Protective Effect of Trillium tschonoskii Maxim Components Against Glutamate-Induced SH-SY5Y Cells Damage Through Regulating Apoptosis

CONTEXT: Among the Tujia people, the root or rhizome of Trillium tschonoskii Maxim.in Bull.Acad (TTM) is considered a miraculous herb for headaches. Previous studies have shown ethyl acetate extract (TTM1) can protect SH-SY5Y cells against glutamate injury. OBJECTIVE: This study clarified TTM1’s mechanism against glutamate-induced cell damage, focusing on the regulation of apoptosis. The compounds were separated, identified, and performed molecular docking with pro-apoptotic proteins. MATERIALS AND METHODS: SH-SY5Y cells were treated with glutamate (2 mM) for 12 hour, and the effect of TTM1 (2.5, 5, 10, and 20 μg/mL) was evaluated with MTT and LDH release assays, taking EGb761(40 μg/mL) as a control. Cell apoptosis was detected with Hoechst 33258 and Annexin V-FITC and measurements of intracellular calcium and caspase-3. The major components were separated and identified by LCMS-IT-TOF and NMR, then the proapoptotic activity of TTM1 was confirmed by molecular docking method. RESULTS: TTM1 protected SH-SY5Y cells by resisting apoptosis, TTM1 (10 and 20 μg/mL) decreased apoptotic bodies and nuclear fragments, increased the proportion of normal cells to 68.38 ± 5.63% and 92.80 ± .88%, decreased VA cells to 4.30 ± .76% and 3.58 ± .45% and caspase-3 to .365 ± .034 and .344 ± .047 ng/mL.TTM1 (10 μg/mL) decreased intracellular free calcium to 2.77 ± .40. Polyphyllin VI and pennogenin 3-O-β-chacotrioside were identified in TTM1 at 15.04% and 2.84%, and had potential anti-apoptosis activities. DISCUSSION AND CONCLUSIONS: Folk records of TTM for headache may be related to its anti-apoptosis of nerve cells. Identification and content determination of index components based on effective extract provides research paradigms for rare and endangered ethnic plants.