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Mechanism of rutin mediated inhibition of insulin amyloid formation and protection of Neuro-2a cells from fibril-induced apoptosis

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Abstract

Many metabolic and neurodegenerative diseases are associated with protein misfolding and aggregation. Insulin a key hormone, under certain conditions aggregates and forms pathological amyloid fibrils. Several polyphenols have been studied extensively to elucidate their inhibitory effect on amyloid formation. In the present study, we used insulin as an amyloid model to test the mechanism and efficacy of rutin as an anti-amyloidogenic molecule. By using electron microscopy, dynamic light scattering and circular dichroism spectroscopy, we show that rutin inhibits the insulin aggregate and fibril formation. Further, rutin interacts with insulin directly and inhibits fibril formation in a dose-dependent manner as demonstrated by micro scale thermophoresis experiments. The molecular docking study predicted the potential binding pocket of rutin at the interface of chain A and chain B of insulin thereby preventing it from forming the aggregates. Since, rutin is a natural anti-oxidant, we studied its role in diminishing amyloid fibril induced cytotoxicity and apoptosis. Rutin, decreases the insulin amyloid fibrils-induced Neuro-2a cytotoxicity by reducing reactive oxygen species (ROS) levels which in turn downregulates Bax and upregulates Bcl-2 and pBad proteins. These findings suggest the potential action of rutin in preventing protein misfolding, cell death, and serves as a lead structure to design novel anti-amyloidosis compounds.

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Abbreviations

Bcl-2:

B-cell lymphoma 2

Bax:

Bcl-2 associated x protein

CD:

Circular dichroism

DAPI:

4′, 6-Diamidino-2-phenylindole

DCFH-DA:

Dichloro-dihydro-fluorescein diacetate

DLS:

Dynamic light scattering

DMEM:

Dulbecco’s modified eagle’s medium

DMSO:

Dimethyl sulfoxide

EGCG:

Epigallocatechin gallate

FBS:

Fetal bovine serum

Kd :

Dissociation constant

MRE:

Mean residue ellipticity

MST:

Microscale thermophoresis

MTT:

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

NMR:

Nuclear magnetic resonance

pBad,phosphor:

Bcl-2 associated death promoter

PBS:

Phosphate buffered saline

PVDF:

Poly-vinylidene difluoride

r-insulin:

Recombinant-insulin

Rh :

Hydrodynamic radii

ROS:

Reactive oxygen species

TEM:

Transmission electron microscope

ThT:

Thioflavin-T

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Acknowledgements

The authors thank SAIF, ICAR-IIHR, Bengaluru for TEM experiment, IISc, Bengaluru for CD analysis, and Dr. Sivaramaiah Nallapeta and Ms. Saji Menon, NanoTemper Technologies, Bengaluru for MST experiments. The authors also thank the Director, CSIR-CFTRI, for the constant support to carry out this work.

Funding

This study was supported by the 12th Five Year Plan Project NUTRI-ARM (BSC0404) of the Council for Scientific and Industrial Research (CSIR), New Delhi. Research fellowship from the University Grants Commission (UGC) is acknowledged.

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Authors and Affiliations

  1. Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute (CFTRI), Mysore, 570020, Karnataka, India

    V. P. Mahendra, K. Yogendra Prasad, P. Ganesan & Ravi Kumar

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    V. P. Mahendra, K. Yogendra Prasad, P. Ganesan & Ravi Kumar

Authors
  1. V. P. Mahendra
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  2. K. Yogendra Prasad
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  3. P. Ganesan
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Contributions

MVP: Carried out most of the experiments, analysed the data and wrote manuscript under the supervision of RK. YPN and MVP: Conducted the experiments on neuronal cytotoxicity with the help of PG and RK. PG: Designed the experiments for neuronal cytotoxicity study and reviewed the manuscript and. RK: Conceived the idea, supervised the project, analysed the data and wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Ravi Kumar.

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The authors declare that they have no conflict of interest.

Ethical approval

The present study is approved by the Institute Animal Ethics Committee (IAEC, CSIR-Central Food Technological Research Institute, Karnataka, India).

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Mahendra, V.P., Yogendra Prasad, K., Ganesan, P. et al. Mechanism of rutin mediated inhibition of insulin amyloid formation and protection of Neuro-2a cells from fibril-induced apoptosis. Mol Biol Rep 47, 2811–2820 (2020). https://doi.org/10.1007/s11033-020-05393-8

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