Comparative Analysis of Autophagy and Apoptosis in Disc Degeneration: Understanding the Dynamics of Temporary-Compression-Induced Early Autophagy and Sustained-Compression-Triggered Apoptosis
<p>(<b>A</b>) In X-ray imaging, compression led to an increase in disc degeneration, with a time-dependent and load-specific influence. The degeneration pattern in both temporary- and sustained-compression groups was similar. However, there was a notable difference in the rate of degeneration (White arrow: Indication of disc status after compression and control). (<b>B</b>) In MRI, observations at different intervals (2, 4, 6, and 12 weeks) post-compression revealed a progressive elevation in disc degeneration over time (White arrow: Indication of disc status after compression and control).</p> "> Figure 1 Cont.
<p>(<b>A</b>) In X-ray imaging, compression led to an increase in disc degeneration, with a time-dependent and load-specific influence. The degeneration pattern in both temporary- and sustained-compression groups was similar. However, there was a notable difference in the rate of degeneration (White arrow: Indication of disc status after compression and control). (<b>B</b>) In MRI, observations at different intervals (2, 4, 6, and 12 weeks) post-compression revealed a progressive elevation in disc degeneration over time (White arrow: Indication of disc status after compression and control).</p> "> Figure 2
<p>(<b>A</b>) H&E staining and (<b>B</b>) Masson’s trichrome staining: The severity of degeneration was directly related to the duration of compression, with SC leading to notably severe central NP cell degeneration, which increased with the duration of compression. Unexpectedly, TC also led to similar but less severe degeneration.</p> "> Figure 2 Cont.
<p>(<b>A</b>) H&E staining and (<b>B</b>) Masson’s trichrome staining: The severity of degeneration was directly related to the duration of compression, with SC leading to notably severe central NP cell degeneration, which increased with the duration of compression. Unexpectedly, TC also led to similar but less severe degeneration.</p> "> Figure 3
<p>(<b>A</b>,<b>B</b>) Beclin-1 expression elevated after both 6 and 12 weeks in all groups compared to the control group. However, the expression was more elevated after 6 weeks than after 12 weeks in the TCD-7, TCD-14, and SC groups. (<b>C</b>,<b>D</b>) LC3 expression was elevated after both 6 and 12 weeks in all groups compared to the control group. However, the expression was more elevated after 6 weeks than after 12 weeks in the TCD-7, TCD-14, and SC groups. (<b>E</b>,<b>F</b>) Cleaved caspase-3 expression was elevated in all groups after 6 and 12 weeks compared to the control group. However, the expression was not significant in the TCD-7 and TCD-14 groups after 12 weeks than after 6 weeks. On the contrary, the expression was peaked and more significant after 12 weeks than after 6 weeks in the SC group (black squares: area is to be magnified, white triangles: indication of the immunopositive area).</p> "> Figure 3 Cont.
<p>(<b>A</b>,<b>B</b>) Beclin-1 expression elevated after both 6 and 12 weeks in all groups compared to the control group. However, the expression was more elevated after 6 weeks than after 12 weeks in the TCD-7, TCD-14, and SC groups. (<b>C</b>,<b>D</b>) LC3 expression was elevated after both 6 and 12 weeks in all groups compared to the control group. However, the expression was more elevated after 6 weeks than after 12 weeks in the TCD-7, TCD-14, and SC groups. (<b>E</b>,<b>F</b>) Cleaved caspase-3 expression was elevated in all groups after 6 and 12 weeks compared to the control group. However, the expression was not significant in the TCD-7 and TCD-14 groups after 12 weeks than after 6 weeks. On the contrary, the expression was peaked and more significant after 12 weeks than after 6 weeks in the SC group (black squares: area is to be magnified, white triangles: indication of the immunopositive area).</p> "> Figure 3 Cont.
<p>(<b>A</b>,<b>B</b>) Beclin-1 expression elevated after both 6 and 12 weeks in all groups compared to the control group. However, the expression was more elevated after 6 weeks than after 12 weeks in the TCD-7, TCD-14, and SC groups. (<b>C</b>,<b>D</b>) LC3 expression was elevated after both 6 and 12 weeks in all groups compared to the control group. However, the expression was more elevated after 6 weeks than after 12 weeks in the TCD-7, TCD-14, and SC groups. (<b>E</b>,<b>F</b>) Cleaved caspase-3 expression was elevated in all groups after 6 and 12 weeks compared to the control group. However, the expression was not significant in the TCD-7 and TCD-14 groups after 12 weeks than after 6 weeks. On the contrary, the expression was peaked and more significant after 12 weeks than after 6 weeks in the SC group (black squares: area is to be magnified, white triangles: indication of the immunopositive area).</p> "> Figure 4
<p>(<b>A</b>) Autophagy-related gene mRNA expression (RT-qPCR): The mRNA expression of beclin-1 was found to be elevated in all groups after both 6 and 12 weeks compared to the control group. However, after 6 weeks, the expression was more elevated than after 12 weeks. LC3 mRNA expression showed an increase after 6 weeks in all groups compared to the control group. However, after 12 weeks, the expression was downregulated compared to the levels observed at 6 weeks and was somewhat lower than the control group. P62 expression was elevated after both 6 and 12 weeks in all groups compared to the control group. Interestingly, after 12 weeks, the expression was more upregulated than at 6 weeks. In the SC group, contrary to expectations, the expression was more downregulated after 12 weeks compared to the TCD groups (level of significance is * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001). (<b>B</b>) Apoptosis-related gene mRNA expression (RT-qPCR): The mRNA expression of PARP was elevated in all groups after both 6 and 12 weeks compared to the control group. Notably, after 12 weeks, a more elevated expression was observed than after 6 weeks. In the TCD-7 and TCD-14 groups, there was no significant upregulation, whereas the SC group exhibited statistically significant upregulation after 12 weeks compared to 6 weeks. Caspase-3 mRNA expressions showed an elevation after both 6 and 12 weeks in all groups compared to the control group. However, after 12 weeks, a more upregulated expression was identified than at 6 weeks. The SC group exhibited the highest expression compared to the TCD-7 and TCD-14 groups (level of significance is * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001) (ns: No significance).</p> "> Figure 4 Cont.
<p>(<b>A</b>) Autophagy-related gene mRNA expression (RT-qPCR): The mRNA expression of beclin-1 was found to be elevated in all groups after both 6 and 12 weeks compared to the control group. However, after 6 weeks, the expression was more elevated than after 12 weeks. LC3 mRNA expression showed an increase after 6 weeks in all groups compared to the control group. However, after 12 weeks, the expression was downregulated compared to the levels observed at 6 weeks and was somewhat lower than the control group. P62 expression was elevated after both 6 and 12 weeks in all groups compared to the control group. Interestingly, after 12 weeks, the expression was more upregulated than at 6 weeks. In the SC group, contrary to expectations, the expression was more downregulated after 12 weeks compared to the TCD groups (level of significance is * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001). (<b>B</b>) Apoptosis-related gene mRNA expression (RT-qPCR): The mRNA expression of PARP was elevated in all groups after both 6 and 12 weeks compared to the control group. Notably, after 12 weeks, a more elevated expression was observed than after 6 weeks. In the TCD-7 and TCD-14 groups, there was no significant upregulation, whereas the SC group exhibited statistically significant upregulation after 12 weeks compared to 6 weeks. Caspase-3 mRNA expressions showed an elevation after both 6 and 12 weeks in all groups compared to the control group. However, after 12 weeks, a more upregulated expression was identified than at 6 weeks. The SC group exhibited the highest expression compared to the TCD-7 and TCD-14 groups (level of significance is * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001) (ns: No significance).</p> "> Figure 5
<p>Visualization of different events of autophagy activation after 2 weeks of compression: (<b>A</b>) phagophore formation, (<b>B</b>) membrane elongation, (<b>C</b>) autophagosome, (<b>D</b>) autolysosome, and (<b>E</b>) degradation (White arrow: Indication of different events of autophagy).</p> "> Figure 6
<p>Compression plan as well as post-operative and pre-operative stages of rats: (<b>A</b>) temporary-compression plan, (<b>B</b>) sustained-compression plan, (<b>C</b>) SD rat with compression, and (<b>D</b>) SD rat without compression considered as control.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Temporary and Sustained Compression Induced Radiologically Different Degrees of Disc Degeneration
2.2. Temporary and Sustained Compression Induced Different Degrees of NP Cell Degradation
2.3. Temporary Compression Induced Pronounced Autophagy-Mediated NP Cell Degradation
2.4. Sustained Compression Induced Different Degrees of Apoptosis-Mediated NP Cell Degradation
3. Discussion
4. Materials and Methods
4.1. Animal Preparation
4.2. Radiography (X-ray)
4.3. MRI
4.4. Transmission Electron Microscope
4.5. Paraffin-Embedded Tissue Preparation
4.6. H&E Staining
4.7. Masson’s Trichrome Staining
4.8. Immunohistochemistry (IHC)
4.9. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)
4.10. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Name | Primer Sequence |
---|---|
LC3 | Forward 5′-CCTGCTGCTGGCCGTAGT-3′ Reverse 5′-TGATGAAGTCTTCCTGCCAAAA-3′ |
Beclin-1 | Forward 5′-TTCAAGATCCTGGACCGAGTGAC-3′ Reverse 5′-AGACACCATCCTGGCGAGTTTC-3′ |
P62 | Forward 5′-TCCCTGTCAAGCAGTATCC-3′ Reverse 5′-TCCTCCTTGGCTTTGTCTC-3′ |
Caspase-3 | Forward 5′-GTGGAACTGACGATGATATGGC-3′ Reverse 5′-CGCAAAGTGACTGGATGAACC-3′ |
PARP | Forward 5′-CGGCACGAGAGGGAGGATGG-3′, Reverse 5′-TGTCAGGCTGCCGGATGGAGT-3′ |
GAPDH | Forward 5′-GTATCGGACGCCTGGTTAC-3′ Reverse 5′-CTTGCCGTGGGTAGAGTCAT-3′ |
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Khaleque, M.A.; Kim, J.-H.; Lee, H.-H.; Kim, G.-H.; You, W.-Y.; Lee, W.-J.; Kim, Y.-Y. Comparative Analysis of Autophagy and Apoptosis in Disc Degeneration: Understanding the Dynamics of Temporary-Compression-Induced Early Autophagy and Sustained-Compression-Triggered Apoptosis. Int. J. Mol. Sci. 2024, 25, 2352. https://doi.org/10.3390/ijms25042352
Khaleque MA, Kim J-H, Lee H-H, Kim G-H, You W-Y, Lee W-J, Kim Y-Y. Comparative Analysis of Autophagy and Apoptosis in Disc Degeneration: Understanding the Dynamics of Temporary-Compression-Induced Early Autophagy and Sustained-Compression-Triggered Apoptosis. International Journal of Molecular Sciences. 2024; 25(4):2352. https://doi.org/10.3390/ijms25042352
Chicago/Turabian StyleKhaleque, Md Abdul, Jae-Hoon Kim, Hwan-Hee Lee, Ga-Hyun Kim, Whang-Yong You, Woo-Jin Lee, and Young-Yul Kim. 2024. "Comparative Analysis of Autophagy and Apoptosis in Disc Degeneration: Understanding the Dynamics of Temporary-Compression-Induced Early Autophagy and Sustained-Compression-Triggered Apoptosis" International Journal of Molecular Sciences 25, no. 4: 2352. https://doi.org/10.3390/ijms25042352