Mitochondrial kinetic mechanism by which triptolide alleviates hydrogen peroxide-induced apoptosis in SH-SY5Y cells

Abstract

BACKGROUND: Previous studies from our group have shown that triptolide exerts protective effects on nerve cells and alleviates symptoms of neurodegenerative diseases. However, whether it acts by improving mitochondrial dynamic abnormalities requires further investigation. OBJECTIVE: To explore the effect and mechanism of triptolide in regulating the mitochondrial fusion-fission balance to mitigate hydrogen peroxide (H₂O₂)-induced apoptosis in SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured and divided into three groups: control group, model group (200 μmol/L H₂O₂), and triptolide group (2.5 nmol/L triptolide + 200 μmol/L H₂O₂). After 24 hours of intervention, oxidative stress markers (superoxide dismutase activity and malondialdehyde levels), mitochondrial membrane potential, and apoptosis levels were measured. Western blot was used to detect the expression of apoptosis-related proteins, mitochondrial dynamics-related proteins, and respiratory chain-related proteins. Immunofluorescence staining was used to detect the expression of phosphorylated dynamin-related protein 1, optic atrophy protein 1, cytochrome C oxidase 1, and ATP synthase F1 subunit alpha. RESULTS AND CONCLUSION: Compared with the control group, the model group showed significantly decreased superoxide dismutase activity, mitochondrial membrane potential, anti-apoptotic protein Bcl-2, mitochondrial fusion proteins 1 and 2, optic atrophy protein 1, and oxidative phosphorylation complex proteins (NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, ubiquinol-cytochrome c reductase core protein 2, cytochrome c oxidase 1, succinate dehydrogenase B, ATP synthase F1 subunit alpha) (P < 0.05). Meanwhile, malondialdehyde levels, pro-apoptotic proteins Bax and Caspase-3, mitochondrial fission protein 1, phosphorylated dynamin-related protein 1 expression, and apoptosis rate were significantly increased (P < 0.05). Compared with the model group, triptolide intervention reduced malondialdehyde levels, increased superoxide dismutase activity and mitochondrial membrane potential, promoted fusion protein expression, inhibited fission protein expression, increased oxidative phosphorylation complex protein levels, and decreased apoptosis rate (P < 0.05). These results confirm that triptolide can regulate mitochondrial dynamic imbalance to alleviate H₂O₂-induced apoptosis in SH-SY5Y cells.

1. Introduction

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis share common features of progressive deterioration and loss of central neuronal function due to multiple etiologies, leading to cognitive, behavioral, and motor impairments, with no effective clinical treatments currently available [1-2]. Studies have shown that oxidative stress and mitochondrial dysfunction are key factors causing neuronal damage [3]. Mitochondria are the 'powerhouses' of cells, converting nutrients into ATP through oxidative phosphorylation and producing a certain amount of reactive oxygen species, which are usually neutralized by antioxidant enzymes such as superoxide dismutase. In the cytoplasm, mitochondria must maintain normal physiological functions through highly dynamic processes of fission and fusion [4]. The brain has high energy demands for neural activity, relying almost entirely on mitochondrial oxidative energy supply, and the brain's high lipid content and relatively weak antioxidant capacity make it particularly sensitive to oxidative stress, so mitochondrial dysfunction is often a pathological cause of abnormal neuronal apoptosis [5-6].

Neurodegenerative diseases are closely related to mitochondrial dynamics abnormalities. Studies have found that in Alzheimer's disease patients, mitochondrial fission-related proteins are abnormally elevated, while fusion-related proteins are decreased, manifesting as abnormal mitochondrial fusion [7], and mitochondrial dysfunction appears in the nervous system [8]. In Parkinson's disease research, mitochondrial dysfunction and neuroinflammation are closely related to its pathological changes [9], and recent studies have found that mitochondrial transplantation therapy has significant efficacy in improving Parkinson's disease symptoms [10]. Thus, early intervention in mitochondrial dynamic imbalance is of great significance for the treatment of neurodegenerative diseases, and research on drugs that regulate mitochondrial dynamics balance is of great value.

Traditional Chinese medicine has advantages such as multi-component, multi-target, and low toxicity, and can exert effects through multiple points and pathways. In drug research for neurodegenerative diseases, triptolide has been found to repair damaged nerve cells by regulating autophagy [11], and to minimize amyloid plaque deposition by inhibiting intracellular signaling pathways and related factor expression, increasing insulin-degrading enzyme activity, and inhibiting amyloid precursor protein enzyme activity [12-14]. Nuclear factor E2-related factor 2 is an important transcription factor that inhibits cellular oxidative stress pathways. Triptolide can also reduce inflammatory responses and oxidative stress damage to neurons by activating nuclear factor E2-related factor 2 and its downstream factors [15]. Triptolide is a diterpenoid lactone compound extracted from the traditional Chinese medicine Tripterygium wilfordii, with anti-inflammatory, anti-oxidative stress, immunomodulatory, anti-tumor, and neuroprotective effects.