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

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).


Introduction
The pain-relieving effects of morphine and other opioid drugs have been known for centuries. Nevertheless, the administration of high doses or prolonged treatment with morphine can cause adverse effects. Therefore, the search for new pain therapies is critically needed [1]. There are three major types of opioid receptors (ORs): µ-OR, δ-OR, and κ-OR, which serve as the primary molecular target for opioid drugs. These receptors belong to the rhodopsin family of G protein-coupled receptors (GPCRs) and are known to inhibit adenylyl cyclase (AC) activity in a pertussis toxin-dependent manner by activation of the Gi/Go class of trimeric G proteins [2,3].
This study found an unequivocal desensitization of µand δ-OR-stimulated G protein response in the plasma membrane-enriched fraction prepared from the cerebral cortex of rats exposed to increasing doses of morphine (10-50 mg/kg) for 10 days [4]. Furthermore, using immunoblot analysis, we have observed a specific up-regulation of AC I (8-fold)

Morphine and Peptide Treatment of Male Wistar Rats
All animal procedures were approved by the Animal Care and Use Committee of the Institute of Physiology of the Czech Academy of Sciences IPHYS (license number 46/2019) to be in agreement with Animal Protection Law of the Czech Republic as well as the European Communities Council Directive (86/609/EEC). Male Wistar rats (weighting 150-200 g) were kept on 12/12 light/dark cycle and had ad libitum access to food and water. The first experiment was performed with groups of rats (n = 3) which were injected subcutaneously for 7 consecutive days with 3 mg/kg per day of morphine (group 1) or enkephalin analogs LYS739 (group 2) and LYS744 (group 3) dissolved in 0.9% NaCl. Control animals (group 4) (n = 3) received 0.9% NaCl for 7 consecutive days. The second experiment was performed in the following manner: the morphine (group 5) and enkephalin analogs LYS739 (group 6) and LYS744 (group 7) (n = 4) were injected subcutaneously for 7 consecutive days with 10 mg/kg per day of these OR ligands dissolved in 0.9% NaCl. Control animals (group 8) (n = 4) received 0.9% NaCl for 7 consecutive days.

Preparation of Samples 2.3.1. Preparation of Post-Nuclear Supernatant (PNS) Fraction from Rat Brain Cortex and Hippocampus
We used the post-nuclear supernatant (PNS) fraction for all proteomic analyses according to our previous experimental experience [13,14,18]. Rats were sacrificed by decapitation under ether narcosis, cortices and hippocampi dissected, washed from remaining blood by ice-cold saline, frozen in liquid nitrogen, and stored at −80 • C until use. Pooled tissue pieces (2 g w.w. per 10 mL) were diluted in STEM medium (250 mM sucrose, 20 mM Tris-HCl, 3 mM MgCl2, 1 mM EDTA, pH 7.6) containing complete protease inhibitor cocktail and fresh 1 mM phenylmethylsulfonyl fluoride (PMSF), homogenized in Teflon-glass homogenizer for 7 min at 1800 rpm, and centrifuged for 7 min at 1200× g. The PNS fractions were snap frozen in liquid nitrogen and stored at −80 • C until use.

Isolation of Lymphocytes from Spleen; Preparation of Post-Nuclear Supernatant (PNS) Fraction
Isolation of lymphocytes from spleens was performed as described by Cechova et al. [19]. Briefly, spleens were dissected, and the single-cell suspension was prepared in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% (v/v) heatinactivated Fetal Bovine Serum (FBS) GibcoTM (Thermo Fischer Scientific, Waltham, MA, USA). Subsequently, the single-cell suspension was layered on the top of Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA) and centrifuged for 35 min at 800× g at 19 • C. The lymphocyte fraction was washed twice in RPMI-1640 medium containing 5% FBS and twice in PBS. Finally, lymphocytes were resuspended in 0.5 mL of PBS containing complete protease inhibitor cocktail, snap frozen in liquid nitrogen, and stored at −80 • C until use.
When preparing PNS fraction, suspension of lymphocytes was diluted in 50 mM Tris-HCl (pH 7.6), 3 mM MgCl2, 1 mM EDTA (TME buffer) containing fresh 1 mM PMSF and complete protease inhibitor cocktail, and homogenized in tight Teflon-glass homogenizer for 7 min at 1400 rpm on ice. The cell homogenates were centrifuged for 7 min at 500× g. The PNS fractions were snap frozen in liquid nitrogen and stored at −80 • C until use.

Label-Free Quantification (LFQ)
LFQ data were evaluated in two rounds (the effect of 3 mg/kg-round 1, the effect of 10 mg/kg-round 2) for 12 samples in three technical replicates of each round: lymphocytes samples (LMOR 1-3, LLYS739 1-3, LLYS744 1-3, LCTR 1-3), cortical samples (CMOR 1-3, CLYS739 1-3, CLYS744 1-3, CCTR 1-3) and hippocampal samples (HMOR 1-3, HLYS739 1-3, HLYS744 1-3, HCTR 1-3). Sample preparation was performed as described before in Ujcikova et al. [13,14]. Eluting peptide cations converted to gas-phase ions were analyzed on Thermo Orbitrap Fusion (Thermo ScientificTM) mass spectrometer. Raw data were analyzed and quantified by using the MaxQuant software. The false discovery rate (FDR) was set to 1% for proteins and peptides (the minimum of peptide length was 7 amino acids). The MS/MS spectra search against the Uniprot Rattus norvegicus database of protein sequences (https://www.uniprot.org, accessed on 3 November 2021) was done by using the Andromeda search engine (enzyme specificity: C-terminal to arginine and lysine; fixed modification: carbamidomethylation of cysteine; variable modifications: N-terminal protein acetylation, methionine oxidation). Quantifications with the label-free algorithm were performed in MaxQuant according to Cox et al. [17], data were analyzed using Perseus 1.6.15.0 software. Binary logarithms of intensity ratios were then median calculated for each group, and the difference between control and treated samples was determined. Only at least 2-fold significant differences calculated for at least two measured values from 3 replicates were taken into consideration. p-values (p ≤ 0.05) were calculated via GraphPad Prism 8.3.0 software (San Diego, CA, USA).

Results and Discussion
3.1. LFQ Analysis of Rat Spleen Lymphocytes after 7-Day Treatment with Morphine, LYS739, and LYS744: The Dose-Effect of 3 mg/kg The study had some limitations. The first one is that we had to work with small amounts of peptides LYS739 and LYS744. For that reason, each testing group included only three of four animals. Second, this study was not designed to search for differences between male and female rats and only included male rats as in our previous proteomic analyses [13][14][15]18]. The third one is connected with mass spectrometry-based data. Some measurements may result in technical failures. Therefore, three technical replicates of each sample enabled us to perform statistical analysis. Label-free quantification (LFQ) analysis revealed 27 altered proteins in rat spleen lymphocytes after 7-day morphine treatment (3 mg/kg). The minimum of 2-fold expression differences were calculated for at least 2 measured values from 3 technical replicates, Table S1a. The 12 proteins were upregulated, and 15 proteins were downregulated. Their subcellular localization and functional significance unveiled most proteins of cytoplasmic origin related to the cytoskeletal changes, Figure 1a,b (upper panels). These proteins were associated with actin filament bundle assembly: beta-adducin (Add2, ↑4.0-fold), spectrin beta chain (Sptbn2, ↑3.9-fold); microtubule polymerization: tubulin polymerization-promoting protein family member 3 (Tppp3, ↑2.6-fold), tubulin beta-3 chain (Tubb3, ↓5.1-fold), or intermediate filament organization: vimentin (Vim, ↓2.8-fold), Table S1a.
, and Ddx21 (↓2.0-fold) were found dysregulated after morphine administration and LYS739 treatment as well, see above. Volcano plots representing significantly altered proteins identified in rat brain cortex after treatment with 3 mg/kg of morphine, LYS739, and LYS744 are depicted in Figure 2B. Hierarchical heatmap clustering of all identified protein expression profiles in the rat brain cortex is presented in Figure 3. LFQ analysis indicated 14 proteins with changed expression levels at least 2-fold after 7-day treatment with morphine. This amount is similar to the number of alterations in the rat brain cortex (15) but lower twice in comparison with spleen lymphocytes (27). Eight proteins were upregulated, 6 had decreased levels, Table S1c. According to the current annotations in the Uniprot database, these proteins were mainly associated with the regulation of transcription and translation: methyl-CpG-binding protein 2 (Mecp2, ↑2.6-fold), general transcription factor II-I (Gtf2i, ↑2.2-fold), CXXC-type zinc finger protein 1 (Cxxc1, ↑2.2-fold), 60S ribosomal protein L29 (Rpl29, ↑2.0-fold), and cytoskeletal changes: vasodilator-stimulated phosphoprotein (Vasp, ↑4.1-fold), transgelin (Tagln, ↑2.7-fold), alphainternexin (Ina, ↓2.2-fold), Figure 5a,b (upper panels). Interestingly, in our previous study, the level of Ina was detected as hypophosphorylated (↓2.3-fold) in rat hippocampal samples after 3-month morphine withdrawal [15].
Treatment with LYS744 revealed 42 alterations in protein expression levels, which is more than in the brain cortex (31) and similar to spleen lymphocytes (45). Twenty-four proteins were upregulated and 18 proteins were downregulated, Table S1c. RNA processing (9) and transport (9) were the main functional categories, Figure 5a,b (lower panels). Mecp2 (↑2.6-fold) and Rpl29 (↑2.0-fold) were found significantly upregulated after morphine treatment as well, see above. Among upregulated proteins, four of these were associated with the change in energy metabolism: electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial (Etfdh, ↑2.6-fold), ATPase inhibitor, mitochondrial (Atpif,↑2.4-fold), complex I-B17 (Ndufb6, ↑2.4-fold), and cytochrome c oxidase subunit 2 (Mtco2, ↑2.3-fold). We previously reported that the majority of altered hippocampal proteins were related to energy metabolism after both chronic morphine treatment (10-50 mg/kg, 10 days) and subsequent drug withdrawal (3 weeks, 3 months, 6 months) [14][15][16]. Volcano plots repre-senting significantly altered proteins identified in rat hippocampus after treatment with 3 mg/kg of morphine, LYS739, and LYS744 are depicted in Figure 2C. Hierarchical heatmap clustering of all identified protein expression profiles in rat hippocampus is presented in Figure 3.   In the second round of our experiments, we increased the dose of morphine and enkephalin analogs from 3 mg/kg to 10 mg/kg. We applied this amount of OR drugs to rats for 7 consecutive days to test the effect of increased doses on protein changes in spleen lymphocytes, cortex, and hippocampus. This scheme was modified according to our previously established protocols [4,5,13,14,18]. LFQ analysis revealed 45 altered proteins (at least 2-fold) in spleen lymphocytes after morphine treatment. Upregulation was found for 29 proteins; 16 proteins were downregulated, Table S2a. According to the current annotations in the Uniprot database, their molecular functions and biological processes were mostly involved in transport (9), immunity (8), apoptosis (8), and RNA processing (7), Figure 6a,b (upper panels). When compared with the effect of a lower dose, the number of proteins associated with immune responses and apoptotic processes was increased, suggesting the state of cell dysfunction and neuroinflammation after chronic morphine administration. Among upregulated apoptotic proteins were interferon-induced protein with tetratricopeptide repeats 3 (Ifit3, ↑4.9-fold), sialic acid-binding Ig-like lectin 1 (Siglec1, ↑3.8-fold), interferon-induced protein with tetratricopeptide repeats 2 (Ifit2, ↑3.4-fold), MAP kinase-activating death domain protein (Madd, ↑2.8-fold), galectin-5 (Lgals5, ↑2.3-fold), and interferon activated gene 204 (Mnda, ↑2.2-fold).
The treatment with LYS744 caused the most significant differences in protein expression levels of spleen lymphocytes. Four times more proteins were altered (182 proteins, 88 upregulated, 94 downregulated) in comparison with the lower dose of LYS744, Table S2a. Identified proteins were primarily associated with RNA processing (58), transport (25), immune responses (16), metabolic changes (13), cytoskeletal changes (13), and apoptotic processes (12), Figure 6 a,b (lower panels). Vav1 (↑3.7-fold), Mnda (↑3.4-fold), Mpeg1 (↑2.6-fold), and Btk (↑2.5-fold) were identified in samples treated with LYS739 as well, see above. Both immunosuppressive and immuno-stimulating effects have been described after opioid administration [30][31][32]. Recent studies have shown that Vav proteins are essential for the homeostasis of the central nervous, cardiovascular, and immune systems. They are considered potential therapeutic targets for several pathological conditions [33,34]. Volcano plots representing significantly altered proteins identified in rat spleen lymphocytes after treatment with 10 mg/kg of morphine, LYS739, and LYS744 are depicted in Figure 7A. Hierarchical heatmap clustering of all identified protein expression profiles in rat spleen lymphocytes is presented in Figure 8. LYS739 treatment resulted in the change of 119 proteins (51 proteins were upregulated, 68 downregulated). The number of altered proteins was six times higher than after the dose of 3 mg/kg, Table S2a. The majority of identified proteins were related to RNA processing (40), transport (16), immunity (11), metabolism (10), and cytoskeletal changes (10), Figure 6a,b (middle panels). Seven proteins that participated in immune responses were upregulated: Ig gamma-2C chain C region (N/A, ↑27.5-fold), proto-oncogene vav (Vav1, ↑9.5-fold), tyrosine-protein kinase (Btk, ↑3.4-fold), Mnda (↑2.8-fold), macrophage- are considered potential therapeutic targets for several pathological cond Volcano plots representing significantly altered proteins identified in rat sp cytes after treatment with 10 mg/kg of morphine, LYS739, and LYS744 ar Figure 7A. Hierarchical heatmap clustering of all identified protein expressi rat spleen lymphocytes is presented in Figure 8.
LFQ analysis revealed 26 altered proteins in the brain cortex after LYS744 treatment. This is a similar number to morphine-induced alterations (31) and, noticeably, less than after the administration of the lower dose of LYS744 (31). Six proteins were upregulated and 20 were downregulated, Table S2b. According to the current annotations in the Uniprot database, these proteins were associated with RNA processing (7), DNA processing (5), cytoskeletal changes (4), transport (3), brain development (3), metabolism (3), apoptosis (2), aging (2), cell cycle (1), immunity (1), protein folding (1), cell adhesion (1), and protein localization (1), Figure 9a,b (lower panels). S100a9 was the most downregulated protein (↓5.4-fold), which was found to decrease in cortical samples after morphine administration (↓13.2-fold) and treatment with LYS739 (↓13.4-fold) as well. Apart from its role in inflammation and apoptosis [25], Toleikis et al. [38] described its involvement in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their data have shown that even small concentrations of S100a9 resulted in the aggregation and formation of α-synuclein (Snca) fibrils. Snca is the main protein found in amyloid plaques in the brains of patients suffering from Parkinson's disease. Volcano plots representing significantly altered proteins identified in rat brain cortex after treatment with 10 mg/kg of morphine, LYS739, and LYS744 are depicted in Figure 7B. Hierarchical heatmap clustering of all identified protein expression profiles in rat brain cortex is presented in Figure 8.  Table S2c. This is three times more than the effect of a lower morphine dose (14). The functional significance of hippocampal proteins was mainly related to RNA processes (8), transport (8), protein folding (6), cytoskeletal changes (4), brain development (4), metabolic changes (4), immunity (3), and cell adhesion (3), Figure 10a,b (upper panels).
The effect of a higher LYS744 dose on hippocampal proteome was surprisingly decreased from 42 to 26 proteins (6 were upregulated, 20 downregulated) when compared with the lower dose, Table S2c. The functional significance of these proteins was associated with RNA processing (8), cytoskeletal changes (5), brain development (3), metabolism (2), transport (2), immunity (2), DNA processing (2), aging (2), protein ubiquitination (1), signal transduction (1), and cell cycle (1), as shown in Figure 10a,b (lower panels). Cytoskeletal proteins: Myh11 (↓15.0-fold), Vim (↓10.4-fold), Tagln (↓4.6-fold), Flna (↓2.6-fold), and Cald1 (↓2.3-fold) were found dysregulated after LYS739 treatment as well, see above. Volcano plots representing significantly altered proteins identified in rat hippocampus after treatment with 10 mg/kg of morphine, LYS739, and LYS744 are depicted in Figure 7C. Hierarchical heatmap clustering of all identified protein expression profiles in the rat hippocampus is presented in Figure 8. LFQ analysis revealed 27 altered proteins in rat spleen lymphocytes after 7-day treatment of male rats with 3 mg/kg of morphine. The 12 proteins were upregulated, downregulation was detected for 15 proteins. Their subcellular localization and functional significance unveiled most proteins to be of cytoplasmic origin and functionally related to the organization of cytoskeleton. Fewer proteins were altered by LYS739, 5 proteins were upregulated, 15 downregulated. LYS744 induced more alterations than morphine, 29 proteins were upregulated, and 16 proteins were downregulated. More than half of these proteins were functionally related to actin cytoskeleton organization (11), RNA processing (8), and transport (6).
In the brain cortex, morphine administration resulted in the change of 15 proteins; 5 proteins were upregulated, 10 proteins were downregulated. Contrary to the spleen lymphocytes, LYS739 treatment resulted in a greater number of protein alterations than morphine, with 7 proteins upregulated and 13 downregulated. As in the case of spleen lymphocytes, treatment with LYS744 exhibited the highest number of alterations in cortical proteome-11 proteins were upregulated, 20 proteins were downregulated.
In the hippocampus, LFQ analysis revealed 14 proteins altered by morphine, 8 proteins were upregulated, 6 were downregulated. This change was similar to that found in the brain cortex (15) but lower twice in comparison with spleen lymphocytes (27). The effect of LYS739 on rat hippocampal protein profile was stronger than that of morphine with 8 proteins upregulated and 28 downregulated. Treatment with LYS744 unveiled 42 alterations in protein expression levels, which is more than in the brain cortex (31) and similar to that found in spleen lymphocytes (45); 24 proteins were upregulated and 18 proteins were downregulated. 3.7.2. Effects of Higher Doses of Morphine, LYS739, and LYS744 (10 mg/kg) Proteomic analysis revealed 45 altered proteins in spleen lymphocytes after morphine treatment. Upregulation was found for 29 proteins; 16 proteins were downregulated. Their molecular functions and biological processes were mostly involved in transport (9), immunity (8), apoptosis (8), and RNA processing (7). When compared with the effect of a lower dose, the number of proteins associated with immune responses and apoptotic processes was increased, suggesting the state of cell dysfunction and neuroinflammation after chronic morphine administration. LYS739 treatment resulted in the change of 119 proteins, 51 proteins were upregulated, 68 downregulated. Thus, the number of altered proteins was six times higher than that obtained after the administration of a lower dose. The majority of altered proteins were related to RNA processing (40), transport (16), immunity (11), metabolism (10), and cytoskeletal changes. Higher dose of LYS744 caused the most significant change in protein expression levels of spleen lymphocytes. Four times more proteins were altered (182 proteins, 88 were upregulated, 94 downregulated) in comparison with the lower dose of LYS744. The identified proteins were primarily associated with RNA processing (58), transport (25), immune responses (16), metabolic changes (13), cytoskeletal changes (13), and apoptotic processes (12).

Conclusions
Overall, 7-day treatment with 3 mg/kg of LYS744 caused the most significant effect on altered proteins (at least 2-fold) in spleen lymphocytes, rat brain cortex, and hippocampus in comparison with morphine and LYS739 treatment. When administered a higher dose (10 mg/kg), the number of alterations in lymphocytes was increased by treatment with both analogs, LYS739 and LYS744. The higher dose of LYS739 caused extensive protein alterations in cortical samples and slightly increased the number of altered proteins in the rat hippocampus. Considering its specific multifunctional activities (µ-OR/δ-OR agonism and κ-OR partial antagonism), our results indicate the possible involvement of multiple opioid receptors in protein regulations in these brain areas. However, the higher dose of LYS744 did not increase the number of altered proteins in the brain cortex and hippocampus. For the same reason, the κ-OR antagonism of LYS744 seems to be related to the reduced number of protein alterations in the central nervous system areas. Interestingly, the effect of κ-OR antagonism of LYS744 was opposite in the peripheral region (spleen lymphocytes).