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Silibinin suppresses TGFβ2-induced lens epithelial cell migration and epithelial–mesenchymal transition

Abstract

Growth factor-induced migration of lens epithelial cell (LEC) toward the posterior of lens capsule bag and their epithelial–mesenchymal transition (EMT) is the key process involved in the pathogenesis of posterior capsular opacification (PCO). Silibinin, a natural flavonolignan, confers therapeutic effects to different cells by regulation of signalling pathways; however, its role in the prevention of migration and EMT of LECs is yet to be analysed. In this study, the inhibitory capabilities of silibinin on migration and EMT were analysed in response to TGFβ2 stimulation in HLE B-3 cells. The anti-migratory effect of silibinin was analysed using wound healing assay. Transcriptional and translational expression of genes related to LEC migration, EMT, and transcription factors related to EMT were studied by quantitative real-time PCR and Western blotting. Immunofluorescence analysis was utilized to study the localization of fibronectin. Silibinin reduced the viability of LECs in a concentration-dependent manner and inhibited the wound healing capacity of LECs induced by TGFβ2. Silibinin also suppressed alteration in the EMT-related markers such as cytoskeletal proteins, cell adhesion markers, extracellular matrix molecules, and transcription factors. Analysis of downstream signalling revealed that treatment with silibinin decreased phosphorylated Akt (Ser473, Thr308), PDK1 (Ser241), PTEN (Ser380), c-Raf (Ser259), and GSK3β (Ser9) in TGFβ-stimulated cells. The effect of silibinin treatment on phosphorylated Akt resembled that of the PI3K inhibitor LY294002. Our results suggest that silibinin can suppress LEC migration and EMT, which involves the inactivation of the PI3K-Akt signalling pathway. Silibinin might be a good candidate for PCO prevention; however, functional evaluation of silibinin using in vivo models is a pre-requisite.

Graphical abstract

Inhibitory effect of silibinin on TGFβ2-induced migration and EMT of LECs.

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Abbreviations

Akt:

protein kinase B

BSA:

bovine serum albumin

COX1/2:

cyclooxygenase 1/2

CTGF:

connective tissue growth factor

DABCO:

1,4-diazabicyclo(2.2.2)octane

DAPI:

4′,6-diamidino-2-phenylindone

ECL:

enhanced chemiluminescence

EGF:

epidermal growth factor

EMT:

epithelial–mesenchymal transition

Erk:

extracellular signal-regulated kinase

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

GSK3β:

glycogen synthase kinase 3 beta

HGF:

hepatocyte growth factor

HRP:

horseradish peroxidase

IC50 :

half maximal inhibitory concentration

IOL:

intraocular lens

IOP:

intraocular pressure

JNK:

C-jun N-terminal kinase

LEC:

lens epithelial cell

MAPK:

mitogen-activated protein kinase

MMP9:

matrix metalloproteinase 9

MTT:

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NdYAG:

neodymium-doped yttrium aluminum garnet

pAkt:

phosphor Akt

PBS:

phosphate-buffered saline

PCO:

posterior capsular opacification

PDK1:

3-phosphoinositide-dependent protein kinase 1

PI3K:

phosphatidylinositol 3-kinase

PTEN:

phosphatase and tensin homolog

qRT PCR:

quantitative real-time polymerase chain reaction

SDS:

sodium dodecyl sulfate

TBS:

tris-buffered saline

TBST:

tris-buffered saline plus tween 20

TGFβ2:

transforming growth factor-beta 2

VEGF:

vascular endothelial growth factor

ZEB:

zinc finger E-box binding homeobox

ZO1/TJP1:

zona occludens/tight junction protein 1

αSMA:

alpha smooth muscle actin

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Acknowledgements

This work was a part of PBM’s PhD programme, Iladevi Cataract and IOL Research Centre, Ahmedabad, India, and Manipal Academy of Higher Education, Manipal, India, and the authors acknowledge Indian Council of Medical Research, India, for providing Senior Research Fellowship (BMS/Nan/2020_6123) to PBM.

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Correspondence to Kaid Johar SR.

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Communicated by Dipankar Nandi.

Corresponding editor: Dipankar Nandi

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Malaviya, P.B., Shukal, D.K., Sharma, T. et al. Silibinin suppresses TGFβ2-induced lens epithelial cell migration and epithelial–mesenchymal transition. J Biosci 47, 51 (2022). https://doi.org/10.1007/s12038-022-00286-3

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  • DOI: https://doi.org/10.1007/s12038-022-00286-3

Keywords

  • Epithelial–mesenchymal transition
  • lens epithelial cells
  • PI3K-Akt pathway
  • posterior capsular opacification
  • silibinin
  • TGFβ