Electroacupuncture inhibits the corneal ROS/TXNIP/NLRP3 signaling pathway in a rat model of dry eye syndrome

2021 ◽  
pp. 096452842110392
Author(s):  
Yanting Yang ◽  
Dan Zhang ◽  
Lijie Wu ◽  
Ji Zhang ◽  
Danyan Wu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Dry Eye ◽  
Clinical Symptoms ◽  
Dry Eye Syndrome ◽  
Receptor Protein ◽  
Sprague Dawley ◽  
Corneal Injury ◽  
Caspase 1 ◽  
Des Model

Background: Electroacupuncture (EA) treatment has been found to ameliorate clinical symptoms in patients with dry eye, but its mechanisms are still not entirely clear. Objective: To study the regulation of EA on ocular surface function and the corneal reactive oxygen species (ROS)/thioredoxin-interacting protein (TXNIP)/Nod-like receptor protein 3 (NLRP3) inflammatory signaling pathway in dry eye syndrome (DES) model rats. Methods: Male Sprague-Dawley (SD) rats were randomly divided into five groups: Normal, Model, Model + EA, Model + NAC (N-actetylcysteine) and Model + NS (normal saline). The DES model was developed by subcutaneous injection of scopolamine hydrobromide with exposure to an air draft in the latter four groups. After intervention, the Schirmer I test (SIT), tear film break-up time (BUT) and ROS content were measured, the histopathological changes of corneal tissues were observed, and the mRNA and protein expression levels of TXNIP, NLRP3, apoptosis-associated Speck-like protein containing CARD (ASC), caspase-1, interleukin (IL)-1β and IL-18 were detected. Results: Compared with the Model group, the SIT and BUT increased significantly in the Model + EA group after intervention (p < 0.05), and the corneal injury was improved. Corneal ROS content declined in both Model + EA and Model + NAC groups (p < 0.05), and mRNA expression of TXNIP, NLRP3, ASC and caspase-1 also decreased (p < 0.01). Corneal protein expression of TXNIP, NLRP3, IL-1β and IL-18 decreased significantly in the Model + EA group (p < 0.01). Conclusion: Inhibiting the ROS/TXNIP/NLRP3 signaling pathway may be the mechanism underlying the role of EA in improving corneal injury in DES model rats.

CLINICAL AND HISTOPATHOLOGICAL FEATURES OF SCOPOLAMINE HYDROBROMIDE INDUCED DRY EYE SYNDROME IN SPRAGUE-DAWLEY RATS

2021 ◽  
pp. 1-8
Author(s):  
Sze-Min Chan ◽  
Hui-Wen Chang ◽  
Pei-Shiue Tsai ◽  
Chian-Ren Jeng ◽  
Hao Lee ◽  
...  
Keyword(s):  
Dry Eye ◽  
Histopathological Examination ◽  
Tear Film ◽  
Dry Eye Syndrome ◽  
Therapeutic Interventions ◽  
Sprague Dawley ◽  
Corneal Surface ◽  
Fluorescein Staining ◽  
Scopolamine Hydrobromide ◽  
Volume Assessment

Dry eye syndrome (DES), is one of the most common and irritating ocular diseases in humans and animals due to deficits in quantities or/and quality of tear film. In this study, a rat model of experimental DES has been developed using the cholinergic inhibitor, scopolamine hydrobromide (SCOP), at the dose of 25[Formula: see text]mg/rat/day via subcutaneous injection, for a consecutive 21 days without low humidity environment. Clinical ophthalmic evaluations were performed by tear volume assessment using endodontic paper point, slit-lamp biomicroscope, and fluorescein staining at day 0, 7, 14, and 21 post-inductions. The results of ophthalmic examination showed that rats with SCOP treatment reduced about 40% of tear secretion. Half of the SCOP-treated rats exhibited diffuse corneal fluorescein staining involving 80% of the corneal surface, minimal keratoconjunctivitis, roughened corneal surface and thin corneal epithelium under histopathological examination. About 30% of the rats showed variable infiltration of lymphocytes in between the tubular acinar glands. This animal model with significant reduction of tear production and diffuse corneal fluorescein staining in rats could be used for the preclinical assessment of therapeutic interventions.

scholarly journals Utilizing Network Pharmacology to Explore the Possible Mechanism of Coptidis Rhizoma in Kawasaki Disease

2021 ◽  
Vol 9 ◽  
Author(s):  
Xue Fan ◽  
Xin Guo ◽  
Ying Li ◽  
Mingguo Xu
Keyword(s):  
Kawasaki Disease ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Drug Targets ◽  
Molecular Mechanisms ◽  
Enrichment Analysis ◽  
Receptor Protein ◽  
Network Pharmacology ◽  
Control Group ◽  
Coptidis Rhizoma

Background: The purpose of the research is to identify the main active ingredients in Coptidis Rhizoma (CR) and explore the possible molecular mechanisms in the treatment of Kawasaki disease (KD).Materials and Methods: A total of 58 children with KD were randomly divided into a control group and a Berberine treatment group. The therapeutic indicators of the two groups before and after treatment were compared. Then, compounds and drug targets of CR from the TCMSP, SWISS, SEA, and the STITCH were collected, and targeted KD genes were retrieved from the DisGeNET, DrugBank, and GeneCards databases. The network pharmacology approach involved network construction, target prediction, and module analysis. GO and KEGG enrichment analysis were performed to investigate the possible pathways related to CR for KD treatments. Finally, protein expression was determined to verify the core targets using Western blotting in the cell experiment.Results: In total, nine compounds, 369 relative drug targets, and 624 KD target genes were collected in the above database. The network analysis revealed that 41 targets might be the therapeutic targets of CR on KD. GO and KEGG enrichment analysis revealed that the biological processes, namely, response to hormone, response to inorganic substance, and enzyme-linked receptor protein signaling pathway, and Pathways in cancer, Toll-like receptor signaling pathway, and Pancreatic cancer are the most significant. Protein expression of CASP3, PTGS2, and SRC was upregulated and AKT1 and ERK were downregulated.Conclusion: We provided useful resources to understand the molecular mechanism and the potential targets for novel therapy of KD.

scholarly journals The Effects of Exogenous Lactate Administration on the IGF1/Akt/mTOR Pathway in Rat Skeletal Muscle

2020 ◽  
Vol 17 (21) ◽  
pp. 7805
Author(s):  
Sunghwan Kyun ◽  
Choongsung Yoo ◽  
Hun-Young Park ◽  
Jisu Kim ◽  
Kiwon Lim
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Protein Expression ◽  
Lactate Concentration ◽  
Ring Finger ◽  
Receptor Protein ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Expression Levels ◽  
Igf Receptor

We investigated the effects of oral lactate administration on protein synthesis and degradation factors in rats over 2 h after intake. Seven-week-old male Sprague–Dawley rats were randomly divided into four groups (n = 8/group); their blood plasma levels of lactate, glucose, insulin, and insulin-like growth factor 1 (IGF1) were examined following sacrifice at 0, 30, 60, or 120 min after sodium lactate (2 g/kg) administration. We measured the mRNA expression levels of protein synthesis-related genes (IGF receptor, protein kinase B (Akt), mammalian target of rapamycin (mTOR)) or degradation-related genes (muscle RING-finger protein-1 (MuRF1), atrogin-1) and analyzed the protein expression and phosphorylation (activation) of Akt and mTOR. Post-administration, the plasma lactate concentration increased to 3.2 mmol/L after 60 min. Plasma glucose remained unchanged throughout, while insulin and IGF1 levels decreased after 30 min. The mRNA levels of IGF receptor and mTOR peaked after 60 min, and Akt expression was significantly upregulated from 30 to 120 min. However, MuRF1 and atrogin-1 expression levels were unaffected. Akt protein phosphorylation did not change significantly, whereas mTOR phosphorylation significantly increased after 30 min. Thus, lactate administration increased the mRNA and protein expression of protein-synthesis factors, suggesting that it can potentially promote skeletal muscle synthesis.

scholarly journals Activation of M3 Muscarinic Acetylcholine Receptors Delayed Cardiac Aging by Inhibiting the Caspase-1/IL-1β Signaling Pathway

2018 ◽  
Vol 49 (3) ◽  
pp. 1249-1257 ◽  
Author(s):  
Shu Wang ◽  
Yanan Jiang ◽  
Jingling Chen ◽  
Changliang Dai ◽  
Dandan Liu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Muscarinic Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptors ◽  
Cardiac Aging ◽  
Expression Levels ◽  
Muscarinic Acetylcholine ◽  
Caspase 1 ◽  
Age Related

Background/Aims: Because the prevalence of age-related cardiac impairment increases as the human lifespan increases, it is important to combat the effects of aging. Recently, the cardiac M3 muscarinic acetylcholine receptor (M3-mAChR) has been demonstrated to play important roles in cardiac development and in the pathogenesis of cardiac diseases. However, the role of M3-mAChR in aging remains largely unknown. Therefore, the aim of this study was to investigate the involvement of M3-mAChR in the progression of cardiac aging. Methods: We established a cardiac aging model in mice through subcutaneous injection with D-galactose at a dose of 100 mg/kg/day for 6 weeks. D-galactose was also used to induce aging in primary cultured neonatal mouse cardiomyocytes. The myocardium from mice was stained with hematoxylin and eosin for histological analysis. The protein expression levels of p53 and p21 were determined using western blotting. The mRNA and protein expression levels of M3-mAChR, caspase-1, and interleukin (IL)-1β were determined using real-time PCR, immunohistochemical staining, and western blotting. Results: The expression of M3-mAChR was down-regulated in the myocardium from aged mice and D-galactose-treated mice, while the expression levels of caspase-1 and its downstream molecule IL-1β were significantly increased. The M3-mAChR agonist choline reduced the increase in caspase-1 in cardiomyocytes induced by D-galactose, which was reversed by the M3-mAChR antagonist 4-DAMP. Moreover, 4-DAMP promoted D-galactose-induced cardiomyocyte aging, which was attenuated by a caspase-1 inhibitor. Conclusion: Activation of M3-mAChR delayed cardiac aging by inhibiting the caspase-1/IL-1β signaling pathway.

scholarly journals PO-144 Intermittent Exercise Activates NRG1-SERCA2a Pathway to Improve Cardiac Function in Myocardial Infarction Rats

2018 ◽  
Vol 1 (4) ◽  
Author(s):  
Wenyan Bo ◽  
Dagang Li ◽  
Zhenjun Tian
Keyword(s):  
Myocardial Infarction ◽  
Cardiac Function ◽  
Protein Expression ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Intermittent Exercise ◽  
Critical Role ◽  
Interval Training ◽  
Sprague Dawley ◽  
Neuregulin 1

Objective  Intermittent exercise can improve cardiac function in rats with myocardial infarction. The Neuregulin-1(NRG1)/SERCA2a palys a critical role in maintain cardiac function. We want to investigate the effect of Neuregulin-1 (NRG1) on NRG1-SERCA2a signaling pathway activated by intermittent exercise and on improves cardiac function in rats with MI. Methods 32 male sprague-dawley rats were randomly divided into four groups (n=8): Sham-operated group (S), sedentary MI group (MI), MI with interval training group (ME), ME with inhibitor AG1478 group (MA). ME and MA model after the MI model was established by ligation of the left anterior descending coronary artery, and began training 1 week after MI surgery. The S model only by threading without ligation. Rats in ME and MA model taken one week adaptive training, then began 8-week interval training. MA model were injected with inhibitor AG1478, once every two days. The 24h after training, rats were anesthetized, the LVSP, LVEDP, ±dp/dt max were tested by carotid artery intubation which in order to evaluate cardiac function. The protein expression of NRG1, PI3K, Akt, eNOS, PKG, PLN, SERCA2a in myocardium were measured by Westernblotting, themRNA expression of serca2a were tested by RT-qPCR. Results Compared with S, the protein expression of NRG1, PKG, peNOS, pAkt, pPLN, pPI3K and SERCA2a decreased, serca2a mRNA expression decreased, LVSP and ±dp/dt max significantly decreased, LVEDP significantly increased; Compared with MI, the protein expression of NRG1, PKG, peNOS, pAkt, pPLN, pPI3K and SERCA2a increased, serca2a mRNA expression increased, LVSP and ±dp/dt max significantly increased, LVEDP decreased, and the effect of exercise were weaken by inhibitor AG1478. Correlation analysis showed that the myocardial pPLN and SERCA2a protein expression both were positively correlated with LVSP, ±dp/dtmax, and negatively correlated with LVEDP. Conclusions Intermittent exercise can increased myocardial NRG1 protein expression and activates NRG1-SERCA2a signaling pathway, improve myocardial infarction cardiac function.

scholarly journals Effect of Tripterygium wilfordii Polycoride on the NOXs-ROS-NLRP3 Inflammasome Signaling Pathway in Mice with Ulcerative Colitis

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Ma Fangxiao ◽  
Ke Yifan ◽  
Zhong Jihong ◽  
Shen Yan ◽  
Liu Yingchao
Keyword(s):  
Ulcerative Colitis ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Nlrp3 Inflammasome ◽  
Receptor Protein ◽  
Tripterygium Wilfordii ◽  
Expression Levels ◽  
Caspase 1 ◽  
Model Control ◽  
Mrna Expression Levels

Objective. To explore the effect of Tripterygium wilfordii polycoride (TWP) on the NADPH oxidases (NOXs)-reactive oxygen species (ROS)-NOD-like receptor protein 3 (NLRP3) inflammasome signaling pathway and the possibility of using TWP to treat ulcerative colitis (UC). Methods. BALB/c mice were randomly divided into five groups: model control, low TWP, middle TWP, high TWP, and normal control groups. A UC model was established with dextran sulfate sodium. The determination of ROS was carried out by using the fluorescent probe DCFH-DA, and NOXs activity was detected based on the NADPH consumption rate. The mRNA expression levels of NLRP3, ASC, and caspase-1 in the colon tissues and neutrophils were assessed via real-time PCR. Results. The colon tissues were abnormal with different degrees in TWP groups with disease activity index and histopathological scores lower than those in the model group. In TWP groups, ROS generation, NOXs activity, and the mRNA expression levels of NLRP3, ASC, and caspase-1 in the colon tissues and colon-isolated neutrophils were remarkably lower than those in the model control group (P<0.05) and higher than those in the normal group (P<0.05). The results of pairwise comparison for the efficacy of TWP administration showed that the above indexes were statistically significant with the lowest expression in the high TWP group (P<0.05) and the highest expression in the low TWP group (P<0.05). Conclusion. TWP demonstrated anti-inflammatory effects on UC by decreasing the expression of proinflammatory factors in the NOXs-ROS-NLRP3 signaling pathway.

scholarly journals Functional estrogen receptor signaling pathway activity in high-grade serous ovarian carcinoma as compared to estrogen receptor protein expression by immunohistochemistry

2021 ◽  
Author(s):  
Phyllis van der Ploeg ◽  
Laura A. M. van Lieshout ◽  
Anja van de Stolpe ◽  
Steven L. Bosch ◽  
Marjolein H. F. M. Lentjes-Beer ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Target Genes ◽  
Estrogen Therapy ◽  
Receptor Protein ◽  
Mrna Levels ◽  
High Grade ◽  
Ovarian Carcinomas ◽  
Pathway Activity

Abstract Purpose Anti-estrogen therapy may be used as a palliative treatment option in high-grade serous ovarian carcinomas (HGSC). However, clinical implementation is limited as the use of estrogen receptor (ER) protein expression by immunohistochemistry remains insufficient in predicting therapy response. To determine the accuracy of ER protein expression as a marker for ER signaling pathway activity, we aimed to correlate ER protein expression to functional ER signaling pathway activity in HGSC. Methods Immunohistochemical ER protein expression was visually scored using total percentages of stained tumor cells and histoscores. Subsequently, mRNA was extracted, and RT-qPCR analysis was performed. Functional ER pathway activity was assessed by a computational Bayesian model inferring ER signaling pathway activity from mRNA levels of ER-specific target genes. Results Our analysis of 29 HGSCs shows that neither total percentage of ER protein expression, nor ER histoscores are significantly correlated to ER signaling pathway activity (respectively, p = 0.473 and p = 0.606). Classification of HGSC into three groups based on ER histoscores 0–100 (n = 6), 101–200 (n = 15) and 201–300 (n = 8) resulted in comparable mean ER signaling pathway activity among the groups (p = 0.356). Several samples in the higher ER histoscore groups had low ER signaling pathway activity, indicating that nuclear ER protein expression is not sufficient to describe transcriptional ER activation. Conclusion Positive immunohistochemical ER staining is not always indicative of an active ER signaling pathway and is, therefore, a poor predictor of anti-estrogen response. Further research is needed to prove the predictive value of ER signaling pathway activity regarding anti-estrogen sensitivity in HGSC patients.

Long-chain PUFA ameliorate ETEC-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in IPEC-1 cells

2021 ◽  
pp. 1-36
Author(s):  
Kan Xiao ◽  
Yang Yang ◽  
Yang Zhang ◽  
Qingqing Lv ◽  
Feifei Huang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Signaling Pathways ◽  
Cell Injury ◽  
Intestinal Inflammation ◽  
Fluorescein Isothiocyanate ◽  
Receptor Protein ◽  
Phosphoglycerate Mutase ◽  
Death Domain ◽  
Caspase 1

Abstract This study was aimed to investigate whether eicosapentaenoic acid (EPA) and arachidonic acid (ARA), the representative n-3 or n-6 polyunsaturated fatty acids (PUFA), could alleviate enterotoxigenic Escherichia coli (ETEC) K88-induced inflammation and injury of intestinal porcine epithelial cells 1 (IPEC-1) by modulating pyroptosis and necroptosis signaling pathways. IPEC-1 cells were cultured with or without EPA or ARA in the presence or absence of ETEC K88. EPA and ARA reduced ETEC K88 adhesion and endotoxin content in the supernatant. EPA and ARA increased transepithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-labeled dextran (FD4), and increased membrane protein expression of occludin, ZO-1 and claudin-1, and relieved disturbed distribution of these proteins. EPA and ARA also reduced cell necrosis ratio. EPA or ARA reduced mRNA and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8, and decreased mRNA abundances of intestinal toll-like receptors 4 (TLR4) and its downstream signals. Moreover, EPA and ARA downregulated mRNA expression of nod-like receptor protein 3 (NLRP3), caspase 1 and IL-18, and inhibited protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D and caspase-1. Finally, EPA and ARA reduced mRNA expression of fas-associated death domain protein (FADD), caspase 8, receptor interacting protein kinase (RIP) 1, mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase 5 (PGAM5), motility related protein 1 (Drp1) and high mobility protein 1 (HMGB1), and inhibited protein expression of phosphorylated-RIP1 (p-RIP1), p-RIP3, p-MLKL and HMGB1. These data demonstrate that EPA and ARA prevent ETEC K88-induced cell inflammation and injury, which is partly through inhibiting pyroptosis and necroptosis signaling pathways.

scholarly journals Protective Effects of Microrna-22 Against Endothelial Cell Injury by Targeting NLRP3 Through Suppression of the Inflammasome Signaling Pathway in a Rat Model of Coronary Heart Disease

2017 ◽  
Vol 43 (4) ◽  
pp. 1346-1358 ◽  
Author(s):  
Wei-Qiang Huang ◽  
Peng Wei ◽  
Ri-Qi Lin ◽  
Feng Huang
Keyword(s):  
Coronary Heart Disease ◽  
Heart Disease ◽  
Endothelial Cell ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lumen Formation ◽  
Caspase 1 ◽  
Mrna And Protein Expression

Background/Aims: This study aimed to identify the role of microRNA-22 (miR-22) in endothelial cell (EC) injury in coronary heart disease (CHD) by targeting NLRP3 through the inflammasome signaling pathway. Methods: A total of 24 healthy male Sprague-Dawley (SD) rats were divided into normal and atherosclerosis groups. The atherosclerosis rats were assigned into blank, negative control (NC), miR-22 mimic, miR-22 inhibitor and miR-22 inhibitor + siNLRP3 groups. A luciferase reporter gene assay was used to detect the relationship between miR-22 and NLRP3. MiR-22 expression as well as NLRP3 and caspase-1 mRNA and protein expression were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The activity and apoptosis of coronary arterial endothelial cells (CAECs) were determined by MTT and Hoechst 33258. CAEC lumen formation was detected by a lumen formation assay. An enzyme-linked immunosorbent assay (ELISA) was used to detect IL-1β, IL-6, IL-10 and IL-18 levels. Results: The results indicated that the atherosclerosis group significantly decreased miR-22 expression but increased NLRP3 and caspase-1 mRNA and protein expression. The cell survival rate was significantly increased in the miR-22 mimic group and significantly reduced in the miR-22 inhibitor group. The miR-22 mimic group displayed a lower apoptosis rate and more cells with obvious lumen walls and numerous tubular structures, while cells in the miR-22 inhibitor group were unable to form lumen walls and had a scattered distribution compared to the blank group. The ELISA showed that IL-1β, IL-6 and IL-18 levels were markedly decreased, while IL-10 was clearly increased in the miR-22 mimic group. In contrast, in the miR-22 inhibitor group, IL-1β, IL-6 and IL-18 levels were significantly increased, and IL-10 levels were decreased. Conclusion: Our findings indicated that miR-22 could lower the levels of pro-inflammatory cytokines by inhibiting the NLRP3 inflammasome pathway, which suppresses CAEC apoptosis and protects CAECs in rats with CHD.

scholarly journals The Protective Effect of Aesculin Eye Drops on Dry Eye Syndrome Mice

2020 ◽  
Author(s):  
Xinquan Liu ◽  
Jing Su ◽  
Yue Fang ◽  
Dahu Wang ◽  
Dan Jiang ◽  
...  
Keyword(s):  
Treatment Group ◽  
Dry Eye ◽  
Treatment Method ◽  
Dry Eye Syndrome ◽  
Eye Drops ◽  
The Novel ◽  
Murine Models ◽  
Corneal Injury ◽  
Tnf Α ◽  
Ifn Γ

Abstract Background: The objective of this study was to test the effectiveness of the novel aesculin (AES) eye drops on treating dry eye syndrome (DES), using murine model. Methods: Dry eye was induced in murine eye with the use of an intelligent controlled environmental system (ICES). High-flow air which had been desiccated within the ICES, induced the DES. Treatment included Aesculin eye drops for 14 days. After expiration, corneal fluorescein staining was examined and stained with an endothelial maker created for tracking of lymph: Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Subsequently, real-time PCR and ELISA were completed to quantify secretion of TNF-α，IFN-γ，IL-1β and IL-8 in serum, ocular surface and lacrimal gland. Results: Aesculin eye drops significantly repaired ICES-induced corneal injury in murine models Statistically significant increase in lymph angiogenesis was observed at day 14 of observation. Consequently, DES in treatment group was reduced using aesculin eye drops. aesculin eye drops can significantly inhibit lymphangiogenesis and reduce inflammatory cytokines levels in mice. The percentage of Th1 and Th17 cells were decreased after treating with aesculin eye drops, while the percentage of Treg was increased. Injection of VEGF-C into mice can reduce the efficacy of aesculin eye drops in ICES-induced DES. Conclusion: as treatment group of murine models indicated significant reduction of inflammation in the cornea associated with DES, treatment of DES with aesculin eye drops, should be considered a viable treatment method for DES.

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