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. 2022 Aug 14;10(8):1969.
doi: 10.3390/biomedicines10081969.

The Dose-Dependent Effects of Multifunctional Enkephalin Analogs on the Protein Composition of Rat Spleen Lymphocytes, Cortex, and Hippocampus; Comparison with Changes Induced by Morphine

Hana Ujcikova  1 , Lenka Roubalova  1 , Yeon Sun Lee  2 , Jirina Slaninova  1 , Jana Brejchova  1 , Petr Svoboda  1
Affiliations

The Dose-Dependent Effects of Multifunctional Enkephalin Analogs on the Protein Composition of Rat Spleen Lymphocytes, Cortex, and Hippocampus; Comparison with Changes Induced by Morphine

Hana Ujcikova et al. Biomedicines. .

Abstract

This work aimed to test the effect of 7-day exposure of rats to multifunctional enkephalin analogs LYS739 and LYS744 at doses of 3 mg/kg and 10 mg/kg on the protein composition of rat spleen lymphocytes, brain cortex, and hippocampus. Alterations of proteome induced by LYS739 and LYS744 were compared with those elicited by morphine. The changes in rat proteome profiles were analyzed by label-free quantification (MaxLFQ). Proteomic analysis indicated that the treatment with 3 mg/kg of LYS744 caused significant alterations in protein expression levels in spleen lymphocytes (45), rat brain cortex (31), and hippocampus (42). The identified proteins were primarily involved in RNA processing and the regulation of cytoskeletal dynamics. In spleen lymphocytes, the administration of the higher 10 mg/kg dose of both enkephalin analogs caused major, extensive modifications in protein expression levels: LYS739 (119) and LYS744 (182). Among these changes, the number of proteins associated with immune responses and apoptotic processes was increased. LYS739 treatment resulted in the highest number of alterations in the rat brain cortex (152) and hippocampus (45). The altered proteins were functionally related to the regulation of transcription and cytoskeletal reorganization, which plays an essential role in neuronal plasticity. Administration with LYS744 did not increase the number of altered proteins in the brain cortex (26) and hippocampus (26). Our findings demonstrate that the effect of κ-OR full antagonism of LYS744 is opposite in the central nervous system and the peripheral region (spleen lymphocytes).

Keywords: chronic pain treatment; label-free quantification; morphine; multifunctional enkephalin analogs; proteomic analysis; rat brain; rat spleen lymphocytes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Subcellular localization (a) and function (b) of significantly altered proteins (at least 2-fold) identified in rat spleen lymphocytes after 7-day treatment with 3 mg/kg of morphine (MOR, upper panels), LYS739 (middle panels), and LYS744 (lower panels); according to the current annotations in the Uniprot database (https://www.uniprot.org, accessed on 28 January 2022). The numbers represent proteins found within each subcellular localization and functional category, *1, **2.
Figure 2
Figure 2
Volcano plots indicating significantly altered proteins identified in rat spleen lymphocytes (A), brain cortex (B), and hippocampus (C) after 7-day treatment with 3 mg/kg of morphine (MOR, left panels), LYS739 (middle panels), and LYS744 (right panels). Significantly altered proteins are labeled in red (the star symbols); performed with Perseus software version 1.6.15.0. L739, LYS739; L744, LYS744.
Figure 3
Figure 3
Heatmap showing hierarchical clustering performed on LFQ intensity values (with Perseus software version 1.6.15.0) of all 4717 proteins identified in rat spleen lymphocytes (L), brain cortex (C), and hippocampus (H) after 7-day treatment with 3 mg/kg of morphine (MOR), LYS739, and LYS744. Significantly upregulated proteins are labeled in red, significantly downregulated proteins are labeled in green. L739, LYS739; L744, LYS744.
Figure 4
Figure 4
Subcellular localization (a) and function (b) of significantly altered proteins (at least 2-fold) identified in rat brain cortex after 7-day treatment with 3 mg/kg of morphine (MOR, upper panels), LYS739 (middle panels), and LYS744 (lower panels); according to the current annotations in the Uniprot database (https://www.uniprot.org, accessed on 28 January 2022). The numbers represent proteins found within each subcellular localization and functional category, *1.
Figure 5
Figure 5
Subcellular localization (a) and function (b) of significantly altered proteins (at least 2-fold) identified in rat hippocampus after 7-day treatment with 3 mg/kg of morphine (MOR, upper panels), LYS739 (middle panels), and LYS744 (lower panels); according to the current annotations in the Uniprot database (https://www.uniprot.org, accessed on 28 January 2022). The numbers represent proteins found within each subcellular localization and functional category, *1.
Figure 6
Figure 6
Subcellular localization (a) and function (b) of significantly altered proteins (at least 2-fold) identified in rat spleen lymphocytes after 7-day treatment with 10 mg/kg of morphine (MOR, upper panels), LYS739 (middle panels), and LYS744 (lower panels); according to the current annotations in the Uniprot database (https://www.uniprot.org, accessed on 22 April 2022). The numbers represent proteins found within each subcellular localization and functional category, *1 or *2.
Figure 7
Figure 7
Volcano plots indicating significantly altered proteins identified in rat spleen lymphocytes (A), brain cortex (B), and hippocampus (C) after 7-day treatment with 10 mg/kg of morphine (MOR, left panels), LYS739 (middle panels), and LYS744 (right panels). Significantly altered proteins are labeled in red (the star symbols); performed with Perseus software version 1.6.15.0. L739, LYS739; L744, LYS744.
Figure 8
Figure 8
Heatmap showing hierarchical clustering performed on LFQ intensity values (with Perseus software version 1.6.15.0) of all 6228 proteins identified in rat spleen lymphocytes (L), brain cortex (C), and hippocampus (H) after 7-day treatment with 10 mg/kg of morphine (MOR), LYS739, and LYS744. Significantly upregulated proteins are labeled in red, significantly downregulated proteins are labeled in green. L739, LYS739; L744, LYS744.
Figure 9
Figure 9
Subcellular localization (a) and function (b) of significantly altered proteins (at least 2-fold) identified in rat brain cortex after 7-day treatment with 10 mg/kg of morphine (MOR, upper panels), LYS739 (middle panels), and LYS744 (lower panels); according to the current annotations in the Uniprot database (https://www.uniprot.org, accessed on 22 April 2022). The numbers represent proteins found within each subcellular localization and functional category, *1, **2.
Figure 10
Figure 10
Subcellular localization (a) and function (b) of significantly altered proteins (at least 2-fold) identified in rat hippocampus after 7-day treatment with 10 mg/kg of morphine (MOR, upper panels), LYS739 (middle panels), and LYS744 (lower panels); according to the current annotations in the Uniprot database (https://www.uniprot.org, accessed on 22 April 2022). The numbers represent proteins found within each subcellular localization and functional category, *1.
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