Mediated Anti-Aging Effects Of Cistanche Extract

EC culture

Human umbilical vein endothelial cells were obtained from the American Type Culture Collection (Manassas VA, USA). Cells were cultured at 37'C with 5% CO2 endothelial growth medium-2 (EGM-2; Lonza, Walkerville, MD, USA) supplemented with 10% fetal bovine serum (FBS). ECs were cultured for 48 h at 37'C with 5%CO, in EGM-2 (control) or HG 30 mM medium, with or without the addition of different concentrations of Cistanche (1040 ug/mL).

Cell viability assay

Cells were cultured at 37'C for 72 h in an EGM-2 medium supplemented with 2% FBS and various concentrations of Cistanche, and were treated with MTT solution for 4 h. Resulting formazan deposits were dissolved with dimethylsulfoxide, where the absorbance was measured at 570 nm using a VersaMax ELISA microplate reader (MolecularDevices, Sunnyvale, CA, USA).

Scratch-wound migration assay

Cells were wounded, and then culture media was replaced with fresh media containing various concentrations of Cistanche. Cells were maintained for 24 to 48 h. When at full migration, cells were imaged using a microscope(Olympus Optical Co, Ltd., Tokyo, Japan). The migrated cells were counted using an optical microscope at 200xmagnification and quantified manually

SA-P-gal staining

To determine the number of senescent cells, SA-B-assays were performed using the SA-P-gal kit (Abcam)according to the manufacturer's instructions. Cells were fixed for 5 min in -gal fixative, washed with PBS, and then stained using -gal fixative solution at 37°C. This process was performed until p-gal staining was visible in either the experimental or control plate. SA-B-gal positive cells were observed via microscopy, with >500 cells counted using three independent fields.

Western blot analysis

Cells were harvested and lysed in protein-extraction solution (Intron Biotechnology, Inc, Gyeonggi, Korea) containing protease and phosphatase inhibitors (10 min at4'C). Total protein concentrations in the supernatants were measured using Bradford assays. After heating at 95 °C for 5 min, protein samples (30 ugs) were separated by 8~12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred onto polyvinylidene fluoride membranes (MilliporeBedford, MA, USA) at 100 V for 60 min. Membranes were incubated in 5% bovine serum albumin (BSA) inTris-buffered saline (TBS) solution supplemented with0.05% TBS with Tween-20 (TBST) (30 min at room temperature), then incubated in 5% BSA in TBST supplemented with primary antibodies (1:200 to l,000) (overnight at 4'C). Next, membranes were inoculated with either HRP-conjugated goat anti-rabbit or anti-mouse antibodies (1:5,000) for h, and protein bands were detected using an enhanced chemiluminescence detection kit (Intron Biotechnology, Inc.) and a LAS-1000 Imager(Fuji Film Corp., Tokyo, Japan). Semi-quantitative analysis of the densitometry results was performed using Image J (National Institutes of Health, Bethesda, MD USA).

Measurement of NO production NO concentrations were determined by detecting the con-centration of nitrite using NO assay kits, according to to manufacturer's instructions. Optical densities were determined at 560 nm using a microplate reader, and concentrations were calculated using a calibration curve

Statistical analysis

Results are expressed as mean+standard deviation (SD)Statistical significance was determined by one-way analysis of variance (ANOVA). Post hoc comparisons between groups were performed using Bonferroni multiple-comparisons tests. All calculations were performed using SPSS in Windows (v.23.0; IBM Corp, Armonk, NYUSA), and P-values <0.05 were considered statistically significant.

RESULTS AND DISCUSSION

Cistanche cytotoxicity

To determine the cytotoxicity of Cistanche on ECs, cells were incubated with various concentrations of Cistanche (0 to 100ug/ml) for 72 h. Cistanche showed significant cytotoxicity (P<0.05) at concentrations >50 ug/ml at all-time points of treatment (Fig. 1). Therefore, Cistanche concentrations of <50ug/ml were considered non-toxic to ECs and were used in subsequent experiments.

The mitogen-activated protein kinase (MAPK) pathway regulates key processes involved in vascular EC development, such as cell proliferation, invasion, metastasis and survival, and angiogenesis (Pišlar et al., 2015). The MAPK involves ERK, c-Jun N-terminal kinase, and p38 (Kim and Choi, 2010). ERK and p38 are important signaling proteins involved in wound healing, whereas ERK plays an important role in controlling cell migration, proliferation, differentiation, and survival (Roskoski, 2012). In a recent study, the p38 MAPK signaling pathway was suggested to be involved in wound healing (Loughlin and Artlett, 2011; Kim et al., 2019; Wang et al., 2019). In the present study, we examined the protein expression of ERK and p38 to identify the signaling pathways involved in Cistanche-regulated EC migration. Cistanche treatment (40 g/mL) increased EC migration 1.5-fold relative to the control (P<0.05) (Fig. 2A). Western blot analysis showed that Cistanche treatment (20 to 40 g/mL) significantly increased phosphorylation of ERK in ECs (P<0.05). Furthermore, Cistanche increased phosphorylation of p38 in a concentration-dependent manner; in particular, treatment with 40 g/mL Cistanche, increased activation of p38 >2.5-fold higher than the control (P<0.01) (Fig. 2B). These results show that 

Fig. 1. Effects of Cistanche (Cistanche) on cell viability. Cells were treated with Cistanche (0 to 100 g/mL) for 72 h. Cell viability was calculated as the percentage of viable cells cultured in the absence of Cistanche. Results show mean±SD. *P<0.05.