Protective effects of flavonoids from Gynostemma pentaphyllum on oxidative damage in LLC-PK1 cells / 中国中药杂志

Four flavonoids were isolated from Gynostemma pentaphyllum by chromatography methods and their structures were identified by MS and NMR spectra data as quercetin-3-O-( 2″,6″-di-α-L-rhamnosyl)-β-D-galactopyranoside( 1),quercetin-3-O-( 2″,6″-di-α-L-rhamnosyl)-β-D-glucopyranoside( 2),quercetin-3-O-( 2″-α-L-rhamnosyl)-β-D-galactopyranoside( 3),and quercetin-3-O-( 2″-α-L-rhamnosyl)-β-D-glucopyranoside( 4). Among them,compounds 1-3 were obtained from the Cucurbitaceae family for the first time.The four flavonoids showed potent antioxidant effects against the DPPH,·OH and ■radicals in vitro,especially for DPPH radical scavenging activity with the IC50 values of 71. 4,29. 5,48. 3 and 79. 2 μmol·L~(-1),respectively. Moreover,the four flavonoids displayed strong cytoprotection against AAPH-induced oxidative damage in LLC-PK1 cells by suppressing the increase of malondialdehyde( MDA) and the decrease of the superoxide dismutase( SOD) and glutathione( GSH). Since further research is needed to prove its efficacy in vivo and clinical trial,the study may provide four potential antioxidants from G. pentaphyllum.

Nephrotoxicity Of Polymyxin B: Experimental Study In Cells And Implications For Nursing Practice / La Nefrotoxicidad De Polimixina B: Estudio Experimental En Las Células Y Implicaciones Para La Práctica De Enfermería / Nefrotoxicidade da polimixina B: estudo experimental em células e implicações para a prática de enfermagem

The aim of the study was to characterize the cell damage mechanisms involved in the pathophysiology of cytotoxicity of polymyxin B in proximal tubular cells (LLC - PK1) and discuss about the nurses interventions to identify at risk patients and consider prevention or treatment of nephrotoxicity acute kidney injury. This is a quantitative experimental in vitro study, in which the cells were exposed to 375μM polymyxin B sulfate concentration. Cell viability was determined by exclusion of fluorescent dyes and morphological method with visualization of apoptotic bodies for fluorescence microscopy. Cells exposed to polymyxin B showed reduced viability, increased number of apoptotic cells and a higher concentration of the enzyme lactate dehydrogenase. The administration of polymyxin B in vitro showed the need for actions to minimize adverse effects such as nephrotoxicity.
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El objetivo del estudio fue caracterizar los mecanismos de daño celular implicado en la fisiopatología de la citotoxicidad de la polimixina B en las células tubulares proximales (LLC-PK1) y discutir las propuestas de intervención de enfermería para identificar a los pacientes de riesgo y considerar la prevención o el tratamiento de la lesión renal aguda nefrotóxica. Corresponde a un estudio experimental cuantitativo in vitro, en el cual las células fueron expuestas a sulfato de polimixina B. La viabilidad celular se determinó por exclusión de los colorantes fluorescentes y el método morfológico con la visualización de cuerpos apoptóticos a la microscopía de fluorescencia. Las células expuestas a polimixina B demostraron reducción de la viabilidad, aumento de células apoptóticas y mayor concentración de la enzima lactato deshidrogenasa. La administración de polimixina B in vitro demostró la necesidad de realizar acciones en la práctica clínica para minimizar los efectos adversos como la nefrotoxicidad.

O objetivo do estudo foi caracterizar os mecanismos de lesão celular envolvidos na fisiopatologia da citotoxicidade da polimixina B em células tubulares proximais (LLC-PK1) e discutir as proposições de intervenção do enfermeiro para identificar os pacientes de risco e considerar a prevenção ou o tratamento para lesão renal nefrotóxica. Estudo experimental in vitro , onde as células foram expostas ao sulfato de polimixina B. A viabilidade celular foi determinada pela exclusão dos corantes fluorescentes e o método morfológico com visualização de corpos apoptóticos à microscopia de fluorescência. As células expostas à polimixina B apresentaram redução de viabilidade, aumento do número de células em apoptose e maior concentração da enzima desidrogenase láctea. A administração de polimixina B in vitro demonstrou a necessidade de ações na prática clínica para minimizar os efeitos adversos como a nefrotoxicidade.

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Antioxidative effects of fermented sesame sauce against hydrogen peroxide-induced oxidative damage in LLC-PK1 porcine renal tubule cells

BACKGROUND/OBJECTIVES: This study was performed to investigate the in vitro antioxidant and cytoprotective effects of fermented sesame sauce (FSeS) against hydrogen peroxide (H2O2)-induced oxidative damage in renal proximal tubule LLC-PK1 cells. MATERIALS/METHODS: 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (*OH), and H2O2 scavenging assay was used to evaluate the in vitro antioxidant activity of FSeS. To investigate the cytoprotective effect of FSeS against H2O2-induced oxidative damage in LLC-PK1 cells, the cellular levels of reactive oxygen species (ROS), lipid peroxidation, and endogenous antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) were measured. RESULTS: The ability of FSeS to scavenge DPPH, *OH and H2O2 was greater than that of FSS and AHSS. FSeS also significantly inhibited H2O2-induced (500 microM) oxidative damage in the LLC-PK1 cells compared to FSS and AHSS (P < 0.05). Following treatment with 100 microg/mL of FSeS and FSS to prevent H2O2-induced oxidation, cell viability increased from 56.7% (control) to 83.7% and 75.6%, respectively. However, AHSS was not able to reduce H2O2-induced cell damage (viability of the AHSS-treated cells was 54.6%). FSeS more effectively suppressed H2O2-induced ROS generation and lipid peroxidation compared to FSS and AHSS (P < 0.05). Compared to the other sauces, FSeS also significantly increased cellular CAT, SOD, and GSH-px activities and mRNA expression (P < 0.05). CONCULUSIONS: These results from the present study suggest that FSeS is an effective radical scavenger and protects against H2O2-induced oxidative damage in LLC-PK1 cells by reducing ROS levels, inhibiting lipid peroxidation, and stimulating antioxidant enzyme activity.

Polimixina B: efeito dose e tempo dependente na nefrotoxicidade in vitro / Polymyxin B: dose and time dependent nephrotoxicity effect in vitro

OBJETIVO: Caracterizar a toxicidade da polimixina B (PmxB) em células renais em dosagem e tempos diferentes. MÉTODOS: Células LLC-PK1, cultivadas em placas multiwell de 12 poços, foram divididas nos seguintes grupos: Controle (CTL) - células mantidas em meio DMEM suplementado a 5%; G1 - células expostas à concentração de 75mM de PmxB; G2 - células expostas à concentração de 375mM de PmxB. Cada grupo foi avaliado nos tempos de 24, 48 e 72 horas quanto à viabilidade celular (Acridine Orange/Brometo de Etídio) e apoptose (Hoechst 33342). RESULTADOS: Os dados demonstraram a viabilidade celular e a apoptose à exposição de três doses de PmxB em três intervalos de tempo, com um aumento significativo da toxicidade à elevação das doses e ao maior tempo de permanência no antibiótico para apoptose. CONCLUSÃO: A citotoxicidade pela PmxB, no modelo de cultivo celular, se mostrou tempo e dose dependente, aumentando com a maior exposição e maior dose de antibiótico.

OBJECTIVE: To characterize the toxicity of polymyxin B (PmxB) in renal cell in different dosage and times. METHODS: LLC-PK1 cells grown in 12 well multiwell plates were divided into the following groups: Control (CTL) - cells maintained in DMEM supplemented with 5%; G1 - cells exposed to concentration of 75µM PmxB G2 - cells exposed to concentration of 375µM PmxB. Each group was assessed at 24,48 and 72 hours as for cell viability (Acridine orange/ethidium bromide) and apoptosis (Hoechst 33342). RESULTS: The data demonstrate the cell viability and apoptosis exposure of three doses of PmxB in three time intervals, with a significant increase in toxicity to high doses and longer duration of stay in the antibiotic to apoptosis. CONCLUSION: Cytotoxicity by PmxB in cell culture model, showed to be time and dose dependent, increasing with increased exposure and higher dose of antibiotic.

Gentamicin-induced preconditioning of proximal tubular LLC-PK1 cells stimulates nitric oxide production but not the synthesis of heat shock protein

Nephrotoxicity is the main side effect of antibiotics such as gentamicin. Preconditioning has been reported to protect against injuries as ischemia/reperfusion. The objective of the present study was to determine the effect of preconditioning with gentamicin on LLC-PK1 cells. Preconditioning was induced in LLC-PK1 cells by 24-h exposure to 2.0 mM gentamicin (G/IU). After 4 or 15 days of preconditioning, cells were again exposed to gentamicin (2.0 mM) and compared to untreated control or G/IU cells. Necrosis and apoptosis were assessed by acridine orange and HOESCHT 33346. Nitric oxide (NO) and endothelin-1 were assessed by the Griess method and available kit. Heat shock proteins were analyzed by Western blotting. After 15 days of preconditioning, LLC-PK1 cells exhibited a significant decrease in necrosis (23.5 ± 4.3 to 6.5 ± 0.3%) and apoptosis (23.5 ± 4.3 to 6.5 ± 2.1%) and an increase in cell proliferation compared to G/IU. NO (0.177 ± 0.05 to 0.368 ± 0.073 ìg/mg protein) and endothelin-1 (1.88 ± 0.47 to 2.75 ± 0.53 pg/mL) production significantly increased after 15 days of preconditioning compared toG/IU. No difference in inducible HSP 70, constitutive HSC 70 or HSP 90 synthesis in tubular cells was observed afterpreconditioning with gentamicin. The present data suggest that preconditioning with gentamicin has protective effects on proximal tubular cells, that involved NO synthesis but not reduction of endothelin-1 or production of HSP 70, HSC 70, or HSP 90. We conclude that preconditioning could be a useful tool to prevent the nephrotoxicity induced by gentamicin.

Sphingosine mediates FTY720-induced apoptosis in LLC-PK1 cells

FTY720, a synthetic sphingoid base analog, was examined as a new sphingosine kinase inhibitor, which converts endogenous sphingosine into its phosphate form. With 20 micrometer of FTY720, sphingosine accumulated in the LLC-PK1 cells in a time- and dose-dependent manner. The FTY720 treated cells showed a high concentration of fragmented DNA, a high caspase-3 like activity and TUNEL staining cells. It was also found that the sphingosine and sphinganine level increased in a time- and dose-dependent manner within 12 h after the FTY720 treatment. The sphingosine kinase activity was reduced by FTY720 as much as other sphingosine kinase inhibitors, N, N-dimethylsphingosine (DMS), dl-threo-dihydrosphingosine (DHS). The fragmented DNA content as a result of the 20 micrometer of FTY720 treatment and by 5 micrometer of the exogenously added BSA-sphingosine complex indicated typical apoptosis. Under similar conditions, the accumulated sphingosine concentration in all the cells was almost identical even though the sphingosine distribution inside the cells was somewhat different. These results indicate that the FTY720 induced apoptosis is associated with the inhibition of the sphingosine kinase activity and is strongly associated with the successive accumulation of sphingosine.

The Study on the Mechanism of Cyclosporine A-induced Nephroptoxicity: The Effects of Ceramide and Phospholipase A2 on Cyclosporine A-induced Apoptosis of Renal Tubular Epithelial Cells / 대한이식학회지

PURPOSE: In order to elucidate the mechanisms mediating cyclosporine A (CsA)-induced renal tubular cell injury, we examined the effects of ceramide, second messenger derived from sphingolipid breakdown, and phospholipase A2 (PLA2), enzyme responsible for release of arachidonic acid, on CsA-induced apoptosis of cultured LLC-PK1 renal tubular cell line. METHODS: The apoptosis was evaluated by flow cytometric analysis and DNA gel electrophoresis. The activities of cytosolic (c)- and secretory (s)-PLA2 were measured by ELISA methods and Western blotting of cPLA2 was also investigated. RESULTS: The exposure to CsA (10microgram/mL) significantly increased the percentage of cells displaying annexin-V binding from 5.1+/-2.0% in control to 24.7+/-6.5% (P<0.05), indicating apoptosis. The addition of ceramide (10 micromol/mL) significantly inhibited the increase of apoptosis induced by CsA (24.7+/-6.5% vs. 14.7+/-6.0). The treatment with PLA2 (5 U/mL) also decreased CsA (5microgram/mL)-induced apoptosis (20.4+/-5.7% vs. 13.6+/-5.2, P<0.05). But there was no dose-dependent further protectective effect of ceramide or PLA2. Concerning the changes of PLA2 activities, cPLA2 activity after CsA exposure (10microgram/mL) was increased from 3.92+/-2.01 nmol/min/mL in control to 9.81+/-3.07 (P<0.05), and the addition of ceramide (5 micro mol/mL) significantly inhibited the increase of cPLA2 activity after CsA exposure (4.64+/-1.52). The Western blotting of cPLA2 (110 kD) also showed similar results. Meanwhile there was no significant changes of sPLA2 activitiy which was markedly low. CONCLUSION: The apoptosis of renal tubular epithelial cell following CsA expsoure appears to be mediated by membrane phospholipid breakdown via sphingomyelinase and PLA2. The mechanism(s) mediating the protective effect of ceramide and PLA2 on CsA-induced apoptosis should be further elucidated.

The Study on the Mechanism of FK506 (Tacrolimus)-Induced Apoptosis in Renal Tubular Epithelial Cells / 대한이식학회지

PURPOSE: One of the major limiting factors in the use of FK506 (Tacrolimus) is nephrotoxicity, but the mechanisms of nephrotoxicity are not fully understood. In order to elucidate the pathogenesis of FK506 tubulotoxicity, we examined mechanisms of cellular injury induced by FK506 in cultured LLC-PK1 renal tubular cell line. METHODS: The 3H-thymidine uptake and flow cytometric analysis of apoptosis following FK506 treatment were evlauated. The changes of the Fas and Bcl-2 protein in FK506-induced renal tubular cell injury were also investigated by Western blotting. RESULTS: FK506 treatment significantly decreased 3H-thymidine uptake (M+/-S.D., 3591.6+/-274.3 cpm/well vs 2217.6+/-79.7 at 1 microgram/mL, p<0.05), in a dose dependent manner upto 50microgram/mL, indicating that DNA damage is a sensitive indicator of FK506- induced nephrotoxicity. The addition of FK506 (50 microgram/mL) for 96 hours also induced a significant increase in the percentage of cells displaying annexin-V binding from 3.9+/-2.2% in control cells to 34.9+/-8.5% in treated cells (P<0.05), indicating early apoptotic cellwith an increase of Bcl-2 protein levels up to 6.2 fold (mean) on Western blot analysis, and the dose-dependent further increase of Bcl-2 protein was observed at a dose above 5microgram/mL. death. This finding was supported by electrophoretic analysis of the DNA extracted from FK506-treatedcells, where a series of bands correspondingto integer multiples of 180 to 200 base pairs was visualized. The treatment with FK506 at a concentration higher than 1 microgram/mL for 96 hours was seen to cause a significant 3.7 (mean) fold elevation in the expression of the 45 kD Fas protein by Western blot analysis. The exposure to FK506 at dose of 5microgram/mL for 96 hours was also associated with an increase of Bcl-2 protein levels up to 6.2 fold (mean) on Western blot analysis, and the dose-dependent further increase of Bcl-2 protein was observed at a dose above 5 microgram/mL. CONCLUSION: FK506 is directly toxic to renal tubular cells with inhibiting DNA synthesis and inducing cell death in the form of apoptosis. The changes of Fas antigen system and Bcl-2 protein may play roles in mediating FK506-induced apoptosis of renal tubular cells.

Gentamicin Induced Apoptosis of Renal Tubular Epithelial (LLC-PK1) Cells

Nephrotoxicity is a major limiting factor in the use of aminoglycoside antibiotics, the mechanisms for which are still speculative. To clarify the mechanisms of renal tubular cell death induced by aminoglycosides, we examined the renal proximal tubule-like cell line, LLC-PK1, after inducing apoptosis through a chronic treatment with gentamicin (GM). Changes in the expression of the Fas were also investigated. On flow cytometric analysis, 5.7 +/- 3.3% of the control cells appeared in a region of decreased forward light scatter and increased side light scatter, where both indices represent the characteristics of apoptotic cell death. Compared to the control, treatment with 10 mM of GM for 15 days significantly increased the proportion of cells in the apoptotic region to 23.9 +/- 8.5%. This finding was supported by electrophoretic analysis of the DNA extracted from the GM-treated cells, where a series of bands corresponding to integer multiples of 180 to 200 base pairs was visualized. However, the 15-day GM treatment did not cause a significant elevation in the expression of the 45 kD Fas protein, the cell surface molecule that stimulates apoptosis, by Western blot analysis. In conclusion, long-term exposure to GM induces apoptosis of the renal tubular epithelial cells, and this process may contribute to some of the aminoglycoside nephrotoxicities. Further studies are needed on the mechanism(s) of apoptosis induced by GM.