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Selective inhibition of PKR by C16 accelerates diabetic wound healing by inhibiting NALP3 expression in mice

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Abstract

Objective and design

To understand the expression of dsRNA-dependent protein kinase R (PKR) in impaired diabetic wounds, hyperglycemia was induced in C57/BL6 mice with streptozotocin. Murine macrophage cell line, Raw 264.7, stimulated with high glucose and LPS was used to mimic diabetic wound environment in in-vitro.

Materials

Macrophages stimulated with HG + LPS, in presence and absence of PKR inhibitor (C16) and wound tissue samples from topically treated mice with C16, were analyzed for the expression of PKR, NALP3, active caspase-1, mature IL-1β and phosphorylation of PKR and eIF2α. Wounds tissues were also analyzed for inflammatory cell infiltration by immunohistochemistry, angiogenesis by CD31 staining, collagen expression by western blotting, expression of CD206+ macrophages by flow cytometry and wound strength by texture analyzer.

Results

PKR and NALP3 were found to be upregulated in macrophages stimulated with HG + LPS as well as in impaired diabetic wounds. PKR inhibition using C16 ameliorated expression of NALP3, caspase-1, IL-1β and phosphorylation of PKR and eIF2α, in macrophages and also in diabetic wounds. Treatment with C16 promoted the wound healing in diabetic mice by increasing collagen synthesis, reducing infiltration of F4/80+ macrophages and MPO+ neutrophil cells, increased angiogenesis, and increased number of M2 macrophages.

Conclusion

PKR inhibition using C16 accelerates the wound healing process in diabetic mice by decreasing NALP3-mediated IL-1β maturation.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

PKR:

Double-stranded RNA-dependent protein kinase

NALP3:

NACHT, LRR and PYD domains containing protein 3

MPO:

Myeloperoxidase

CD31:

Cluster of differentiation 31 also named as PECAM-1

PECAM-1:

Platelet endothelial cell adhesion molecule

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Acknowledgements

The authors would like to thank Central Analytical (CAL) facility at BITS-PILANI, Hyderabad campus for providing the access to use laser confocal microscopy (Leica DMi8), BD FACS Aria™ III and HPLC.

Funding

This study was funded by Department of Science and technology, India (DST-SERB) [YSS/2015/001969] to O.P.K. O.P.K also received funds from BITS-PILANI, Hyderabad campus (Additional research initiation grant).

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Authors and Affiliations

  1. Department of Pharmacy, Birla Institute of Technology and Science-Pilani, Hyderabad Campus, Hyderabad, India

    Kalyani Karnam, Kavitha Sedmaki, Pravesh Sharma, Venkata Vamsi Krishna Venuganti & Onkar Prakash Kulkarni

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  1. Kalyani Karnam
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  2. Kavitha Sedmaki
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  3. Pravesh Sharma
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  4. Venkata Vamsi Krishna Venuganti
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  5. Onkar Prakash Kulkarni
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Contributions

OPK conceived the idea and OPK, VVVK and KK designed the experiments. KK, SK and PS performed the experiments and analyzed the data. KK and OPK completed the manuscript writing. All the authors approved the final version.

Corresponding author

Correspondence to Onkar Prakash Kulkarni.

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The authors declare no conflicts of interest.

Ethical approval

The animal studies performed in this report were approved by institute animal ethical committee (BITS-HYD/IAEC/2016/02) and (BITS-HYD/IAEC/2018/12).

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Responsible Editor: Mauro Teixeira.

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Karnam, K., Sedmaki, K., Sharma, P. et al. Selective inhibition of PKR by C16 accelerates diabetic wound healing by inhibiting NALP3 expression in mice. Inflamm. Res. 72, 221–236 (2023). https://doi.org/10.1007/s00011-022-01667-y

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