Nicandra physalodes Extract Exerts Antiaging Effects in Multiple Models and Extends the Lifespan of Caenorhabditis elegans via DAF-16 and HSF-1.

The development of safe and effective therapeutic interventions is an important issue for delaying aging and reducing the risk of aging-related diseases. Chinese herbal medicines for the treatment of aging and other complex diseases are desired due to their multiple components and targets. Through screening for effects on lifespan of 836 Chinese herbal medicine extracts, Nicandra physalodes extract (HL0285) was found to exhibit lifespan extension activity in Caenorhabditis elegans (C. elegans). In further experiments, HL0285 improved healthspan, enhanced stress resistance, and delayed the progression of neurodegenerative diseases in C. elegans. Additionally, it ameliorated senescence in human lung fibroblasts (MRC-5 cells) and reversed liver function damage and reduced senescence marker levels in doxorubicin- (Dox-) induced aging mice. In addition, the longevity effect of HL0285 in C. elegans was dependent on the DAF-16 and HSF-1 signaling pathways, as demonstrated by the results of the mutant lifespan, gene level, and GFP level assays. In summary, we discovered that HL0285 had an antiaging effect in C. elegans, MRC-5 cells, and Dox-induced aging mice and deserves to be explored in the future studies on antiaging agents.

Conditional ablation of protein tyrosine phosphatase receptor U in midbrain dopaminergic neurons results in reduced neuronal size.

Protein tyrosine phosphatase receptor U (PTPRU) is involved in midbrain patterning during early stages of development and is continuously expressed in the adult midbrain areas where dopaminergic neurons reside in. However, whether PTPRU is also involved in the maintenance and survival of midbrain dopaminergic (mDA) neurons during the late stages of development or in the adult midbrain remains largely unknown. In the present study, Ptpru was ablated by crossing a floxed Ptpru mouse strain with tyrosine hydroxylase (TH)-Cre mice that express Cre recombinase in postmitotic mDA neurons. Conditional ablation of Ptpru in postmitotic mDA neurons resulted in a reduction of somatic and nuclear size in adulthood. However, TH-immunoreactivity of Ptpru-ablated mDA neurons and their projections to the striatum appeared undisturbed. We also investigated the maintenance of several mDA neuronal markers following Ptpru ablation and found no significant changes. Taken together, these findings suggest that PTPRU is involved in regulating the neuronal size of mDA neurons and provided mechanistic insights into the development and maintenance of mDA neurons.

Crotamiton derivative JM03 extends lifespan and improves oxidative and hypertonic stress resistance in Caenorhabditis elegans via inhibiting OSM-9.

While screening our in-house 1072 marketed drugs for their ability to extend the lifespan using Caenorhabditis elegans (C. elegans) as an animal model, crotamiton (N-ethyl-o-crotonotoluidide) showed anti-aging activity and was selected for further structural optimization. After replacing the ortho-methyl of crotamiton with ortho-fluoro, crotamiton derivative JM03 was obtained and showed better activity in terms of lifespan-extension and stress resistance than crotamiton. It was further explored that JM03 extended the lifespan of C. elegans through osmotic avoidance abnormal-9 (OSM-9). Besides, JM03 improves the ability of nematode to resist oxidative stress and hypertonic stress through OSM-9, but not osm-9/capsaicin receptor related-2 (OCR-2). Then the inhibition of OSM-9 by JM03 reduces the aggregation of Q35 in C. elegans via upregulating the genes associated with proteostasis. SKN-1 signaling was also found to be activated after JM03 treatment, which might contribute to proteostasis, stress resistance and lifespan extension. In summary, this study explored a new small molecule derived from crotamiton, which has efficient anti-oxidative, anti-hypertonic, and anti-aging effects, and could further lead to promising application prospects.

Characterization of cellular senescence in doxorubicin-induced aging mice.

With a rise in the need to develop anti-aging drugs, a growing number of in vivo studies evaluating the efficacy of potential drug candidates have used doxorubicin-induced aging mice. However, changes in the biomarkers of senescent cells have not been reported in detail in these animals. To lay a foundation for the use of doxorubicin-induced aging mice, we examined the biomarkers of hepatic and renal senescent cells in these mice. We found that the 5 mg/kg doxorubicin dose is optimal to induce cellular senescence in mice. Subsequently, using this dose, we found that doxorubicin-induced an increase in senescence-associated ß-galactosidase (SA-ß-gal) positive cells in the kidney and lipofuscin accumulation in the liver. Some markers of senescent cells (p21WAF1/CIP1, p16INK4A, and γH2AX) were also significantly upregulated by doxorubicin and then counteracted by metformin treatment. These preliminary findings support the application of doxorubicin-induced aging mice as an animal model to evaluate the efficacy of anti-aging drug candidates.

Anti-aging effects of chlorpropamide depend on mitochondrial complex-II and the production of mitochondrial reactive oxygen species.

Sulfonylureas are widely used oral anti-diabetic drugs. However, its long-term usage effects on patients' lifespan remain controversial, with no reports of influence on animal longevity. Hence, the anti-aging effects of chlorpropamide along with glimepiride, glibenclamide, and tolbutamide were studied with special emphasis on the interaction of chlorpropamide with mitochondrial ATP-sensitive K+ (mitoK-ATP) channels and mitochondrial complex II. Chlorpropamide delayed aging in Caenorhabditis elegans, human lung fibroblast MRC-5 cells and reduced doxorubicin-induced senescence in both MRC-5 cells and mice. In addition, the mitochondrial membrane potential and ATP levels were significantly increased in chlorpropamide-treated worms, which is consistent with the function of its reported targets, mitoK-ATP channels. Increased levels of mitochondrial reactive oxygen species (mtROS) were observed in chlorpropamide-treated worms. Moreover, the lifespan extension by chlorpropamide required complex II and increased mtROS levels, indicating that chlorpropamide acts on complex II directly or indirectly via mitoK-ATP to increase the production of mtROS as a pro-longevity signal. This study provides mechanistic insight into the anti-aging effects of sulfonylureas in C. elegans.

Antiaging Effects of Vicatia thibetica de Boiss Root Extract on Caenorhabditis elegans and Doxorubicin-Induced Premature Aging in Adult Mice.

The roots of Vicatia thibetica de Boiss are a kind of Chinese herb with homology of medicine and food. This is the first report showing the property of the extract of Vicatia thibetica de Boiss roots (HLB01) to extend the lifespan as well as promote the healthy parameters in Caenorhabditis elegans (C. elegans). For doxorubicin- (Doxo-) induced premature aging in adult mice, HLB01 counteracted the senescence-associated biomarkers, including P21 and γH2AX. Interestingly, HLB01 promoted the expression of collagen in C. elegans and mammalian cell systemically, which might be one of the essential factors to exert the antiaging effects. In addition, HLB01 was also found as a scavenger of free radicals, thereby performing the antioxidant ability. Lifespan extension by HLB01 was also dependent on DAF-16 and HSF-1 via oxidative stress resistance and heat stress resistance. Taken together, overall data suggested that HLB01 could extend the lifespan and healthspan of C. elegans and resist Doxo-induced senescence in mice via promoting the expression of collagen, antioxidant potential, and stress resistance.

Verapamil extends lifespan in Caenorhabditis elegans by inhibiting calcineurin activity and promoting autophagy.

Previous evidence has revealed that increase in intracellular levels of calcium promotes cellular senescence. However, whether calcium channel blockers (CCBs) can slow aging and extend lifespan is still unknown. In this study, we showed that verapamil, an L-type calcium channel blocker, extended the Caenorhabditis elegans (C. elegans) lifespan and delayed senescence in human lung fibroblasts. Verapamil treatment also improved healthspan in C. elegans as reflected by several age-related physiological parameters, including locomotion, thrashing, age-associated vulval integrity, and osmotic stress resistance. We also found that verapamil acted on the α1 subunit of an L-type calcium channel in C. elegans. Moreover, verapamil extended worm lifespan by inhibiting calcineurin activity. Furthermore, verapamil significantly promoted autophagy as reflected by the expression levels of LGG-1/LC3 and the mRNA levels of autophagy-related genes. In addition, verapamil could not further induce autophagy when tax-6, calcineurin gene, was knocked down, indicating that verapamil-induced lifespan extension is mediated via promoting autophagy processes downstream of calcineurin. In summary, our study provided mechanistic insights into the anti-aging effect of verapamil in C. elegans.

First-generation species-selective chemical probes for fluorescence imaging of human senescence-associated ß-galactosidase.

Human senescence-associated ß-galactosidase (SA-ß-gal), the most widely used biomarker of aging, is a valuable tool for assessing the extent of cell 'healthy aging' and potentially predicting the health life span of an individual. Human SA-ß-gal is an endogenous lysosomal enzyme expressed from GLB1, the catalytic domain of which is very different from that of E. coli ß-gal, a bacterial enzyme encoded by lacZ. However, existing chemical probes for this marker still lack the ability to distinguish human SA-ß-gal from ß-gal of other species, such as bacterial ß-gal, which can yield false positive signals. Here, we show a molecular design strategy to construct fluorescent probes with the above ability with the aid of structure-based steric hindrance adjustment catering to different enzyme pockets. The resulting probes normally work as traditional SA-ß-gal probes, but they are unique in their powerful ability to distinguish human SA-ß-gal from E. coli ß-gal, thus achieving species-selective visualization of human SA-ß-gal for the first time. NIR-emitting fluorescent probe KSL11 as their representative further displays excellent species-selective recognition performance in biological systems, which has been herein verified by testing in senescent cells, in lacZ-transfected cells and in E. coli-ß-gal-contaminated tissue sections of mice. Because of our probes, it was also discovered that SA-ß-gal content in mice increased gradually with age and SA-ß-gal accumulated most in the kidneys among the main organs of naturally aging mice, suggesting that the kidneys are the organs with the most severe aging during natural aging.

Lactose induced redox-dependent senescence and activated Nrf2 pathway.

Lactose is a disaccharide found in milk and thus a part of our daily food intake. Upon ingestion, it is hydrolyzed to glucose and galactose by the enzyme lactase and absorbed in the small intestine. People who suffer from lactose intolerance are unable to completely digest it due to deficiency of lactase, leading to intestinal problems such as diarrhoea, and bloating. Various studies have focused on treating these symptoms. However, the effects of lactose that diffuses passively into cells, on cellular senescence have largely remained unknown. Thus, the present study investigated the effects and mechanisms of lactose on senescence both in vitro and in vivo. The study was conducted in MRC-5 cells. The cellular senescence was estimated by determining the expression of SA-ß-gal and p16ink4a. The cell viability of MRC-5 cells was determined by the CCK-8 Assay. Activity of intracellular reactive oxygen species was estimated by measuring the levels of superoxide dismutase (SOD), glutathione (GHS), and reactive oxygen species (ROS). The mechanism of lactose on cellular senescence was explored by western blotting. We also studied the effect of lactose on the lifespan of Caenorhabditis elegans. Increased activities of SA-ß-gal and p16ink4a revealed the ability of lactose to induce senescence in MRC-5 cells. The elevated intracellular ROS level and decreased GSH and SOD levels in these cells were indicative of cellular oxidative stress induced by lactose. Furthermore, western blotting analysis of Nrf2 and mRNA expression of its downstream genes suggested the Nrf2/ARE pathway was involved in the oxidative stress induced by lactose. These results were further validated by the shortened lifespan of C. elegans after lactose supplement. Moreover, the lactose-induced senescence could be alleviated by an antioxidant, N-Acetyl-L-cysteine (NAC), both in vitro and in vivo. The present study observed a positive correlation between lactose and cellular oxidative stress, suggesting the latter to be an underlying mechanism of lactose-induced senescence.

Osteopontin-enriched formula feeding improves the T-cell-dependent humoral immune responses in infant rats.

Previous studies have shown that osteopontin (OPN) can enhance infant resistance to infection. However, the underlying mechanisms remain to be explored. Here, we studied the effects of OPN on the development and functions of immune cells in infant rats fed with OPN-enriched formula (OF) compared with regular formula (RF). After 21 days feeding, the proportion of infant rats' CD3+ T cells of lymph nodes in the OF group is significantly increased compared with the RF group. The proportion of CD4+ and CD8+ T cells of lymph nodes in the OF group is closer to the breast feeding (BF) group than to the RF group. Upon immunisation with the thymus-dependent antigen ovalbumin (OVA), the concentration of OVA-specific IgG in the OF group was significantly higher than that in the RF group. Altogether OPN-enriched infant formula feeding can promote the differentiation of CD3+ T cells and improve the T-cell-dependent humoral immune responses in infant rats.

Effects of sleeve gastrectomy on lipid and energy metabolism in ZDF rats via PI3K/AKT pathway.

Sleeve Gastrectomy (SG), as the most effective bariatric surgery, has been using to chronically lose weight and control glucose metabolism in Type 2 diabetes mellitus patients. However, the underlining mechanism is still unclear. In this study, we performed SG on Zucker diabetes fatty (ZDF) rat and investigated visceral lipid metabolism and energy metabolism. After performance of SG, weight, food intake, fasting plasma glucose (FPG) and oral glucose tolerance teat (OGTT) of rats were measured. Furthermore, whole-body metabolic parameters were obtained through TSE LabMaster. Blood lipid and renal function were analyzed by serum from rats' tail vein. Furthermore, the renal genes expression was either detected by real-time PCR, while western blotting was employed to detect the AKT/PI3K proteins level in rats' kidney. Compared to control groups, body weight of ZDF rats treated with SG were significantly reduced, simultaneously with glucose homeostasis and energy metabolism improved including RER (P<0.05), energy expenditure (P<0.05) at night and activity of animal. Meanwhile, serum lipid of ZDF rats after SG was decreased, and renal function recovered. Histology analysis confirmed that the size of perirenal adipose from SD treated ZDF rats obviously decreased (P<0.001), effectively stimulating up-regulation of lipogenesis genes (P<0.05), while adipogenesis genes (P<0.05) in kidney was down-regulated. In addition, phosphorylation of PI3K (p-PI3K) and AKT (p-AKT) in rats kidney were significantly decreased in SG group (P<0.05). Weight loss, food intake, fasting plasma glucose and glucose tolerance in SG surgery rats were improved, which were coincident with energy metabolism changes. In conclusion, SG improves lipid and energy metabolism in ZDF rats model due to activating PI3K/Akt signaling pathway, which was contributed to the mechanism of bariatric surgery toward kidney.

DSTYK kinase domain ablation impaired the mice capabilities of learning and memory in water maze test.

DSTYK (Dual serine/threonine and tyrosine protein kinase) is a putative dual Ser/Thr and Tyr protein kinase with unique structural features. It is proposed that DSTYK may play important roles in brain because of its high expression in most brain areas. In the present study, a DSTYK knockout (KO) mouse line with the ablation of C-terminal of DSTYK including the kinase domain was generated to study the physiological function of DSTYK. The DSTYK KO mice are fertile and have no significant morphological defects revealed by Nissl staining compared with wildtype mice. Open field test and rotarod test showed there is no obvious difference in basic motor and balance capacity between the DSTYK homozygous KO mice and DSTYK heterozygous KO mice. In water maze test, however, the DSTYK homozygous KO mice show impaired capabilities of learning and memory compared with the DSTYK heterozygous KO mice.

Knockdown of protein tyrosine phosphatase receptor U inhibits growth and motility of gastric cancer cells.

Protein tyrosine phosphatase receptor U (PTPRU) has been shown to be a tumor suppressor in colon cancer by dephosphorylating ß-catenin and reducing the activation of ß-catenin signaling. Here, we investigate the expression of PTPRU protein in gastric cancer cell lines, gastric cancer tissues and respective adjacent non-cancer tissues and find that the 130 kDa nuclear-localized PTPRU fragment is the main PTPRU isoform in gastric cancer cells, whereas the full-length PTPRU is relatively lowly expressed. The level of the 130 kDa PTPRU is higher in gastric cancer tissues than in adjacent non-cancer tissues. Knockdown of endogenous PTPRU in gastric cancer cells using lentivirus-delivered specific shRNA results in the attenuation of cell growth, migration, invasion and adhesion. Knockdown of PTPRU also inhibits tyrosine phosphorylation and transcriptional activity of ß-catenin as well as levels of focal adhesion proteins and lysine methylation of histone H3. These results indicate that PTPRU is required for gastric cancer progression and may serve as a potential therapeutic target.

Androglobin knockdown inhibits growth of glioma cell lines.

Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of glioma cell lines determined by MTT assay and colony formation assay. ADGB knockdown also increased the apoptosis of glioma cell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro.

Protein tyrosine phosphatase receptor U (PTPRU) is required for glioma growth and motility.

The membrane protein tyrosine phosphatase receptor U (PTPRU) has been shown to function as a negative regulator of adhesion and proliferation in certain cancer cell types, primarily through its dephosphorylation of ß-catenin and inhibition of subsequent downstream signaling. In the present study, we set out to characterize the role of PTPRU in glioma and found that, while the expression of full-length PTPRU protein is low in these tumors, a number of non-full-length PTPRU isoforms are highly expressed. Among these isoforms, one in particular is localized to the nucleus, and its expression is increased in glioma tissues in a manner that positively correlates with malignancy grade. Short hairpin RNA knockdown of endogenous PTPRU in human and rat glioma cell lines suppressed proliferation, survival, invasion, migration, adhesion and vasculogenic tube formation in vitro, as well as intracranial tumor progression in vivo. In addition, knocking down PTPRU reduced tyrosine phosphorylation (pY) and transcriptional activity of ß-catenin, and we were able to specifically rescue the cell migration defect by expressing a LEF1-ß-catenin fusion protein in PTPRU-depleted cells. PTPRU knockdown also led to increased tyrosine pY of the E3 ubiquitin ligase c-Cbl and to the destabilization of several focal adhesion proteins. Taken together, our findings demonstrate that endogenous PTPRU promote glioma progression through their effect on ß-catenin and focal adhesion signaling.

Inactivation of ß-catenin results in the reduction of postnatal body weight gain.

Arcuate nucleus of hypothalamus (ARH) is the core component in the regulation circuits of food intake and energy homeostasis. ARH projections to other parts of the hypothalamus and to extrahypothalamic areas are established in the postnatal two weeks, which is a pivotal stage for individual development. ß-Catenin, a cell adhesion protein and also the mediator of canonical Wnt signaling pathway, plays an important role in embryonic development and adult homeostasis. However, whether ß-catenin plays any roles in the development of hypothalamus is not clear. Here, we report that perinatal conditional knockout of ß-catenin by CamKIIα-Cre in forebrain reduces body weight gain from P8 and dramatically shortens life span. Quantitative PCR and in situ hybridization results showed the expression of NPY mRNA in the ARH of ß-catenin CKO mice at P15 is obviously increased compared with that of littermate controls, whereas the expression of POMC mRNA is significantly decreased, which suggested the reduction of postnatal body weight gain might be due to the deficiency of food intake. Together, ß-catenin might play an important role in the regulation of food intake and postnatal body weight gain probably through affecting the development of ARH circuits.

ZFX knockdown inhibits growth and migration of non-small cell lung carcinoma cell line H1299.

ZFX (zinc finger transcription factor, X chromosome-linked) contributes to the maintenance of different types of stem cells and the progression of various cancers. We have previously reported that ZFX knockdown inhibits proliferation of glioma in vitro and in vivo. Since overexpression of ZFX in lung cancer tissue correlates with lymph node metastasis, we hypothesized that ZFX may play a role in lung cancer. In this study, we identified ZFX as a promoter of lung cancer growth and migration in a NSCLC (non-small cell lung carcinoma) cell line H1299. ZFX knockdown caused proliferation inhibition determined by MTT assay and colony formation assay, G0/G1 arrest of cell cycle and slightly increased proportion of apoptotic cells assessed by flow cytometry assay, decreased population of migrating cells showed by wound-healing assay, increased cell senescence evidenced by senescence-associated ß-galactosidase staining. ZFX knockdown also led to decreased proportion of tumor bearing mice and reduced mean tumor volume in a subcutaneous tumor model. In addition, western blot showed that ZFX knockdown down regulated a set of proteins involved in proliferation, survival and motility. Altogether, these results suggest that ZFX may be a potential therapeutic target for NSCLC.

Delayed but not loss of gliogenesis in Rbpj-deficient trigeminal ganglion.

Somatosensory ganglia including dorsal root ganglion (DRG) and trigeminal ganglion (TG) are derived from a common pool of neural crest stem cells (NCCs), and are good systems to study the mechanisms of neurogenesis and gliogenesis. Previous studies have reported that deletion of Rbpj, a critical integrator of activation signals from all Notch receptors, in NCCs and their derived cells resulted in the delayed gliogenesis at early stage and a loss of glial cells at later stage in the DRG. But the phenotypes in the TG have not been described. Here we reported although the gliogenesis was also delayed initially in Rbpj-deficient TG, it was recovered as the development progressed, as shown by the presence of large number of glial cells in the TG at later stages. However, neuronal reduction was observed in Rbpj-deficient TG, which is similar to what observed in Rbpj-deficient DRG. Taken together, our data indicate the function of Rbpj is diversified and context dependent in the gliogenesis of somatosensory ganglia.

ZFX regulates glioma cell proliferation and survival in vitro and in vivo.

The zinc finger transcription factor ZFX functions as an important regulator of self-renewal in multiple stem cell types, as well as a sex determinant of mammals. Moreover, ZFX expression is abnormally elevated in several cancers, and correlates with malignancy grade. To investigate its role in the pathogenesis of gliomas, we used lentivirus-mediated RNA interference (RNAi) to knockdown ZFX expression in human glioma cell lines. Our results demonstrate that ZFX plays a crucial role in glioma proliferation and survival, confirming recent reports. We also show for the first time that ZFX knockdown decreases the in vivo growth potential of U87 glioma xenografts in both subcutaneous and intracranial models in nude mice. We conclude that lentivirus-mediated RNAi targeting of ZFX may serve as a promising strategy for glioma therapy.

Notch-Rbpj signaling is required for the development of noradrenergic neurons in the mouse locus coeruleus.

The locus coeruleus (LC) is the main source of noradrenaline in the brain and is implicated in a broad spectrum of physiological and behavioral processes. However, genetic pathways controlling the development of noradrenergic neurons in the mammalian brain are largely unknown. We report here that Rbpj, a key nuclear effector in the Notch signaling pathway, plays an essential role in LC neuron development in the mouse. Conditional inactivation of Rbpj in the dorsal rhombomere (r) 1, where LC neurons are born, resulted in a dramatic increase in the number of Phox2a- and Phox2b-expressing early-differentiating LC neurons, and dopamine-ß-hydroxylase- and tyrosine-hydroxylase-expressing late-differentiating LC neurons. In contrast, other neuronal populations derived from the dorsal r1 were either reduced or unchanged. In addition, a drastic upregulation of Ascl1, an essential factor for noradrenergic neurogenesis, was observed in dorsal r1 of conditional knockout mice. Through genomic sequence analysis and EMSA and ChIP assays, a conserved Rbpj-binding motif was identified within the Ascl1 promoter. A luciferase reporter assay revealed that Rbpj per se could induce Ascl1 transactivation but this effect was counteracted by its downstream-targeted gene Hes1. Moreover, our in vitro gene transfection and in ovo electroporation assays showed that Rbpj upregulated Ascl1 expression when Hes1 expression was knocked down, although it also exerted a repressive effect on Ascl1 expression in the presence of Hes1. Thus, our results provide the first evidence that Rbpj functions as a key modulator of LC neuron development by regulating Ascl1 expression directly, and indirectly through its target gene Hes1.