Cordycepin induces apoptosis in human bladder cancer T24 cells through ROS-dependent inhibition of the PI3K/Akt signaling pathway.

Cordycepin, a derivative of nucleoside adenosine, is one of the active ingredients extracted from the fungi of genus Cordyceps, which have been used for traditional herbal remedies. In this study, we examined the effect of cordycepin on the proliferation and apoptosis of human bladder cancer T24 cells and its mechanism of action. Cordycepin treatment significantly reduced the cell survival rate of T24 cells in a concentration-dependent manner, which was associated with the induction of apoptosis. Cordycepin activated caspase-8 and -9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. Additionally, cordycepin increased the Bax/Bcl-2 ratio and truncation of Bid, and destroyed the integrity of mitochondria, which contributed to the cytosolic release of cytochrome c. Moreover, cordycepin effectively inactivated the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, while LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of cordycepin. Cordycepin further enhanced the intracellular levels of reactive oxygen species (ROS), while the addition of N-acetyl cysteine (NAC), a ROS inhibitor, significantly diminished cordycepin-induced mitochondrial dysfunction and growth inhibition, and also blocked the inactivation of PI3K/Akt signaling pathway. Furthermore, the presence of NAC significantly attenuated the enhanced apoptotic cell death and reduction of cell viability by treatment with cordycepin and LY294002. Collectively, the data indicate that cordycepin induces apoptosis through the activation of extrinsic and intrinsic apoptosis pathways and the ROS-dependent inactivation of PI3K/Akt signaling in human bladder cancer T24 cells.

Anti-inflammatory effects of Daehwangmokdantang, a traditional herbal formulation, in lipopolysaccharide-stimulated RAW 264.7 macrophages.

Daehwangmokdantang (DHMDT) is a traditional polyherbal formulation that has known antidiarrheal and anti-inflammatory activities. However, the underlying mechanisms of these activities are poorly understood. In the present study, the inhibitory effects of DHMDT on the production of proinflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were investigated. The inhibitory effects of DHMDT on LPS-induced nitric oxide (NO), prostaglandin (PG)E2, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß production were examined using Griess reagent and ELISA detection kits. The effects of DHMDT on the expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2, IL-1ß and TNF-α, and their upstream signal proteins, including nuclear factor (NF)-κB, mitogen-activated protein kinases (MAPKs) and RAC-α serine/threonine-protein kinase (Akt), a phosphatidylinositol 3-kinase (PI3K) downstream effector, were investigated using western blotting and immunofluorescence staining. The results revealed the pretreatment with DHMDT significantly inhibited the LPS-induced production of NO, PGE2, TNF-α, and IL-1ß, and expression of iNOS, COX-2 TNF-α, and IL-1ß, without any significant cytotoxicity. DHMDT also efficiently prevented the translocation of the NF-κB subunit p65 into the nucleus by interrupting the activation of the upstream mediator inhibitor of NF-κB kinase α/ß. Furthermore, the anti-inflammatory effects of DHMDT were associated with the suppression of LPS-induced phosphorylation of Akt and MAPKs in RAW 264.7 macrophages. Therefore, the results of the present study indicate that DHMDT exhibited anti-inflammatory activity via the inhibition of proinflammatory mediators and cytokines, in which the inactivation of NF-κB, PI3K/Akt, and MAPKs may be involved. These results suggest that DHMDT may be a potential anti-inflammatory drug candidate.

Ethanol extracts of Aster yomena (Kitam.) Honda inhibit adipogenesis through the activation of the AMPK signaling pathway in 3T3-L1 preadipocytes.

The leaves of Aster yomena (Kitam.) Honda have long been used as a traditional herb for treating disorders including coughs, asthma, and insect bites. According to recent studies, A. yomena leaf extracts have several pharmacological properties, including anti-inflammatory, antioxidant, and anti-asthmatic activities. However, little information is available regarding their anti-obesity effect. In this study, we investigated the inhibitory effect of the ethanol extracts of A. yomena leaves (EEAY) on adipocyte differentiation and adipogenesis using 3T3-L1 preadipocytes. When 3T3-L1 preadipocytes were treated with various concentrations of EEAY (ranging from non-toxic), the number of lipid droplets, lipid content, and triglyceride production, the typical characteristics of adipocytes, were suppressed in a concentration-dependent manner. During this process, EEAY significantly reduced the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein α and ß, and sterol regulatory element-binding protein-1c. In addition, EEAY was also found to potently inhibit the expression of adipocyte-specific genes, including adipocyte fatty acid-binding protein and leptin. In particular, EEAY treatment effectively enhanced the activation of the AMP-activated protein kinase (AMPK) signaling pathway; however, the co-treatment with compound C, an inhibitor of AMPK, significantly restored the EEAY-induced inhibition of pro-adipogenic transcription factors and adipocyte-specific genes. These results indicate that EEAY may exert an anti-obesity effect by controlling the AMPK signaling pathway, suggesting that the leaf extract of A. yomena may be a potential anti-obesity agent.

An ethanol extract of Aster yomena (Kitam.) Honda inhibits lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages.

Aster yomena (Kitam.) Honda has been widely used as a traditional herbal medicine for centuries to treat cough, asthma, insect bites, etc. Recent reports indicate that A. yomena possesses a wide spectrum of pharmacological activities; however, few experiments have described its anti-inflammatory properties. The present study examined the anti-inflammatory effects of an ethanol extract of A. yomena leaves (EEAY) on lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. Treatment with EEAY significantly reduced the secretion of pro-inflammatory molecules, such as nitric oxide and interleukin-1ß, in LPS-stimulated RAW 264.7 cells, without incurring any significant cytotoxicity. These protective effects were accompanied by a marked reduction in the expression of regulatory genes at the transcription level. Treatment with EEAY also inhibited the DNA-binding activity of nuclear factor-κB (NF-κB) by suppression of nuclear translocation of NF-κB and by degradation of the inhibitor of NF-κB; these effects were associated with suppression of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways. The EEAY treatment also potently suppressed LPS-induced toll like receptor (TLR) 4 expression and attenuated the binding of LPS to the macrophage cell surface. In addition, EEAY treatment markedly inhibited LPS-induced accumulation of intracellular reactive oxygen species in RAW 264.7 macrophages. Therefore, the inhibitory effects of EEAY on LPS-stimulated inflammatory responses in RAW 264.7 macrophages were apparently associated with suppression of the TLR-mediated NF-κB signaling pathway. More work is needed to fully understand the critical role and clinical usefulness of EEAY treatment, but the findings of the present study provide some insights into the potential of EEAY as a therapeutic agent for treatment of inflammatory disorders.

An exploration of the antioxidant effects of garlic saponins in mouse-derived C2C12 myoblasts.

In this study, we aimed to confirm the protective effects of garlic saponins against oxidative stress-induced cellular damage and to further elucidate the underlying mechanisms in mouse-derived C2C12 myoblasts. Relative cell viability was determined by 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide assay. Comet assay was used to measure DNA damage and oxidative stress was determined using 2',7'-dichlorofluorescein diacetate to measure intracellular reactive oxygen species (ROS) generation. Western blot analysis and small interfering RNA (siRNA)-based knockdown were used in order to investigate the possible molecular mechanisms. Our results revealed that garlic saponins prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular ROS. We also observed that garlic saponins prevented H2O2-induced comet tail formation and decreased the phosphorylation levels of γH2AX expression, suggesting that they can prevent H2O2-induced DNA damage. In addition, garlic saponins increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme associated with the induction and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the translocation of Nrf2 from the cytosol into the nucleus. However, the protective effects of garlic saponins on H2O2-induced ROS generation and growth inhibition were significantly reduced by zinc protoporphyrin Ⅸ, an HO-1 competitive inhibitor. In addition, the potential of garlic saponins to mediate HO-1 induction and protect against H2O2­mediated growth inhibition was adversely affected by transient transfection with Nrf2-specific siRNA. Garlic saponins activated extracellular signal­regulated kinase (ERK) signaling, whereas a specific ERK inhibitor was able to inhibit HO-1 upregulation, as well as Nrf2 induction and phosphorylation. Taken together, the findings of our study suggest that garlic saponins activate the Nrf2/HO-1 pathway by enabling ERK to contribute to the induction of phase Ⅱ antioxidant and detoxifying enzymes, including HO-1 in C2C12 cells.

Ethanol extract of Kalopanax septemlobus leaf induces caspase-dependent apoptosis associated with activation of AMPK in human hepatocellular carcinoma cells.

The Kalopanax septemlobus leaf (Thunb.) Koidz. has been used as a traditional medicine herb for the treatment of various human diseases for hundreds of years. In this study, we investigated the mechanism underlying the inhibitory effects of an ethanol extract of K. septemlobus leaf (EEKS) on proliferation of HepG2 hepatocellular carcinoma cells. For this study, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, DAPI (4,6-diamidino-2-phenylindole) staining, agarose gel electrophoresis, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether HepG2 cell death occurred by apoptosis. Treatment of HepG2 cells with EEKS concentration-dependently reduced cell survival while significantly increasing the ratio of apoptotic cells. EEKS treatment increased the levels of the death receptors (DRs), DR4 and DR5, and activated caspases, as well as promoting proteolytic degradation of poly(ADP-ribose)-polymerase associated with the downregulation of protein expression of members of the inhibitor of apoptosis protein family. Treatment with EEKS also caused truncation of Bid, translocation of pro-apoptotic Bax to the mitochondria, and loss of mitochondrial membrane permeabilization, thereby inducing the release of cytochrome c into the cytosol. However, treatment of HepG2 cells with a pan-caspase inhibitor reversed EEKS-induced apoptosis and growth suppression, indicating that EEKS appears to induce apoptosis though a caspase-dependent mechanism involving both intrinsic and extrinsic apoptotic pathways. In addition, the phosphorylation level of AMP-activated protein kinase (AMPK) was elevated when cells were exposed to EEKS. A specific inhibitor for AMPK attenuated the EEKS-induced activation of caspases, and consequently prevented the EEKS-induced apoptosis and reduction in cell viability. Overall, our findings suggest that EEKS inhibits the growth of HepG2 cells by inducing AMPK-mediated caspase-dependent apoptosis, suggesting the potential therapeutic application of EEKS in the treatment or prevention of cancers.

Sargassum horneri methanol extract rescues C2C12 murine skeletal muscle cells from oxidative stress-induced cytotoxicity through Nrf2-mediated upregulation of heme oxygenase-1.

BACKGROUND: Sargassum horneri, an edible marine brown alga, is typically distributed along the coastal seas of Korea and Japan. Although several studies have demonstrated the anti-oxidative activity of this alga, the regulatory mechanisms have not yet been defined. The aim of the present study was to examine the cytoprotective effects of S. horneri against oxidative stress-induced cell damage in C2C12 myoblasts. METHODS: We demonstrated the anti-oxidative effects of a methanol extract of S. horneri (SHME) in a hydrogen peroxide (H2O2)-stimulated C2C12 myoblast model. Cytotoxicity was determined using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl-tetrazolium assay and mode of cell death by cell cycle analysis. DNA damage was measured using a comet assay and expression of phospho-histone γH2A.X (p-γH2A.X). Levels of cellular oxidative stress as reactive oxygen species (ROS) accumulation were measured using 2',7'-dichlorofluorescein diacetate. The involvement of selected genes in the oxidative stress-mediated signaling pathway was explored using Western blot analysis. RESULTS: SHME attenuated H2O2-induced growth inhibition and exhibited scavenging activity against intracellular ROS that were induced by H2O2. The SHME also inhibited comet tail formation, p-γH2A.X expression, and the number of sub-G1 hypodiploid cells, suggesting that it prevents H2O2-induced cellular DNA damage and apoptotic cell death. Furthermore, the SHME significantly enhanced the expression of heme oxygenase-1 (HO-1) associated with induction of nuclear factor-erythroid 2 related factor 2 (Nrf2) in a time- and concentration-dependent manner. Moreover, the protective effect of the SHME on H2O2-induced C2C12 cell damage was significantly abolished by zinc protoporphyrin IX, a HO-1 competitive inhibitor, in C2C12 cells. CONCLUSIONS: These findings suggest that the SHME augments cellular antioxidant defense capacity through both intrinsic free radical scavenging activity and activation of the Nrf2/HO-1 pathway, protecting C2C12 cells from H2O2-induced oxidative cytotoxicity.

Induction of apoptosis in MDA-MB-231 human breast carcinoma cells with an ethanol extract of Cyperus rotundus L. by activating caspases.

Cyperus rotundus L. belongs to the Cyperaceae family and is a well documented traditional medicinal herb. Its rhizome has been reported to possess wide spectrum pharmacological activities including anti-inflammatory and antioxidant activity. However, the cellular and molecular mechanisms of the anticancer activity have not been elucidated. In the present study, we investigated the pro-apoptotic effects of C. rotundu rhizomes in a human breast carcinoma MDA-MB-231 cell model. Treatment of MDA-MB-231 cells with an ethanol extract of C. rotundu rhizomes (EECR) and a methanol extract of C. rotundu rhizomes (MECR), but not a water extract of C. rotundu rhizomes, resulted in potent antiproliferative activity. The activity of the EECR was higher than that of the MECR and was associated with the induction of apoptosis. The induction of apoptosis by the EECR was associated with upregulation of death receptor 4 (DR4), DR5 and pro-apoptotic Bax, as well as downregulation of anti-apoptotic survivin and Bcl-2. EECR treatment also downregulated Bid expression and activated caspase-8 and -9, the respective initiator caspases of the extrinsic and intrinsic apoptotic pathways. The increase in mitochondrial membrane depolarization was correlated with activation of effector caspase-3 and cleavage of poly(ADP-ribose) polymerase, a vital substrate of activated caspase-3. Blockage of caspase activation by pretreatment with a pan-caspase inhibitor consistently inhibited apoptosis and abrogated growth inhibition in EECR-treated MDA-MB-231 cells. Although reactive oxygen species (ROS) increased following treatment with the EECR, inhibiting ROS with a ROS scavenger did not attenuate EECR-induced apoptosis. Furthermore, inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling pathways failed to reverse EECR-induced apoptosis and growth inhibition. These results suggest that the pro-apoptotic activity of the EECR may be regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways that is not associated with ROS generation or the PI3K/Akt and MAPK pathways.

Anti-inflammatory potential of peat moss extracts in lipopolysaccharide-stimulated RAW 264.7 macrophages.

The aim of the present study was to identify the anti-inflammatory and anti-oxidative effects of peat moss aqueous extract (PME) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. To demonstrate the anti-inflammatory and antioxidant effects of PME, the levels of nitric oxide (NO) and cytokines were measured using Griess reagent and cytokine ELISA kits, respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis were conducted to evaluate the expression of genes and proteins. Immunofluorescence was used to measure the expression and translocation of transcription factors. Pre-treatment with PME inhibited the production of prostaglandin E(2) and NO by suppressing the gene expression of cyclooxygenase-2 and inducible NO synthase, respectively. The LPS-stimulated gene expression and the production of tumor necrosis factor-α and interleukin-1ß were significantly reduced by PME. In the LPS-stimulated RAW 264.7 cells, nuclear factor­κB (NF-κB) translocated from the cytosol to the nucleus, while pre-treatment with PME induced the sequestration of NF-κB in the cytosol through the inhibition of IκBα degradation. In the same manner, PME contributed to the inhibition of the activation of mitogen-activated protein kinases. In addition, the PME-treated RAW 264.7 cells facilitated the activation of nuclear factor-like 2 (Nrf2) , and in turn, enhanced heme oxygenase-1 (HO-1) expression. These results indicate that PME exerts anti-inflammatory and antioxidant effects, and suggest that PME may neutralize inflammation and prevent cellular damage by oxidative stress.

N-benzyl-N-methyldecan-1-amine, a phenylamine derivative isolated from garlic cloves, induces G2/M phase arrest and apoptosis in U937 human leukemia cells.

Epidemiological studies indicate that components of garlic (Allium sativum) have anti-proliferative effects against various types of cancer. In the present study, we investigated the effect of newly isolated phenylamine derivative N-benzyl-N-methyldecan-1-amine (NBNMA) from garlic cloves on the inhibition of the growth and apoptosis of human leukemia U937 cells and its potential anticancer mechanism. NBNMA exhibited an antiproliferative effect in U937 cells by inducing cell cycle arrest at the G2/M phase and apoptotic cell death. Western blot analyses revealed that NBNMA decreased the expression of the regulator genes of G2/M phase progression, cyclin dependent kinase (Cdk) 2 and Cdc2 and elevated the expression of the Cdk inhibitor p21WAF1/CIP1 in a p53-independent manner. In addition, NBNMA activated caspase-8 and caspase-9, initiator caspases of the extrinsic and intrinsic pathways of apoptosis, respectively, which led to activation of executioner caspase-3 along with degradation of poly(ADP-ribose) polymerase. NBNMA-induced apoptosis was observed in parallel with an increased ratio of pro-apoptotic Bax and Bad/anti-apoptotic Bcl-2 and Bcl-xL, and inhibition of inhibitor of apoptosis protein (IAP) family members XIAP and cIAP-1. Furthermore, NBNMA-treated cells displayed enhanced release of cytochrome c from the mitochondria into the cytosol concomitant with a loss of mitochondrial membrane potential and downregulation of Bid, suggesting that NBNMA-induced apoptosis occurred via the extrinsic and intrinsic apoptotic pathways with a possible link to Bid protein activity between the two pathways. These results indicate that NBNMA has promising potential to become a novel anticancer agent for the treatment of leukemia. We provide new insight into the mechanisms underlying the anticancer effect of NBNMA.

[6]-Gingerol Prevents Disassembly of Cell Junctions and Activities of MMPs in Invasive Human Pancreas Cancer Cells through ERK/NF- κ B/Snail Signal Transduction Pathway.

To study the effects of [6]-gingerol, a ginger phytochemical, on tight junction (TJ) molecules, we investigated TJ tightening and signal transduction pathways in human pancreatic duct cell-derived cancer cell line PANC-1. The following methods were utilized: MTT assay to determine cytotoxicity; zymography to examine matrix metalloproteinase (MMP) activities; transepithelial electrical resistance (TER) and paracellular flux for TJ measurement; RT-PCR and immunoblotting for proteins related to TJ and invasion; and EMSA for NF- κ B activity in PANC-1 cells. Results revealed that TER significantly increased and claudin 4 and MMP-9 decreased compared to those of the control. TJ protein levels, including zonula occludens (ZO-) 1, occludin, and E-cadherin, increased in [6]-gingerol-treated cells, which correlated with a decrease in paracellular flux and MMP activity. Furthermore, NF- κ B/Snail nuclear translocation was suppressed via downregulation of the extracellular signal-regulated kinase (ERK) pathway in response to [6]-gingerol treatment. Moreover, treatment with U0126, an ERK inhibitor, completely blocked NF- κ B activity. In conclusion, these findings demonstrate that [6]-gingerol regulates TJ-related proteins and suppresses invasion and metastasis through NF- κ B/Snail inhibition via inhibition of the ERK pathway. Therefore, [6]-gingerol may suppress the invasive activity of PANC-1 cells.

The ethyl alcohol extract of Hizikia fusiforme inhibits matrix metalloproteinase activity and regulates tight junction related protein expression in Hep3B human hepatocarcinoma cells.

We tested the correlation between the tightness of tight junctions (TJs) and the anti-invasive activity of the ethyl alcohol extract of Hizikia fusiforme (EHF) in Hep3B human hepatocarcinoma cells. EHF inhibited cell proliferation, motility, and invasiveness, which were associated with increased TJ tightness, as demonstrated by an increase in transepithelial electrical resistance. EHF dose-dependently decreased the secretion of matrix metalloprotease-2 and -9, which correlated with a decrease in mRNA and protein expression, but increased tissue inhibitor of metalloproteinase-1 and -2 mRNA levels. Additionally, immunoblotting results indicated that EHF suppressed the major components of TJ, claudins (-1, -3, and -4), which play a key role in the control and selectivity of paracellular transport. These data indicate that EHF may inhibit cancer cell invasion through the tightening of TJs, which may counteract the up-regulation of claudins. Furthermore, EHF treatment decreased the expression of insulin-like growth factor-1 receptor proteins, while concurrently increasing that of thrombospondin-1 and E-cadherin. In conclusion, these results suggest that EHF treatment may inhibit tumor metastasis and invasion and therefore act as a dietary resource for decreasing the risk of developing cancer.

The water extract of adlay seed (Coix lachrymajobi var. mayuen) exhibits anti-obesity effects through neuroendocrine modulation.

To find out whether the immunohistochemical expression of neuropeptid Y (NPY) and leptin receptor (LR) in the rat hypothalamus is influenced by adlay seed water extract (adlay), obesity in rats was induced by high fat diet (HFD) for 8 weeks; these rats were injected with 50 mg/100 g body weight adlay daily for 4 weeks. The results showed that the optical density of NPY immunoreactivity in paraventricular nucleus of rats increased approximately by 3.4 fold in HFD group compared to the normal diet group. Conversely, that of HFD + adlay group was about 2.6 fold lower than HFD group. The pattern of LR expression was similar to that of NPY. Both of NPY and LR mRNA levels, determined by real time PCR, in HFD + adlay group were decreased compared to those of HFD group, but there were no significant changes in the level of LR. These results suggest that adlay may regulate neuroendocrine activity in the brain. Accordingly, administration of adlay may be considered for therapies targeting obesity.

Hypolipidemic effects of crude extract of adlay seed (Coix lachrymajobi var. mayuen) in obesity rat fed high fat diet: relations of TNF-alpha and leptin mRNA expressions and serum lipid levels.

To find out whether the expressions of these adipocyte markers are influenced by oriental medicine, obesity rats induced by high fat diet (HFD) for 8 weeks were injected with 50 mg/100 g body weight adlay seed crude extract (ACE), daily for 4 weeks. The results are summarized as follows: HFD + ACE group significantly reduced food intakes and body weights. Weights of epididymal and peritoneal fat were dramatically increased in HFD groups compared with those of normal diet (ND) group but significantly decreased more in HFD + ACE group than those of HFD + saline group (sham). Those of brown adipocytes were increased in HFD + ACE group compared to ND and sham groups but there was no significant difference. The sizes in white adipose tissue (WAT) by microscope were markedly larger in HFD groups than ND group but considerably reduced in HFD + ACE group compared with sham group. The levels of triglyceride, total-cholesterol and leptin in blood serum were significantly decreased in HFD + ACE group compared to those of sham group. Leptin and TNF-alpha mRNA expressions in WAT of rats were remarkably increased more in sham group than in those of ND group. Those of HFD + ACE group were significantly decreased compared with those of sham group, especially. TNF-alpha mRNA expression in HFD + ACE group was declined more than that of ND group. In conclusion, treatments of ACE modulated expressions of leptin and TNF-alpha and reduced body weights, food intake, fat size, adipose tissue mass and serum hyperlipidemia in obesity rat fed HFD. Accordingly, the oriental medicine extract, adlay seed crude extract, can be considered for obesity therapies controlling.