Neuroprotective effects of Camellia nitidissima Chi leaf extract in hydrogen peroxide‐treated human neuroblastoma cells and its molecule mechanisms

Abstract Camellia nitidissima Chi (CNC) is a famous medicinal and edible plant with the name of “Tea for Longevity” in Guangxi province of China. In present study, we determined the protective effect of extract from CNC leaves on H2O2‐induced cell injury and its underlying mechanisms in human neuroblastoma (SH‐SY5Y) cells. The ethyl acetate fraction of CNC leaves (CLE, 50–200 μg/ml) treatment significantly increased the cell viability of H2O2‐treated SH‐SY5Y cells and reduced the leakage of LDH in a reversed “U”‐shape manner. It was confirmed by Hoechst 33,342 staining that CLE attenuated H2O2‐induced apoptosis in SH‐SY5Y cells. The CLE (100 and 150 μg/ml) treatment significantly relieved H2O2‐induced oxidative stress by decreasing intracellular ROS level, and increasing the activities of superoxide dismutase (SOD) and catalase (CAT). Western blot analysis demonstrated that the CLE treatment reserved H2O2‐induced decrease of pCREB (Ser133) expression, and its downstream protein BDNF. In addition, 37 phenolic compounds in CLE were identified by UPLC‐TOF MS/MS, and the main active phytochemicals seemed to be catechins, quercetin, kaempferol, and their derivatives. In conclusion, the data analysis showed that the neuroprotective effect of CNC leaves might be achieved via synergistically boosting endogenous antioxidant defenses and neurotrophic signaling pathway. These results suggest that CNC leaves are valuable resources for functional foods and beverages.

attracted increasing attention for their bioactivities and potentials as functional food and drug (Hou et al., 2018;Wang et al., 2016).
As the elder population increasing, brain aging and its related neurodegenerative diseases, such as Alzheimer's disease has become a major burden of public health (Cutler, 2015). Among therapeutic and preventative remedies, one of the most prospective strategies is to boost brain's endogenous defenses by active phytochemicals.
Excessive oxidative stress is considered to be a key player in age-related brain pathology. The break of equilibrium between reactive oxygen species (ROS) and endogenous antioxidant defenses in brain has been reported to induce dystrophy and death of neurons, ultimately leading to the degeneration of brain function (Fagan-Murphy, Hachoumi, Yeoman, & Patel, 2016;Halliwell, 1992).
In addition, loss of brain function in aging is attributed to weakening neuronal signaling that normally alleviate damage to brain cell (Poulose, Carey, & Shukitt-Hale, 2007). cAMP-response element binding protein (CREB) and its downstream target molecule brain-derived neurotrophic factor (BDNF) are downregulated during oxidative stress damage in brain. It has been well documented that CREB and its downstream signaling pathway are impaired in brain aging conditions and neurodegenerative disorders (Gass & Riva, 2007;Saura & Valero, 2011;Zhu, Lau, Liu, Wei, & Lu, 2004). BDNF, one of the most important neurotrophic molecules, is considered as a key regulator involved in neuronal survival, differentiation, neurogenesis, as well as learning and memory (Lonze & Ginty, 2002). The decreased level of BDNF and pCREB was documented in both H 2 O 2induced SH-SY5Y cells in vitro and the brain of D-galactose induced aged rats in vivo, while many phytochemicals such as polyphenols ameliorated these deficits Qi et al., 2017;Shen, Xu, Qu, Sun, & Zhang, 2018;Yoo, Lee, Sok, Ma, & Kim, 2017). Increasing evidence suggests that CREB-BDNF signaling pathway may be one of the common mechanisms boosting the brain's endogenous defenses by active phytochemicals.
In previous studies, we found that the ethanolic extract of CNC leaves demonstrated significant cytoprotection against H 2 O 2 -induced SH-SY5Y cells, which is one of the mostly validated cell models in neuroprotective effect evaluation (Feng et al., 2016;Park et al., 2015).
However, the phytochemicals and mechanisms underlying the neuroprotective effect of CNC leaves are still unclear. Thus, in present study, we focus on further investigation of the neuroprotective effects of the CNC leaves, its mechanisms in endogenous defenses, as well as identifying the active compounds by UPLC-TOF MS/MS.

| Materials
The leaves of Camellia nitidissima Chi (CNC) were collected in November 2016 from Fangchenggang, Guang xi, China, and authenticated by Mr. All samples were frozen at −80°C for analysis.
All solvents were of HPLC grade, and all chemicals were of analytical reagent grade.

| Preparation of Camellia nitidissima Chi (CNC) leaf extract
The crushed leaves of CNC (200 g) were refluxed with 75% ethanol/ water (4 L × 3) at 70°C for 1 hr, then combined and evaporated in a rotary evaporator at 45°C to yield the ethanolic extract (13.55 g).
Phenolic contents and antioxidant activity measurement were described in Supporting Information.

| Determination of Hoechst 33,342 staining
To observe cell apoptosis, cells (1 × 10 5 cells/ml) were seeded in 6-well plates. After 24 hr, the cells were treated with different concentrations of CLE for 12 hr. Subsequently, the cells were treated with H 2 O 2 for 6 hr, and then the medium was replaced with Hoechst 33,342 solution and incubated at 37°C for 10 min. It was then washed 3 times with PBS to remove excess dye. Cells were observed under a fluorescence microscope (Axioplan 2 imaging E, Carl Zeiss, Germany). Apoptotic cells exhibit strong blue fluorescence and atrophic nuclei, while nonapoptotic cells exhibit weak blue fluorescence and normal nuclei.

| Determination of extracellular Lactate dehydrogenase (LDH) activity
The cell damage was judged by the level of LDH in the cell culture medium. The SH-SY5Y cells were cultured in 24-well plates, and after the treatment, the supernatants were collected. The amount of LDH in the medium was determined by the LDH assay kit.

| Determination of intracellular ROS
The intracellular ROS level was determined by DCFH-DA that will be oxidized by intracellular ROS. SH-SY5Y cells 1 × (10 5 cells/ml) were seeded in black 96-well plates. After the H 2 O 2 (200 μM) treatment, the cells were washed with PBS and incubated with 100 μl of DCFH-DA (20 μM) in dark at 37°C for 40 min. The cells were washed with PBS for 3 times, and fluorescence was measured by a fluorescence microplate reader (INFINITE M1000 P120, Tecan, Mannedorf, Switzerland) at an excitation wavelength of 500 nm and an emission wavelength of 530 nm. The ROS levels were expressed as a relative percentage of the control.

| Determination of SOD and CAT activity
The activity of antioxidant enzymes, SOD and CAT, was measured according to the manufacturer's instructions. After the treatment, cells

| Western blot analysis
Cells were collected, washed with PBS, lysed using whole cell lysates (RIPA buffer with protease inhibitor cocktail and phosphatase inhibitors), homogenized and centrifuged. The protein concentration was determined by BCA assay kit. For Western blot analysis, samples (70 μg protein) were separated in F I G U R E 1 Effect of H 2 O 2 on SH-SY5Y cell viability by MTT assay. Each column is expressed as mean ± S.E.M (n = 6). Data are expressed as a relative percentage of the control. One-way ANOVA followed by Dunnett's test was used for statistical analysis, **p < .01, compared with control group. Results are representative of at least three individual experiments SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% defatted milk at room temperature for 2 hr and then incubated with the following primary antibodies of rabbit anti-BDNF (1:1,000) or anti-pCREB (1:1,000) at 4°C overnight. The membranes were then washed with 0.1 M Tris-buffered (10 min × 3) and incubated with secondary antibody HRP-conjugated goat anti-rabbit IgG (1:2,500) for 2 hr at room temperature. The protein band was developed by a Super Signal kit, and the chemiluminescence signal was transformed into a digital image. β-Actin was used as a control marker.

| UPLC-TOF MS/MS
The ethyl acetate fraction of CNC leaves (CLE) was used for phenolic compounds identification by UPLC-QTOF-MS/MS method.
The full scan mass spectra data were collected at 100-1,000 m/z and fragments mass spectra 50-1,000 m/z under the negative ion scanning condition mode. The mass and mass-mass data were processed with the Masshunter software (Agilent Technologies).

| Data analysis
Results were expressed as means ± standard deviation (SD) and analyzed by ANOVA followed by Dennett's t test for intergroup comparisons (GraphPad Prism 6.0, GraphPad software Inc., San Diego, CA). A value of p < .05 was considered to be significant.
A similar result was obtained for BDNF expression (Figure 5b).
These results suggest that CLE treatment could produce potent neurotrophic effects and upregulate the CREB-BDNF signaling in SH-SY5Y cells.
The relative contents of active compounds in CLE were determined by area nomalization method. The result (showed in Table 1) demonstrated the main phytochemicals were catechin, quercetin, kaempferol, and its derivatives.

| CON CLUS ION
It is well-known that natural products are important sources of neu-

Accumulated evidence revealed that overproduction of ROS
disrupted not only the balance between oxidation and antioxidant defenses, but also protective neuronal signaling, and ultimately to disrupt structures and function of brain cells. Among the overproduction of free radicals, H 2 O 2 is considered to be one of the most important ROS species that cause these damage (Yang et al., 2016).  (Ding, Ma, Man, & Lv, 2017;Mi et al., 2017). Therefore, the neuroprotective effect of CNC leaves might be a combined effect of these active compounds.
In conclusion, our study demonstrated the neuroprotective effects of phytochemicals from Camellia nitidissima Chi leaves in H 2 O 2 -treated SH-SY5Y cells, and the phytochemicals of catechin, procyanidin, quercetin, kaempferol, and their derivatives might work synergistically to enhance endogenous defenses.

| E THI C AL RE VIE W
This study does not involve any human or animal testing.