Gastrodin alleviates mitochondrial dysfunction by regulating SIRT3-mediated TFAM acetylation in vascular dementia.

BACKGROUND: Mitochondrial dysfunction is key to the pathogenesis of vascular dementia (VaD). Sirtuin-3 (SIRT3), an essential member of the sirtuins family, has been proven to be a critical sirtuin in regulating mitochondrial function. The phenolic glucoside gastrodin (GAS), a bioactive ingredient from Gastrodiae Rhizome (known in Chinese as Tian ma) demonstrates significant neuroprotective properties against central nervous system disorders; however, the precise mechanisms through which GAS modulates VaD remain elusive. PURPOSE: This study aims to investigate whether GAS confers a protective role against VaD, and to figure out the underlying molecular mechanisms. METHODS: A bilateral common carotid artery occlusion (BCCAO)-mediated chronic cerebral hypoperfusion (CCH) VaD rat model and a hypoxia model using HT22 cells were employed to investigate pharmacological properties of GAS in mitigating mitochondrial dysfunction. A SIRT3 agonist resveratrol (RES), a SIRT3 inhibitor 3-TYP and SIRT3-knockdown in vitro were used to explore the mechanism of GAS in association with SIRT3. The ability of SIRT3 to bind and deacetylate mitochondrial transcription factor A (TFAM) was detected by immunoprecipitation assay, and TFAM acetylation sites were further validated using mass spectrometry. RESULTS: GAS increased SIRT3 expression and ameliorated mitochondrial structure, mitochondrial respiration, mitochondrial dynamics along with upregulated TFAM, mitigating oxidative stress and senescence. Comparable results were noted with the SIRT3 agonist RES, indicating an impactful neuroprotection played by SIRT3. Specifically, the attenuation of SIRT3 expression through knockdown techniques or exposure to the SIRT3 inhibitor 3-TYP in HT22 cells markedly abrogated GAS-mediated mitochondrial rescuing function. Furthermore, our findings elucidate a novel facet: SIRT3 interacted with and deacetylated TFAM at the K5, K7, and K8 sites. Decreased SIRT3 is accompanied by hyper-acetylated TFAM. CONCLUSION: The present results were the first to demonstrate that the SIRT3/TFAM pathway is a protective target for reversing mitochondrial dysfunction in VaD. The findings suggest that GAS-mediated modulation of the SIRT3/TFAM pathway, a novel mechanism, could ameliorate CCH-induced VaD, offering a potentially beneficial therapeutic strategy for VaD.

Membrane permeability-guided identification of neuroprotective components from Polygonum cuspidatun.

CONTEXT: Polygonum cuspidatum Sieb et Zucc. (Polygonaceae) possesses various pharmacological activities and has been widely using as one of the most popular and valuable Chinese herbal medicines in clinics. Its usage has increasingly attracted much of our attention and urges investigation on its bioactive components. OBJECTIVE: To establish a rapid and valid approach for screening potential neuroprotective components from P. cuspidatum. MATERIALS AND METHODS: Potential neuroprotective components from P. cuspidatum were screened utilizing liposome equilibrium dialysis followed by high-performance liquid chromatography (HPLC) analysis. Their neuroprotective effects on modulation of protein expression of α7 nAChR, α3 nAChR and synaptophysin (SPY) on SH-SY5Y human neuroblastoma cell line (SH-SY5Y) were evaluated by means of Western blotting. RESULTS: Two potential compounds, polydatin (C1) and emodin-8-O-ß-D-glucoside (C2), were detected and identified in our study. The biological tests showed that both compounds C1 and C2, respectively, at concentrations of 0.1 and 0.25 mg/mL significantly increased protein expression of α7 and α3 nicotinic acetylcholine receptors (nAChRs) in SH-SY5Y cells. Moreover, C1 and C2 at 0.1 mg/mL significantly reversed the Aß1₋42-induced decrease of α7 and α3 nAChRs protein expression in SH-SY5Y cells. In addition, C2 at 0.1 mg/mL significantly increased protein expression of SPY in SH-SY5Y cells and Aß11₋42-induced SH-SY5Y cells whereas C1 did not provide any positive effects. DISCUSSION AND CONCLUSION: In conclusion, our approach utilizing liposome equilibrium dialysis combined with HPLC analysis and cell-based assays is a prompt and useful method for screening neuroprotective agents.

Pretreatment of SH-SY5Y cells with dicaffeoylquinic acids attenuates the reduced expression of nicotinic receptors, elevated level of oxidative stress and enhanced apoptosis caused by ß-amyloid peptide.

OBJECTIVES: This in vitro investigation was designed to examine potential neuroprotection by dicaffeoylquinic acids (diCQAs) extracted from a traditional Chinese medicinal herb herba erigerontis and their effects against the toxicity induced by ß-amyloid peptide (Aß25-35 ). METHODS: The neuroblastoma SH-SY5Y cell line was treated with Aß or 3, 4-diCQA, 3, 5-diCQA or 4, 5-diCQA. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction was assayed by spectrophotometrical method, lipid peroxidation (malondialdehyde) on the basis of the level of thiobarbituric acid-reactive substance, the activity of superoxide dismutase by the xanthine oxidase procedure, the frequency of apoptosis by flow cytometry, and the levels of α3 and α7 nicotinic acetylcholine receptor (nAChR) subunit proteins by Western blotting. KEY FINDINGS: When the cells were exposed to Aß25-35 , MTT reduction declined, oxidative stress and apoptosis were enhanced, and the expression of α3 and α7 nAChR subunit proteins was lowered. Expression of the α7 nAChR subunit protein was increased by all three diCQAs, and the level of α3 was increased by 3, 5-diCQA and 4, 5-diCQA. Significantly, pretreatment with diCQAs attenuated the neurotoxic effects of Aß25-35 , a neuroprotective effect in which the upregulation of α7 and α3 nAChR may be involved. CONCLUSION: The diCQAs exert a protective effect on Aß-induced neurotoxicity in SH-SY5Y cells and a potential underlying mechanism involving stimulation of nAChRs.

Rapid screening and identification of caffeic acid and its esters in Erigeron breviscapus by ultra-performance liquid chromatography/tandem mass spectrometry.

Caffeic acid and its esters (CAEs) are widely distributed in the plant kingdom and have been reported to elicit a wide range of exceptional biological activities. Present methods for screening and characterization of CAEs normally need the use of liquid chromatography diode-array detection/multistage mass spectrometry (LC-DAD/MS(n)). In this report, a rapid and efficient method coupling ultra-performance liquid chromatography (UPLC) with fragment-targeted multi-reaction monitoring (MRM) has been developed for screening CAEs in a crude extract of Erigeron breviscapus, while a UPLC/quasi-MS(n) method has been applied in the structural identification of these compounds. Furthermore, a simple quasi-UPLC/MS/MS method based on in-source collision-induced dissociation (CID) has been proposed for rapid identification of the CAEs. As a result, a total of more than 34 CAEs were detected and their structures characterized. Nine of them were reported from E. breviscapus for the first time. Applications of these strategies in the chemical investigation of an injection of E. breviscapus resulted in the identifications of 16 CAEs. These strategies, if appropriate modifications are made, will be very useful in screening and characterization not only of CAEs, but of other structural types of compounds in various complex matrices.

[Protection of Tianshen Yizhi Recipe against low expression of nicotinic receptor and neurotoxicity induced by beta-amyloid peptide].

OBJECTIVE: To investigate the inhibition effects of Tianshen Yizhi Recipe (TSYZR), a compound traditional Chinese herbal medicine, on decreased expression of nicotinic acetylcholine receptor (nAChR) and the neurotoxicity as well as lipid peroxidation induced by beta-amyloid peptide (Abeta) in human SH-SY5Y neuroblastoma cells. METHODS: The SH-SY5Y cells were treated by a certain concentration of TSYZR, and then exposed to Abeta(25-35). Methyl thiazolyl tetrazolium reduction assay was carried out to understand the influences of the drugs on cellular viability. Expressions of nAChR subunits (alpha3 and alpha7) at protein and mRNA levels were detected by Western-blotting and reverse transcription polymerase chain reaction, respectively. Lipid peroxidation was measured by thiobarbituric acid to observe the capacity of antioxidant of the drugs. RESULTS: TSYZR at a safe concentration could increase alpha7 protein in the cells, inhibit decreased expressions of alpha3 and alpha7 nAChR subunit proteins, prevent lower expression of alpha7 mRNA in SH-SY5Y cells induced by Abeta, reduce the neurotoxicity and lipid peroxidation resulting from Abeta, but had no significant effect on the lower expression of alpha3 mRNA. CONCLUSIONS: TSYZR can up-regulate the expression of alpha7 nAChR subunit protein and prevent decreased expressions of nAChRs and neurotoxicity as well as lipid peroxidation induced by Abeta. This drug may play an important therapeutic role in treatment of Alzheimer disease.

[Influence of beta-amyloid peptide on cell membrane lipids and cholinergic receptors in human neuroblastoma SH-SY5Y cells].

OBJECTIVE: To study the effects of beta-amyloid peptide (Abeta) on cell membrane lipids and cholinergic receptors of human neuroblastoma cells. METHODS: Human SH-SY5Y neuroblastoma cells were treated with different concentrations of Abeta(1-42) with and without pretreatment of vitamin E. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction, lipid peroxidation, protein oxidation and phospholipids were measured by spectrophotometry. Levels of cholesterol and unbiquinone were determined by high-performance liquid chromatography (HPLC). The numbers of cholinergic receptor binding sites were determined by receptor binding assay and the protein levels of nicotinic receptor alpha3 and alpha7 subunits were studied by Western blotting. RESULTS: SH-SY5Y cells showed decreased reduction rates of MMT and phospholipids, and increased lipid peroxidation and protein oxidation after exposure to Abeta (0.1 micromol/L) as compared to the control. The number of cholinergic receptor binding sites, the protein level of nicotinic receptor alpha3 and alpha7 subunits and the content of ubiquinone decreased in cells treated with high dose of Abeta (1 micromol/L). Although the level of cholesterol was not changed in any way, vitamin E partially prevented the neurotoxic effects of Abeta. CONCLUSION: beta-amyloid peptide reduces the level of cell membrane lipids and cholinergic receptors in human SH-SY5Y neuroblastoma cells, likely through the induction of an enhanced oxidative stress.