Necrostatins regulate apoptosis, necroptosis, and inflammation in cisplatin-induced nephrotoxicity in LLC-PK1 cells.

Acute kidney injury (AKI) is a common clinical problem that is associated with high mortality due to multiple complex mechanisms. Cisplatin is the most important and highly effective chemotherapeutic agent used for the treatment of various solid tumors; however, it is associated with dose-dependent adverse effects, particularly in the kidney where it can cause severe nephrotoxicity. The pathophysiological basis of cisplatin-induced nephrotoxicity has been investigated over the last few decades, and the key pathological occurrences in cisplatin nephrotoxicity include renal tubular cell injury and death. Necrostatin-1 (Nec-1) has been confirmed to act as a specific and potent small-molecule inhibitor of necroptosis. However, the effects of three structurally distinct necrostatins on cisplatin-induced nephrotoxicity remain ambiguous. The aim of this study was to determine if three types of necrostatins (Nec-1, Nec-3-A, and/or Nec-3-B) can exert protective effects in regard to the AKI induced by cisplatin. Our results indicated that necrostatins can prevent cisplatin induced nephrotoxicity via modulating apoptotic pathways through the suppression of cleaved caspase-3 and also by influencing the function of mitogen-activated protein kinase pathway members, including extracellular signal-regulated kinases, c-Jun N-terminal kinases, and p38, in the renal tubular epithelial cell line LLC-PK1. These findings suggest that necrostatins exert beneficial anti-apoptotic effects in the context of AKI induced by cisplatin.

Phytochemicals from the flowers of Prunus persica (L.) Batsch: Anti-adipogenic effect of mandelamide on 3T3-L1 preadipocytes.

Flowers of Prunus persica (L.) Batsch (Rosaceae), known as peach blossoms, have been reported to exert anti-obesity effects by improving hepatic lipid metabolism in obese mice. However, little is known regarding the anti-adipogenic effects of the phenolic compounds isolated from P. persica flowers. This study investigated the inhibitory effects of compounds extracted from P. persica flowers (PPF) on adipogenesis in 3T3-L1 murine preadipocytes using adipogenic differentiation assays. Additionally, we compared the anti-adipogenic effects of the phenolic compounds isolated from PPF, such as prunasin amide (1), amygdalin amide (2), prunasin acid (3), mandelamide (4), methyl caffeate (5), ferulic acid (6), chlorogenic acid (7), benzyl α-l-xylpyranosyl-(1 â†’ 6)-ß-d-glucopyranoside (8), prunin (9), naringenin (10), nicotiflorin (11), astragalin (12), afzelin (13), and uridine (14), on adipogenesis in 3T3-L1 murine preadipocytes. PPF and compounds 4-7 and 10 significantly inhibited adipogenesis. Among them, mandelamide (4) exhibited the maximum inhibitory activity with an IC50 of 36.04 ± 1.82 µM. Additionally, mandelamide downregulated the expression of key adipogenic markers, such as extracellular signal-regulated kinase, c-Jun-N-terminal kinase, P38, CCAAT/enhancer-binding protein α, CCAAT/enhancer-binding protein ß, peroxisome proliferator activated receptor γ, and glucocorticoid receptor. These results indicate that mandelamide is an active ingredient of PPF possessing anti-obesity properties.

Bioactivity-based analysis and chemical characterization of anti-inflammatory compounds from Curcuma zedoaria rhizomes using LPS-stimulated RAW264.7 cells.

Inflammation is not only a self-defense response of the innate immune system, but also the pathogenesis mechanism of multiple diseases such as arthritis, neurodegeneration, and cancer. Curcuma zedoaria Roscoe (Zingiberaceae), an indigenous plant of India, has been used traditionally in Ayurveda and folk medicine. As part of our ongoing efforts to screen traditional medicinal plants exhibiting pharmacological potential and to characterize the compounds involved, we examined the anti-inflammatory effects of the MeOH extract of C. zedoaria rhizomes using lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage cells and found that MeOH extract inhibited the synthesis of nitric oxide (NO) in a dose-dependent manner (IC50: 23.44 ±â€¯0.77 µg/mL). In our efforts to characterize the compounds responsible for these anti-inflammatory effects, bioactivity-guided fractionation of the MeOH extract and chemical investigation of its active hexane-soluble fraction led to the successful isolation of five sesquiterpenes (1-5), the structures of which were elucidated by NMR spectroscopic analysis and LC/MS analysis. Among them, curcuzedoalide (5) exhibited potent inhibitory effects on NO synthesis (IC50: 12.21 ±â€¯1.67 µM) and also suppressed pre-inflammatory protein expression of iNOS and COX-2. Curcuzedoalide (5) was thus determined to be a contributor to the anti-inflammatory effect of C. zedoaria rhizomes and could be a potential candidate for therapeutic applications.

Protective effect of hypoxylonol C and 4,5,4',5'-tetrahydroxy-1,1'-binaphthyl isolated from Annulohypoxylon annulatum against streptozotocin-induced damage in INS-1 cells.

We evaluated the protective effects of hypoxylonol C and 4,5,4',5'-tetrahydroxy-1,1'-binaphthyl (BNT) isolated from Annulohypoxylon annulatum on pancreatic ß-cell apoptosis, using the ß-cell toxin streptozotocin (STZ). Hypoxylonol C and BNT restored the STZ-induced decrease in INS-1 cell viability in a dose-dependent manner. In addition, treatment of INS-1 cells with 50 µM STZ resulted in an increase in apoptotic cell death, which was observed as annexin V fluorescence intensity. Apoptotic cell death was decreased by co-treatment with 100 µM hypoxylonol C and 100 µM BNT. Similarly, STZ caused a marked increase in the expression of cleaved caspase-8, caspase-3, Bax, and poly (ADP-ribose) polymerase (PARP), as well as a decrease in the expression of B-cell lymphoma 2 (Bcl-2), which was reversed by co-treatment with 100 µM hypoxylonol C and 100 µM BNT. These findings suggest that hypoxylonol C and BNT play an important role in protecting pancreatic ß-cells against apoptotic damage.

Eupatilin inhibits angiogenesis-mediated human hepatocellular metastasis by reducing MMP-2 and VEGF signaling.

Metastasis is responsible for the great majority of deaths in cancer patients. Matrix metalloproteinases (MMPs) have critical functions in cancer metastasis. Especially, MMP-2 and MMP-9 play a major role in tumor-cell migration and invasion. Therefore, to first find out the inhibitory effect of eupatilin on expression of MMPs in SNU182 cells, we used quantitative real-rime PCR to measure MMP-2 and MMP-9 mRNA levels. Eupatilin suppressed transcription of MMP-2 in SNU182 cells more than did the corresponding controls. Also, eupatilin significantly blocked tube formation when treated with a concentration of 3.125 or 6.25 µg/mL on human umbilical vein vascular endothelial cells (HUVECs). Eupatilin induced significant anti-angiogenic potential associated with down-regulation of hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), and phosphorylated Akt expression. Thus, tube-formation inhibition and MMP-2-mediated migration are likely to be important therapeutic targets of eupatilin in hepatocellular carcinoma metastasis.

Chemical Identification of Isoflavonoids from a Termite-Associated Streptomyces sp. RB1 and Their Neuroprotective Effects in Murine Hippocampal HT22 Cell Line.

Insect-associated bacteria have been recognized as a very promising natural resource for discovering bioactive secondary metabolites with diverse pharmacological effects. One new isoflavonoid glycoside, termisoflavone D (1), together with seven known isoflavonoids (2⁻8), were identified from MeOH extracts of the fungus-growing termite-associated Streptomyces sp. RB1. The chemical structure of the new compound 1 was elucidated using comprehensive spectroscopic methods including 1D and 2D NMR, along with LC/MS analysis. The existence of two rhamnose moieties in 1 was determined with comparative NMR analysis, and the absolute configuration was elucidated using chemical reactions. The neuroprotective activities of compounds 1⁻8 were thoroughly investigated using the murine hippocampal HT22 cell line. Compound 5 prevented glutamate-induced HT22 cell death by blocking intracellular reactive oxygen species (ROS) accumulation. The present study provides the first experimental evidence for the potential use of isoflavonoids from termite-associated bacteria as lead compounds that can prevent neuronal damage induced by glutamate.

Beneficial Effects of Deoxyshikonin on Delayed Wound Healing in Diabetic Mice.

Shiunko ointment is composed of five ingredients including Lithospermi Radix (LR), Angelicae Gigantis Radix, sesame seed oil, beeswax, and swine oil. It is externally applied as a treatment for a wide range of skin conditions such as eczema, psoriasis, hair loss, burns, topical wounds, and atopic dermatitis. Deoxyshikonin is the major angiogenic compound extracted from LR. In this study, we investigated the efficacy of LR extract and deoxyshikonin on impaired wound healing in streptozotocin (STZ)-induced diabetic mice. Treatment with LR extract elevated tube formation in human umbilical vein endothelial cells (HUVECs) and exerted antioxidant activity. An open skin wound was produced on the backs of diabetic mice and was then topically treated with deoxyshikonin or vehicle. In addition, deoxyshikonin promoted tube formation in high glucose conditions exposed to HUVECs, and which may be regulated by increased VEGFR2 expression and phosphorylation of Akt and p38. Our results demonstrate that deoxyshikonin application promoted wound repair in STZ-induced diabetic mice. Collectively, these data suggest that deoxyshikonin is an active ingredient of LR, thereby contributing to wound healing in patients with diabetes.

Beneficial Effects of Bioactive Compounds in Mulberry Fruits against Cisplatin-Induced Nephrotoxicity.

Mulberry, the fruit of white mulberry tree (Morus alba L., Moraceae), is commonly used in traditional Chinese medicines as a sedative, tonic, laxative, and emetic. In our continuing research of the bioactive metabolites from mulberry, chemical analysis of the fruits led to the isolation of five compounds, 1-5. The compounds were identified as butyl pyroglutamate (1), quercetin 3-O-ß-d-glucoside (2), kaempferol 3-O-ß-d-rutinoside (3), rutin (4), and 2-phenylethyl d-rutinoside (5) by spectroscopic data analysis, comparing their nuclear magnetic resonance (NMR) data with those in published literature, and liquid chromatography-mass spectrometry analysis. The isolated compounds 1-5 were evaluated for their effects on anticancer drug-induced side effects by cell-based assays. Compound 1 exerted the highest protective effect against cisplatin-induced kidney cell damage. This effect was found to be mediated through the attenuation of phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, p38, mitogen-activated protein kinase, and caspase-3 in cisplatin-induced kidney cell damage.

Protective effect of α-mangostin against iodixanol-induced apoptotic damage in LLC-PK1 cells.

Radiographic contrast media facilitate the visibility of internal body structures, but its use to patients with lowered renal function needs to be careful because of severe side effect in kidney. The present study aims to evaluate potential protective effect and mechanism of Alpha mangostin (α-mangostin) against contrast-induced apoptotic damage in LLC-PK1 cells. As a result, α-mangostin in non-toxic concentrations improved the viability of the iodixanol-treated cells up to 90.42% against contrast-induced damage in LLC-PK1 cells. Iodixanol treatment increased the phosphorylation of p38, ERK and cleavage of caspase-3 in LLC-PK1 cells, which were significantly decreased by co-treatment with α-mangostin (2.5 and 5µM). The protective effect of α-mangostin on contrast-induced apoptotic damage was mediated by the inhibition of MAPKs and caspase activation.

Estriol blunts postprandial blood glucose rise in male rats through regulating intestinal glucose transporters.

Despite increased total food intake in healthy, late-stage pregnant women, their peak postprandial blood sugar levels are normally much lower than the levels seen in healthy nonpregnant women. In this study, we sought to determine whether estriol (E3), an endogenous estrogen predominantly produced during human pregnancy, contributes to the regulation of the postprandial blood glucose level in healthy normal rats. In vivo studies using rats showed that E3 blunted the speed and magnitude of the blood glucose rise following oral glucose administration, but it did not appear to affect the total amount of glucose absorbed. E3 also did not affect insulin secretion, but it significantly reduced the rate of intestinal glucose transport compared with vehicle-treated animals. Consistent with this finding, expression of the sodium-dependent glucose transporter 1 and 2 was significantly downregulated by E3 treatment in the brush-border membrane and basolateral membrane, respectively, of enterocytes. Most of the observed in vivo effects were noticeably stronger with E3 than with 17ß-estradiol. Using differentiated human Caco-2 enterocyte monolayer culture as an in vitro model, we confirmed that E3 at physiologically relevant concentrations could directly inhibit glucose uptake via suppression of glucose transporter 2 expression, whereas 17ß-estradiol did not have a similar effect. Collectively, these data showed that E3 can blunt the postprandial glycemic surge in rats through modulating the level of intestinal glucose transporters.

Synthesis of apoptotic chalcone analogues in HepG2 human hepatocellular carcinoma cells.

Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was significantly higher in compound 5-treated cells than in control cells. Exposure to compound 5 for 24h induced cleavage of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). Our findings suggest that compound 5 is the active chalcone analogue that contributes to cell death in HepG2 cells via the extrinsic apoptotic pathway.

Mechanism of Ascorbate-Induced Cell Death in Human Pancreatic Cancer Cells: Role of Bcl-2, Beclin 1 and Autophagy.

The present study investigates the anticancer effect of ascorbate in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions. We showed that ascorbate suppresses the growth of human pancreatic cancer cells via the induction of oxidative stress and caspase-independent cell death. Ascorbate induces the formation of autophagosomes and the presence of autophagy inhibitors suppresses ascorbate-induced cell death. These data suggest that the induction of autophagosome formation contributes to ascorbate-induced pancreatic cancer cell death.

Protective effect of tetrahydrocurcumin against cisplatin-induced renal damage: in vitro and in vivo studies.

The adverse effects of anticancer drugs can prompt patients to end their treatment despite the efficacy. Cisplatin is a platinum-based molecule widely used to treat various forms of cancer, but frequent and long-term use of cisplatin is limited due to severe nephrotoxicity. In the present study, we investigated the protective effect and mechanism of tetrahydrocurcumin on cisplatin-induced kidney damage, oxidative stress, and inflammation to evaluate its possible use in renal damage. Cisplatin-induced LLC-PK1 renal cell damage was significantly reduced by tetrahydrocurcumin treatment. Additionally, the protective effect of tetrahydrocurcumin on cisplatin-induced oxidative renal damage was investigated in rats. Tetrahydrocurcumin was orally administered every day at a dose of 80 mg/kg body weight for ten days, and a single dose of cisplatin was administered intraperitoneally (7.5 mg/kg body weight) in 0.9 % saline on day four. The creatinine clearance levels, which were markers of renal dysfunction, in cisplatin-treated rats were recovered nearly back to normal levels after administration of tetrahydrocurcumin. Moreover, tetrahydrocurcumin exhibited protective effects against cisplatin-induced oxidative renal damage in rats by inhibiting cyclooxygenase-2 and caspase-3 activation. These results collectively provide therapeutic evidence that tetrahydrocurcumin ameliorates renal damage by regulating inflammation and apoptosis.

Inhibitory effects of ginseng sapogenins on the proliferation of triple negative breast cancer MDA-MB-231 cells.

Because of poor prognosis, clinical treatment of triple-negative (TN) breast cancer remains the most challenging factor in cancer treatment. Extensive research into alternative cancer therapies includes studying the naturopathic effects of the medicinal herb ginseng. This study investigates the anti-neoplastic properties of ginseng sapogenins and the derivatives: (1) (20(S)-protopanaxadiol (PPD), (2) 20(S)-protopanaxatriol), (3) (20(S)-dihydroprotopanaxadiol, and (4) 20(S)-dihydroprotopanaxatriol). These compounds were found to prevent the proliferation of MDA-MB-231 human breast cancer cells. PPD was the most potent inhibitor, exhibiting an IC50 (5.87 µM) comparable to that of the chemotherapeutic drug taxol. Furthermore, PPD induced dose-dependent cleavage of caspase-8, caspase-3, and PARP in MDA-MB-231 cells. Thus, we propose that PPD acts as anti-cancer agent by stimulating caspase-dependent apoptosis in breast cancer cells.

Chinese prescription kangen-karyu ameliorates the development of diabetic hepatic damages via regulating oxidative stress and inflammation in the liver of db/db mice.

The prevention and treatment of diabetic complications are considered to be the most important for the general care of diabetic patients. We have been conducting pre-clinical animal experiments related to diabetes using kangen-karyu, a Chinese prescription, to examine its therapeutic potential. In the present study, we further studied the anti-diabetic mechanism of kangen-karyu, especially on the regulation of hyperglycemia-induced hepatic oxidative stress and inflammation in db/db mice. Kangen-karyu (100 or 200 mg/kg body weight/day, per os (p.o.) was administered every day for 18 weeks to db/db mice, and its effect was compared with vehicle-treated db/db and m/m mice. The administration of kangen-karyu decreased the elevated serum and hepatic glucose concentration in db/db mice. The elevated expressions of p22(phox) and Nox-4 proteins (reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits) were significantly decreased after kangen-karyu treatments. The oxidative stress-related markers in hepatic tissue (reactive oxygen species, reduced glutathione/oxidized glutathione ratio, and thiobarbituric acid-reactive substance) were also significantly ameliorated by the kangen-karyu treatments. The db/db mice exhibited the up-regulation of nuclear factor-κBp65, cyclooxygenase-2, and inducible nitric oxide synthase levels in the liver; however, kangen-karyu treatment significantly reduced those expressions. Taking these into consideration, our findings support the therapeutic evidence for kangen-karyu ameliorating the development of diabetic hepatic damages via regulating oxidative stress and inflammation.

Growth-stimulatory effect of resveratrol in human cancer cells.

Earlier studies have shown that resveratrol could induce death in several human cancer cell lines in culture. Here we report our observation that resveratrol can also promote the growth of certain human cancer cells when they are grown either in culture or in athymic nude mice as xenografts. At relatively low concentrations (

Beneficial effect of 17ß-estradiol on hyperglycemia and islet ß-cell functions in a streptozotocin-induced diabetic rat model.

The modulating effect of estrogen on glucose homeostasis remains a controversial issue at present. In this study, we sought to determine the beneficial effect of 17ß-estradiol (E2) on hyperglycemia and islet ß-cell functions in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were injected i.p. with STZ to induce a relatively mild diabetic condition. The rats were then treated with E2 orally at 500 µg/kg body weight/day for 15 days to evaluate the modulating effect on hyperglycemia, insulin secretion, and islet ß-cell proliferation. E2 administration for 10 days significantly lowered plasma glucose levels, increased plasma insulin levels, and improved glucose tolerance by attenuating insulin response to oral glucose loading. These beneficial effects of E2 were accompanied by increases in islet number and volume, rate of islet cell proliferation, and the amount of insulin secreted. The growth-stimulatory effect of E2 on islet cells was linked to the functions of the estrogen receptor α. Notably, these protective effects of E2 on diabetic conditions were basically not observed when the STZ-treated rats had a more severe degree of islet damage and hyperglycemia. Taken together, we conclude that E2 can promote the regeneration of damaged pancreatic islets by stimulating ß-cell proliferation in diabetic rats, and this effect is accompanied by improvements in glucose tolerance and a decrease in plasma glucose levels. These findings suggest that oral administration of E2 may be beneficial in diabetic patients with an accelerated loss of islet ß-cells.

Effects of heat-processed ginseng and its active component ginsenoside 20(S)-Rg3 on the progression of renal damage and dysfunction in type 2 diabetic Otsuka Long-Evans Tokushima Fatty rats.

The effects of heat-processed ginseng (HPG) and ginsenoside 20(S)-Rg(3) on the progression of renal damage in type 2 diabetic rats were investigated. Twenty-two-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were divided into 4 orally administered groups: vehicle (diabetic control), HPG water extract (100 mg/kg) and 20(S)-Rg(3) (5, 10 mg/kg). Non-diabetic Long-Evans Tokushima Otsuka (LETO) rats were used as a normal group. OLETF rats showed markedly higher blood glucose, triglyceride, and total cholesterol levels than those of LETO rats. The elevated blood glucose level of OLETF rats was significantly lowered by 20(S)-Rg(3) administration. The elevated serum triglyceride and total cholesterol levels were significantly reduced by the administrations of HPG and 20(S)-Rg(3). The serum levels of thiobarbituric acid-reactive substance, an index of lipid peroxidation, were markedly increased in OLETF compared to LETO rats, but it was significantly reduced by HPG and 20(S)-Rg(3) administrations. The urinary protein level, an indicator of advanced diabetic nephropathy, of OLETF rats was 4.4 times higher than in LETO rats, but it was reduced significantly by the administrations of HPG and 20(S)-Rg(3). Creatinine clearance of OLETF rats was significantly increased after HPG and 20(S)-Rg(3) administrations. The elevation of inducible nitric oxide synthase and N(epsilon)-(carboxymethyl)lysine protein expressions in renal tissues of OLETF rats was prevented by 20(S)-Rg(3) administration. This study provides scientific evidence that 20(S)-Rg(3) prevents the progression of renal damage and dysfunction in type 2 diabetic rats via inhibiting oxidative stress and advanced glycation endproduct formation.

Caffeic acid phenethyl ester, a coffee polyphenol, inhibits DNA methylation in vitro and in vivo.

DNA methylation represents an important epigenetic regulation of the genome. Earlier studies have suggested that dietary phenolic compounds including those contained in coffee, tea and soy products may modulate the level of DNA methylation. In this study, we first characterize the effect of caffeic acid phenethyl ester (CAPE) and other dietary phenolic compounds on DNA methylation in vitro. The IC50 values of CAPE, daidzein, isorhamnetin and genistein are 7.6, 6.9, 6.2, and 4.3 µM, respectively, in an in-vitro enzymatic assay system. Computational analysis indicates that CAPE, daidzein, isorhamnetin and genistein can bind inside the DNA substrate-binding site in human DNMT1 with a favorable binding energy. In an animal study, we find that maternal CAPE treatment shifts the coat color distribution of the 21-day-old Avy/a offspring towards the yellow phenotype, indicating that CAPE inhibits the methylation of the agouti gene promoter sequence in vivo. The results from this study may shed light on the potential epigenetic effect in the offspring resulting from maternal intake of certain coffee phenolics during pregnancy.