Antioxioxidant and antiapoptotic effects of Thymosin β4 in Aβ-induced SH-SY5Y cells via the 5-HTR1A/ERK axis

Abstract

Alzheimer's disease (AD) is a common amnestic cognitive impairment characterised by β-amyloid (Aβ) plaques deposit in the brain of the elderly. AD is a yet incurable disease due to its unknown exact pathogenesis and unavailability of effective remedies in clinical application. Thymosin β4 (Tβ4) is a housekeeping protein that plays important role in cell proliferation, migration and differentiation. It has the ability to protect and repair neurons however it is still unclear involvement in AD. Therefore, the aim of this study is to elucidate the role and mechanism of Tβ4 in mediating the improvement of AD. AD-like cell model was constructed in neuroblastoma cell line SH-SY5Y treated with Aβ. Overexpression of Tβ4 were done using lentivirus infection and downregulation through siRNA transfection. We performed western blot and flow cytometry to study the apoptosis and standard kits to measure the oxidative stress-associated biomarkers. There is significant increased in viability and decreased apoptosis in Tβ4 overexpression group compared to control. Furthermore, overexpression of Tβ4 suppressed the expression of pro-apoptotic markers such as Caspase-3, Caspase-8, and Bax meanwhile upregulated the expression of anti-apoptotic gene Bcl-2. Tβ4 alleviated oxidative damage by reducing MDA, LDH and ROS and increasing SOD and GSH-PX in Aβ-treated SH-SY5Y cells. We found that Tβ4 inhibit ERK/p38 MAPK pathway and intensify the expression of 5-HTR1A. Additionally, we showed that upregulation of 5-HTR1A dampened the Tβ4 to activate ERK signalling. In conclusion, our study revealed the neuroprotective role of Tβ4 in AD which may open up new therapeutic applications in AD treatment.

Fig 1. Neuroprotective effects of Tβ4 on Aβ-induced cytotoxicity in SH-SY5Y cells.

(A) SH-SY5Y cells were treated with different doses of Aβ and the cell viability was analyzed. (B, C) SH-SY5Y cells were transduced with lentivirus empty vector or lentivirus vector overexpressing Tβ4. The relative Tβ4 mRNA expression (B) and protein expression (C) in SH-SY5Y cells were analyzed by qPCR and western blot 48 hours later. (D) SH-SY5Y cells were transduced with lentivirus empty vector or lentivirus vector overexpressing Tβ4, with or without 25 μM Aβ treatment for 48 h. The neuroprotective effect of Tβ4 on Aβ-induced cytotoxicity was analyzed by cell viability. Data are presented as mean ± SD (n = 3). NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 2. Effects of Tβ4 on MDA, SOD, LDH, GSH-PXand ROS in Aβ-treated SH-SY5Y cells.

SH-SY5Y cells transduced with Lentivirus empty vector or Lentivirus-Tβ4 were treated with Aβ at the concentration of 25 μM Aβ, or left untreated for 48 hours. The levels of MDA (A), SOD (B), LDH (C), and GSH-PX (D) in SH-SY5Y cells were analyzed by using commercial kits. (E) Cells were stained with DHE, observed under a fluorescence microscope, detected by a fluorescence microplate reader and expressed as fold of non-treated normal group (F). (G) The fluorescence intensity in different cells was analyzed by flow cytometry. The data are shown as mean ± SD of three independent experiments each in triplicate. Scale bars, 100 μm. NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 3. Effects of Tβ4 on Aβ-induced apoptosis and apoptosis-associated biomarkers in SH-SY5Y cells.

SH-SY5Y cells with Lentivirus empty vector or Lentivirus-Tβ4 transduction were treated with 25 μM Aβ, or left untreated for 48 hours. Then cells were stained with Annexin V/PI and cell apoptosis was analyzed by flow cytometry (A) and expressed as mean ± standard deviation (B). Caspase-3, Caspase-8, Bax, and Bcl-2 proteins were detected by Western blotting (C, D). β-actin was used as the loading control. The data are shown as mean ± SD of three independent experiments each in triplicate. NS, not significant; **, p < 0.05; ***, p < 0.001.

Fig 4. Effects of Tβ4 on ERK/p38 MAPK signaling in Aβ-treated SH-SY5Y cells.

SH-SY5Y cells with or without overexpression of Tβ4 were treated with Aβ at a concentration of 25 μM for 48 h prior to western blot analysis to measure the levels of (A) p-p38/p38, (B) p-JNK/JNK and (C) p-ERK/ERK. β-actin was used as an internal control. The bars represent the mean ± SD in the different experimental groups (n = 3). NS, not significant; **, p < 0.01; ***, p < 0.001.

Fig 5. Effects of Tβ4 on 5-HTR1A and 5-HTR2C expression in Aβ-treated SH-SY5Y cells.

SH-SY5Y cells with or without overexpression of Tβ4 were treated with Aβ at a concentration of 25 μM for 48 h prior to western blot analysis to measure the expression of 5-HTR1A and 5-HTR2C. β-actin was used as an internal control. The bars represent the mean ± SD in the different experimental groups (n = 3). NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 6. Tβ4 inhibits ERK activation through up-regulating 5-HTR1A in Aβ-treated SH-SY5Y cells.

(A) SH-SY5Y cells were transfected with 5-HTR1A siRNAs for 48 h before the mRNA level of 5-HTR1A was quantified by qPCR. (B and C) Lentivirus-T4β transduced SH-SY5Y cells were transfected with 5-HTR1A siRNA and subsequently subjected to Aβ treatment at 25 μM for 48 h prior to western blot analysis to measure the expression of JNK/pJNK, p38/pp38 and ERK/pERK. β-actin was used as an internal control. The bars represent the mean ± SD in the different experimental groups (n = 3). NS, not significant; **, p < 0.01; ***, p < 0.001.