Neuroprotective effect of hydroxy-α-sanshool against β-amyloid-induced damage in differentiated PC12 cells.

To the Editor: Alzheimer's disease (AD) is a chronic neurodegenerative disorder that accounts for approximately 70% of global dementia cases and leads to cognitive and behavioral impairments.[1] Neuritic plaques and neurofibrillary tangles are the pathological hallmarks of AD and are related to the accumulation of β-amyloid (Aβ) peptides in cerebral tissues and cytoskeletal changes, which arise from the hyperphosphorylation of microtubule-associated tau proteins in neurons. Oxidative stress is among the earliest events in AD pathogenesis. The accumulation of Aβ could promote the production of reactive oxygen species (ROS) and consequently lipid peroxidation, protein oxidation, and tau hyperphosphorylation, exerting toxic effects on synapses and neurons. In turn, oxidative stress could increase Aβ production. Therefore, antioxidant therapy is becoming a popular strategy for treating AD. Zanthoxylum piperitum (Z. piperitum), which is commonly known as Szechuan pepper, is widely used among Asians and native Americans as a traditional medicine and culinary supplement. Extracts from Z. piperitum possess wide-ranging biological activities, such as anti-inflammatory and analgesic effects, antioxidant and antitumor effects, antibacterial and antifungal effects, and regulatory effects on the gastrointestinal and nervous systems.[2] Chemical compounds have been isolated and identified from Z. piperitum, and alkaloids were determined to be the key characteristic components.[3] Alkaloids from Z. piperitum were reported to have potential therapeutic effects against several neurodegenerative diseases, including AD. Hydroxy-α-sanshool (HαSS) is the main alkaloid isolated from the pericarps of Szechuan peppers and was found to potentiate neuronal activity and stimulate neurite outgrowth from rat adrenal pheochromocytoma PC12 cells.[4] Nevertheless, the direct effect of HαSS against Aβ-induced neuronal damage has not been documented. To investigate the neuroprotective effect of HαSS, PC12 cells were differentiated with 50 μg/L nerve growth factor (NGF), and the morphology and molecular signatures of ubiquitin carboxy terminal hydrolase-1, neurofilament genes, and microtubule-associated protein 2 were verified [Supplementary Figure 1, https://links.lww.com/CM9/B280]. To establish an AD model, differentiated PC12 cells were incubated with 20 μmol/L Aβ1–42 for 24 h. Cell viability was determined by a cell counting kit-8 (CCK-8) kit. Apoptosis was analyzed by flow cytometry (annexin V-propidium Iodide annexin V-PI) and Western blotting (proapoptotic Bax/antiapoptotic Bcl-2). HαSS exposure for various durations and concentrations demonstrated that treatment with 10 μmol/L HαSS for 24 h followed by 20 μmol/L Aβ1–42 incubation for another 24 h was optimal for further mechanistic investigations [Supplementary Figure 2, https://links.lww.com/CM9/B281]. Supplementary Figure 3, https://links.lww.com/CM9/B282 shows that 10 μmol/L HαSS significantly reduced the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), and nitric oxide (NO) and decreased the intracellular ROS level, as indicated by the fluorescent dye 2’,7’-Dichlorodihydrofluorescein diacetate DCFH-DA (P < 0.05). Hoechst 33342 staining showed that the percentage of apoptotic cells was markedly reduced by HαSS. In addition, HαSS significantly prevented the Aβ1–42-induced reduction in mitochondrial membrane potential (MMP), as suggested by a decrease in the aggregation of MMP marker JC-1 and corresponding green fluorescence. To determine the effects of HαSS on the apoptotic pathway, the expression levels of Bax and Bcl-2 were investigated by performing Western blotting [Figure 1]. HαSS significantly decreased Bax expression and increased Bcl-2 expression (P < 0.05). Additionally, HαSS significantly reduced Aβ-induced apoptosis, as shown by flow cytometry and annexin V-PI staining. To analyze the effect of HαSS on tau protein hyperphosphorylation, a marker of AD pathogenesis, the levels of phospho-tau and tau protein were determined. Figure 1B shows that Aβ increased the ratio of phospho-tau/tau (P < 0.05), which was significantly inhibited by HαSS (P < 0.05). Furthermore, HαSS significantly increased the expression of nuclear factor-E2-related factor 2 (Nrf2) and p21, which are key factors in regulating oxidative stress and the cell cycle (P < 0.05), suggesting that the antioxidant activity of HαSS was mediated via the Nrf2 signaling pathway.Figure 1: HαSS altered Aβ1–42-induced Bax, Bcl-2, Nrf2, p21, and p-Tau/tau protein expressions in PC12 cells. Western blotting analysis and expression quantifications of Bax and Bcl-2 (A), p-Tau and Tau (B), Nrf2 and p21 (C) after pre-treatment with or without HαSS for 24 h and incubated with or without Aβ1–42 for another 24 h. HαSS ameliorated Aβ1–42-induced apoptosis and the apoptosis rate of PC12 cells was determined with annexin V-FITC and PI double labeling (D). ∗ P < 0.05, † P < 0.01, all vs. Aβ group. Aβ: β-amyloid; HαSS: Hydroxy-α-sanshool; Nrf2: Nuclear factor-E2-related factor 2; PI: propidium Iodide.To further explore the mechanism of HαSS, the expression levels of the autophagy markers light chain 3 (LC3) and p62 were examined. Pre-treatment with HαSS significantly reduced the expression of LC3 II (P < 0.05) but insignificantly increased the p62 level. Further exploration revealed that HαSS decreased Beclin-1 levels (P < 0.05), an essential factor for autophagosome formation, while autophagy related gene 5 Atg5 expression was not distinctly altered [Supplementary Figure 4, https://links.lww.com/CM9/B283]. These combined results indicated that HαSS might inhibit Aβ1–42-induced apoptosis by blocking autophagy. Inhibiting Aβ-mediated ROS generation and oxidative damage is an effective strategy for treating AD. NGF-induced PC12 cells have morphologic and physiologic features similar to those of neural cells and are extensively used as an AD model. In this original research, HαSS improved cell viability, and reduced nuclear damage, LDH release, NO levels, and oxidative stress (ROS and MDA levels). The ratio of p-Tau/Tau and Bax expression was decreased by HαSS, while the levels of Bcl-2 and Nrf2 were increased. In addition, HαSS inhibited apoptosis and prevented MMP loss. These findings strongly supported the view that HαSS was able to protect Aβ-induced apoptotic injury by inhibiting tau hyperphosphorylation and the activation of antioxidative stress pathways. Previous studies have provided evidence that Aβ can induce apoptosis in neurons.[5] Accumulating evidence suggests that Aβ contributes to AD pathogenesis and progression by various mechanisms, including deoxyribonucleic acid fragmentation, Tau protein phosphorylation, mitochondrial dysfunction, and oxidative stress. While oxidative stress is an important driver of neurodegenerative diseases, this study showed that pre-treatment with 10 μmol/L HαSS effectively attenuated oxidative stress and affected the expression of apoptotic markers. Upregulation of pro-apoptotic Bax could release cytochrome c and change MMP, causing mitochondrial cell death. This study demonstrated that HαSS decreased the rate of apoptosis and mitochondrial disruption. In addition, HαSS also increased Nrf2 levels, indicating that a mitigating effect occurred on oxidative stress. In addition, HαSS could increase p21 expression, suggesting that HαSS may inhibit apoptosis in multiple ways. Nrf2 can directly or indirectly regulate changes in autophagy. In this study, HαSS decreased LC3, p62, and Beclin-1 expression and inhibited autophagy. There are two major limitations in this study that could be addressed in future research. First, the study used PC12 cells to construct an AD model, rather than using human nerve cells for mechanistic exploration, and subsequent studies require to be further investigated in human nerve cells or glial cells. Second, animal experiments are necessary to explore the effects and mechanisms of HαSS on AD. In conclusion, the results from this study demonstrated that HαSS alleviated Aβ-induced intracellular oxidative stress, inhibited autophagy, and reduced hyperphosphorylation of tau, supporting the neuroprotective effects of HαSS. Funding The study was supported by grands from the Department of Science and Technology of Sichuan Province (No. 2021YJ0156) and the Active Health and Aging Technologic Solutions Major Project of National Key R&D Program (No. 2020YFC2006300). Conflicts of interest None.