Berberine ameliorates cellular senescence and extends the lifespan of mice via regulating p16 and cyclin protein expression

2.2. Yeast growth conditions

Yeast was incubated on a standard liquid SD medium (1% yeast extract, 1% Bacto Peptone, 2% glucose) on a rotary shaker at 250 rpm at 30°C. For lifespan experiments, all strains were incubated overnight in a liquid culture (OD was kept below 0.8) and diluted 4 hr before acquisition so that cultures were in the logarithmic phase at the time of imaging.

2.3. Analysis of yeast replicative lifespan (RLS)

Mother cells were monitored for two days by repeated microscopic imaging as described (Xie et al., 2012). Micro‐posts integrated into the microfluidic device were used to clamp mother cells in place while daughter cells were washed away by hydrodynamically controlled flow of the surrounding liquid medium. Survival curves are drawn based on data pooled from multiple experiments with accompanying controls; mean RLS and number of mother cells scores are shown for each curve. Survival curves and cell‐cycle curves were plotted using Matlab.

2.4. Cell lines and cell culture

Human diploid fibroblast 2BS and WI38 cells, isolated from female fetal lung fibroblast tissue, have been fully characterized previously (Tang, Zhang, Zheng, Corbley, & Tong, 1994). These cell lines were originally established at National Institute of Biological Products. 2BS cell line was kindly granted by professor Tong of Peking University. WI38 was purchased from National Institute of Biological Products. Cells were grown in Dulbecco's Modification Eagle's Minimum Essential Medium (DMEM, Life Technologies Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco), 2 mm glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere with 5% CO₂ at 37°C. These cells were considered as young at PD30 or below and fully senescent at PD50 or above.

2.5. Cell cytotoxicity and growth assays

The CCK‐8 assay kit (Dojindo) was used for testing cytotoxicity in vitro. At PD45, 2BS or WI38 cells were seeded into flat‐bottomed 96‐well microplates at a density of 5 × 10³ cells/0.2 ml per well. After 20 hr, when the cells reached a subconfluent state, the cells were transferred to a special culture medium containing various concentrations of BBR for further growth, at 37°C in 5% CO₂ up to 24 hr. Then, the CCK‐8 solution (diluted 0.1 times with 10% FBS DMEM) was added to each well and the cells were incubated for 1 hr at 37°C. The absorbance values of each well were determined spectrophotometrically at 490 nm using a microplate reader (Biotek, MQX200).

Cell viability (%) = (OD _{treatment group} − OD _blank)/ (OD_{control group} − OD _blank) × 100.

Cell proliferation was assayed using the CCK‐8 method. Cells were seeded into 96‐well plates at 2.5 × 10³ cells/0.2 ml per well and incubated with different concentrations of BBR (0, 0.3125, 1.25, and 5 μg/ml) for one week. The absorbance values of each well were measured at day 0 (4 hr after plating) and on days 1, 2, 3, 4, 5, and 6. The medium containing BBR or DMSO was refreshed every 24 hr. At the indicated points, cells were harvested with 10% CCK‐8 for one hour, and the absorbance was measured at 490 nm. Each data point was measured five times, and each curve was repeated more than three times.

2.6. Cell‐cycle analysis

cell‐cycle analysis was conducted according to a previously described procedure (Duan, Zhang, & Tong, 2001). Briefly, WI38 or 2BS cells, grown to approximately 85% confluency at PD45, were split in the ratio of 1:2. One of them was resuspended in DMEM containing different concentrations of BBR (0, 0.3125, 0.625, 1.25, and 2.5 μg/ml) and incubated at 37°C in 5% CO₂ for 72 hr, while the other one was resuspended in normal DMEM and incubated under similar conditions. The DNA content of the cells was measured by fluorescence‐activated cell sorting method using a Becton‐Dickinson FACScan Flow Cytometry System (BD). The data were analyzed using CellFIT software.

2.7. Senescence‐associated β‐galactosidase (SA‐β‐gal) assay

2BS cells (PD45) grown in DMEM with/without different BBR concentrations (0, 0.3125, 0.625, 1.25, and 2.5 μg/ml) were stained according to the method of Dimri with minor modifications (Dimri et al., 1995). Cells grown on plates were washed with PBS, fixed in 4% formaldehyde for 5 min at room temperature, and washed again with PBS. Then, cells were incubated overnight at 37°C without CO₂ in a freshly prepared staining buffer (1 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactopyranoside (X‐gal), 40 mm citric acid/ sodium phosphate, pH 6.0, 5 mm potassium ferrocyanide, 5 mm potassium ferricyanide, 150 mm NaCl, and 2 mm MgCl₂) for 16 hr before being examined according to the instruction manual (CST, 9860S). The average value of the percentage of SA‐β‐gal‐positive cells for each set of samples was calculated based on three independent experiments.

2.8. Western blot analysis

Total protein was extracted using RIPA lysis buffer containing protease inhibitor cocktail (Roche). Total protein was measured by BCA (Pierce) assay, and equal amounts of proteins were subjected to 10% SDS‐PAGE and then transferred to polyvinylidene difluoride membranes (Millipore Corp.). The membranes were blocked and then incubated with primary antibodies against ACTIN (ABclonal Technology), P16, Cyclin D1, CDK4, pRB, and RB (ProteinTech) at 4°C overnight with gentle agitation. This was followed by incubation in the presence of goat anti‐rabbit IgG or goat anti‐mouse IgG (Santa Cruz) labeled secondary antibodies at room temperature for 45 min. Each sample was washed thrice in TBS‐T (10 min each) after labeling with the secondary antibody. Blots were developed with an ECL Plus kit (Amersham Biosciences). The images were scanned using an Epson scanning system, and the data were expressed as the values relative to the control. While probing for multiple targets, stripping and re‐probing a single membrane were required. The same membrane was incubated in a stripping solution (Applygen) at room temperature with gentle agitation for 20 min, followed by a 5 min wash in TBS‐T. Then, the membrane was blocked and incubated in the next primary antibody. Data were acquired as detailed above.

2.9. In vivo experiments

All young and natural aged mice were purchased from SPF Biotechnology Co., Ltd, Beijng, China. C57BL/6J male mice, used as wild‐type (WT) mice (8 weeks/18 months/22 months of age), were fed a normal diet (ND) with free access to water. The mice were kept in an environment with minimum stress along with standard conditions, that is, constant temperature and 12:12 hr light/dark cycle. The animals were allocated at random to a control group and treatment groups. The animals in the treatment group were administered 50 mg/kg of BBR in saline, a dose that was chosen according to a previous study (Zhao et al., 2013). The BBR was administrated orally for four consecutive months (once a day). Animals in the control group were administered the same volume of saline.

An accelerated aging mice model was prepared by administering a common chemotherapeutic drug, doxorubicin, intraperitoneally which can induce senescence in both rodents and humans. The drug was administered at the 10 mg/ml dose, twice weekly.

2.10. Rotarod test

Rotarod test was performed using the Rotarod apparatus. Specifically, 23 males (12 WT and 11 BBR treated) before and after BBR treated for four months were trained with three rounds of rotarod tests for 3 days, and at the 4th day, data from three successive experiments were recorded. Final data are the average of the three experiments performed the 4th day (Nobrega‐Pereira et al., 2016). The investigator was blind to the animal grouping.

2.11. Statistical analyses

All the experiments were performed at least five times. All results are represented as the mean ± SEM. Data involving only two groups were analyzed by one‐ or two‐way ANOVA test. A p‐value of < .01 (two stars) was considered as statistically significant.

3.2. Effects of berberine on cell growth and morphology

We further studied the effects of low concentration BBR on replicative senescence and its mechanism using human fetal lung diploid fibroblast cells (2BS and WI38). These cell lines are considered as young at PD30 or below and fully senescent at PD55 or above. Cell proliferation was used to evaluate the aging state of the cultured cells. First, the optimum concentration of BBR was determined by evaluating the effects of different concentrations of BBR on growth and proliferation of 2BS (PD45) and WI38 (PD45) cells (Figure ​(Figure2a,b,2a,b, respectively). For comparison, the effect on young cells is shown in Figure S1b. The optimum concentration was found to be 0.3125 μg/ml (≈0.929 μm). Above that, BBR (>5 μg/ml ≈ 14.865 μm) inhibited cell proliferation. This result was in agreement with previous studies showing that BBR at concentrations of 10 μm or higher inhibited cell proliferation in a dose‐dependent manner in cancer cells (Sefidabi, Mortazavi, & Hosseini, 2017). However, there are no reports on the effects of low concentrations berberine on cell proliferation. Herein, we observed that berberine, at a concentration of 0.3125 μg/ml, promoted proliferation of 2BS (PD45) and WI38 (PD45) cells during seven days of incubation (Figure ​(Figure22c,d).

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Figure 2

Effects of berberine on cell growth and morphology. (A–B) Effect of BBR at different concentrations on the proliferation of PD45 2BS cells & WI38 cells measured by MTT‐assay. (C–D) Time course of the effect of BBR on proliferation of 2BS & WI38 cells. BBR at 0.3125 ug/ml promoted cell proliferation most. (E) Cell state or morphology of early PD 2BS cells (a) and very late PD 2BS cells (b). (F) SA‐β‐gal staining of early PD 2BS cells (a), late PD 2BS cells (b), late PD 2BS cells grown in DMEM supplemented (c) with or (d) without 0.3125 μg/ml BBR. SA‐β‐gal staining was significantly reduced after BBR treatment. (G) Quantification of SA‐β‐gal‐positive cell rates of 2BS cells. ^*** p < .001 vs. PD30, n = 3–5. ^###p < .001 vs. PD30, n = 3–5. Statistical significance was assessed using one‐way ANOVA test for all experiments. Bar represents the mean ± SEM. ^* p < .05; ^** p < .01; ^*** p < .001 vs. control, n = 3–5

Furthermore, we investigated cell morphology and senescence‐associated markers, such as SA‐β‐gal. Young 2BS cells (PD30) had a flat, spread‐out appearance, with uniform spacing (Figure ​(Figure2Ea),2Ea), while the untreated cells in late PD level (PDL) cells showed characteristics of senescence with an accumulation of granular cytoplasmic inclusions (Figure ​(Figure2Eb).2Eb). The positive rate of cells for senescence‐associated β‐galactosidase (SA‐β‐gal) is usually used as a marker of population senescence in culture (Dimri et al., 1995). Only 3.5% SA‐β‐gal‐positive 2BS cells were seen in PD30 cells (Figure ​(Figure2Fa),2Fa), while 60% were observed in late PDL cells (colored in blue in Figure ​Figure2Fb).2Fb). In BBR‐treated 2BS cells (PD45), the SA‐β‐gal‐positive cell rate reverted to the level of young cells (16%, Figure ​Figure2Fc),2Fc), as compared to the control group (56.5%, Figure ​Figure2Fd).2Fd). Quantification analysis is shown in Figure ​Figure2g.2g. These results suggest that BBR either delayed or reversed the population senescence of 2BS and WI38 cells. However, at late PD the senescent phenotype of the cells could be alleviated but not reversed completely.

3.3. Berberine promotes G1/S transition in aged cells

To understand the mechanism involved in delayed senescence by BBR, we evaluated the level of markers related to replicative senescence and the cell‐cycle profiles. The WI38 (PD45) and 2BS (PD45) cell‐cycle profiles were analyzed using flow cytometry (Figure ​(Figure3a3a and Figure S2). The percentage of WI38 (PD45) and 2BS (PD45) cells in G₁/S/G₂‐M phase without/with BBR at different concentrations were quantified (Figure ​(Figure3b).3b). The percentage of cells in S/G₂‐M phase at 0.3125 μg/ml of BBR were higher by ~18% than at other concentrations, indicating that low concentration of BBR (0.3125 μg/ml) could promote the entry of aged WI38/2BS cells from G₀ or G₁ phase to S/G₂‐M phase. This was consistent with earlier findings in the yeast cells.

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Figure 3

Effect of BBR on cell cycle and cell cycle‐related proteins. (a) Phase analysis of cell cycle in WI38 cells and BBR‐treated cells by flow cytometry. The fraction of S phase cells reached maximum at 0.3125 μg/ml of BBR. (b) Abundance of different cell‐cycle phases of WI38 & 2BS cells on different BBR concentration. ^*** p < .001 vs. WI38 control, n = 3–5. ^###p < .001 vs. 2BS control, n = 3–5. G₁‐S phase transition was observed statistically from 0 to 0.3125 μg/ml. (c) Changes in the expression of p16, cycline D1, CDK4, pRB, RB, and E2F as determined by Western blot. Statistical significance was assessed using the two‐way ANOVA test for all experiments. Bar represents the mean ± SEM. ^* p < .05; ^** p < .01; ^*** p < .001 vs. control, n = 3–5

Next, we evaluated the levels of p16, cyclin D, CDK4, cyclin E, RB/pRB, and E2F proteins in WI38 (55PD) cells using Western blot (Figure ​(Figure3c).3c). When cells were treated with BBR (0.3125 μg/ml), the level of p16 decreased and that of cyclin protein and cyclin‐dependent kinases, such as cyclin D1 and CDK4, increased. Meanwhile, the level of phosphorylated retinoblastoma protein (pRB), which plays an important role in anti‐aging, was up‐regulated. The retinoblastoma protein (RB) is a tumor suppressor protein that prevents excessive cell growth by inhibiting cell‐cycle progression until a cell is ready to divide. Phosphorylation of RB to pRB induces the release of E2F1 that promotes the synthesis of DNA and allows cell‐cycle progression. In fact, the cyclin D1 interacts with its specific kinases (CDK4 and/or CDK6) resulting in cell‐cycle development through RB phosphorylation (Bienvenu et al., 2010). Previous studies have illustrated that cyclinD1/CDK4 interaction during G₁/S phase results in a complex formation which consequently regulates the proliferation of aging cells (Beumer, Roepers‐Gajadien, Gademan, Kal, & Rooij, 2000). Hence, any disruption in cyclin D1 and CDK4 expressions would remarkably impact the cell‐cycle arrest during cellular proliferation. Together, this experimental data indicated that BBR ameliorated the replicative cellular senescence by up‐regulating cyclin D1 and CDK4 expressions.

3.4. Berberine counteracts doxorubicin‐induced chemotoxicity in vitro and in vivo

Off‐target toxicity limits the maximum tolerated dose of chemotherapeutic drugs and causes long‐term health problems in cancer survivors, including accelerated aging (Henderson, Ness, & Cohen, 2014). Since chemotherapy can induce senescence (Ewald, Desotelle, Wilding, & Jarrard, 2010), we explored whether BBR could rescue cells after chemotherapy. Doxorubicin (Dox), a chemotherapeutic drug known to induce senescence (Cahu, Bustany, & Sola, 2012) and liver toxicity in rodents and humans (Damodar, Smitha, Gopinath, Vijayakumar, & Rao, 2014), was used as a model drug. Dox (0.1 μm) was used to induce senescence in WI‐38 in vitro, evident by elevated SA‐β‐GAL activity. Then, we tested whether BBR could affect premature, stress‐induced cellular senescence caused by Dox. The accelerated aged cell model samples were treated with 0.3125, 1.25, and 5 μg/ml BBR after Dox addition followed by evaluation of the level of senescence using SA‐β‐gal. We found BBR was able to rejuvenate premature senescence in Dox‐treated WI38 cells in a dose‐dependent manner (Figure ​(Figure4a,b).4a,b). Next, we performed in vivo experiments at 10 mg/kg dose in mice. Treatment with Dox reduced the total body weight and exercise performance as well as lifespan in 8‐week‐old mice (Figure ​(Figure4c–e).4c–e). Next, we treated the mice with both BBR (100 mg/kg) and Dox through a procedure as shown in Figure ​Figure4f.4f. The effective dose of BBR used in these experiments was chosen based on a previous study (Mehrzadi et al., 2018). We used slightly lower dose because of longer treatment period. Strikingly, the median lifespan of the treatment group was extended by ~52% (Figure ​(Figure4g)4g) and the coordination measured based on retention time on rotarod of the BBR group was significantly longer than those of the control group (Figure ​(Figure4h),4h), indicating better physical condition. Taken together, BBR was effective in reducing doxorubicin‐induced senescence in vitro and alleviated the doxorubicin‐induced loss in body weight and the reduction in coordination. Thus, BBR may be used to treat cytotoxicity resulting from chemotherapeutic drugs.

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Figure 4

Berberine counteracts doxorubicin‐induced senescence and chemotoxicity in vitro and in vivo. (A) SA‐β‐gal staining assay in early PD (P30) WI38 cells (a), doxorubicin‐induced P30 WI38 cells (b), BBR (0.3125 μg/ml (c), 1.25 μg/ml (d), 5 μg/ml (e)) treated doxorubicin‐induced P30 WI38 cells. (B) Quantification of SA‐β‐gal‐positive cell rates of WI38 cells in (A). ***p < .001, n = 3–5. ^#p < .05, n = 3–5. (C) Survival curve of mice treated with 10 mg/kg doxorubicin (p‐value < .001 using log‐rank test). (D) The change in body weight in mice treated with 10 mg/kg doxorubicin. (E) Rotarod test on doxorubicin accelerated aging mice. (F) Schematic timeline of experiments in (G–H). Doxorubicin: twice i.p. at dose of 10 mg/kg. (G) Survival curve of accelerated aging mice treated with 100 mg/kg BBR first (p‐value < .0001 using log‐rank test). (H) Rotarod test on accelerated aging mice treated with 100 mg/kg BBR first. Statistics significance was assessed using the two‐way ANOVA test for all experiments. Bar represents the mean ± SEM. ^* p < .05; ^** p < .01; ^*** p < .001 vs. control, n = 3–5

The authors would like to thank Dr. Hong Zhou and Min Li for their kind discussion and suggestions. We are grateful to Dr. Tanjun Tong from the School of Basic Medical Sciences, Peking University for giving 2BS cells and the National Institutes for Food and Drug Control for providing WI‐38 cells. We further thank Dr. Qi Ouyang from the Center for Quantitative Biology, Peking University for their invaluable assistance and experimental facilities.