Tianma extract combined with isorhynchophylline inhibits MPP+- induced apoptosis in PC12 cells through mitochondrial pathway
Parkinson’s disease (PD) is a degenerative disease of the central nervous system accompanied by symptoms of motor deficits. More than 90% of PD is sporadic, and about 10% of PD is hereditary. The main pathological and biochemical features of Parkinson’s disease are progressive loss of dopaminergic neurons in the substantia nigra, leading to a decrease in striatal dopamine levels. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has inhibitory effects on mitochondrial complex I, and upon entering the body, it generates active 1-methyl-4-phenylpyridine ions (MPP+). The free radicals produced by MPP+in mitochondria cause mitochondrial damage. Mitochondrial mediated apoptosis of dopaminergic neurons is an important pathological mechanism of Parkinson’s disease. In the brain tissue of PD patients after death, some dying neurons exhibit morphological features of apoptosis. The research on PD therapy targeting mitochondrial damage has received widespread attention from both basic and clinical medical researchers. Since Cotzias et al. first successfully applied levodopa in the clinical treatment of PD in 1967, dopamine replacement therapy has been the main means of treating PD. However, this therapy can only alleviate clinical symptoms to a certain extent and cannot fundamentally delay the disease progression. Traditional Chinese medicine has the advantages of stable and long-lasting therapeutic effects and minimal toxic side effects in treating PD. With the deepening of research on the mechanism of apoptosis in the occurrence and development of PD, the combination of effective ingredients of traditional Chinese medicine has shown significant advantages in the neuroprotective treatment of PD. Our previous research found that gastrodin and isocrocetine both exert neuroprotective effects in Parkinson’s disease models in vitro and in vivo. PD is a complex disease caused by genetic and environmental factors, and multi-target combination therapy for PD is receiving increasing attention. The aim of this study is to investigate the protective effect and mechanism of the combination of gastrodin and isocrocetine on MPP+- induced apoptosis in PC12 cells.

Dopamine replacement therapy, which focuses on improving motor symptoms, is still the cornerstone of PD treatment, but is limited by long-term efficacy and complications. Neuroprotection has become an important treatment strategy for delaying the progression of Parkinson’s disease. PD is a complex disease caused by multiple factors, and the synergistic effect between biological targets induced by combination therapy may be an effective means to improve the complex pathology of PD. The traditional Chinese medicine compound Tianma Gouteng Yin can significantly improve the symptoms of PD patients. In our previous research, we found that gastrodin and isocrocetine are the main active ingredients of Tianma and Gouteng in anti PD.

Many apoptotic neurons and oxidative stress have been detected in the brains of PD patients, and oxidative stress is an important factor in initiating the apoptotic signaling pathway. Excessive apoptosis of dopaminergic neurons plays a key role in the progression of PD, and various causes of PD can ultimately lead to the onset of PD through the common pathway of cell apoptosis. MPTP is taken up and oxidized by glial cells through the blood-brain barrier to generate MPP+. Dopaminergic neurons in the midbrain take up MPP+through dopamine transporters and inhibit mitochondrial complex I activity in the mitochondria, leading to apoptosis of dopaminergic neurons in the substantia nigra. MPTP/MPP+is an effective drug for inducing PD models. This study showed that MPP+increased apoptosis and caspase-3/7 activity in PC12 cells, while gastrodin or isorhynchophylline alone could significantly inhibit apoptosis and caspase-3/7 activity in PC12 cells. The inhibitory effect of gastrodin and isorhynchophylline combined was significantly stronger than that of both alone.

In recent years, with the in-depth study of mitochondrial structure and function, it has been found that the progressive development of PD is closely related to mitochondria. Mitochondrial dysfunction leads to increased permeability of the mitochondrial outer membrane, release of Cyt-c into the cytoplasm, and binding with apoptotic protease activator 1 to form a multimer, activating caspase-9 and caspase-3, resulting in activation of resting endonucleases, ultimately causing DNA breakage and cell apoptosis. Dopaminergic neuron apoptosis can lead to the occurrence of Parkinson’s disease. This study showed that MPP+reduced the red/green ratio of JC-1 in PC12 cells and increased the content of cyct-C in cell culture supernatant. The combined application of gastrodin and isorhynchophylline had a significantly stronger effect on the increase of JC-1 red/green ratio and the decrease of cyct-C in cell culture supernatant than their individual application, suggesting that the neuroprotective effect of gastrodin and isorhynchophylline combined application is related to the improvement of mitochondrial dysfunction. The ERK1/2, Akt, and GSK-3 β signaling pathways play important roles in the neuroprotective effects of the combination of gastrodin and isorhynchophylline.

Niacin can be converted into NAD+and NADP+under the action of enzymes after entering the body. NAD+is one of the important components of the respiratory chain, participating in redox reactions in organisms. Increasing the content of NAD+/NADH in cells can alleviate neurodegenerative diseases. The Akt signaling pathway is an important intracellular survival promoting signaling pathway, and an increase in Akt phosphorylation levels can counteract neuronal apoptosis and promote neuronal survival. Akt has been shown to be involved in the pathogenesis of Parkinson’s disease. Studies have shown that Akt levels in the substantia nigra pars compacta are reduced in PD patients. This study showed that MPP+reduced the NAD+content and NAD+/NADH ratio in PC12 cells, while the combination of gastrodin and isorhynchophylline increased the NAD+content and NAD+/NADH ratio in PC12 cells. Isocrocetine alone or in combination with gastrodin increased Akt phosphorylation levels in PC12 cells. Akt plays an important role in the combined application of gastrodin and isorhynchophylline to increase cellular NAD+content and NAD+/NADH ratio.

In summary, this study has confirmed that the combination of gastrodin and isorhynchophylline has a synergistic effect, which can better inhibit PD related neuronal apoptosis. The mechanism may be related to improving mitochondrial function, but the synergistic neuroprotective effect of the combination in vitro needs to be verified in vivo experiments, and the molecular mechanism of its synergistic neuroprotection needs further research.