Epigallocatechin-3-Gallate Protects H₂O₂-Induced Nucleus Pulposus Cell Apoptosis and Inflammation by Inhibiting cGAS/Sting/NLRP3 Activation

Abstract

Background

Intervertebral disc degeneration (IDD) is the most common diagnosis of patients with lower back pain. IDD is the underlying lesion of many spinal degenerative diseases; however, the role of cGAS/Sting/NLRP3 pathway and epigallocatechin gallate (EGCG) in the development of IDD remained unclear.

Methods

The expressions of cGAS, Sting and NLRP3 mRNA of intervertebral disc (IVD) samples from IDD patients and controls were detected by RT-PCR. The nucleus pulposus cells (NPCs) were induced by hydrogen peroxide (H₂O₂) and used as an in-vitro model. Both 5 μM and 25 μM EGCG treatment were used to detect the effect of EGCG on the in-vitro model. Cell viability was detected by the MTT method, and cell apoptosis and cell cycle would be detected by flow cytometry. Western blot was used in the detection of the expression of cGAS/Sting/NLRP3 as well as apoptosis-related protein level. ELISA was used in the detection of pro-inflammatory factors, including IL-1β, TNF-α, IL-6 and IL-10.

Results

The expressions of cGAS, Sting and NLRP3 mRNA were significantly increased in the IVD samples from IDD patients and NLRP3 was associated with cGAS and Sting. Advanced in-vitro study showed that H₂O₂ significantly increased the expression of cGAS, Sting and NLRP3 protein levels. Advanced experiments showed that EGCG treatment demonstrated significant protective effects in cell viability, apoptosis, cell cycle arrest and inflammatory status through down-regulation of cGAS/Sting/NLRP3 pathway.

Conclusion

It was shown that the cGAS, Sting and NLRP3 up-regulation was associated with the incidence of IDD. Our findings also suggest that EGCG treatment would provide anti-apoptosis, anti-inflammation and promote cell viability in H₂O₂ treatment-incubated NPCs through inhibiting cGAS/Sting/NLRP3 pathway.

Keywords:

• intervertebral disc degeneration
• nucleus pulposus cells
• epigallocatechin gallate
• cGAS
• sting
• NLRP3

Acknowledgments

We acknowledged Dr. Wei Zhu from Tongji University in the in-vitro model development in this study.

Author Contributions

YXT and ZHB performed all experiments and analyzed the data. YXT and HLY prepared the original manuscript. YXT, YMJ, XM, HLY revised the manuscript. All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no competing financial interests.