Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca2+ level through GRP75

Objective To investigate the effects of damaged nerve-derived mitochondrial DNA (mtDNA) on GRP75 protein expression, mitochondrial Ca2+ level and apoptosis in human glioblastoma cell line U-87MG (U87) cells, and its mechanism of inducing pain sensitization in mice. Methods Chronic constriction injury (CCI) of the sciatic nerve was used to establish a mouse model of neuropathic pain (NeP). A total of 20 male C57BL/6 mice (6~8 weeks old, weighing 20~30 g) were randomly divided into sham group, and CCI-7, -14 and CCI-21 d groups. Paw withdrawal mechanical threshold (PWMT) was measured, and the changes in mtDNA content in the spinal cord were detected with quantitative real-time PCR (qPCR). After mtDNA was extracted from human neuroblastoma cells (SH-SY5Y) treated with H2O2, U87 cells were treated with mtDNA at a dose of 0~1 ng/μL. CCK-8 assay was utilized to detect cell viability, and the appropriate concentration was selected according to the results. Then U87 cells were divided into: control group, mtDNA group, mtDNA+GRP75 inhibitor (MKT-077 1 μg/mL) group, mtDNA+calcium chelator (BAPTA-AM 10 μmol/L) group, and mtDNA+MKT-077+BAPTA-AM group. MKT-077 and BAPTA-AM were pre-treated in 2 h before mtDNA treatment, respectively. Western blotting was employed to assess the expression of glucose-regulated protein 75 (GRP75) protein, and endoplasmic reticulum-mitochondrial colocalization staining was conducted to observe the endoplasmic reticulum-mitochondrial junction. Calcium ion fluorescent probe was used to measure mitochondrial Ca2+ levels. The oxidative stress and function of mitochondria were evaluated by reactive oxygen species (ROS) and mitochondrial membrane potential. PI fluorescent labeling was employed to examine apoptotic rate of U87 cells. Results Compared with the sham group, the cytochrome C oxidase I (CO1) and nicotinamide adenine dinucleotide dehydrogenase 1 (ND1), which were utilized to measure mtDNA content in the spinal cord of mice in the CCI-21 group, were increased (1.01±0.20 vs 2.22±0.26, P