J Cancer Prev 2022; 27(1): 42-49
Published online March 30, 2022
© Korean Society of Cancer Prevention
Jong Su Kang1,** , Xin Yi Zhao2,** , Jeong Ho Lee3 , Jeong-Sang Lee2,* , Young-Sam Keum1,*
1College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University, Goyang, 2Department of Biotechnology and Functional Foods, College of Medical Sciences, Jeonju University, Jeonju, 3Sunchang Research Institute of Health and Longevity, Sunchang, Korea
Correspondence to :
Jeong-Sang Lee, E-mail: firstname.lastname@example.org, https://orcid.org/0000-0002-2080-2065
Young-Sam Keum, E-mail: email@example.com, https://orcid.org/0000-0002-9348-466X
*These authors contributed equally to this work as co-correspondence authors.
**These authors contributed equally to this work as co-first auhtors.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chaenomeles sinensis is known to inhibit the development and progression of many age-related diseases, but the underlying molecular mechanisms are largely unclear. In the present study, we observed that the ethanol extract of Chaenomeles sinensis scavenged 2,2’-diphenylpicrylhydrazyl and 2,2’-azinobis diammonium radicals in vitro. The ethanol extract of Chaenomeles sinensis activated antioxidant response element-luciferase activity and induced expression of NRF2 target genes in HaCaT cells. The ethanol extract of Chaenomeles sinensis also suppressed LPS-induced expression of COX-2 and iNOS proteins, and mRNA expression of TNF-α and IL-2 in RAW264.7 cells. Finally, the ethanol extract of Chaenomeles sinensis significantly suppressed testosterone propionate-induced benign prostatic hyperplasia in mice. Together, our study provides the evidence that the ethanol extract of Chaenomeles sinensis inhibits the development of benign prostatic hyperplasia by exhibiting anti-oxidant and anti-inflammatory effects.
Keywords: Benign prostatic hyperplasia, Chaenomeles sinensis, NF-E2-related factor 2, Lipopolysaccride, Oxidative stress
Benign prostatic hyperplasia (BPH) is a common disease among old-aged men . Epidemiological studies have demonstrated that various etiological factors are responsible for increasing the risk of the development and the progression of BPH . BPH occurs when both stromal and epithelial cells of the prostate in the transitional zone proliferate by cellular processes that are thought to be influenced by sex hormones and inflammation, resulting in the lower urinary tract syndromes (LUTS) . Treatment options for men with BPH include the oral administration of 5α-reductase inhibitors and α1-adrenergic receptor antagonists . Finasteride and durasteride are clinically available 5α-reductase inhibitors, which attenuate the development of BPH by suppressing the conversion of testosterone into dihydrotestosterone . On the other hand, doxazocin and tamsulosin are widely used α1-adrenergic receptor antagonists, which relieve the LUTS by relaxing the neck of the bladder and smooth muscles in the prostate . However, the use of these medications is restricted due to the adverse effects, such as erectile dysfunction, loss of libido, dizziness, and upper respiratory infection .
Plants have been the most utilized natural sources of pharmacologically active products due to the abundance and the accessibility . Therefore, exploring plant ingredients or plant extracts has been proposed as a strategy to inhibit or delay many age-related diseases .
HaCaT and RAW264.7 cells were purchased from the American Type Cell Collection (ATCC, Manassas, VA, USA). Testosterone propionate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol extract of
DPPH and ABTS assays were conducted as previously described [15,16].
Establishment of human keratinocyte HaCaT-ARE-GFP-luciferase cells were previously described . In brief, HaCaT-ARE-luciferase cells were seeded in six-well plates, cultured until 70% confluence, and exposed to ethanol extract of
HaCaT cells were plated in 96-well culture plates. After exposure of ethanol extract of
After acquisition, samples were incubated with 200 μL RIPA buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and protease inhibitors cocktail) for 1 hour on ice. Lysates were collected by centrifugation, and protein concentrations were measured by use of the BCA protein assay kit (Thermo Fisher, Pittsburgh, PA, USA). Equal amounts of lysates were resolved by SDS-PAGE and transferred to PVDF membrane. The membrane was incubated in blocking buffer (5% skim milk in 1x PBS-0.1% Tween-20, PBST) for 1 hour and hybridized with appropriate primary antibodies in 1x PBS containing 3% bovine serum albumin (BSA) overnight at 4°C. After washing three times with 1x PBST for 30 minutes, the membrane was hybridized with appropriate HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 hour at room temperature and washed three times with 1x PBST solution for 30 minutes. The membrane was visualized using an enhanced chemiluminescence (ECL) detection system. The image of β-actin was used as a control for equal loading of samples.
Total RNA was isolated using Hybrid-R RNA extraction kit (GeneAll, Seoul, Korea). One μg of total RNA was subject to cDNA synthesis, using PrimeScript RT-PCR kit (TaKaRa Korea, Seoul, Korea). Real-time PCR was performed on CFX96 instrument (Bio-Rad, Hercules, CA, USA) using EvaGreen Supermix (Bio-Rad). The primer sequences used for the quantitation of target genes are illustrated in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (human) or β-actin (mouse) was used as an internal control.
The animal experiment was carried under the Institutional Animal Care and Use Committee-approved Protocol (IACUC-2019-002-1) of Dongguk University (Seoul, Korea). Six-week-old Balb/c mice were purchased from Daehan Biolink (Eumseong, Korea), housed in the sterile filter-capped microisolator cages, and provided with water and diet ad libitum. Mice were separated and intraperitoneally injected with testosterone alone for 35 days to induce BPH or together with intraperitoneal injection of finasteride (positive control) or with oral administration of ethanol extract of
At sacrifice, mouse prostate tissues were excised and fixed in 10% formalin solution overnight. Tissue dehydration was performed by serially immersing the tissues into 75%, 80%, 85%, 90%, 95%, 100% ethanol, and xylene solution for 1 hour at each step. Dehydrated tissues were embedded in the paraffin block. Paraffin-embedded tissues were sectioned at 5 μm, mounted on the slide, and deparaffinized. Tissues were stained with Mayer’s hematoxylin solution for 5 minutes at room temperature and rinsed in tap water until the water becomes clear. In the bluing step, the tissues were stained with repeated cycles of eosin Y ethanol solution for 70 seconds, 5 dips in 95% ethanol, and 5 dips in 100% ethanol at room temperature. The tissues were rinsed with distilled water and the images were taken on the microscope (Olympus, Tokyo, Japan).
Tissues on the slide were incubated with 1% BSA blocking solution for 30 minutes. After washing three times with 1x PBS, the tissues were hybridized with primary antibodies overnight at 4°C. The slides were washed with 1x PBS three times and incubated with anti-rabbit and anti-mouse UltraTEk HRP antibodies (ScyTek Inc., Logan, UT, USA). Development of the slides was performed with 3’,3′-diaminobenzidine (DAB) (GBI Labs, Bothell, WA, USA). The slides were then sealed with mounting medium and the images were taken on the microscope (Olympus).
Statistical analyses were performed using Microsoft Exel (Microsoft, Redmond, WA, USA). Statistical analysis was conducted using Student’s
Aberrant production of reactive oxygen species (ROS) is responsible for the generation of oxidative stress . Oxidative stress can be relieved by treatment with antioxidants or by the induction of cellular phase II cytoprotective enzymes , the latter of which is under the control of transcription factor, NRF2 . In a normal condition, NRF2 is constantly poly-ubiquitinated by Kelch-like ECH-associated protein 1 (KEAP1), an adaptor of Cullin 3 E3 ubiquitin ligase complex. Upon exposure to oxidants or electrophiles, NRF2 translocates into the nucleus and activates the ARE, a cis-acting DNA element located in the promoter of NRF2 target genes . We first examined whether ethanol extract of
We next examined whether ethanol extract of
Because oxidative stress is closely associated with pro-inflammatory responses , we examined whether ethanol extract of
We examined whether ethanol extract of
Lee and colleagues  have characterized phytochemicals existing in the twig of
In the present study, we have demonstrated that ethanol extract of
This work was supported by the grant of the Sunchang Research Institute of Health and Longevity.
No potential conflicts of interest were disclosed.
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