Mechanisms by which mitochondria-endoplasmic reticulum interaction stress mediates activation of inflammatory vesicles in nerve roots of lumbar intervertebral disc herniation rabbits modulated by acupotomy

Abstract

BACKGROUND: Acupotomy, as one of the representative therapies of minimally invasive interventional therapy, has been applied to the clinical treatment of lumbar disc herniation, which can antagonize nerve root inflammatory response in lumbar disc herniation with precise curative effects, but its potential mechanism of action remains to be explored. OBJECTIVE: To investigate how acupotomy intervention affects the mitochondrial-mitochondria-associated endoplasmic reticulum membranes-endoplasmic reticulum interaction stress-mediated NLRP3 inflammasome activation in the microenvironment of nerve roots in the model rabbits of lumbar disc herniation. METHODS: Forty healthy adult New Zealand white rabbits were randomly divided into a blank control group (10 rabbits) and a model group (30 rabbits). Animal models were established in the model group through a standard autologous nucleus pulposus transplantation method. Once the model was successfully established, the model rabbits were randomly divided into model control group (10 rabbits), electroacupuncture intervention group (10 rabbits), and acupotomy intervention group (10 rabbits). One week after modeling, acupotomy and electroacupuncture interventions were performed. After 3 weeks of intervention, rabbit dorsal root ganglion cells were isolated and cultured. TUNEL staining was used to detect cell apoptosis. Western blot was used to detect the expression levels of NLRP3, MAMs-related proteins, endoplasmic reticulum stress marker protein GRP78, specific marker protein CHOP, and TXNIP protein in the key PERK axis. Mito Tracker and ER Tracker fluorescent probe staining and transmission electron microscopy were used to observe the structural coupling of mitochondria and endoplasmic reticulum. Flow cytometry was used to analyze Ca2+ levels. Reactive oxygen species probe staining was used to detect reactive oxygen species content. RESULTS AND CONCLUSION: Compared with the model control group, the apoptosis rate in the acupotomy intervention group was significantly decreased (P=0.000 2); the protein expressions of NLRP3 (P=0.014 4), IP3R (P=0.013 2), GRP75 (P=0.009 9), VDAC1 (P=0.000 3), GRP78 (P=0.006 5), CHOP (P=0.008 5), and TXNIP (P=0.001 5) were significantly downregulated, while MFN2 (P=0.010 8) protein expression was significantly upregulated; Ca2+ level (P < 0.000 1) and reactive oxygen species level (P=0.039 2) were significantly decreased. Mito Tracker and ER Tracker fluorescent probe staining results showed that the co-localization level of mitochondria-endoplasmic reticulum was highest in the model control group, lowest in the normal control group, and higher in the acupotomy intervention group than in the normal control group but lower than in the model control group. Transmission electron microscopy observation showed that the contact/association between mitochondria and endoplasmic reticulum was enhanced in the model control group, less in the normal control group, and enhanced in the acupotomy intervention group compared with the normal control group but lower than in the model control group. These results indicate that acupotomy intervention can regulate the interaction stress between mitochondria and endoplasmic reticulum, modulate mitochondria-associated endoplasmic reticulum membranes, reduce Ca2+ influx, reactive oxygen species generation, and mitochondrial dysfunction, thereby inhibiting the formation of NLRP3 inflammasome. This elucidates the upstream mechanism by which acupotomy inhibits NLRP3 inflammasome assembly by regulating mitochondria-endoplasmic reticulum interaction stress, reveals the deep-level therapeutic targets of acupotomy for lumbar disc herniation, and provides a theoretical basis for acupotomy treatment of lumbar disc herniation.

1. Introduction

Lumbar disc herniation is a common disease in orthopedics and traumatology [1-3]. Epidemiological studies show that 60%-80% of people will experience varying degrees of low back and leg pain in their lifetime, and lumbar disc herniation is the main cause of pain [4-5]. Lumbar disc herniation is characterized by low back pain, radiating leg pain, numbness, and weakness, and in severe cases, may be accompanied by bowel and bladder dysfunction [3-4], significantly reducing the quality of life of patients. In recent years, its incidence has shown a trend of younger age, and it has become a focus of clinical prevention and treatment.

The nerve root inflammatory response caused by lumbar disc herniation is one of the main causes of low back and leg pain [6]. It is characterized by the nucleus pulposus breaking through the annulus fibrosus, releasing pro-inflammatory cytokines leading to local inflammation and oxidative stress, exacerbating nerve root irritation and damage. Acupotomy therapy is a minimally invasive interventional treatment that has been widely used clinically for lumbar disc herniation [7-11]. Previous basic research has shown that the biological mechanism of acupotomy in treating lumbar disc herniation mainly lies in inhibiting inflammatory responses, thereby alleviating lumbar disc herniation symptoms.