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F U L L T E X T S O U R C E : Aging Cell
Abstract
Although aging and senescence have been extensively studied in the past few decades, however, there is lack of clinical treatment available for anti‐aging. This study presents the effects of berberine (BBR) on the aging process resulting in a promising extension of lifespan in model organisms. BBR extended the replicative lifespan, improved the morphology, and boosted rejuvenation markers of replicative senescence in human fetal lung diploid fibroblasts (2BS and WI38). BBR also rescued senescent cells with late population doubling (PD). Furthermore, the senescence‐associated β‐galactosidase (SA‐β‐gal)‐positive cell rates of late PD cells grown in the BBR‐containing medium were ~72% lower than those of control cells, and its morphology resembled that of young cells. Mechanistically, BBR improved cell growth and proliferation by promoting entry of cell cycles from the G0 or G1 phase to S/G2‐M phase. Most importantly, BBR extended the lifespan of chemotherapy‐treated mice and naturally aged mice by ~52% and ~16.49%, respectively. The residual lifespan of the naturally aged mice was extended by 80%, from 85.5 days to 154 days. The oral administration of BBR in mice resulted in significantly improved health span, fur density, and behavioral activity. Therefore, BBR may be an ideal candidate for the development of an anti‐aging medicine.
1 INTRODUCTION
Aging is characterized by a progressive loss of physiological integrity resulting in impaired function along with increased vulnerability to diseases and consequently death. This deterioration is the primary risk factor related to major human chronic noncommunicable diseases including cancer, diabetes, cardiovascular, and neurodegenerative diseases (Lopez‐Otin, Blasco, Partridge, Serrano, & Kroemer, 2013).
The discovery of the first long‐lived mutant strain in Caenorhabditis elegans (Son, Altintas, Kim, Kwon, & Lee, 2019) was a breakthrough in aging research. This finding aimed to find appropriate means to extend the lifespan of eukaryotic species, from single‐celled yeast to humans. Several approaches were demonstrated to prolong the lifespan both in vitro as well as in vivo, such as clearance of senescent cells (Baar et al., 2017), parabiosis (Villeda et al., 2014), and application of Yamanaka factors (Ocampo et al., 2016). However, it is difficult to translate these methods of extending the lifespan in humans into therapy. Oral drugs present a convenient medium for lifespan intervention. Previously, dasatinib + quercetin (Xu et al., 2018), fisetin (Yousefzadeh et al., 2018), metformin (Zakeri et al., 2019), rapamycin (Harrison et al., 2009), nicotinamide mono‐nucleotide (NMN) (Zhang et al., 2016), etc., were reported to extend lifespan in mice. However, more drugs are required to overcome the safety and cost issues.
Cellular senescence is one of the most important in vivo mechanisms related to aging. Senescent cells impair tissue function by irreparable cell damage resulting from acute stress or natural aging, consequently restricting the lifespan (Childs, Durik, Baker, & Deursen, 2015). Cellular senescence can be categorized into two groups. The replicative senescence, seen after approximately sixty rounds of cell division in cultures (Hayflick's limit) (Hayflick, 1965), results from the progressive erosion of telomeres following each division. This progressive erosion leads to telomere dysfunction and irreversible cell‐cycle arrest. The second category is defined as premature cellular senescence. It is unrelated to telomere shortening but is related to persistent cellular stress. Thus, replicative stress caused by oxidative DNA damage, activation of oncogenes, and loss of tumor suppressor genes also results in premature senescence. Furthermore, premature senescence includes irreversible impairment of tumor cell reproductive capability via chemotherapy or radiotherapy‐induced apoptosis which is defined as a drug or radiation‐induced senescence. The in vivo stress‐induced premature senescence of normal cells is considered to be a critical mechanism affecting organismal aging and longevity (Davalli, Mitic, Caporali, Lauriola, & D'Arca, 2016).
Berberine (BBR), a natural alkaloid found in Coptis chinensis, has a long history of medicinal use in both Ayurvedic and traditional Chinese medicine. It is commonly used as a dietary supplement for treating diarrhea. Furthermore, BBR possesses anti‐cancer (Ortiz, Lombardi, Tillhon, & Scovassi, 2014), anti‐inflammatory (Li et al., 2018), and anti‐neurodegenerative (Ahmed et al., 2015) properties. Although the biological properties of BBR are well‐documented (Cicero & Baggioni, 2016), there is little evidence of its role in anti‐aging processes. It was previously observed that BBR inhibited mTOR/S6 signaling concurrent with the reduction in the level of endogenous oxidants and constitutive DNA damage response (Zhao, Halicka, Li, & Darzynkiewicz, 2013). BBR also prolonged lifespan and improved viability of the wild‐type Drosophila melanogaster pupae, and the climbing activity of adult insects at higher temperatures is known to accelerate aging in wild‐type flies (Navrotskaya, Oxenkrug, Vorobyova, & Summergrad, 2014).
Thus, it was hypothesized that BBR, with its potential anti‐aging effects, could treat the senescence in aging cells. Yeast and human fetal lung diploid fibroblasts (2BS and WI38) were chosen as model systems to investigate the effects of BBR on anti‐aging in vitro, while naturally aged and chemo‐treated mice were used for in vivo studies.
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3 RESULT
3.1 Effects of berberine on yeast lifespan
The development of anti‐aging drugs is a tedious and time‐consuming process because the long‐term lifespan experiments need to be performed to achieve meaningful results. Saccharomyces cerevisiae (Xie et al., 2015) is one of commonly used model organisms for aging research. The budding yeast S. cerevisiae shows progressive aging similar to mammalian dividing cells (Gershon & Gershon, 2000). A microfluidic chip‐based device has been developed to automate the lifespan assay of yeast (Jo, Liu, Gu, Dang, & Qin, 2015), and this technology provides medium‐throughput screening results within three days. Since the aging pathways in yeast and other model organisms are highly conserved, it was proposed to first screen the anti‐aging compounds in yeast before testing them in higher species.
The SD media was used in the experiments as yeast has a shorter lifespan in this media as compared to the YEPD media, enabling quicker data acquisition. Cells were taken from solid YEPD media and incubated in the SD media for 20 hr and harvested and loaded onto the chip, where the cells were continuously visualized by continuous microscopic imaging for over two days. BBR was added to SD media at the concentration of 0, 5, 20, and 80 μg/ml, and the lifespan of yeast in the presence of BBR was evaluated. It was found that BBR at 20μg/ml (≈ 59.46 μm) extends the lifespan of yeast by 28% (Figure 1a). Additionally, the number of cells with long cell‐cycle durations was significantly reduced when treated with 20 μg/ml of BBR (green‐colored cells in Figure 1b). Thus, BBR reduced the heterogeneity of cell‐cycle length, resulting in a longer lifespan (other concentrations are shown in Figure S1a). Therefore, at low concentrations, BBR showed potential anti‐aging effect.
Fig. 1
Effects of BBR at low concentrations on the lifespan of yeast. (a) BBR‐treated mother cells showed prolonged replicative lifespan as compared to wild‐type cells in SD medium (number of cells [n]: WT, 80; 5 μg/ml, 80; 20 μg/ml, 80; 80 μg/ml, 80. p‐value for difference between 20 μg/ml and WT < 0.001). (b) Mother cell budding profiles of wild‐type and BBR‐treated cells (20 μg/ml), showing cell‐cycle duration and heterogeneity (see exponential color scale; cell cycles with durations 1.4hr or less were colored in purple). The x‐axis displays individual mother cells shown as vertical bars, with budding events indicated as horizontal white division. Mean lifespan for each group is presented in the upper‐left corner of the plot
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 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 2c,d).
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