate (186 ?M) and 1.2 mL
sodium pyrophosphate buffer (0.052 mmol, pH 7.0). Lung homogenate was prepared in
10 mL ice-coldlysis buffer (50 mM phosphate buffer with 1 m Methylene diamine tetraacetic
acid (EDTA) per g tissue and centrifuged twice (once at 1500?×?g for 10 min, and the
supernatant centrifuged again at 10,000?×?g for 15 min). An aliquot (0.3 mL) of the
resulting supernatant was added to the reaction mixture. The enzymatic reaction was
initiated by adding 0.2 mL NADH (780 ?mol) and stopped after 1 min by adding 1 mL
glacial acetic acid. The amount of chromogen formed was measured by absorbance at
560 nm. Results are expressed in units/mg protein.

Measurement of malondialdehyde (MDA)

Lung tissue homogenates from control and experimental groups were prepared in 0.1 M
Tris–HCl buffer (pH 7.4) at 4 °C. The resulting tissue homogenates were used for all
biochemical measurements unless otherwise indicated. MDA content was determined by
a colorimetric assay with a commercially available kit (Jiancheng Bioengineering Institute,
Nanjing, China) according to the manufacturer’s instructions. Briefly, MDA content
in the lung was measured according to the thiobarbituric acid method based on the
formation of a red complex when MDA reacted with thiobarbituric acid. The absorbance
was spectrophotometrically measured at 532 nm.

Measurement of GSH and GSSG in lung tissues

Lung tissues were homogenised with 10 mL ice-cold buffer (50 mM phosphate buffer containing
1 mM EDTA per gram tissue). After centrifugation at 10,000?×?g for 15 min at 4 °C,
the supernatants were removed, deproteinated with 7.5 ?l 5 M KOH per 500 ?l volume
and then stored at ?20 °C until assayed. Total GSH and GSSG levels were determined
with a GSH Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the
manufacturer’s protocol.

Hydroxyproline assay

Collagen content was determined based on hydroxyproline (HYP) levels 25]. Briefly, 10 mg lung tissue was minced in 1 mL 6 M HCl, hydrolysed and then incubated
overnight at 120 °C. Five milliliters 0.5 M acetic acid was added and the pH adjusted
to between 6.0 and 6.5 with 0.2 M NaOH. Chloramine T solution (1 mL, 0.05 M) was added
and the mixture was incubated for 20 min at room temperature. Aldehyde-perchloric
acid (1 mL of a 0.5 M solution) was added and the mixture incubated at 60 °C for 15 min.
Absorbance was then recorded at 550 nm. Results are expressed as ?g/mg wet lung weight
with HYP values read from a standard curve.

Histopathology

Whole lungs were inflated in situ with PBS-buffered formalin (4.5 %, pH 7.0) (Roth, Darmstadt, Germany) and then carefully
immersed in more PBS-buffered formalin. Lung tissue samples were paraffin-embedded
and 5-?m-thick sections were prepared and stained with haematoxylin/eosin (H/E) and
Masson’s or Elastica-van-Gieson staining. The degree of lung fibrosis was assessed
in lung samples as described by Ashcroft and colleagues 26] with a numerical scaling system. In the scaling system, 0 indicates normal lung and
eight indicates total fibrous obliteration of the field. Mean lung fibrosis degree,
as defined by Ashcroft et al. 26], was calculated from individual scores of~15 microscopic fields analysed per mouse
lung.

Statistical analysis

All values were expressed as the means?±?standard deviation (SD) or means?±?standard
error of the mean (SEM), as indicated in the individual figure legends. A one-way
ANOVA followed by the Student-Newman-Keuls test was used to compare the differences
among multiple groups. Pulmonary fibrosis scores were compared using nonparametric
methods. The significant level was defined by a P value of 0.05. The SPSS 13.0 software package (SPSS, Inc., Chicago, IL, USA) was
used for statistical analyses.