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Molecular and Cellular Endocrinology xxx (2017) 1e13

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier .com/locate/mce

Quercetin inhibited epithelial mesenchymal transition in diabetic rats,high-glucose-cultured lens, and SRA01/04 cells through transforminggrowth factor-b2/phosphoinositide 3-kinase/Akt pathway

Lei Du a, 1, Meng Hao a, 1, Chengcheng Li a, Wenya Wu a, WenwenWang a, Zhongxuan Ma a,Tingting Yang b, Nan Zhang a, Adelusi Temitope Isaac a, Xia Zhu a, Ying Sun a, Qian Lu a, **,Xiaoxing Yin a, *

a Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou 221004, Chinab Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, China

a r t i c l e i n f o

Article history:Received 20 December 2016Received in revised form9 May 2017Accepted 9 May 2017Available online xxx

Keywords:QuercetinDiabetic cataractLensTGF-b2AktEMT

* Corresponding author. Department of Clinical Phmacy, Xuzhou Medical University, NO. 209. Tongs221004, China.** Corresponding author. Department of Clinical Phmacy, Xuzhou Medical University, NO. 209. Tongs221004, China.

E-mail addresses: prairy@126.com (Q. Lu), yinxx@1 Lei Du and Meng Hao contributed equally to this

Please cite this article in press as: Du, L., etlens, and SRA01/04 cells through transEndocrinology (2017), http://dx.doi.org/10.1

a b s t r a c t

Diabetic cataract (DC), an identified life-threatening secondary complication of diabetes mellitus, hasproven to be a dilemma because of its multifactorial caused and progression. An increasing number ofstudies have shown that in addition to the maillard reaction, enhanced polyol pathway, and oxidativeinsults, epithelial mesenchymal transition (EMT) is related to the prevalence of DC. Quercetin, a classicflavonoid with multiple pharmacological effects has been reported to possess therapeutic efficacy in themanagement and treatment of this disease. However, the mechanism underlying its therapeutic efficacyin EMT of lens epithelial cells (SRA01/04) and contribution to resolving DC remains a mystery. Therefore,in this study, we investigated the effects of quercetin on EMT of SRA01/04 and high-glucose (HG)-induced lens opacity accompanied by lens fibrosis induced by type-1 diabetes. Furthermore, we soughtto clarify the specific mechanisms underlying these effects. At week 14 after streptozotocin (STZ)intraperitoneal administration, diabetic rats showed lens opacity accompanied with diminished anti-oxidant function, enhanced polyol pathway activity, and non-enzymatic glycation. Western blottingconfirmed EMT in rat SRA01/04 cells with significantly increased a-smooth muscle actin (a-SMA) anddecreased E-cadherin expressions. Treatment of the lens with quercetin ameliorated the oxidative stress,inhibited aldose reductase (AR) activation, reduced advanced glycation end product (AGE) production,and finally suppressed EMT in the early stages. Our in vitro results showed that high-glucose activatedthe transforming growth factor-b2/phosphoinositide 3-kinase/protein kinase B (TGF-b2/PI3K/Akt) sig-nalling and EMT in SRA01/04 cells. Further, induced oxidative stress, activation of aldose reductase, andaccumulation of advanced glycation end products were also involved in this process. Quercetin waspotent enough to effectively ameliorate the high glucose (HG)-induced EMT of SRA01/04 cells byinhibiting the activation of TGF-b2/PI3K/Akt, enhancing the antioxidant capacity, inhibiting AR activity,and reducing AGE production. From the whole animal to tissues, and finally the cellular level, our resultsprovide considerable evidence of the therapeutic potential of quercetin for DC. This might be due to itsinhibition of EMT mediated through inhibition of the TGF-b/PI3K/Akt pathway.

armacology, School of Phar-han Road, Xuzhou, Jiangsu

xzhmu.edu.cn (X. Yin).manuscript (co-first author).

al., Quercetin inhibited epithforming growth factor-b2/p016/j.mce.2017.05.011

1. Introduction

Hyperglycaemia is a risk factor for numerous diabetic compli-cations including diabetic cataract (DC). DC, which is characterizedby lens opacity, is one of the earliest secondary complications ofdiabetes mellitus associated with hyperglycaemia (Pollreisz andSchmidt-Erfurth, 2010; Albulescu and Zolog, 2011). Chronichyperglycaemia induces oxidative insults, osmotic damage, and

elial mesenchymal transition in diabetic rats, high-glucose-culturedhosphoinositide 3-kinase/Akt pathway, Molecular and Cellular

L. Du et al. / Molecular and Cellular Endocrinology xxx (2017) 1e132

non-enzymatic glycation processes in optical lens, which are theprimary reasons for DC progression (Pollreisz and Schmidt-Erfurth,2010). Thus, the decreased activity of antioxidant enzymes andreducing substances, excessive activation of aldose reductase (AR),and the accumulation of advanced glycation end products (AGEs)were considered as signs of the occurrence of DC in previousstudies (Sadi et al., 2012; Richer et al., 2015; Kandarakis et al., 2014).Current evidence supports the view that there is a close relation-ship between cataract and epithelial to mesenchymal transition(EMT), which is the transdifferentiation of epithelial cells intomesenchymal cells. The EMT of lens epithelial cells (SRA01/04)describe a series of events during which epithelial cells lose severalepithelial characteristics, such as E-cadherin, and acquire proper-ties typical of mesenchymal cells, such as a-smooth muscle actin(a-SMA) (Kim et al., 2012). Although, growing evidence has impli-cated this process as a major pathway leading to the generation ofcataract, it is not yet known if the prevalence of diabetic cataract isrelated to EMT of lens epithelial cells.

Many factors could lead to EMT including the induction oftransforming growth factor-b (TGF-b), which has been recognizedto be one of the strongest cell cytokines involved in the regulationof lens epithelial EMT (Katsuno et al., 2013). Among the three ho-mologous isoforms of TGF-b (TGF-b1, TGF-b2, and TGF-b3), TGF-b2has the highest expression with the highest activity in the orbitaltissue. Moreover, TGF-b2 can induce abnormal proliferation, dif-ferentiation, and apoptosis in lens epithelial cells, which also con-tributes to cataract formation (Meng et al., 2013). Phosphoinositide3-kinase (PI3K) is an important downstream signalling molecule ofTGF-b, which is involved in the regulation of cell division, differ-entiation, and apoptosis. Protein kinase B (PKB), also known as Akt,plays an important role in regulating cell survival, and can beactivated by PI3K (Zhang et al., 2013). Studies have shown thattreatment of HLEB-3 human lens epithelial cells with TGF-b2 leadsto morphological changes, inhibition of cell connexion, and fibro-nectin expression with increased binding protein expression,thereby leading to EMT. Activation of the PI3K/Akt pathway plays akey role in the TGF-b2-induced HLEB-3 EMT process (Yao et al.,2008). High blood glucose stimulates the production of TGF-b2 byreleasing of reactive oxygen species (ROS) in the cell (Tang and Lai,2012). Therefore, it is reasonable to propose that high glucose (HG)-induced EMT in lens epithelial cells is mediated by the TGFb2/PI3K/Akt pathway, which can be activated by ROS (Abboud, 2012).

Some natural Chinese medicine extracts that express little or noside effects have gradually been recognized by researchers as po-tential candidates in the prevention/treatment of diabetic cataract.Quercetin, 3,30,4,5,7- pentahydroxy flavone is one of the mostimportant flavonoids in the human diet and idwidely distributed innumerous plant leaves, flowers, and fruits. It possesses numerouspharmacological activities including antioxidant, anti-inflammatory, anti-aging, and anti-fibrotic properties (Yoon et al.,2012). A study by Yin et al. (2013) showed that quercetin inhibi-ted the activity of catalase and increased the glutathione (GSH) andglutathione disulphide (GSSG) ratio, thereby abating DNA damageand immune inflammatory response. It was also reported thatquercetin effectively inhibited hydrogen peroxide (H2O2)-inducedlens opacity (Sanderson et al., 1999). Kador and Kinoshita (1984)showed that quercetin has a strong inhibitory effect on rat lensaldose reductase (Kinoshita, 1990). Another study by Kim et al.(2007) showed that quercetin inhibited the formation of AGEs bytrapping active carbonyl compounds (Li et al., 2014). In addition, arecent study found that quercetin inhibited the TGF-b signallingpathway in human corneal cells, thereby reducing fibrosis andextracellular matrix deposition (McKay et al., 2015). Therefore, withthis considerable body of published supporting the immensepharmacological potency of quercetin, it would not be an

Please cite this article in press as: Du, L., et al., Quercetin inhibited epithlens, and SRA01/04 cells through transforming growth factor-b2/pEndocrinology (2017), http://dx.doi.org/10.1016/j.mce.2017.05.011

exaggeration to suggest that quercetin's preventive efficacy againstDC might emanate from its actions on multiple therapeutic targets.

It is well known that the surgical removal of opaque lens is thecommon clinical treatment for DC. However, some patients find itchallenging to accept surgical treatment involving the removal ofopaque lens, which infers that early drug treatment would bepreferable for preventing DC. Therefore, to evaluate the effects ofquercetin and clarify its nutraceutical and therapeutic potential inthe prevention and treatment of DC, we designed this project toexamine its effect on antioxidant enzymes, AGEs levels, AR activity,and EMT of lens epithelial cells in vivo and in vitro.

2. Materials and methods

2.1. Cells and animals

SRA01/04, the SV40 T-antigen-transformed human lensepithelial cell line, was obtained from Cancer Institute of the Chi-nese Academy of Medical Sciences. All experiments and animalhandling in this study were performed in accordance with theGuidelines and Principles for Care and Use of Laboratory Animals ofXuzhou Medical University. Adult male Sprague-Dawley rats(weighing approximately 200 g) were provided by the LaboratoryAnimal Center of Xuzhou Medical University (Xuzhou, China). Theanimals were housed under standard laboratory conditions (23 �Cand 12-h light/dark cycle) with food and water given ad libitum. Allthe rats were subjected to ophthalmic examination and those withany ophthalmic abnormalities were excluded from the study.

2.2. Reagents

The quercetin (�98% purity) used in the cell-based studies andstreptozotocin (STZ) were purchased from Sigma-Aldrich (St. Louis,MO, USA) while the quercetin (�98% purity) used in the animalexperiments was purchased from Shaanxi Huike Plant Develop-ment Co., Ltd. The pirenoxine sodium (PS) eye drops were obtainedfrom EBE Pharmaceutical Co., Ltd. The LY294002 used in cells waspurchased from Selleck (St. Houston, TX, USA). T-superoxide dis-mutase (T-SOD) and GSH kits were purchased from JianchengBioengineering Institute (Nanjing, China) while the GSH/GSSG,catalase (CAT), glutathione peroxidase (GSH-Px) and malondial-dehyde (MDA) kits were purchased from JieMei BioengineeringInstitute (Shanghai, China). Tropicamide eye drops was purchasedfrom Wuhan Wujing Medicine Co., Ltd. Medium 199 (M-199,M3769) was purchased from Sigma-Aldrich, (St. Louis, MO, USA).The E-cadherin, a-SMA, and TGF-b2 antibodies were purchasedfrom Abcam (Cambridge, UK) while Akt, phosphorylated (p)-Akt,and b-actin antibodies were purchased from Bioworld Technology(St. Louis, MO, USA).

2.3. Experimental design

To detect the protective role of quercetin on DC, we establishedan in vivo STZ-induced DC Sprague-Dawley rat model, and in vitroexperiments were performed in rat and human lens epithelial cells.The specific experimental groups and details are illustrated inTables 1e3 (see Fig. 1).

In vivo experimentsIn vitro experiments

2.4. Establishment of animal models and evaluation of cataractprogression in diabetic rats

The rat lenses were examined using slit lamp microscopy andthose without lens defect were included for induction of DC. Fifty

Fig. 1. STZ injection can induce diabetic cataract in SD rats.

Table 1Groups of SD rats.

Groups n Treatment

Normal control (NC) 8 equal volume 1% CMC-Na solutionDiabetic cataract (DC) 8 equal volume 1% CMC-Na solutionLow dosage of quercetin (QL) 9 30 mg/kg, ig.Moderate dosage of quercetin (QM) 10 60 mg/kg, ig.High dosage of quercetin (QH) 9 120 mg/kg, ig.Pirenoxine sodium (PS) 9 2 drops/time, 2 times/day

Table 2Groups of rat lens.

Groups n Treatment

Normal control (NC) 6 2.40 mmol/L glucose M199High glucose (HG) 6 50 mmol/L glucose M199Low dosage of quercetin (QL) 6 HGþ10 mg/ml quercetinModerate dosage of quercetin (QM) 6 HGþ20 mg/ml quercetinHigh dosage of quercetin (QH) 6 HGþ 40 mg/ml quercetinPirenoxine sodium (PS) 6 HGþ 0.5 mmol/L pirenoxine sodium

Table 3Groups of human lens epithelial cells.

Groups n Treatment

Normal control (NC) 3 5.56 mmol/L glucose MEMHigh glucose (HG) 3 50 mmol/L glucose MEMLow dosage of quercetin (QL) 3 HGþ 1 mmol/L quercetinModerate dosage of quercetin (QM) 3 HGþ 2 mmol/L quercetinHigh dosage of quercetin (QH) 3 HGþ 4 mmol/L quercetinPirenoxine sodium (PS) 3 HGþ 4 mmol/L pirenoxine sodiumPI3K inhibitor LY294002 (LY) 3 HGþ20 mmol/L LY294002

L. Du et al. / Molecular and Cellular Endocrinology xxx (2017) 1e13 3

rats were fasted overnight and then intraperitoneally injected with65 mg/kg STZ (freshly dissolved in pH 4.5 citrate buffers beforeadministration). Eight normal rats (NC group) received the sameamount of buffer only. The fasting blood glucose (FBG) wasmeasured 3 days after STZ administration and rats withlevels � 13.88 mmol/L were considered diabetic. The diabetic ratswere subsequently randomly divided into the following fivegroups, which were treated as indicated: 1% sodium carboxymethylcellulose (CMC-Na) solution (DC group, n ¼ 8); 30, 60, and 120 mg/kg quercetin (QL, QM, and QH groups, n¼ 9,10, and 9, respectively);and PS group (n ¼ 9) two drops one time and twice a day.Furthermore, the rats in the NC group were administered 1% CMC-Na solution at the same volume (Table 1). The animals wereallowed free access to food and water ad libitum and wereeuthanised 14 weeks later. The body weight and blood glucosewere measured before and after treatment.

The development of rat cataract was examined using slit lampobservation by dilating the pupils with 0.25% tropicamide. At least5 min later, the rats were anaesthetised by intraperitoneallyinjecting 10% chloral hydrate at a dose of 0.35 mL/100 g bodyweight. The cataract was scored on a scale of IeV according to thedescription of Azuma et al. (1990). Grade I was a normal clear lens,grade II showed subcapsular opacity, grade III exhibited nuclearcataract, grade IV showed a strong nuclear cataract, and grade Vexhibited dense opacity involving the entire lens. Relevant imageswere captured using a 66Vision Tech YZ5T900 imaging system.

2.5. Rat lens organ culture and analysis of lens opacity

The lens culture method used was previously described by Luet al. (2014). Briefly, the lenses were incubated at 37 �C underconditions of 95% air and 5% CO2 for 24 h. Then, the selected 36transparent lenses were divided into the following six groups andtreated as indicated: normal group (NC, 5.56 mmol/L glucose plus44.44 mmol/L mannitol); HG group (HG, 50 mmol/L glucose plus0.5‰ dimethyl sulphoxide, DMSO); low-, moderate-, and high-dosequercetin groups (QL, QM, and QH, administered 10, 20, and 40 mg/mL quercetin, respectively plus 50 mmol/L glucose each); and PSgroup (50 mmol/L glucose plus 0.5 mmol/mL PS eye drops, Table 2).

All the lenses were observed under an optical microscope andphotographed on a black background. The photos were scannedinto a computer and the ImageJ image processing software (Na-tional Institutes of Health, NIH, Bethesda, MD, USA) was used tocalculate the grey value of the image and analyse the degree of lensopacity. The analytical method of lens opacity determination usedwas previously described by Olofsson et al. (2005). The photo-graphic images were converted to grey scale, and each image pixelwas automatically assigned one of 256 grey scale levels. The stan-dard deviation (SD) and mean density (MD) of the values obtainedfor each pixel within a central circular 1/mm2 area of the lens werecalculated. The development of cortical cataract would show moregrey and less black/white pixels and, thereby, lower the SD. TheMDis only slightly affected by cortical cataract, but a nuclear opacifi-cation would act as a filter, making the pixels darker and the MDhigher. The highest SD obtained for a clear lens in the material andthe MD for a completely opaque lens were used to construct aformula to quantify the percentage lens opacification in arbitraryunits (a.u.): (1-SD/59.4) � (MD/255) � 100.

2.6. Cell culture and cytomorphology

The cells were cultured in minimum essential medium (MEM,with 5.56 mmol/L glucose) containing 10% foetal bovine serum(FBS), supplemented with 100 U/mL penicillin and 100 mg/mLstreptomycin at 37 �C in a humidified 5% CO2 atmosphere. Cellswere grown in the normal medium until they were 60%e80%confluent. The cultured cells were divided into the followinggroups: NC, 5.56 mmol/L glucose plus 44.44 mmol/L mannitol; HG,50 mmol/L glucose plus 0.5‰ DMSO; QL, QM, and QH 1, 2, and4 mmol/L quercetin, respectively plus 50 mmol/L glucose each; PS,50 mmol/L glucose plus 4 mmol/L PS eye drops; and PI3K inhibitorgroup (LY, 50 mmol/L glucose plus 20 mmol/L LY294002, Table 3).After a 24-h incubation, the cell morphology was observed using anOlympus IX53 microscope (Tokyo, Japan) and the cells were har-vested and stored at �20 �C for subsequent analysis.

2.7. Analysis of lens epithelial cell E-cadherin and a-SMAexpression using immunofluorescence microscopy

SRA01/04 cells cultured on glass coverslips were washed thricewith cold phosphate-buffered saline (PBS) and fixed with cold

methanol at �20 �C for 20 min. After washing thrice with PBS, thecells were blocked with 5% bovine serum albumin (BSA) for 1 h at25 �C and incubated with anti-E-cadherin or anti-a-SMA antibodiesfor 2 h at room temperature, followed by incubation with a sec-ondary antibody conjugated with DyLight 594 (Earthox, Millbrae,CA, USA) at 37 �C for 1 h. Subsequently, the cells were stained with40,6-diamidino-2-phenylindole (DAPI, Beyotime Institute ofBiotechnology, Nantong, China) to detect the cell nuclei. The cov-erslips weremounted onto glass slides and the images were viewedusing an Olympus BX43F fluorescence microscope (Tokyo, Japan).

2.8. Haematoxylin and eosin (H&E) staining andimmunohistochemistry of rat lens epithelial cells

For the haematoxylin and eosin (H&E) staining, the rats wereeuthanised at the end of week 14, the lenses were harvested, andthen fixed in 4% formaldehyde for more than 24 h after paraffin-embedding. Then lens were subsequently dehydrated using afully automated dewateringmachine, with the following sequentialsteps: 75% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, 100%ethanol I, 100% ethanol II, xylene I, xylene II, paraffin I, paraffin II,and paraffin III, each run for 1 h. Next, the rat eyeballs of each groupwere embedded in paraffin and treated sequentially for 10min eachwith xylene and xylene II dewaxing, followed by 100%, 95%, 80%,and 70% ethanol, and then after 5 min they were placed in distilledwater. Finally, the lens were treated according to the followingprotocol: haematoxylin staining for 3 min; tap water rinse for5 min, which was then change to distilled water; 1% hydrochloricacid alcohol treatment for 10 s; tap water wash for 5 min; 1%lithium carbonate Portland treatment for 1 min; 80% ethanol for2 min; staining for 50 s; sequential dehydration, 95%, 95%, 100%,and 100% ethanol for 2 min each; xylene and xylene II dewaxing for1 min; and then transparent neutral gum mounting.

For the immunohistochemistry, the rat lens samples were fixedin 4% paraformaldehyde and embedded in paraffin. Sections (3-mMthick) were cut perpendicularly to the long axis of the lenses forimmunohistochemistry. The paraffin embedded lens sections weredeparaffinized in xylene, hydrated in graded concentrations ofalcohol and water, and then subsequently placed in 3% H2O2 toeliminate endogenous peroxidase activity. Then, the sections wereblocked with normal goat serum, followed by incubationwith anti-E-cadherin and anti-a-SMA antibodies overnight at 4 �C. The sec-tions were stained using a polymer horseradish peroxidase (HRP)detection system (ZSGB-BIO, Beijing, China), counterstained withhaematoxylin, and the examined using an Olympus BX43Fmicroscope.

Table 4Effects of quercetin on the fasting blood glucose of diabetic rats.

Group n Fasting blood glucose (mmol/L)

Week0 Week14

NC 8 4.6 ± 2.1 6.3 ± 2.5DC 8 21.1 ± 3.9## 26.6 ± 3.3##

QL 9 20.1 ± 3.3 23.1 ± 7.1QM 10 21.0 ± 5.5 27.2 ± 2.7QH 9 20.2 ± 4.9 21.9 ± 5.8PS 9 20.6 ± 1.3 26.6 ± 3.7

Data are expressed as mean ± SD, 8e10 rats in each group, ##P < 0.01, comparedwith the NC group.

2.9. Determination of AGE levels in lenses usingfluorospectrophotometry

Rats were euthanised at the end of week 14, their lenses weredissected, homogenized in pre-chilled 0.9% normal saline, theresultant homogenate was centrifuged at 4 �C at 10,000 � g for15 min, and then the supernatant and pellets were stored at�80 �Cuntil further analysis. The supernatant was collected to determineprotein concentrations, AR activity, and GSH while the pellets wereharvested to measure the AGEs levels. The levels of AGEs in the ratlens tissues and organs were determined using fluorospectropho-tometry as previously reported (Bhattacharyya et al., 2007) andexpressed as the enzymatic activity of type I collagenase permilligram of protein (U/mg prot).

2.10. Measurement of lens AR activity usingfluorospectrophotometry

The AR activity of the rat lenses and lens organs was detectedusing spectrophotometry according to Chaudhry (Chaudhry et al.,1983). The enzymatic reaction system adopted was in a 200-mLvolume consisting of 135 mmol/L phosphate buffer, 100 mmol/Llithium sulphate, 150 mmol/L nicotinamide adenine dinucleotidephosphate (NADPH), 5 mL lens homogenate, and 1.0 mmol/L DL-glyceraldehyde. The fluorescence was measured using a spectro-fluorophotometer (Ex/Em-360 nm/460 nm, GloMax-Multijr,Promega Co., Ltd., USA), and expressed as the unit of AR enzymeactivity per gram of protein (U/g prot).

2.11. Measurement of MDA, GSH/GSSG, CAT, GSH-Px and total SOD(T-SOD)

The levels of MDA, CAT, GSH-Px and GSH/GSSG in the tissuesand SRA01/04 cells were measured using a spectrophotometerusing kits from JieMei Bioengineering Institute (Shanghai, China).The levels of T-SOD in the lenses, tissues, and SRA01/04 cells weremeasured using a spectrophotometer using kits from JianChengBioengineering Institute (Nanjing, China).

Specific steps indicated in the kit manual were as follows.Sample pretreatment: rat lens tissue and rat lens organs/cellsweight by weight (g) were added to 9 times their volume of 0.9%saline volume (ML) at a 1:9 ratio in an ice water bath. Then, themechanical homogenatewas prepared as a 10% homogenate, whichwas centrifuged at 2500 � g for 10 min, and the supernatant wasmeasured. We mixed the test sample and kit reagents homoge-neously, incubated the mixture in the 96-well microplate at thetemperature indicated by the kit instructions, and then the OD wasestimated using the microplate reader at different wavelengths fordifferent indicators.

2.12. Determination of TGF-b2, E-cadherin, and a-SMA expressionand Akt phosphorylation using western blotting in HG-culturedhuman lens epithelial cells

Cultured SRA01/04 cells were harvested in lysis buffer con-taining 50 mmol/L Tris (pH 7.6), 150 mmol/L sodium chloride(NaCl), 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), 1 mmol/Lsodium orthovanadate (Na3VO4) and 20 mmol/L sodium fluoride(NaF0), and then lysed on ice for 30 min with vortexing every10 min. The supernatant was decanted after centrifugation at12,000 � g at 4 �C for 15 min. The protein concentration wasdetermined using the bicinchoninic acid (BCA) protein assay kit(Pierce Thermo-Scientific, Rockford, IL, USA) according to themanufacturer's instructions. For immunoblotting, an equal amount

of protein (40e60 g) was separated using 8% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and thentransferred onto polyvinylidene difluoride membranes (Millipore,Bedford, MA, USA). Themembranes were blocked in PBS containing3% BSA for 1 h at room temperature, followed by incubation withprimary antibodies overnight at 4 �C. The primary antibodies usedwere against E-cadherin, a-SMA, TGF-b2, and b-actin. The mem-branes were colorimetrically developed using the BCIP/NBT alka-line phosphatase color development kit (Beyotime Institute of

Table 5Effects of quercetin on the body weight of diabetic rats.

Group n Weight (g)

Week 0 Week 4

NC 8 191.2 ± 15.3 231.6 ± 21.4DC 8 195.2 ± 13.8 174.4 ± 20.8##

QL 9 197.0 ± 12.9 177.2 ± 12.7QM 10 199.9 ± 16.0 186.1 ± 21.8QH 9 201.4 ± 17.9 176.8 ± 28.4PS 9 211.8 ± 22.8 177.0 ± 11.4

Data are expressed as mean ± SD, 8e10 rats in each group, ##P < 0.01, compared with t

Fig. 2. Effects of quercetin on the lens opacity of diabetic rats (A) and high glucose culturedglucose cultured lens (D, n ¼ 6) and effects of quercetin on the activities of AGEs in diabeticas mean ± SD. ##P < 0.01, compared with NC group, **P < 0.01, compared with HG group. NCrat lens cultured in 50 mol/L glucose medium; QL: diabetic cataract rats treated with 30diabetic cataract rats treated with 60 mg/kg of quercetin, high glucose cultured lens treatedhigh glucose cultured lens treated with 40 mg/ml quercetin; PS: diabetic cataract rats treatelens treated with 0.5 mmol/ml pirenoxine sodium eye drop.

Biotechnology, Nantong, China). The quantification involvedmeasuring the signal intensity using the ImageJ software.

2.13. Statistical analysis

The data are presented as the mean ± standard deviation (SD),and the statistical analysis was performed using the statisticalpackage for the social sciences (SPSS) software version 13.0 (SPSS,Inc., Chicago, IL, USA). Intergroup differences were compared using

Week 8 Week 12 Week 14

260.2 ± 12.5 298.4 ± 12.1 305.2 ± 19.1176.7 ± 27.3## 184.4 ± 37.5## 172.11 ± 37.5##

174.9 ± 15.5 180.3 ± 24.4 164.7 ± 27.8182.0 ± 18.2 188.0 ± 33.1 177.6 ± 28.7180.1 ± 26.6 183.6 ± 41.1 163.5 ± 30.9176.9 ± 11.9 187.7 ± 23.4 173.1 ± 22.1

he NC group.

rat lens (B), effects of quercetin on the activities of AR in diabetic rats (C, n ¼ 6) and highrats (E, n ¼ 6) and high glucose cultured lens (F, n ¼ 6). In all panels, data are presented: normal control group; DC: diabetic cataract rats treated with 1% CMC-Na solution; HG:mg/kg of quercetin, high glucose cultured lens treated with 10 mg/ml quercetin; QM:with 20 mg/ml quercetin; QH: diabetic cataract rats treated with 120 mg/kg of quercetin,d with pirenoxine sodium eye drop twice a day, a drop at a time, high glucose cultured

Table 6Effects of quercetin on the lens opacity of diabetic rats.

Group n Cataract grades(percentage)

Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ

NC 16 16 0 0 0 0DC ## 16 0 0 3(18.75%) 1(6.25%) 12(75.00%)QL 18 0 2(11.11%) 3(16.67%) 0 13(72.22%)QM* 20 0 2(10.00%) 9(45.00%) 1(5.00%) 8(40.00%)QH** 18 0 3(16.67%) 10(55.56%) 1(5.55%) 4(22.22%)PS** 18 0 7(38.89%) 6(33.33%) 1(5.56%) 4(22.22%)

Data are expressed as mean ± SD, ##P < 0.01, compared with NC group, *P < 0.05,compared with DC group, **P < 0.01, compared with DC group.

Table 7Effects of quercetin on the lens opacity of high glucose cultured lens.

Groups N Opacity (au)

NC 6 4.80 ± 1.59HG## 6 13.35 ± 3.74QL 6 11.82 ± 2.26QM** 6 9.81 ± 3.08QH** 6 8.60 ± 3.23PS** 6 4.74 ± 2.09

Data are expressed as mean ± SD, ##P < 0.01, compared with NC group, *P < 0.05,compared with DC group, **P < 0.01, compared with DC group.

a one-way analysis of variance (ANOVA) followed by the Dunnett'stest. The cataracts were graded using the Wilcoxon signed-ranktest, and the statistical significance was defined as P < 0.05.

3. Results

3.1. Effects of quercetin on FBG and body weight of diabetic rats

The FBG and body weight of the rats were examined aftertreatment with quercetin for 14 weeks. Compared with the rats inthe normal group, the FBG level of the rats in the diabetic groupssignificantly increased, and treatment with quercetin had no effect(Table 4). In addition, measurement of the body weights showed asignificant decline after STZ administration while quercetinadministration had no effect on the body weights of diabetic rats(Table 5).

3.2. Quercetin delayed the development of DC in diabetic rats andhigh glucose cultured rat lens

The change in lens opacity is the most intuitive indicator of lensstatus. We used STZ-induced 14-week diabetic cataract rats as thein vivo model and HG-cultured rat lenses in vitro to evaluate theeffect of quercetin on the lens opacity induced by HG. After the ratshad been fed for 14 weeks, the eyes were examined using a slitlamp biomicroscope. The progression of lenticular opacity wasassessed according to the description of Azuma et al. (1990). The slitlamp findings indicated that the lenses of the NC rats remainedclear and transparent without any turbidity. In contrast, the rats inthe DC group presented cloudy lenses with nuclear opacity within arange of grades IIIeV. Most of the lenses of the QL and QM grouprats were grade V and II (75.00 and 45.00%, respectively), butnumerous lenses were also at grade V (40.00%). Compared with thelenses of the QM group, those of the QH group showed an increasein the proportion of grade III (55.56%) lenses, and those in grade V(22.22%) reduced. Most of the lenses of the PS group were in gradeII (38.89%), which was higher than the values of any other group.Collectively, comparedwith the DC group, the lenses of the QM, QH,and PS groups showed a significant improvement in lens opacity.

The in vivo model was used to examine the effect of quercetin onlens opacity, and the change in glucose-induced lens opacity wasmonitored microscopically. After 5 days, all the lenses in the NCgroup appeared clear while most lenses of the HG group rats werecovered with opaque rings. After treatment with quercetin, the QMand QH group showed a significant reduction in HG-induced lensopacity levels compared with that of the HG group. Moreover, PS asthe positive control also reduced the lens opacity (Fig. 2A and B;Tables 6 and 7).

AR and AGEs are two important indicators of DC. AR is a keyenzyme in the polyol pathway, which can be activated in diabeticconditions and plays a crucial role in the pathogenesis of DC(Wilhelm et al., 2007). AGEs are generated by the non-enzymaticglycation reaction and accumulate in the lens, which leads to theopacification (Ding et al., 2007). The AR activities and AGE level ofthe DC-HG groupwere significantly increased comparedwith thoseof the NC group (P < 0.01). The diabetic rats in the QL, QM, and QHgroups showed weaker AR activity, and lower AGEs levels thanthose in the DC group did (P < 0.01). PS therapy also significantlyreduced the AR activity and AGE level of the DC rat lenses (P < 0.01).The administration of quercetin and PS decreased AR activity andAGE level to different degrees in vivo and in vitro. The above resultssuggest that quercetin significantly improved the internal DCcrystal aldose reductase abnormalities and AGE accumulation inrats, whereas quercetin high-dose showed better effects than PSdid (Fig. 2CeF).

3.3. Quercetin inhibited oxidative stress of lens epithelial in vivoand in vitro

An array of antioxidant defence mechanisms expressed by thecell plays a vital role in maintaining the physiological redox statusand protecting the cells against the harmful effect of toxic drugmetabolites and free radicals. These include GSH, GSSG, GSH-Pxand ratio of GSH/GSSG, which reflect the oxidation-reductionstate of cells, enzymatic antioxidants such as SOD and CAT, andthe lipid peroxidation product MDA. A change in these indicatorsreflects the strength of the cellular antioxidant capacity and thedegree of oxidative damage. SRA 01/04 cells and excluded lenswere exposed to NC (5.56 mmol/L in cells, 2.40 mmol/L in the lens)or HG (50 mmol/L) for 24 h and 5 days respectively. The normal andSTZ-induced DC rats were fed under specific pathogen-free (SPF)conditions for 14 weeks. The GSH and T-SOD were determined inHG-cultured lenses while the GSH/GSSG ratio, T-SOD, CAT, GSH-Pxand MDA were detected in SRA 01/04 cells and 14-week STZ-induced DC rats lenses. The results revealed that HG down-regulated the levels of GSH in cells, lenses, and DC rats(Fig. 3AeC), GSH/GSSG in cells and DC rats (Fig. 3F and G), the ac-tivity of T-SOD in cells, lenses, and DC rats (Fig. 3JeL), GSH-PX andCAT in cells and DC rats (Fig. 3H, I, M and N) compared with thecells/lenses of the NC group. Conversely, increased levels of MDAand GSSG were observed in HG-cultured cells and diabetic rats(Fig. 3O, P, D and E). The administration of quercetin and PSincreased the GSH level, GSH/GSSG ratio, and the T-SOD, GSH-Pxand CAT activity under HG condition to different degrees in vivoand in vitro. Moreover, quercetin alleviated the accumulation ofMDA and GSSG caused by HG in the HG-cultured cells and diabeticrats. Taken together, these data suggest that quercetin alleviatesoxidative stress induced by HG in vivo and in vitro.

3.4. Quercetin inhibited morphological change and EMT of lensepithelial cell of diabetic rats and HG-cultured SRA01/04 cells

Under normal conditions, the SRA01/04 human lens epithelialcell morphology was regular and showed a typical cobblestone

Fig. 3. Effects of quercetin on the level of GSH (A, B, C), GSSG (D, E) and MDA (O, P) and the activities of T-SOD (JeL), CAT (M, N) and GSH-Px (H, I) in diabetic rats (C, E, G, I, L, N, P) (n ¼ 6), high glucose cultured lens (B, K) (n ¼ 6), and HLECs(A, D, F, H, J, M, O) (n ¼ 3). In all panels, data are presented as mean ± SD. ##P < 0.01, compared with NC group, *P < 0.05, compared with HG group, **P < 0.01, compared with DC or HG group. NC: normal control group; DC: diabeticcataract rats treated with 1% CMC-Na solution; HG: rat lens or HLECs cultured in 50 mol/L glucose medium; QL: diabetic cataract rats treated with 30 mg/kg of quercetin, high glucose cultured lens treated with 10 mg/ml quercetin, highglucose cultured HLECs treated with 1 mmol/L quercetin; QM: diabetic cataract rats treated with 60 mg/kg of quercetin, high glucose cultured lens treated with 20 mg/ml quercetin, high glucose cultured HLECs treated with 2 mmol/Lquercetin; QH: diabetic cataract rats treated with 120 mg/kg of quercetin, high glucose cultured lens treated with 40 mg/ml quercetin, high glucose cultured HLECs treated with 4 mmol/L quercetin; PS: diabetic cataract rats treated withpirenoxine sodium eye drop twice a day, a drop at a time, high glucose cultured lens treated with 0.5 mmol/ml pirenoxine sodium eye drop, high glucose cultured HLECs treated with 4 mmol/L pirenoxine sodium eye drop.

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Fig. 4. Effects of quercetin on the morphological change of high glucose cultured HLECs (A) and HE staining in rat lens epithelial cells (B). NC: normal control group; DC: diabeticcataract rats treated with 1% CMC-Na solution; HG: HLECs cultured in 50 mol/L glucose medium; QL: diabetic cataract rats treated with 30 mg/kg of quercetin, high glucose culturedHLECs treated with 1 mmol/L quercetin; QM: diabetic cataract rats treated with 60 mg/kg of quercetin, high glucose cultured HLECs treated with 2 mmol/L quercetin; QH: diabeticcataract rats treated with 120 mg/kg of quercetin, high glucose cultured HLECs treated with 4 mmol/L quercetin; PS: diabetic cataract rats treated with pirenoxine sodium eye droptwice a day, a drop at a time, high glucose cultured HLECs treated with 4 mmol/L pirenoxine sodium eye drop.

pattern. Cells cultured under HG conditions for 24 h showed asignificantly elongated fibre-like form compared with the NC groupcells. The shape of cells in the QH and PS groups were turned toregular and exhibited a cobblestone-like appearance. Furthermore,the results showed that HG induced SRA01/04 cell fibrotic changeswhile quercetin, which possesses a high PS content, improved theHG-induced cell fibrotic changes. This observation suggests that thePS content of quercetin improve the HGe-induced human lensepithelial cell morphological changes (Fig. 4A). The H&E stainingalso validated these results in vivo (Fig. 4B).

SRA01/04 cells were exposed to NC (5.56 mmol/L) or HG(50mmol/L) for 24 h. The expression of the epithelial cell marker, E-cadherin, and the mesenchymal cell marker, a-SMA, was deter-mined using western blotting and immunofluorescence. Westernblotting demonstrated that HG down-regulated the expression ofE-cadherin and up-regulated that of a-SMA compared with the NCcells (Fig. 5C and D). In correlation with the findings of the westernblotting, decreased E-cadherin and increased a-SMA expressionswere observed using immunofluorescence (Fig. 5A and B). Inaddition, each concentration of quercetin down-regulated the highexpression of a-SMA in SRA01/04 cells exposed to HG. E-cadherinwas prominently localized at the cellecell junctions in cellscultured under NC conditions, but this distribution was notobserved in the HG-cultured SRA01/04 cells. Treatment of thesecells with quercetin induced the reformation of the cell-cell junc-tion and decreased the expression of a-SMA in the cytoplasmcompared with the HG group cells.

To confirm the effects of quercetin on HG-induced EMT, we usedimmunohistochemistry to detect the expression of E-cadherin anda-SMA in diabetic rats. Similar to the results of the SRA01/04 cellanalysis, HG also induced EMT in diabetic rats in vivo, and quercetin

alleviated this effect by increasing E-cadherin and decreasing a-SMA expressions, simultaneously (Fig. 6A and B). Taken together,these results support the hypothesis that treatment with quercetinreverses the EMT in HG-cultured SRA01/04 cells and diabetic rats.

3.5. Inhibitory effects of quercetin on TGF-b2/PI3K/Akt signalling-mediated EMT of SRA01/04 cells

To investigate whether the TGF-b2/PI3K/Akt signalling pathwayis involved in the EMT of SRA01/04 cells induced by HG, weexamined the expression of TGF-b2 and phosphorylation of Akt atSer473. Compared with the NC group, the expression of TGF-b2 wassignificantly increased in the HG group. Moreover, the phosphor-ylation of Akt (Ser473) was also elevated in HG-cultured SRA01/04 cells (Fig. 7A and B). Treatment with quercetin inhibited the HG-induced overexpression of TGF-b2 and phosphorylation of Akt inSRA01/04 cells, and increased E-cadherin and decreased a-SMAexpressions (see Fig. 8).

Furthermore, administration of LY294002 (the PI3K inhibitor)ameliorated the HG-induced EMT, and the effect was comparablewith that of intermediate doses of quercetin, based on the findingsof the effects on E-cadherin and a-SMA using immunofluorescence(Fig. 7C and D). Collectively, these results indicate that TGF-b2/PI3K/Akt signalling was activated in the HG-mediated EMT ofSRA01/04 cells. Furthermore, quercetin might prevent the EMT ofhuman lens epithelial cell lines by inhibiting TGF-b2/PI3K/Aktsignalling.

4. Discussion

EMT is a critical physiological process that exposes interactive

Fig. 5. Distribution and expression of E-cadherin (A, D) and a-SMA (B, C) in HLECs cells through immunofluorescence and western blotting. In all panels, data are presented asmean ± SD, n ¼ 3. NC: normal control group; HG: HLECs cultured in 50 mol/L glucose medium; QL: high glucose cultured HLECs treated with 1 mmol/L quercetin; QM: high glucosecultured HLECs treated with 2 mmol/L quercetin; QH: high glucose cultured HLECs treated with 4 mmol/L quercetin; PS: high glucose cultured HLECs treated with 4 mmol/L pir-enoxine sodium eye drop.

basement membrane epithelial cells to innumerable biochemicalalterations and, thereby, induces a mesenchymal cell phenotypeassumption. These phenotypic characteristics include invasiveness,

enhanced migratory capacity, resistance to apoptosis, andincreased extracellular matrix (ECM) component production(Kalluri and Neilson, 2003). Additionally, this transition is

Fig. 6. Distribution and expression of E-cadherin (A) and a-SMA (B) in diabetic rats through immunohistochemistry. (A) E-cadherin expression in rat lens epithelial cells throughimmunohistochemistry. (B) a-SMA expression in rat lens epithelial cells through immunohistochemistry. NC: normal control group; DC: diabetic cataract rats treated with 1% CMC-Na solution; QL: diabetic cataract rats treated with 30 mg/kg of quercetin; QM: diabetic cataract rats treated with 60 mg/kg of quercetin; QH: diabetic cataract rats treated with120 mg/kg of quercetin; PS: diabetic cataract rats treated with pirenoxine sodium eye drop twice a day, a drop at a time.

associated with the down-regulation of epithelial cells biomarkersand the up-regulation of mesenchymal biomarkers (Lee et al.,2007). Various pathologies had been linked to this transition pro-cess, and DC, an end-stage diabetic complication that erodes thenative integrity of eye lens, is involved in this scenario. We foundthat HG induces ROS generation and ultimately induces EMT-associated DC by activating the PI3K/Akt pathway. Furthermore,we proved that quercetin, a versatile flavonoid, has the potential toameliorate DC through this pathway. Therefore, to buttress thispoint, we explored whole animal, tissue, and cellular experimentalmodels using STZ-induced DC rats, HG-cultured rat lenses, andHLECs SRA01/04 cells.

First, we demonstrated that HG induced oxidative stress byevaluating the antioxidant enzyme system (the reduced form ofGSH, GSH/GSSG ratio, GSH-Px, T-SOD, and CAT activities) and thecontent of lipid peroxidation products (MDA). The reaction be-tween GSH and H2O2 catalysed by GSH-peroxidase (Px), whichgenerates water (H2O) and GSSG can directly detoxify H2O2 and,therefore, the GSH/GSSG ratio of the lens depicts the level of itsantioxidant capacity. In addition, antioxidant enzymes such as SOD,CAT, and GSH-Px play synergistic roles in the clearance of oxygenfree radical (Ozmen et al., 2002). Therefore, it is obvious the anti-oxidantmechanisms that scavenge the deleterious oxidative insultsare no operational.

Moreover, MDA is a key product of lipid peroxidation that bindsto amino acid residues on the membrane, leading to the instabilityof cell membrane and cell death. Consequently, MDA is the mostrepresentative indicator of oxidative damage. Under HG conditions,GSH, GSH/GSSG ratio, GSH-Px, T-SOD, and CAT are down-regulatedwhile the MDA content accumulates. This indicates that the anti-oxidant status of lenses in the diabetic condition is dysfunctional,which might increase the vulnerability of the cells to oxidativeinsults.

This observation suggests that quercetin protected the antioxi-dant status of the STZ-induced DC and HG-cultured lenses as wellas the SRA01/04 cells. Quercetin also reduced the expression ofaldose reductase and abated AGE production significantly. The keystructural features of quercetin that might be responsible for itsstability in fighting oxidative insults include the B ring o-dihydroxylgroups, the 4-oxo group in conjugation with the 2, 3-alkene, andthe 3- and 5-hydroxyl groups (Hollman and Katan, 1997). Thesefunctional groups can donate electrons to the rings, which in-creases the number of resonance forms available in addition tothose created by the benzene structure (Mariani et al., 2008).

The cellular platform of cataractogenesis is the pathologicalchange in lens epithelial cells, and the exploration of their patho-physiological characteristics might facilitate the elucidation of themechanism of cataract pathogenesis. Furthermore, this strategycould also provide further insight into the development of thera-peutic options that could remedy the condition. EMT of lensepithelial cells is recognized as the pillar that supports the cata-ractogenesis machinery while the PI3K/Akt signalling pathway hasbeen shown to be an important mediator of this process.

Previous studies have shown that HG-cultured rat lenses exhibita significant increase in the expression levels of TGF-b2 mRNA andprotein (Olofsson et al., 2005). In human lens epithelial cells, thePI3K/Akt signalling pathway is involved in TGF-b2-induced EMT(Wilhelm et al., 2007). Thus, TGFb/PI3K/Akt signalling in the DCpathological process could invoke the onset and progression ofEMT. Previous studies found that quercetin inhibits epithelial-mesenchymal transition of PC-3 cell lines in vitro by theepidermal growth factor receptor (EGFR)/PI3K/Akt signal trans-duction pathway (Hollman and Katan, 1997).

To confirm the occurrence of EMT under HG conditions in vivoand in vitro, we examined the expression of E-cadherin and a-SMA,and observed a significant decrease and increase in E-cadherin and

Fig. 8. Activation of TGF-b/PI3K/Akt signalling promoted the progression of EMT and diabetic cataract, and the protective effects of quercetin might be related to inhibition of TGF-b/PI3K/Akt pathway.

Fig. 7. (A) Effects of quercetin on the expression of TGF-b2 in high glucose cultured HLECs (n ¼ 3). (B) Effects of quercetin on the phosphorylation of Akt in high glucose culturedHLECs (n ¼ 3). (C, D) The role of quercetin in PI3K/Akt pathway compared with PI3K inhibitor (LY294002). In all panels, data are presented as mean ± SD. ##P < 0.01, compared withNC group, **P < 0.01, compared with HG group. NC: normal control group; HG: HLECs cultured in 50 mol/L glucose medium; QL: high glucose cultured HLECs treated with 1 mmol/Lquercetin; QM: high glucose cultured HLECs treated with 2 mmol/L quercetin; QH: high glucose cultured HLECs treated with 4 mmol/L quercetin; PS: high glucose cultured HLECstreated with 4 mmol/L pirenoxine sodium eye drop; LY: high glucose cultured HLECs treated with 20 mmol/L LY294002.

Please cite this article in press as: Du, L., et al., Quercetin inhibited epithelial mesenchymal transition in diabetic rats, high-glucose-culturedlens, and SRA01/04 cells through transforming growth factor-b2/phosphoinositide 3-kinase/Akt pathway, Molecular and CellularEndocrinology (2017), http://dx.doi.org/10.1016/j.mce.2017.05.011

a-SMA expressions, respectively in HG-cultured human lensepithelial cells. This observation was accompanied by cellmorphological changes from a neatly arranged cobblestone type tothe shuttle-shaped fibre type. The results showed that the HGenvironment promoted the occurrence of EMT in lens epithelialcells. In addition, TGF-b2 expression and Akt phosphorylationincreased in HG-cultured cells, which suggests that HG induced theEMT of lens epithelial cells by activating TGF-b2/PI3K/Aktsignalling.

The administration of quercetin significantly improved E-cad-herin expression, inhibited a-SMA, and reduced TGF-b2 expressionand Akt activation, which inhibited the EMT of lens epithelial cellsand, thereby, inhibited cataractogenesis. Probably, we could inferthat quercetin had an effect on PI3K inhibition because PI3K is anupstream protein of Akt. There is evidence that quercetin and theinhibitor LY294002 are structural analogues and, therefore, arelikely to have similar roles, including the inhibition of PI3K/Aktsignalling (Mariani et al., 2008).

In DC, a reduction in galactokinase activity and insulin defi-ciency provokes high blood glucose concentration, leading toincreased intraocular aqueous osmotic pressure and swelling of thelens fibre, which eventually sets the stage for lens opacification, acritical hallmark of DC (Pollreisz and Schmidt-Erfurth, 2010). Long-term research studies have proved beyond reasonable scientificdoubt that HG concentration alone might not be sufficient to causeDC. These observations further suggest that HG likely acts in con-cert with other processes such as oxidative stress, abnormal polyolmetabolism, non-enzymatic glycosylation, and alteration in lensepithelial to form a multiple factor that could induce a strongenough response to provoke the onset and progression of DC(Hegde and Varma, 2005; Obrosova et al., 2010).

While some research findings have reported that HG concen-tration and oxidative stress are closely related twin factors thatcannot be separated, other data suggest a mutual relationship be-tween oxidative stress and non-enzymatic glycation. The specifictarget of quercetin might require further investigations; however,our present findings indicate that quercetin inhibited EMT-inducedDC through the TGF-b2/PI3K/Akt pathway.

5. Conclusion

Our results demonstrate that HG induced the generation of ROS,increased TGF-b2 expression and Akt phosphorylation and alteredthe biological markers of EMT to mediate the development of cat-aractogenesis. This observation clarifies that the occurrence of DC isdriven by multiple factors and not just a single mechanism. Quer-cetin, an incredibly versatile flavonoid, was proven to haveconsiderably more effects other than antioxidant activity. This wasbased on its amelioration of the opacification of lenses and cata-ractogenesis by the improvement of antioxidant status, down-regulation of TGF-b2 expression, reduction of Akt phosphoryla-tion, down-regulation of a-SMA, and up-regulation of E-cadherinexpressions. Therefore, the evidence supporting the anti-cataractogenesis potency of quercetin at the cellular, tissue, andwhole animal levels, indicates that this compound could potentiallyqualify as a therapeutic breakthrough agent. Furthermore, it shouldbe considered as nutraceutical that is worth further investigation asa candidate drug to combat this pathology. It was also interesting toreport that its anti-cataractogenesis activity might be mediated bythe inhibition of TGF-b2/PI3K/Akt pathway-induced EMT. There-fore, the three experimental model perspectives of our research(whole animal, tissue, and cellular), quercetin was proven to bepotent enough to ameliorate DC in the early stages, thereby pre-senting a potential therapeutic strategy for its treatment andmanagement.

Acknowledgements

This study was supported by National Natural Foundation ofChina (no. 81173104), the Jiangsu Six Talent Peaks Foundation ofChina (no. 2011-SWYY-019), the Graduate Research and InnovationPlan of Jiangsu Province (no. CXZZ13-0997) and the Priority Aca-demic Program Development of Jiangsu Higher Education In-stitutions (PAPD). We gratefully acknowledge Ying Sun for criticalreading of this manuscript.

References

Abboud, H.E., 2012. Mesangial cell biology. Exp. Cell Res. 318 (9), 979e985.Albulescu, R., Zolog, I., 2011. Specific aspects in diabetic cataract. Oftalmologia 55

(3), 47e52.Azuma, M., Shearer, T.R., Matsumoto, T., David, L.L., Murachi, T., 1990. Calpain II in

two in vivo models of sugar cataract. Exp. Eye Res. 51 (4), 393e401.Bhattacharyya, J., Shipova, E.V., Santhoshkumar, P., Sharma, K.K., Ortwerth, B.J.,

2007. Effect of a single AGE modification on the structure and chaperone ac-tivity of human alphaB-crystallin. Biochemistry 46 (50), 14682e14692.

Chaudhry, P.S., Cabrera, J., Juliani, H.R., Varma, S.D., 1983. Inhibition of human lensaldose reductase by flavonoids, sulindac and indomethacin. Biochem. Phar-macol. 32 (13), 1995e1998.

Ding, Y., Kantarci, A., Hasturk, H., Trackman, P.C., Malabanan, A., Van Dyke, T.E.,2007. Activation of RAGE induces elevated O2- generation by mononuclearphagocytes in diabetes. Leukoc. Biol. 81 (2), 520e527.

Hegde, K.R., Varma, S.D., 2005. Cataracts in experimentally diabetic mouse:morphological and apoptotic changes. Diabetes Obes. Metab. 7 (2), 200e204.

Hollman, P.C.H., Katan, M.B., 1997. Absorption, metabolism and health effects ofdietary flavonoids in man. Biomed. Pharmacother. 51 (8), 305e310.

Kador, P.F., Kinoshita, J.H., 1984. Diabetic and galactosaemic cataracts. Ciba Found.Symp. 106, 110e131.

Kalluri, R., Neilson, E.G., 2003. Epithelial mesenchymal transition and its implica-tions for fibrosis. J. Clin. Invest. 112, 1776e1784.

Kandarakis, S.A., Piperi, C., Topouzis, F., Papavassiliou, A.G., 2014. Emerging role ofadvanced glycation-end products (AGEs) in the pathobiology of eye diseases.Prog. Retin Eye Res. 42, 85e102.

Katsuno, Y., Lamouille, S., Derynck, R., 2013. TGF-b signaling and epithelial-mesenchymal transition in cancer progression. Curr. Opin. Oncol. 25 (1), 76e84.

Kim, Y.S., Kim, N.H., Lee, S.W., Lee, Y.M., Jang, D.S., Kim, J.S., 2007. Effect of proto-catechualdehyde on receptor for advanced glycation end products and TGF-b1expression in human lens epithelial cells cultured under diabetic conditionsand on lens opacity in streptozotocin-diabetic rats. Eur. J. Pharmacol. 569 (3),171e179.

Kim, B., Kim, S.Y., Chung, S.K., 2012. Changes in apoptosis factors in lens epithelialcells of cataract patients with diabetes mellitus. Cataract. Refract. Surg. 38 (8),1376e1381.

Kinoshita, J.H., 1990. A thirty year journey in the polyol pathway. Exp. Eye Res. 50(6), 567e573.

Lee, H., O'Meara, S.J., O'Brien, C., Kane, R., 2007. The role of gremlin, a BMP antag-onist, and epithelial-to-mesenchymal transition in proliferative vitreoretinop-athy. Invest Ophthalmol. Vis. Sci. 48, 4291e4299.

Li, X., Zheng, T., Sang, S., 2014. Quercetin inhibits advanced glycation end productformation by trapping methylglyoxal and glyoxal. Agric. Food Chem. 62 (50),12152e12158.

Lu, Q., Yang, T.T., Zhang, M.Z., Du, L., Liu, L., Zhang, N., Guo, H., Zhang, F., Hu, G.,Yin, X.X., 2014. Preventative effects of Ginkgo biloba extract (EGb761) on highglucose-cultured opacity of rat lens. Phytother. Res. 28 (5), 767e773.

Mariani, C., Braca, A., Vitalini, S., 2008. Flavonoid characterization and in vitroantioxidant activity of Aconitum anthora L. Phytochemistry 69 (5), 1220e1226.

McKay, T.B., Lyon, D., Sarker-Nag, A., Lv, L., 2015. Quercetin attenuates lactate pro-duction and extracellular matrix secretion in keratoconus. Sci. Rep. 5, 9003.

Meng, Q., Guo, H., Xiao, L., Cui, Y., Guo, R., Xiao, D., Huang, Y., 2013. mTOR regulatesTGF-b2-induced epithelial-mesenchymal transition in cultured human lensepithelial cells. Graefes Arch. Clin. Exp. Ophthalmol. 251 (10), 2363e2370.

Obrosova, I.G., Chung, S.S., Kador, P.F., 2010. Diabetic cataracts: mechanisms andmanagement. Diabetes Metab. Res. Rev. 26 (3), 172e180.

Olofsson, E.M., Marklund, S.L., Karlsson, K., Brannstrom, T., Behndig, A., 2005.In vitro glucose-induced cataract in copper zinc superoxide dismutase nullmice. Exp. Eye Res. 81 (6), 639e646.

Ozmen, B., Ozmen, D., Erkin, E., Güner, I., Habif, S., Bayindir, O., 2002. Lens super-oxide dismutase and catalase activities in diabetic cataract. Clin. Biochem. 35(1), 69e72.

Pollreisz, A., Schmidt-Erfurth, U., 2010. Diabetic cataract-pathogenesis, epidemi-ology and treatment. Ophthalmol. 2010, 608751.

Richer, S., Stiles, W., Zargar, P., Rezaei, M., Bone, R., Sardi, B., 2015. The Human Lens:a Living Biometric Indicator of Health Status and Successful Aging. Springer,New York, pp. 155e186.

Sadi, G., Eryilmaz, N., Tutuncuoglu, E., Cingir, S., Güray, T., 2012. Changes inexpression profiles of antioxidant enzymes in diabetic rat kidneys. DiabetesMetab. Res. Rev. 28 (3), 228e235.

Sanderson, J., McLauchlan, W.R., Williamson, G., 1999. Quercetin inhibits hydrogenperoxide-induced oxidation of the rat lens. Free Radic. Biol. Med. 26 (5e6),639e645.

Tang, S.C., Lai, K.N., 2012. The pathogenic role of the renal proximal tubular cell indiabetic nephropathy. Nephrol. Dial. Transpl. 27 (8), 3049e3056.

Wilhelm, J., Smistik, Z., Mahelkova, G., Vyt�asek, R., 2007. Redox regulation of pro-liferation of lens epithelial cells in culture. Cell Biochem. Funct. 25 (3), 317e321.

Yao, K., Ye, P.P., Tan, J., Tang, X.J., Shen Tu, X.C., 2008. Involvement of PI3K/Aktpathway in TGF-b2-mediated epithelial mesenchymal transition in human lensepithelial cells. Ophthalmic Res. 40 (2), 69e76.

Yin, Y., Li, W., Son, Y.O., Sun, L., Lu, J., Kim, D., Wang, X., Yao, H., Wang, L.,Pratheeshkumar, P., Hitron, A.J., Luo, J., Gao, N., Shi, X., Zhang, Z., 2013. Quer-citrin protects skin from UVB-induced oxidative damage. Toxicol. Appl. Phar-macol. 269 (2), 89e99.

Yoon, J.S., Chae, M.K., Jang, S.Y., Lee, S.Y., Lee, E.J., 2012. Antifibrotic effects ofquercetin in primary orbital fibroblasts and orbital fat tissue cultures of Graves'orbitopathy. Invest. Ophthalmol. Vis. Sci. 53 (9), 5921e5929.

Zhang, L., Zhou, F., Dijke, P., 2013. Signaling interplay between transforming growthfactor-beta receptor and PI3K/Akt pathways in cancer. Trends Biochem. Sci. 38(12), 612e620.

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Quercetin lowers plasma uric acid in pre-hyperuricaemic ......quercetin-containing tablet was light green in colour and the placebo tablet was off-white. As quercetin is light yellow,

QUERCETIN AND A REVIEW ON ITS IMPORTANCE

Preparation of Silymarin–quercetin Loaded Nanoparticles by

Quercetin and hydroxytyrosol attenuates xanthine/xanthine

Quercetin attenuates reduced uterine perfusion pressure ...Quercetin could be widely found in vegetables, fruits, and soybeans [9]. Various studies reported the effect of quercetin