Luteolin Protects Pheochromocytoma (PC-12) Cells against Aβ25-35-Induced Cell Apoptosis through the ER/ERK/MAPK Signalling Pathway

The regulatory effect of luteolin on the progression of Alzheimer's disease (AD) remains unclear from the perspective of apoptosis. The present study aimed to investigate the protective effects of luteolin against Aβ25-35-induced cell apoptosis in pheochromocytoma (PC-12) cells. Aβ25-35 was used to induce an in vitro model of AD. Estradiol was used as a positive control. The PC-12 cells were incubated with luteolin alone or in combination with fulvestrant or U0126. The results showed that luteolin treatment significantly prevents Aβ25-35-induced decrease in cell viability and inhibits Aβ25-35-induced cell apoptosis. After the addition of fulvestrant and U0126, the apoptosis rate of PC-12 cells increased significantly. In addition, luteolin treatment significantly upregulated the expression of Bcl-2 and downregulated the expression of Bax and caspase-3, whereas fulvestrant and U0126 partially reversed the effects of luteolin. Moreover, luteolin treatment upregulated the expression of ERβ and p-ERK1/2, whereas fulvestrant blocked the expression of p-ERK1/2. The study showed that luteolin could activate the ER/ERK/MAPK signalling pathway to protect PC-12 cells against Aβ25-35-induced cell apoptosis via selectively acting on ERβ. Thus, luteolin may be considered as a potential novel therapeutic strategy for AD.

Flavonoids are a class of about 4000 naturally occurring compounds [8]. It has been reported that flavonoids exhibit numerous biological effects, including antioxidative, antiinflammatory, and anticancer activity [9,10]. Luteolin is a kind of flavonoid that is found widely in edible plants such as carrot, broccoli, and Perilla leaf [11]. Previous study showed that luteolin improves cognitive dysfunction in AD flies [12]. In addition, study has found that the combination of L-theanine and luteolin may prevent AD-like symptoms, possibly by improving norepinephrine metabolisms and hippocampal insulin signalling and decreasing neuroinflammation [13]. Combining these published evidence, luteolin may be used as a possible therapeutic agent in the treatment of AD. Moreover, it has been shown that luteolin can induce apoptosis of human myeloid leukemia cells, but how luteolin causes this effect has not been definitely elucidated [14]. Most importantly, the regulatory effect of luteolin on the progression of AD remains unclear from the perspective of apoptosis. us, the present study aimed to investigate whether luteolin had protective effects against Aβ 25-35 -induced cell apoptosis in PC-12 cells and explored the possible molecular mechanisms for the first time.

Apoptosis Assay by Flow
Cytometry. Apoptosis was measured using the Annexin V-FITC/PI double staining kit according to the manufacturer's instruction. In brief, the PC-12 cells were trypsinized, washed with phosphatebuffered saline (PBS), and then resuspended in 400 μL binding buffer. After the cells were incubated in the dark at room temperature for 15 min, 5 µL of Annexin V-FITC and 10 µL of PI were added into cells for flow cytometric analysis.

Western Blot.
Western blot was performed to detect the effects of luteolin on the expression of apoptosis factors. e total protein was extracted with RIPA lysate, and the protein was quantified and denatured to obtain a protein sample. Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). e PVDF membrane was blocked in 5% nonfat milk for 2 h at room temperature and subsequently incubated at 4°C overnight with antibodies against Bcl-2, Bax, caspase-3, ERβ, or β-actin (Beijing Boosen Biological Technology Co., Ltd., China). Next, the PVDF membrane was incubated with corresponding horseradish peroxidase-conjugated (HRP)linked secondary IgG antibodies (Wuhan Boster Biological Technology Co., Ltd., China) for 2 h at 37°C. e enhanced chemiluminescence (ECL) kit (Beyotime, Shanghai, China) was used to detect immunoreactive bands, and a lane ID gel analysis system was used to analyse the grey values.

Statistical Analysis.
All statistical analyses were performed by using SPSS version 18.0 (SPSS Institute, IL, USA). Results from each experiment were expressed as the mean ± standard deviation of three separate experiments. e comparison of different groups was performed by oneway analysis of variance (ANOVA) followed by least significant difference-(LSD-) t-test or Tukey's test for multiple comparisons. Statistical significance was set at p < 0.05.
To further verify the effect of luteolin on Aβ 25-35 -induced cell apoptosis, we detected the expression of genes and proteins related to cell apoptosis (Figure 3). e results showed that the expressions of Bax and caspase-3 (apoptosis promoters) at mRNA and protein levels in the model group were significantly higher than those in the blank group (all p < 0.01). In addition, the expressions of Bcl-2 (apoptosis inhibitor) at mRNA and protein levels in the model group were significantly lower than those in the blank group (all p < 0.01). Luteolin and estradiol significantly downregulated the expression of Bax and caspase-3 and upregulated the expression of Bcl-2 (Figures 3(a) and 3(b)), whereas fulvestrant and U0126 partially reversed the effects of luteolin (all p < 0.01, Figures 3(a) and 3(c)). ese results also indicated that ER/ERK/MAPK signalling pathways might be involved in the protective role of luteolin against Aβ 25-35induced cell apoptosis. [25][26][27][28][29][30][31][32][33][34][35] -Induced Cell Apoptosis through the ER/ERK/MAPK Signalling Pathway. To further verify our hypothesis, we detected the expression of proteins related to the ER/ERK/MAPK signalling pathway. As shown in Figure 4(a), the expression level of ERβ and phosphorylated ERK1/2 (p-ERK1/2) in the model group was significantly lower than that in the blank group (p < 0.01). No ERα proteins were detected in any group. In addition, luteolin and estradiol significantly upregulated the expression of ERβ and p-ERK1/2 (Figure 4(a)), whereas fulvestrant blocked the expression of p-ERK1/2 (Figure 4(b)). ese results indicated that luteolin could activate the ER/ERK/MAPK signalling pathway to protect PC-12 cells against Aβ 25-35induced cell apoptosis via selectively acting on ERβ.

Discussion
AD is an age-related progressive neurodegenerative disease which is characterized by loss of learning and memory in the early stages [18]. Aβ [25][26][27][28][29][30][31][32][33][34][35] deposition is the main histopathological feature of AD, which leads to degeneration and apoptosis of nerve cells, further leading to cognitive dysfunction and memory loss [19,20]. us, the regulation of Aβ 25-35 -induced cell apoptosis to prevent the occurrence of AD may be a promising approach. Previous studies have confirmed that luteolin plays an important role in the process of AD [21,22]. However, the underlying mechanism has not been well elucidated. In this study, we established an in vitro model of AD and investigated the possible mechanisms of luteolin against Aβ 25-35 -induced cell apoptosis for the first time.
Luteolin, a naturally occurring flavonoid found in edible plants, had been found to have chemopreventive effects against malignant tumors in vivo without toxic side effects [23,24]. In the present study, we first verified the effect of luteolin on cell viability. e results showed that luteolin could protect PC-12 cells from Aβ 25-35 -related toxicity at different concentrations (10 −2 , 10 −3 , 10 −4 , 10 −5 , and 10 −6 μmol/L). Surprisingly, we found that luteolin showed a significant toxic side effect at a dose of 10 μmol/L, which reminded us that high doses of luteolin may have potential toxicity.
e present study showed that luteolin at the concentration of 10 −4 μmol/L could significantly prevent Aβ 25-35 -induced decrease in cell viability. Similarly, Guo et al. demonstrated that luteolin could significantly attenuate 6-OHDA-induced PC-12 cell viability loss in a dose-dependent manner [25]. Besides, we also detected the effect of luteolin on Aβ 25-35 -induced cell apoptosis. e study performed by Guo et al. had indicated that the increased apoptotic rate induced by 6-OHDA in the PC-12 cell could be significantly inhibited by luteolin (12.5 or 50 µM) [25]. In the present study, we found that luteolin could inhibit Aβ 25-35 -induced cell apoptosis. ese results indicated that Evidence-Based Complementary and Alternative Medicine luteolin could protect PC-12 cells against Aβ 25-35 -related toxicity.
Next, we further investigated the possible mechanism of luteolin against Aβ 25-35 -induced cell apoptosis. Previous studies had demonstrated that Aβ-induced apoptosis could be regulated by triggering an intracellular apoptotic cascade, including the activation of caspase-3 and Bax in hippocampus neurons [26,27]. In the present study, we found that luteolin treatment could significantly upregulate the expression of Bcl-2 and downregulate the expression of Bax and caspase-3. Extracellular signal-regulated kinase 1/2 (ERK1/2) belonged to the mitogen-activated protein kinase (MAPK) family, which plays an important role in the signal cascade and transfers extracellular signals to intracellular targets [28]. Evidence had showed that the bee venom-induced apoptosis in human leukemic cells was through the   (b) Quantitative assessment of the apoptosis rate. Data were presented as the mean ± standard deviation. * * p < 0.01, compared with the blank group; ## p < 0.01, compared with the model group; ++ p < 0.01, compared with the luteolin group. downregulation of the ERK and Akt signal pathway [29]. e present study showed that the addition of fulvestrant (ER antagonist) and U0126 (ERK inhibitor) partially reversed the effects of luteolin on the apoptosis rate and apoptosis-related genes and proteins. ese results indicated that luteolin might inhibit Aβ 25-35 -induced cell apoptosis through ER/ ERK/MAPK signalling pathways.
To further verify our hypothesis, we detected the expression of proteins related to the ER/ERK/MAPK signalling pathway. It was known that the phosphorylation of ERK1/2 activated a series of protein signalling cascades that regulated a variety of cellular processes, such as plasticity of neurons, growth, and survival [30]. e present study showed that luteolin significantly upregulated the expression of p-ERK1/ 2, whereas fulvestrant blocked the expression of p-ERK1/2. Estrogen receptors (ERs) existed in two isoforms: ER-alpha (ERα) and ER-beta (ERβ) [31]. ERα was mainly expressed in reproductive tissues, bone, kidney, liver, and white adipose tissue, while the expression of ERβ was found in the central nervous system, cardiovascular system, male reproductive organs, and the immune system [32]. In the present study, we found that luteolin significantly upregulated the expression of ERβ, whereas no ERα proteins were detected in any group. Several studies had demonstrated the ability of estrogen mediated by the ER to activate the PI3 and MAPK signalling pathway [33]. ese results indicated that luteolin could activate the ER/ERK/MAPK signalling pathway to protect PC-12 cells against Aβ 25-35 -induced cell apoptosis via selectively acting on ERβ.   Evidence-Based Complementary and Alternative Medicine the ER/ERK/MAPK signalling pathway to protect PC-12 cells against Aβ 25-35 -induced cell apoptosis via selectively acting on ERβ. us, luteolin may be considered as a potential novel therapeutic strategy for AD.

Data Availability
All data generated or analysed during this study are included in this published article.
Ethical Approval e experimental protocols were approved by the Ethics Review Committee of the Heilongjiang University of Chinese Medicine.

Disclosure
Han-Rui Wang and Si-Ying Pei are the co-first authors.

Conflicts of Interest
e authors declare that they have no conflicts of interest.