CX was obtained from Shanghai Yuanye Biological Technology Co., Ltd. (S85614-5 mg; Shanghai, China), and dissolved in dimethyl sulfoxide. Primary human aortic endothelial cells (HAECs) were purchased from American Type Culture Collection (ATCC) (Cat No. PCS-100-011; Manassas, VA, USA). Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC. Fetal bovine serum (FBS) (Cat No. 10099141), F-12k medium (Cat No. 21127022), and endothelial cell growth supplement were purchased from Themo Fisher Scientific (Waltham, MA, USA). Trypsin (Cat No. 15090046) and heparin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Malondialdehyde (MDA) (Cat No. BC0025) and superoxide dismutase (SOD) (Cat No. S8410) detection kits were purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China). $\beta$-actin/p21/p53/B-cell lymphoma 2 (Bcl-2) were from ABclonal Technology (Woburn, MA, USA). Anti-Bcl-2-associated X protein (Bax) and anti-GAPDH were obtained from Proteintech Group (Wuhan, China). Total RNA Extraction Kit, Reverse Transcription Kit, Real-Time Fluorescent PCR Kit, and Lipofectamine 3000 Transfection Reagent were purchased from Bao Bioengineering (Dalian) Co., Ltd. (Dalian, China). Endothelin-1 ELISA kit was purchased from Shanghai Youkewei Biotechnology Co., Ltd. (Shanghai, China). DMEM (Dulbecco’s Modified Eagle Medium, Cat No. 12430054) was purchased from Themo Fisher Scientific (Waltham, MA, USA).

Human umbilical vein endothelial cells (HUVECs, Cat No. PCS-100-010) were cultured in F-12K medium containing 5% FBS, 100 U/mL penicillin (Beyotime, Cat.no C0222, Shanghai, China), and 100 µg/mL streptomycin (Beyotime, Cat.no C0222, Shanghai, China) supplemented with 5 ng/mL, L-glutamine: 10 mM, Heparin sulfate: 0.75 Units/mL, Hydrocortisone: 1 µg/mL, Ascorbic acid (Endothelial Cell Growth Kit-BBE PCS-100-040): 50 µg/mL. Subculturing was performed when the cells had grown to logarithmic phase. Primary human aortic endothelial cel (HAEC) cells were cultured in complete DMEM (Cat No. 12430054) containing 5% FBS supplemented with EGF: 5 ng/mL, L-glutamine: 10 mM, Heparin sulfate: 0.75 Units/mL, Hydrocortisone: 1 µg/mL, Ascorbic acid: 50 µg/mL Primary human aortic endothelial cells were purchased form ATCC (Cat No. PCS-100-011). The above cells have been tested accordingly to exclude mycoplasma contamination. HUVECs and HAEC have been authenticated by STR and sequencing.

Aging mice (20-month-old female C57BL/6 mice, 25–30 g) were purchased from Beijing Huafukang Company (Beijing, China). The experimental animals were housed in a room with a 12-h day:night cycle and with controlled temperature (22–23 °C). The experiment was divided into two groups (N: 8 mice per group): control group and experimental group. The experimental mice were administered with CX (20 mg/kg, the CX dose used in the current study was chosen based on previous literature reports [16, 17]) by intragastric gavage for 8 weeks (5 time/week). At the end of the experiment, blood and organ tissues were collected for further analysis. Animal experiments were approved by the Animal Ethics Committee of Zhongshan Hospital, Fudan University (IACUC-20200326).

The cells were divided into different groups according to the requirements of the experiment. Different concentrations of hydrogen peroxide (H${}_2$O${}_2$) were used to establish a cell senescence model. Specifically, cells were treated with H${}_2$O${}_2$ (10–100 µmol/L) for 2 h, after which the cells continued to be cultured for 24 h. The effects of the different H${}_2$O${}_2$ concentrations on cell senescence were evaluated by Sa-$\beta$-gal staining as described below.

Sa-$\beta$-galactosidase is a marker of cellular senescence; thus, it was used to confirm that the cell senescence model had been successfully established. First, cells were seeded in 6-well plates when the cells reached 80% confluence. After three washes with phosphate-buffered saline (PBS), cells were fixed in paraformaldehyde at room temperature for 5 to 15 min. After washing, cells were incubated with Sa-$\beta$-gal staining solution at 37 °C for different time points, and visualized under a microscope.

After the cells (HUVECs, HAEC) were treated with CX, cells were incubated with the DCFH-DA probe in a CO${}_2$ incubator for 2 h. Then the cells were washed twice with PBS and collected, followed by flow cytometry (BD Biosciences, San Diego, CA, USA) to detect reactive oxygen species (ROS).

Nitric oxide (NO) content was detected with the Nitric Oxide Assay Kit (flow cytometry – orange, No. ab219933; Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. In brief, the cells (HUVECs and HAEC) were cultured in 6-well plates. After washing, the cells were stained with NO staining working solution, washed again, and incubated with test solution at 37 °C, followed by flow cytometry to detect NO. The flow cytometer was equipped with adequate filters to measure fluorescence at Ex/Em = 540/590 nm. ELISA kit was used to detect the endothelin-1 content.

HUVECs were seeded and cultured 24 h before transfection. After the cells were cultured to 80% confluence, small interfering RNA (siRNA) targeting Clock1 was transfected into the cells with Lipofectamine 3000 according to the manufacturer’s instructions. After 48 h, cells were lysed and proteins were detected by Western blotting.

Cells were seeded in 6-well plates (5 $\times$ 10${}^4$ cells/well) and cultured for 24 h. The cells were collected, washed twice, and diluted to 1 $\times$ 10${}^5$ cells/mL. Next, 5 µL Annexin V-FITC and 10 µL propidium iodide were added and incubated at room temperature for 20 min in the dark. Then 200 µL binding buffer was added, and the apoptosis rate was analyzed by flow cytometry (BD Biosciences).

To test the effect of CX on MDA and SOD, the cells (HUVECs and HAEC) were stimulated with CX (5 µM) treatment for 24 h, the cells were then collected. The contents of MDA and SOD in the cells were analyzed using the MDA (Cat No. BC0025) and SOD (Cat No. S8410) detection kits (Beijing Solarbio Technology Co., Ltd.), according to the manufacturer’s instructions.

HUVECs and HAECs were collected and lysed by sonication at 4 °C, after which the protein was collected. The protein concentration was detected with the BCA assay. The proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrotransferred to polyvinylidene fluoride (PVDF) membranes, and blocked at room temperature for 2 h. After washing, the membranes were incubated with primary antibodies overnight at 4 °C. After washing the membrane twice, the membranes were incubated with enzyme-labeled secondary antibodies at room temperature for 2 h. $\beta$-actin was used as the internal control.

Tissues from each group were fixed, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Briefly, tissue sections were incubated with hematoxylin staining solution for 8 min, after which they were rinsed with tap water for 15 min and differentiated with hydrochloric acid in ethanol for 30 s at room temperature. Then the sections were rinsed with tap water and counterstained with eosin for 30 s. After rinsing again with tap water, the samples were dehydrated in gradient alcohol, immersed in xylene to make the tissue transparent, and sealed with neutral gum. Finally, the pathological changes of the aortic endothelial vessels were observed under a light microscope.

After the cells were treated with CX, the culture medium was discarded. The cells were washed twice with PBS, and fixed in 4% paraformaldehyde at room temperature for 15 min. After washing with PBS three times for 5 min each, the cells were permeabilized with 0.1% Tritonx-100 for 15 min at room temperature. After washing, the cells were blocked in 5% bovine serum albumin (BSA). After incubation for 1 h at 37 °C, primary antibodies were added and incubated at 4 °C. After a 12 h incubation, the cells were treated with Alexa 488-labeled secondary antibody for 60 min. Then the cells were incubated with DAPI for 5 min at room temperature. After washing, the cells were observed under a confocal microscope (FV3000; Olympus, Tokyo, Japan).

After the cells were treated with CX, equal volumes of JC-1 staining working solution and cell culture solution were added, and the cells were thoroughly mixed and incubated at 37 °C for 20 min in the dark. During the incubation period, the JC-1 staining buffer (pre-cooled JC-1 staining buffer) was added. After the incubation, the staining working solution was discarded. After washing, the samples were detected by flow cytometry. Under normal conditions, when the intracellular mitochondrial membrane potential (MMP) is high, the JC-1 fluorescent probe aggregates in the mitochondrial matrix to form a polymer (red); whereas when the MMP is decreased, the JC-1 fluorescent probe cannot form a polymer, resulting in green fluorescence.

The paraffin sections were soaked twice in xylene (20 min each time) and soaked twice in absolute ethanol, 5 min each time. Then the sections were soaked respectively in 95%, 80%, and 60% ethanol, followed by immersion in a solution containing 3% H${}_2$O${}_2$ for 20 min. After washing, the sections were sealed by incubation in 3% BSA at for 90 min at room temperature. After washing, sections were incubated with primary antibody for 2 h at room temperature. Then the tissue sections were washed again with PBS five times (30 s each), followed by incubation with fluorescently labeled secondary antibody for 30 min at room temperature. After washing with PBS four to five times (30 s each), the cells were observed under a confocal microscope (FV3000; Olympus).

Data are expressed as mean values $\pm$ standard deviation (SD). Statistical analyses were performed with SPSS19.0 software (IBM Corp., Armonk, NY, USA). Differences between two groups were compared using the Student’s t-test. Differences between multiple groups were compared using one-way analysis of variance and Tukey’s post hoc test. p 0.05 was considered statistically significant. * represent as p 0.05, ** represent as p 0.01 and *** represent as p 0.001.

HUVECs were treated with H${}_2$O${}_2$ (10–200 µmol/L) to establish a cell senescence model. According to the results of the pre-experiment, we selected 50 µmol/L H${}_2$O${}_2$ to establish a senescence model of HUVECs (Supplementary Fig. 1). Next, to determine the dosage of CX to use in this study, the cells were stimulated with various concentrations of CX (0.5–100 µM). The results showed that CX promoted cell proliferation, which was blocked after treatment with 50 µM CX (Fig. 1A). Therefore, we chose CX at a concentration of 5 µM for subsequent experiments. The MTT assay showed that the cell viability was significantly decreased when the HUVECs were treated with 50 µmol/L H${}_2$O${}_2$ (Fig. 1B); 50 µmol/L H${}_2$O${}_2$ increased the proportion of Sa-$\beta$-gal-positive cells (Fig. 1C). Western blot analysis showed that the expression of senescent markers was upregulated (Fig. 1D). However, in the CX treatment group, the proportion of Sa-$\beta$-gal-positive cells was significantly decreased (Fig. 1C), and the expression of p16/p21/p53 was also decreased (Fig. 1D). The number of cells in the S phase of the cell cycle was significantly increased (Fig. 1E), and the MMP was also increased in the CX treatment group (Fig. 1F).

Fig. 1.

Effect of canthaxanthin (CX) on cellular senescence. (A) Effects of different concentrations of CX on the human umbilical vein endothelial cells (HUVECs) proliferation. HUVECs were treated with different concentrations of CX for the indicated time points. Cell proliferation was detected by MTT assay. (B) CX enhanced the cell proliferation of the senescent HUVECs. (C) Effect of of CX on SA-$\beta$-gal level. (D) CX reduced the p16/p21/p53 expression. (E) Effect of CX on cell cycle. (F) Mitochondrial membrane potential increased in CX intervention group. (G) CX treatment elevated Ki67 expression. n = 3. *p 0.05, **p 0.01 and ***p 0.001.

Next, we evaluated the effects of CX on the expression of Ki67. CX treatment significantly increased Ki67 expression compared with the aging model group (Fig. 1G). Furthermore, CX treatment also downregulated the cell apoptosis rate (Supplementary Fig. 2). These results suggest that CX alleviated the senescence of HUVECs.

Next, we evaluated the anti-aging effects of CX on HAECs. According to the results of the pre-experiment, we selected 30 µmol/L H${}_2$O${}_2$ to establish the HAEC senescence model. The MTT assay showed that the cell viability was significantly decreased when the HAECs were treated with H${}_2$O${}_2$ (Fig. 2A). Sa-$\beta$-gal-positive cells were clearly increased with 30 µmol/L H${}_2$O${}_2$treatment (Fig. 2B). Western blot analysis showed that the expression of senescence markers, p16 and p21, was clearly upregulated (Fig. 2C). However, in the CX treatment group, the cell viability was significantly increased (Fig. 2A), and the number of Sa-$\beta$-gal-positive cells was clearly increased (Fig. 2B). Furthermore, p16/p21 expression was downregulated in the CX treatment group (Fig. 2C). Flow cytometry analysis showed that the number of cells in the S phase of the cell cycle was also significantly increased (Fig. 2D), and the MMP was also increased in the CX treatment group (Fig. 2E). Next, we evaluated the effect of CX on the expression of Ki67. The results showed that compared with the senescence model group, the CX treatment significantly increased Ki67 expression (Fig. 2F). Furthermore, CX treatment also significantly reduced the cell apoptosis rate (Fig. 2G).

Fig. 2.

CX reduced the cellular senescence. (A) CX enhanced the cell proliferation of the senescent human aortic endothelial cells (HAECs). (B) Sa-$\beta$-gal positive cells was significantly increased in the CX treatment group compared with the control group. (C) CX treatment down-regulated the expression of senescent markers (p16, p21 and p53). (D) Effect of CX on cell cycle. (E) Mitochondrial membrane potential increased in CX intervention group. (F) CX treatment increased Ki67 expression compared to control group. (G) CX treatment was down-regulated the cell apoptosis rate. n = 3. Data are represented as mean values $\pm$ standard deviation (SD). *p 0.05 and ***p 0.001.

We found that CX treatment reduced the levels of tumor necrosis factor alpha (TNF-$\alpha$), interleukin-1$\beta$ (IL-1$\beta$), and IL-6 in HUVECs and HAECs (Fig. 3A). Additionally, CX reduced the expression of inflammatory-related molecule (nuclear factor kappa B) (Fig. 3B), which may be one of the potential molecular mechanisms by which CX relieves inflammation.

Fig. 3.

Effect of CX on cell senescence. (A) CX treatment reduced the levels of the tumor necrosis factor alpha (TNF-$\alpha$), interleukin-1$\beta$ (IL-1$\beta$) and IL-6 in HUVECs and HAEC cells. (B) CX treatment reduced the expression of inflammatory-related molecule (nuclear factor kappa B (NF-$\kappa$B)). Data are represented as mean values $\pm$ standard deviation (SD). *p 0.05, **p 0.01 and ***p 0.001.

A recent study showed that Clock1 is involved in the aging process of stem cells [17]. We found that the expression of Clock1 was significantly downregulated in senescent cells induced by H${}_2$O${}_2$ (Fig. 4A), whereas Clock1 expression was increased after CX treatment (Fig. 4B). Therefore, we speculated that CX exerts its anti-aging effects by regulating Clock1 expression. To test this hypothesis, we treated the vascular endothelial cells with CX, and at the same time, downregulated the expression of Clock1 with siRNA. The results showed that Clock1 knockdown reversed the anti-aging effects of CX, as determined by evaluation of the corresponding senescence markers (Fig. 4B). These findings suggest that CX reduces (at least in part) the aging of the vascular endothelial cell senescence via upregulation of Clock1 expression.

Fig. 4.

Effect of CX on Clock1 expression. (A) The expression of Clock1 was significantly down-regulated in the senescent cells induced by hydrogen peroxide (H${}_2$O${}_2$) (left panel: HUVECs; right panel: HAECs). (B) CX relieved the aging by regulation of Clock1 (left panel: HUVECs; right panel: HAECs). Data are represented as mean values $\pm$ standard deviation (SD). *p 0.05 and ***p 0.001.

NO and endothelin-1 (ET-1) are markers of endothelial cell damage. When vascular endothelial cells are damaged, NO is downregulated and ET-1 is upregulated [18]. Here, we analyzed whether CX may improve endothelial cell function. The results showed that with CX treatment, the level of NO was significantly increased and the level of ET-1 was significantly decreased compared to the H${}_2$O${}_2$ group (Fig. 5A). Indirect immunofluorescence assay (IFA) showed similar results (Fig. 5B).

Fig. 5.

Effect of CX on NO level and ET-1 expression. (A) CX treatment increased the NO level and decreased the level compared to H${}_2$O${}_2$ group. (B) Endothelin-1 (ET-1) expression was down-regulated by Indirect immunofluorescence assay (IFA) analysis (n = 5). Data are represented as mean values $\pm$ standard deviation (SD). **p 0.01 and ***p 0.001.

To further evaluate the effects of CX on endothelial cell damage, we assessed the protective effects of CX on the DNA damage of HUVECs. DNA damage response is one of the consequences of oxidative damage. Gamma-H2AX is considered to be one of the markers of DNA double-strand damage, which is involved in the process of cellular oxidative damage induced by various factors. Western blot analysis showed that the expression level of gamma-H2AX in the H${}_2$O${}_2$ treatment group was significantly higher than that of the control group. Compared with the H${}_2$O${}_2$ group, after CX treatment, the gamma-H2AX expression was significantly reduced (Fig. 6A). In addition, indirect immunofluorescence experiments confirmed this finding (Fig. 6B).

Fig. 6.

Effect of CX on $\gamma$-H2AX expression. (A) CX treatment reduced the expression of $\gamma$-H2AX. (B) $\gamma$-H2AX expression was declined in the CX group (n = 3). Data are represented as mean values $\pm$ standard deviation (SD). ***p 0.001.

We assessed the effects of CX on oxidative damage using two cell models, HUVECs and HAECs. The results showed that intracellular ROS was significantly decreased with CX treatment (p 0.05; Fig. 7A). Additionally, we also evaluated the effects of CX on MDA and SOD. Treatment with 5 µM CX led to a significant increase in intracellular SOD, and a significant decrease in MDA level (Fig. 7B; p 0.05) (Fig. 7B).

Fig. 7.

The effect of CX on oxidative damage of HUVECs and HAEC. (A) The fluorescence intensity of the intracellular reactive oxygen species (ROS) was decreased under the intervention of CX. (B) CX and Ursolic acid treatment enhanced the intracellular superoxide dismutase (SOD) level, and decreased malondialdehyde (MDA) level (n = 5). Data are represented as mean values $\pm$ standard deviation (SD). ns, not significant. *p 0.05 and ***p 0.001.

We evaluated the potential effects of CX on the blood vessels in vivo. We chose a dose of 20 mg/kg to treat 20-month-old mice by oral gavage for 8 weeks. We collected the blood vessels for subsequent analyses, and found that the aging of blood vessels was obvious, which was alleviated by treatment with CX. The levels of p16 and p21 were reduced in the CX treatment group (Fig. 8A). These observations indicated that CX exhibited potential anti-aging effects. Furthermore, we also detected the expression of Ki67 and found that the Ki67 was increased in the CX treatment group (Fig. 8B). In addition, endothelin-1 expression was also reduced (Fig. 8C), suggesting that vascular damage was reduced after CX treatment. The above findings were also confirmed by western-blot analysis (Supplementary Fig. 3).

Fig. 8.

Effect of CX on aging of vascular tissue. (A) The expression of p16 and p21 was down-regulated in CX treatment in vivo. (B) The Ki67 expression was increased. (C) ET-1 expression was reduced (n = 5). Data are represented as mean values $\pm$ standard deviation (SD). ***p 0.001.