Methylenedioxy Piperamide-Derived Compound D5 Regulates Inflammatory Cytokine Secretion in a Culture of Human Glial Cells
Abstract
:1. Highlights
- -
- D5 reduced the level of pro-inflammatory cytokines, through up-regulating PPAR-γ expression, suppressing IKK-β, iNOS, NO production, and NF-κB activation in inflamed astrocytes (SVG) and microglia (CHME3) after 24 h of incubation.
- -
- D5 showed higher efficacy than ASA on the inhibition of the NF-κB pathway leading to neuroinflammation.
- -
- D5 acts as a potent anti-neuroinflammatory agent.
2. Introduction
3. Materials and Methods
3.1. In Vitro Study
3.1.1. Cell Culture
3.1.2. Cell Treatment
3.1.3. Cytoprotective Effect of D5 in the Selected Concentration
3.1.4. NO Quantification
3.1.5. Isolation of Total Proteins
3.1.6. Western Blot Analysis
3.1.7. Enzyme-Linked Immunosorbent Assay (ELISA)
3.1.8. Immunocytochemistry
3.1.9. Total RNA Extraction and Quantitative Real-Time PCR (qPCR)
3.2. The Pharmacological Properties of Methylenedioxy (D5) vs. Hydroxymethyl (D4) Derivatives of Piperamide
3.2.1. In Silico Evaluation of Pharmacological Properties and Potential Inhibitory Nature
- Comparison of the physicochemical and toxicological properties
- b.
- Comparison of pharmacodynamic properties against IKK-β enzyme
3.2.2. In Vitro Druggability Evaluation of D5 vs. D4 on Neuroinflammatory Progress
3.3. Data Presentation and Statistical Analysis
4. Results
4.1. In Vitro Pharmacological Property Evaluation of D5 against LPS-Induced Inflammation in the Glial Cells
4.1.1. Determination of the Drug’s Optimal Concentrations
4.1.2. Drug Safety
4.1.3. Inhibitory Effect of D5 on the iNOS Expression Level and Nitric Oxide Secretion in LPS-Stimulated SVG and CHME3 Cell Lines
4.1.4. Inhibitory Effects of D5 on TNF-α and IL-1β Expression in LPS-Stimulated SVG and CHME3 Cell Lines
4.1.5. Up-Regulation of PPAR-γ Receptor Expression by D5 in LPS-Stimulated SVG and CHME3 Cell Lines
4.1.6. Inhibitory Effects of D5 on NF-κBSignaling in LPS-Stimulated SVG and CHME3 Cell Lines
4.2. Comparison of the Anti-Neuroinflammatory Potential of Methylenedioxy (D5) and Hydroxymethyl (D4) Piperamide Derivatives
4.2.1. In Silico Comparison between Pharmacological Properties of D5 and D4
- Comparison of the physicochemical and toxicological properties of both compounds
- b.
- Comparison of the pharmacodynamic properties of D4 and D5 against IKK-β enzyme
4.2.2. In Vitro Comparison of the Druggability Potential of Methylenedioxy and Hydroxymethyl Piperamide Derivatives on LPS-Induced inflammation in the Glial Cells
5. Discussion
6. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Drug | Amounts | DOSE * | IL-6/24 h | INF-γ/24 h | ||
---|---|---|---|---|---|---|
OD | QUANTIFY (pg/mL) | OD | QUANTIFY (pg/mL) | |||
D4 | ½ EC50 | 140 | 0.32 | 282.11 ± 5.24 | 0.30 | 219.82 ± 10.13 |
EC50 | 280 | 0.22 | 188.07 ± 6.17 | 0.23 | 163.04 ± 4.52 | |
2x EC50 | 560 | 0.17 | 141.04 ± 8.03 | 0.21 | 146.82 ± 5.85 | |
D5 | ½ EC50 | 120 | 0.28 | 259.08 ± 9.22 | 0.31 | 291.37 ± 5.93 |
EC50 | 240 | 0.21 | 194.93 ± 5.92 | 0.20 | 186.97 ± 5.28 | |
2x EC50 | 480 | 0.19 | 170.98 ± 5.40 | 0.16 | 149.33 ± 7.12 | |
ASP | ½ EC50 | 4600 | 0.31 | 283.64 ± 5.13 | 0.33 | 309.09 ± 5.23 |
EC50 | 9100 | 0.27 | 244.04 ± 10.86 | 0.20 | 192.98 ± 6.18 | |
2x EC50 | 18,200 | 0.22 | 206.90 ± 8.05 | 0.14 | 131.92 ± 7.77 | |
CELL + LPS | 0.44 | 402.44 ± 8.29 | 0.43 | 412.54 ± 4.49 | ||
CELL | 0.16 | 144.28 ± 5.99 | 0.12 | 111.68 ± 7.90 |
Physicochemical Properties | Computed Range | Acceptable Range | |
---|---|---|---|
D5 | D4 | ||
SASA | 565.2 | 611.314 | 300–1000 |
FOSA | 196.15 | 179.518 | 0–50 |
FISA | 52.860 | 101.941 | 7–330 |
Volume | 993.086 | 1044.573 | 500–2000 |
donorHB | 1 | 2 | 0–6 |
accptHB | 5.5 | 5.7 | 2–20 |
CNS | 0 | −1 | −2–2 |
QPlogPC16 | 10.272 | 11.365 | 4–18 |
QPlogPoct‡ | 14.632 | 17.079 | 8–35 |
QPlogPw | 9.148 | 10.906 | 4–45 |
QPlogPo/w | 3.232 | 3 | −2–6.5 |
QPlogHERG | −5.44 | −6.108 | <−5 |
QPlogBB | −0.284 | −0.943 | −3–1.2 |
QPPMDCK | 1694.260 | 531.993 | <25 poor, >500 great |
QPlogKp | −0.804 | −1.468 | −8–−1 |
QPlogKhsa | 0.012 | −0.012 | −1.5–1.5 |
HOA | 3 | 3 | 1–3 |
%HOA | 100 | 100 | <25% poor, >80% high |
ROF | 0 | 0 | Max 5 |
ROT | 0 | 0 | Max 3 |
QPlogS | −3.642 | −4.03 | >−5.7 * |
QPPCaco | 3210.48 | 1069.535 | >22 nm/s * |
# Primary Metabolites | 0 | 1 | <7 * |
WOE | Non-carcinogen | Non-carcinogen | |
Rodent Carcinogenicity | Non-carcinogen | Non-carcinogen | |
Rodent Mutagenicity | Non-mutagen | Non-mutagen | |
LD50 | 2.152542 | 4.905009 | g/kg_body_weight |
LC50 (inhalation) | 8.922757 | 8.173971 | mg/m3/h |
TD50 | 0.167370 | 0.196023 | g/kg_body_weight |
EC50 | 0.241032 | 0.560252 | mg/L |
Name | D4 | D5 | Pvalue | Tcrit | Tstat | df | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
SVG | CHME3 | SVG | CHME3 | SVG | CHME3 | SVG | CHME3 | SVG | CHME3 | SVG | CHME3 | |
* No secretion (normalized by LPS group) | 69.9 ± 4.7 | 87.4 ± 1.0 | 55.6 ± 5.0 | 54.3 ± 4.4 | 0.05 | 0.01 | 2.8 | 4.3 | 3.6 | 12.7 | 4 | 2 |
% iNOS secretion (normalized by LPS group) | 59.2 ± 3.9 | 79.2 ± 4.8 | 47.2 ± 2.6 | 36.3 ± 3.2 | 0.05 | 0.01 | 3.2 | 2.8 | 4.5 | 12.9 | 3 | 4 |
% Phospho-IkB-α (normalized by LPS group) | 22.0 ± 1.8 | 32.1 ± 4.7 | 38.8 ± 2.8 | 25.8 ± 3.8 | 0.01 | 0.05 | 3.2 | 2.8 | −8.8 | 3.4 | 3 | 4 |
% TNF-α secretion (normalized by LPS group) | 127.7 ± 5.4 | 100 ± 9.2 | 101.7 ± 7.7 | 101.6 ± 6.0 | 0.01 | -- | 2.8 | -- | 4.8 | -- | 4 | -- |
# INF-γ secretion (normalized by LPS group) | 73.2 ± 0.5 | -- | 46.7 ± 1.3 | -- | 0.01 | -- | 3.2 | -- | 32.4 | -- | 3 | -- |
@ IKK-β gene expression (normalized by LPS group) | 0.062 ± 0.010 | 0.105 ± 0.024 | 0.036 ± 0.003 | 0.098 ± 0.028 | 0.05 | -- | 4.3 | -- | 4.5 | -- | 2 | -- |
@ TNF-α gene expression (normalized by LPS group) | 0.05 ± 0.008 | 0.054 ± 0.012 | 0.025 ± 0.001 | 0.096 ± 0.027 | 0.05 | -- | 4.3 | -- | 5.7 | -- | 2 | -- |
@ PPAR-γ gene expression (normalized by LPS group) | 37.365 ± 1.347 | 25.909 ± 1.792 | 43.133 ± 2.836 | 55.624 ± 2.88 | -- | 0.01 | -- | 3.2 | -- | 15.1 | -- | 3 |
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Shahbazi, S.; Zakerali, T. Methylenedioxy Piperamide-Derived Compound D5 Regulates Inflammatory Cytokine Secretion in a Culture of Human Glial Cells. Molecules 2022, 27, 3527. https://doi.org/10.3390/molecules27113527
Shahbazi S, Zakerali T. Methylenedioxy Piperamide-Derived Compound D5 Regulates Inflammatory Cytokine Secretion in a Culture of Human Glial Cells. Molecules. 2022; 27(11):3527. https://doi.org/10.3390/molecules27113527
Chicago/Turabian StyleShahbazi, Sajad, and Tara Zakerali. 2022. "Methylenedioxy Piperamide-Derived Compound D5 Regulates Inflammatory Cytokine Secretion in a Culture of Human Glial Cells" Molecules 27, no. 11: 3527. https://doi.org/10.3390/molecules27113527
APA StyleShahbazi, S., & Zakerali, T. (2022). Methylenedioxy Piperamide-Derived Compound D5 Regulates Inflammatory Cytokine Secretion in a Culture of Human Glial Cells. Molecules, 27(11), 3527. https://doi.org/10.3390/molecules27113527