Phoenix dactilyfera L. Pits Extract Restored Bone Homeostasis in Glucocorticoid-Induced Osteoporotic Animal Model through the Antioxidant Effect and Wnt5a Non-Canonical Signaling
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Preparation of Date Pits Extract
2.3. Characterization of Date Pits Extract
2.4. Experimental Design
2.5. Collection and Preparation of Blood and Tissues
2.6. Bone Density Test
2.7. Biochemical Analysis and Spectrophotometry Evaluations
2.8. Sandwich ELISA
2.9. Total RNA Isolation and Quantitative Real-Time Reverse Transcription PCR Analysis (qRT-PCR)
2.10. Histopathological Study
2.11. Statistical Analysis
3. Results
3.1. Characterization of Date Pits Extract
3.2. BMD, BMC, and Serum Calcium Level
3.3. Oxidative Stress Indices
3.4. Estimation of MSC Proliferation Markers
3.5. Osteoblast’s Differentiation Indices
3.6. Osteoclast’s Differentiation Markers
3.7. Histopathologic Outcomes
3.8. Heat Map Analysis
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene Name | Accession Number | Primer Sequence | Annealing Temperature (°C) | Number of Cycles | Ref. | |
---|---|---|---|---|---|---|
GAPDH | NM_017008.4 | F | 5′-AGATCCACAACGGATACATT-3′ | 52 | 35 | [36] |
R | 5′-TCCCTCAAGATTGTCAGCAA-3′ | |||||
OPG | NM_012870.2 | F | 5′-GTTCTTGCACAGCTTCACCA-3′ | 54 | 40 | [37] |
R | 5′-AAACAGCCCAGTGACCATTC-3′ | |||||
RANKL | NM_057149.1 | F | 5′-ACCAGCATCAAAATCCCAAG-3′ | 52 | 35 | [37] |
R | 5′-GGCCGCTAATTTCCTCACCA-3′ | |||||
DKK1 | NM_001106350.1 | F | 5′-GCTGCATGAGGCACGCTAT-3′ | 55 | 35 | [38] |
R | 5′-AGGGCATGCATATTCCGTTT-3′ | |||||
Wnt5a | NM_022631.3 | F | 5′-CCATGAAGAAGCCCATTGGAATA-3′ | 60 | 40 | [39] |
R | 5′-GGCCAAAGCCATTAGGAAGAA-3′ | |||||
SOST | NM_030584.2 | F | 5′-GTGCAAGTGCAAGCGCCTCA-3′ | 60 | 40 | [40] |
R | 5′-GCTCCGCCTGGTTGGCTTTG-3′ | |||||
Runx2 | NM_001278483.1 | F | 5′-AGTGTGTGTGTCCGCATGAT-3′ | 56 | 40 | [41] |
R | 5′-CCACTTGGGGTCTAAGAACG-3′ | |||||
Osx | NM_181374.2 | F | 5′-TGAGGAAGAAGCCCATTCAC-3′ | 53.5 | 40 | [42] |
R | 5′-ACTTCTTCTCCCGGGTGTG-3′ | |||||
COL1A1 | NM_053304.1 | F | 5′-CAAGGACTATGAAGTTGATGC-3′ | 43 | 40 | [40] |
R | 5′-ACCAGTAGAGAAATCGCAGT-3′ |
Scavenging Activity (IC50, µg/mL) | |||
---|---|---|---|
DPPH | 112.17 ± 5.11 | ||
H2O2 radical | 44.38 ± 2.56 | ||
Constituents | Concentration | ||
Phytochemicals | µg equivalent/ mg extract | LDPE (150 mg/kg) | HDPE (300 mg/kg) |
Total phenolics | 301.97 ± 5.16 | 45,295 ± 774 | 90,591 ± 1548 |
Total flavonoids | 5.10 ± 2.75 | 765 ± 412.5 | 1530 ± 825 |
HPLC Analysis of Phenolic Compounds | |||
Constituents | (μg/g extract) | LDPE (μg/150 mg/kg) | HDPE (μg/300 mg/kg) |
Quinol | 703.81 ± 8.5 | 105.6 ± 1.3 | 211.14 ± 2.55 |
p-Hydroxybenzoic acid | 1042.01 ± 15.9 | 156.3 ± 2.25 | 312.6 ± 4.77 |
Catechin | 70.76 ± 2.0 | 10.61 ± 0.3 | 21.23 ± 0.6 |
Chlorogenic | 26.67 ± 1.5 | 4.0 ± 0.225 | 8.0 ± 0.45 |
Vanillicacid | 625.43 ± 11.6 | 93.81 ± 1.74 | 187.63 ± 3.48 |
Caffeic acid | 89.58 ± 7.0 | 13.44 ± 1.05 | 26.87 ± 2.1 |
Syringic acid | 119.03 ± 3.9 | 17.85 ± 0.59 | 35.71 ± 1.17 |
p-Coumaric acid | 19.24 ± 1.9 | 2.89 ± 0.29 | 5.77 ± 0.57 |
Benzoic acid | 587.45 ± 17.6 | 88.12 ± 2.64 | 176.24 ± 5.28 |
Ferulic acid | 21.16 ± 3.2 | 3.17 ± 0.48 | 6.35 ± 0.96 |
Rutin | 17.55 ± 2.9 | 2.63 ± 0.44 | 5.27 ± 0.87 |
Ellagic acid | 24.89 ± 5.0 | 3.73 ± 0.75 | 7.74 ± 1.5 |
o-Coumaric acid | 10.72 ± 1.1 | 1.61 ± 0.17 | 3.22 ± 0.33 |
Resveratrol | 822.57 ± 13.7 | 123.39 ± 2.1 | 246.77 ± 4.11 |
Total | 4180.86 ± 18.5 | 627.13 ± 2.78 | 1254.26 ± 5.55 |
Mineral Content | DPE (µg/g Extract) | LDPE (150 mg/kg) | HDPE (300 mg/kg) |
---|---|---|---|
Ca | 44,675.673 | 6701.35 | 13,402.7 |
Cu | 1121.442 | 168.22 | 336.43 |
Fe | 1488.990 | 223.35 | 446.7 |
K | 236,567.019 | 35,485.053 | 70,970.11 |
Mg | 37,368.077 | 5605.21 | 11,210.42 |
Mn | 133.269 | 19.99 | 39.98 |
Na | 35,755.288 | 5363.29 | 10,726.59 |
P | 34,262.933 | 5139.44 | 10,278.88 |
Se | 20.817 | 3.12 | 6.25 |
Zn | 697.163 | 104.57 | 209.15 |
MDA Level (µmol/mg Protein) | NO Level (µmol/mg Protein) | GSH Content (µmol/mg Protein) | GPx Activity (U/mg Protein) | GST Activity (u/mg Protein) | SOD Activity (U/mg Protein) | |
---|---|---|---|---|---|---|
Control | 1.9534 ± 0.231 | 369.22 ± 12.425 | 10.2953 ± 0.483 | 58.1272 ± 8.152 | 0.727 ± 0.041 | 1.337 ± 0.059 |
LDPE | 1.2168 ± 0.055 * | 296.44 ± 50.414 * | 10.9006 ± 0.729 | 54.4612 ± 2.901 | 0.7637 ± 0.044 | 1.3448 ± 0.085 * |
HDPE | 1.063 ± 0.129 * | 235.23 ± 12.130 * | 13.8351 ± 0.492 * | 56.0947 ± 4.179 | 1.3592 ± 0.044 * | 1.6993 ± 0.040 * |
DEXA | 2.2015 ± 0.033 * | 861.26 ± 17.011 * | 6.287 ± 0.290 * | 23.257 ± 1.874 * | 0.389 ± 0.028 * | 0.8987 ± 0.057 * |
LDPE + DEXA | 1.0653 ± 0.074 *# | 365.8 ± 24.901 # | 15.9467 ± 0.646 *# | 72.4151 ± 4.680 *# | 1.0359 ± 0.041 *# | 1.5423 ± 0.062 *# |
HDPE + DEXA | 1.5578 ± 0.084 *# | 338.29 ± 22.747 # | 17.9689 ± 0.637 *# | 57.4594 ± 3.724 # | 1.34 ± 0.067 *# | 1.4421 ± 0.044 *# |
IPRI + DEXA | 1.1083 ± 0.050 *# | 441.28 ± 22.444 *# | 6.608 ± 1.116 * | 55.2588 ± 3.020 # | 1.0398 ± 0.017 #* | 1.336 ± 0.125 # |
Epiphyseal Plate Thickness (µm) | Trabecular Bone Quality Score * | Trabecular Bone Area Score ** | Osteoblast Mean Count/HPF | |
---|---|---|---|---|
Control | 277 | 0 | 0 | 18 |
LDPE | 233 | 0 | 0 | 16 |
HDPE | 229 | 0 | 0 | 16 |
DEXA | 80 | 2 | 3 | 4 |
LDPE + DEXA | 210 | 0 | 1 | 18 |
HDPE + DEXA | 181 | 1 | 1 | 13 |
IPRI + DEXA | 204 | 1 | 1 | 15 |
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Saleh, S.R.; Ghareeb, D.A.; Masoud, A.A.; Sheta, E.; Nabil, M.; Masoud, I.M.; Maher, A.M. Phoenix dactilyfera L. Pits Extract Restored Bone Homeostasis in Glucocorticoid-Induced Osteoporotic Animal Model through the Antioxidant Effect and Wnt5a Non-Canonical Signaling. Antioxidants 2022, 11, 508. https://doi.org/10.3390/antiox11030508
Saleh SR, Ghareeb DA, Masoud AA, Sheta E, Nabil M, Masoud IM, Maher AM. Phoenix dactilyfera L. Pits Extract Restored Bone Homeostasis in Glucocorticoid-Induced Osteoporotic Animal Model through the Antioxidant Effect and Wnt5a Non-Canonical Signaling. Antioxidants. 2022; 11(3):508. https://doi.org/10.3390/antiox11030508
Chicago/Turabian StyleSaleh, Samar R., Doaa A. Ghareeb, Aliaa A. Masoud, Eman Sheta, Mohamed Nabil, Inas M. Masoud, and Adham M. Maher. 2022. "Phoenix dactilyfera L. Pits Extract Restored Bone Homeostasis in Glucocorticoid-Induced Osteoporotic Animal Model through the Antioxidant Effect and Wnt5a Non-Canonical Signaling" Antioxidants 11, no. 3: 508. https://doi.org/10.3390/antiox11030508
APA StyleSaleh, S. R., Ghareeb, D. A., Masoud, A. A., Sheta, E., Nabil, M., Masoud, I. M., & Maher, A. M. (2022). Phoenix dactilyfera L. Pits Extract Restored Bone Homeostasis in Glucocorticoid-Induced Osteoporotic Animal Model through the Antioxidant Effect and Wnt5a Non-Canonical Signaling. Antioxidants, 11(3), 508. https://doi.org/10.3390/antiox11030508