Critical role of NLRP3 in causing paravertebral muscle injury in adolescent idiopathic scoliosis

Dear Editor,

During the development and treatment of adolescent idiopathic scoliosis (AIS), obvious soft tissue injury around the vertebrae and back pain are always present.^1,2 Abnormal paraspinal muscle development can affect the progression and rehabilitation of scoliosis.³ However, there is insufficient attention to the pathogenesis and personalised treatment for addressing the abnormal changes in the paraspinal muscles in patients with AIS. Our study found that the activation of the NLRP3/GSDMD pathway, a key factor in paraspinal muscle injury of patients with AIS, and a nano-drug delivery system we designed can effectively inhibit this damage.

In this study, we enrolled a total of 30 patients with AIS and 15 age- and gender-matched patients with non-AIS (Table S1). Clinical imaging analysis revealed high levels of fat infiltration in AIS paraspinal muscle, but no significant differences were observed between concave and convex sides (Figure S1A,B). Next, we investigated anomalous changes in the paravertebral muscle tissues of patients with AIS, including increased muscle cell pyroptosis and apoptosis, increased inflammatory infiltration and decreased proliferation ability of primary paravertebral muscle cells (Figures 1A–C and S1). Subsequently, RNA sequencing was performed on paravertebral muscle tissues to identify essential genes associated with AIS paraspinal muscle injury. Through various bioinformatic analyses and qPCR validation, we identified NLRP3, an important regulator in pyroptosis pathway,⁴ as a key candidate gene (Figures 1D–G and S2A,B). The sequences of all qPCR primers are listed in Table S2. Subsequently, various validation confirmed that the pyroptosis and apoptosis pathway proteins in paravertebral muscle of AIS were significantly higher than those in non-AIS (Figures 1H–K and S2C–E). Then, we overexpressed NLRP3 in C2C12 cells using lentiviral transfection and found that not only the NLRP3/GSDMD/IL-1β signalling pathway was activated (Figures 2A–F and S3A–C), but also the apoptosis pathway was upregulated (Figures 2E–H and S3D–F). Meanwhile, knocking down NLRP3 expression in primary paravertebral muscle cells of AIS using siRNA transfection significantly inhibited cell pyroptosis and apoptosis (Figure S4). The sequences of all interfering RNAs are presented in Tables S3 and S4. Some studies have reported that the N-terminal peptides of GSDM protein families, such as GSDMD/GSDME, can activate apoptotic pathway through entering the mitochondrial.⁵

[IMAGE OMITTED. SEE PDF]

Therefore, we further explored the mechanism by which NLRP3 simultaneously activates pyroptosis and apoptosis in AIS paraspinal muscle cells. Using Crispr-Cas9 technology to knockout GSDMD expression in C2C12 cells, we found that both the activation of pyroptotic and apoptotic pathway caused by OE-NLRP3 were inhibited (Figures S5 and S6). Moreover, JC1 staining suggested that GSDMD-N activated the cell apoptosis by affecting the mitochondrial membrane potential and increasing the expression of CYTC (Figure 3D–G). In addition, novel material-based drugs have been demonstrated to have promising applications in many diseases, such as aneurysm.^6,7 Here, we designed a nanoparticle (NP) drug containing disulphiram (DSF), a recognised inhibitor of GSDMD.⁸ The NP we constructed contains thioketal bonds, which would break and release DSF under oxidative stress stimulation.⁹ Through multiple analyses, we examined a variety of physicochemical properties of NP (Figure S7).

[IMAGE OMITTED. SEE PDF]

In vitro experiments showed that it could be effectively taken up by muscle cells (Figure 3A,B), and could effectively inhibit the pyroptosis and apoptosis of AIS primary paravertebral muscle cells at appropriate doses (Figure 3C–K). In vivo study, OE-NLRP3 and OE-NLRP3 + NP groups were established via paraspinal muscle injection adenovirus or NP. The results suggested that at the appropriate concentration, NP could effectively inhibited the activation of the pyroptosis pathway and the necrosis of paraspinal muscle fibres caused by OE-NLRP3 (Figure 3L–T). Figure 4 is the schematic diagram of the critical role of NLRP3 in causing paravertebral muscle Injury in patients with AIS.

[IMAGE OMITTED. SEE PDF]

In conclusion, we first demonstrated that the high level of NLRP3 in AIS mediates severe inflammatory events, further inducing pyroptosis and apoptosis through the activation of GSDMD, which is an important pathogenic factor in paraspinal muscle injury in AIS. Moreover, the use of NP can effectively alleviate the paraspinal muscle injury caused by OE-NLRP3, offering potential targets for effective and personalised clinical treatment of AIS and providing new insights for the development of novel drugs.

AUTHOR CONTRIBUTIONS

Conceived and completed the experiment: Zhuo-Tao Liang, Hao Tang and Meng-Jun Li. Data analysis and interpretation: Jia-Ke Li. Assembly and collection of data: Jiong Li and Zhuo-Tao Liang. Administrative and financial support: Hong-Qi Zhang and Chao-Feng Guo. Manuscript writing: Zhuo-Tao Liang. Guidance for writing and submission: Hong-Qi Zhang and Chao-Feng Guo.

ACKNOWLEDGEMENTS

The authors would like to express their gratitude to Rong Rong (Hunan Key Laboratory of Ophthalmology, Changsha, Hunan 410008, P. R. China) for the guidance of nanoparticle design and other technical support. The authors would also like to thank EditSprings (https://www.editsprings.com/) for the expert linguistic services provided. This study was supported by the National Natural Science Foundation of China (81772298, 82072390 and 82270937) and the National Natural Science Foundation of China (82170901).

CONFLICT OF INTEREST STATEMENT

The authors declare they have no conflicts of interest.

ETHICS STATEMENT

The human sample study and animal study were reviewed and approved by the Ethics Committee approval of Xiangya Hospital. Informed written consent was given by all the participants and their legal guardians before study.

References

Lou EH, Hill DL, Raso JV, Moreau M, Hedden D. How quantity and quality of brace wear affect the brace treatment outcomes for AIS. Eur Spine J. 2016; 25 (2): 495-499.

Bartley CE, Yaszay B, Bastrom TP, et al. Perioperative and delayed major complications following surgical treatment of adolescent idiopathic scoliosis. J Bone Joint Surg Am. 2017; 99 (14): 1206-1212.

Kiemel T, Elahi AJ, Jeka JJ. Identification of the plant for upright stance in humans: multiple movement patterns from a single neural strategy. J Neurophysiol. 2008; 100 (6): 3394-3406.

Eggelbusch M, Shi A, Broeksma BC, et al. The NLRP3 inflammasome contributes to inflammation-induced morphological and metabolic alterations in skeletal muscle. J Cachexia Sarcopenia Muscle. 2022; 13 (6): 3048-3061.

Rogers C, Erkes DA, Nardone A, Aplin AE, Fernandes-Alnemri T, Alnemri ES. Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation. Nat Commun. 2019; 10 (1): 1689.

Laurent D, Lucke-Wold B, Dodd WS, et al. Combination release of chemokines from coated coils to target aneurysm healing. J Neurointerv Surg. 2023; 15 (7): 689-694.

Patel D, Dodd WS, Lucke-Wold B, Chowdhury MAB, Hosaka K, Hoh BL. Neutrophils: novel contributors to estrogen-dependent intracranial aneurysm rupture via neutrophil extracellular traps. J Am Heart Assoc. 2023; 12 (21): [eLocator: e029917].

He H, Markoutsa E, Li J, Xu P. Repurposing disulfiram for cancer therapy via targeted nanotechnology through enhanced tumor mass penetration and disassembly. Acta Biomater. 2018; 68 : 113-124.

Zhai Z, Ouyang W, Yao Y, et al. Dexamethasone-loaded ROS-responsive poly(thioketal) nanoparticles suppress inflammation and oxidative stress of acute lung injury. Bioact Mater. 2022; 14 : 430-442.