Efecto De Cistanches Deserticola Extract On Injury Of Rabbit RTECs By H2O2

Abstracto: Rabbit renal tubular epithelial cells were cultured in vitro to study the effect of H2O2 on cell proliferation and Cistanche deserticola extract on the tubular epithelial cells injured by H2O2 ． Cell morphology was used to observe the effect of H2O2 on the proliferation of rabbit renal tubular epithelial cells by MTT method and the protective effect of 2,20 and 200 mg·L － 1 C． deserticola water extract and alcohol extract was studied by LDH release． The results showed that the number of morphological changes of cells were gradually increased． The number of attached cells were gradually reduced． The inhibition rate of cell proliferation and LDH release were increased along with increasing concentration of H2O2 ． High dose of C． deserticola extract could significantly decrease the inhibiting effect of ＲTECs proliferation by H2O2 ( P ＜ 0． 01) ． The water extraction and alcohol extract of C． deserticola could significantly decrease the LDH release( P ＜ 0． 01) ． H2O2 had some effects on the proliferation reaction of ＲTECs of rabbit,while C． deserticola extract has protective effect on ＲTECs injury by H2O2 ．

Clave palabras: Cistanches deserticola; renal tubular epitelial cell; cell damage prefacio

Cistanche deserticola, also known as desert ginseng is a dry, scaly, mesophyll stem of Cistanche deserticola Y.C. Ma or Cistanche tubulosa (Schenk) Wish in the family Orobaceae. It has the effects of tonificing kidney yang, benefiting essence and blood, moistening intestinos, and aliviing estreñimiento. Tradicionalmente usado para riñón yang deficiencia, esencia y sangre deficiencia, impotencia y infertilidad, cintura y rodilla dolor, debilidad de músculos y huesos, y intestinal sequedad y estreñimiento, it es el más frecuentemente usado tonificante riñón yang medicina en historia. 

Cistanche deserticola

H2O2, as a forma de reactivo oxígeno especie, has a fuerte efecto on inductor lípido peroxidación y puede reaccionar con casi cualquier celular componente, causante cadena lípido peroxidación, y leading a célula apoptosis y disfunción [2,3]. El experimento principalmente investiga el efecto de Cistanche deserticola extract on H2O2-induced proliferación inhibición y lesión de conejo renal tubular epitelial células and preliminarmente explora su protector efecto en renal tubular epitelial células.

1 Material y Métodos

1.1 Experimental drug

Cistanche deserticola ma was comprado de Sichuan Chuannan Pharmaceutical Co., Ltd. (lote número 151201).

Desierto vivir cistanche

1.2 Animales

Nuevo Zelanda blanco conejo, macho, 20 días viejo, comprado de el experimental base de de Guizhou Ganado Investigación Instituto, con animal licencia número SCXK (Guizhou) 2012-001.

1.3 Reactivos

DMEM/F-12 (1:1), Gbico Corporation; Fetal bovino serum, Zhejiang Tianhang Biotechnology Co., Ltd; Dimetil sulfóxido (DMSO), Beijing Dingguo Changsheng Biotechnology Co., Ltd; H2O2, Sichuan Jinshan Pharmaceutical Co., Ltd; Trypsin, Amresco Company; Thiazole blue (MTT), Genview; Nonradioactive cytotoxicity testing kit, Promega Corporation; Anhydrous ethanol, Tianjin Fuyu Fine Chemical Co., Ltd.

1.4 Instruments

CO2 incubadora, Tipo 3111, Termo Fisher Científico; Enzima marcador, 1510 tipo, Termo Fisher Científico; Ultra limpio workbench, SW-CJ-1FD, Suzhou Purificación Equipo Co., Ltd; Invertida fluorescencia microscopio, Ningbo Shunyu; Rotatorio evaporador, RE-52AA, Shanghai Yarong Bioquímico Instrumento Fábrica; Freeze dryer, LGJ-12, Beijing Songyuan Huaxing Tecnología Desarrollo Co., Ltd.

1.5 Método

1. 5.1  Preparación de Cistanche deserticola extracto

Take the dried medicinal herbs of Cistanche deserticola and grind them into small pieces. Accurately weigh 100 g of each powder and add 10 times the amount of distilled water and 75 percent ethanol to heat and reflux for extraction 3 times. The first time is 3 hours, and the second time is 1 5 hours, the third time for 1 hour, merge 3 filtrates, reduce pressure and concentrate to the extract, calculate the extract yield as 22 30 percent , ethanol extract 21 70 percent , volatile residual alcohol, a freeze-drying mechanism to obtain water and alcohol extracts from Cistanche deserticola, sealed, dried, and stored in a dark place for future use.

1. 5. 2 Celda Cultivo

Construct a renal tubular epitelial célula modelo usando el método descrito por Zhao Dan et al. [4], aislar and cultivo primario renal tubular epitelial células from 20-}day-old rabbits, and wait for the cells to grow to over 90 percent of the culture dish, and then digest them with trypsin. usando DMEM/F-12 completo cultivo medio conteniendo 1 por ciento fetal bovino suero at a tas de 1 dos × 104 células fueron inoculadas on a 96 pozo placa y cultivadas at 37 grado y 5 por ciento CO2.

1. 5. 3 Establecimiento de un conejo renal tubular epitelial célula lesión modelo inducido por 3 H2O2

Refer to 1 5. 2 Inoculate cells, and after 48 hours of stable cell culture, use H2O2 for damage. Set up an experimental group and a blank control group, with 4 repetitions in each group, repeated three times. Experimental group: Replace the complete culture medium with different concentrations of H2O2, with H2O2 concentrations of 550, 500, 450, 400, and 350, respectively μ Mol · L-1, blank control group: Replace the complete culture medium, continue to cultivate for 24 hours under the condition of 37 degree and 5 percent CO2, perform the microscopic examination, and collect pictures. The MTT [5] method was used to measure the proliferation of cells after the action of H2O2, and the absorbance value (OD value) was measured at 490 nm. Using a nonradioactive cytotoxicity detection kit to determine the changes in cell LDH release after H2O2 treatment, LDH release ( percent )=supernatant LDH amount/(supernatant LDH amount plus lysate LDH amount) × 100 percent . AO/EB fluorescence staining [6]: Take 100 respectively μ G/mL AO solution and 100 μ 100 g/mL EB solution each μ L. Mix well and add 10 to each well μ L. Immediately place it on an inverted phase contrast fluorescence microscope for observation, maintain the observation field of view unchanged within 5 minutes, and collect images under blue light at 450 ± 10 nm and green light at 532 ± 10 nm, respectively. Use a fluorescence collection system for fluorescence synthesis to obtain fluorescence staining images.

1. 5. 4 Efecto de 4 Cistanche extracto polvo on H2O2-induced damage to rabbit renal tubular epithelial cells

Cistanche polvo

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Acordando a 1 5. 2 Inocular células, y después 24 horas de estable célula cultivo, add 200 ul of completa cultivo medio conteniendo Cistanche deserticola extract a each well to pre-protect the cells for 24 hours. Set up an experimental group, a model group, and a blank control group, with three repetitions per group. Experimental grupo y modelo grupo: reemplazar con 400 μ Completo cultivo medio con mol · L-1 H2O2; Blanco control grupo: completo cultivo medio. Continuar a cultivar para 24 horas. Célula proliferación fue medido usando el MTT método, and cambios en célula LDH liberación fueron medidos using a no radiactivos citotoxicidad ensayo kit.

1.6 Estadística procesamiento

El datos resultados son expresados en x ± s using SPSS 19 0 estadístico software was used for statistical analysis. Single-factor analysis of variance was used for inter-group comparisons, and the LSD method was used for inter-group multiple comparisons, with P<0 05 indicating statistical significance.

2 Resultados y Análisis

2.1 Morfológico cambios de conejo renal tubular epitelial células inducido por H2O2

Después de el conejo renal tubular epitelial células fueron tratadas con diferentes concentraciones de H2O2 para 24 horas , la morfología de el conejo renal tubular epitelial células se observó bajo el microscopio, as se muestra en figura 1. con aumento el aumento de aumento de aumento de células el célula brillo disminución, y el tamaño disminución. La concentración de H2O2 alcanzado 550 μ Mol · L-1, axones desaparecer, célula membranas ruptura, cc contracción, celda huecos aumento, y huecos aparecen entre parches de células.

Fig. 1   Efecto de H2O2 concentración on morfología de conejo RTEC( 100 × )

2.2 El efecto de 2.2 H2O2 on la proliferación de conejo renal tubular epitelial células

Uso 350, 400, 450, 500, y 550 respectivamente μ Mol · L-1 H2O2 tratado conejo renal tubular epitelial células for 24 horas, and the absorbance was medd using the MTT method. los resultados mostrados que diferentes concentraciones de H2O2 tenía an inhibidor efecto sobre la proliferación de conejo renal tubular epitelial células, con an IC50 valor de 439 07 μ mol·L － 1, 500 μ El inhibidor efecto de H2O2 con mol · L-1 on célula proliferación puede alcanzar sobre 50 percent después 24 horas, so esta concentración fue seleccionada para subsiguiente investigación.

2.3 El efecto de 2.3 H2O2 on la liberación de LDH en conejo renal tubular epitelial células.

Después de 24 horas de H2O2 tratamiento en conejo renal tubular epitelial células , el OD valores de LDH reacciones en célula sobrenadante célula lisado fueron medidos usando no radiactivos citotoxicidad detección métodos, y la liberación de LDH de células fue calculado. Los resultados se muestran en Tabla 1. Diferentes concentraciones de H2O2 puede causa an aumento en LDH liberación de conejo renal tubular epitelial células, y el mayor el H2O2 concentración, el mayor el LDH liberación en a concentración-dependiente manera. H2O2 at a concentración de 400 mmol · L-1 causas cilla LDH liberación a exceder el blanco grupo por más que 50 por ciento , resultante en grave célula daño.

Tab. 1 Effects of H2O2 on LDH release from rabbit RTECs

<0 05; ＊＊ P ＜ 0. 01

2.4 AO/EB fluorescencia tinción resultados de conejo renal tubular epitelial células tratado con 2.4 H2O2

Después de el conejo renal tubular epitelial células fueron tratadas con H2O2 para 24 horas , las células fueron teñidas con AO/EB fluorescencia, as mostradas en figura 2. fue preliminarmente juzgado eso el mecanismo H2O2 en conejo renal tubular epitelial células fue principalmente a causa necrosis. Diferentes concentraciones de H2O2 puede causa variar grados de necrosis en conejo renal tubular epitelial células, con H2O2 concentraciones menos de 400 μ At mol · L-1, el nuclear cromatina encoge encoja en a círculo con verde color, que es principalmente juzgado as temprano apoptosis. Con el aumento de H2O2 concentración, it es más que 450 μ At mol · L-1, the cells mosted an orange-red nonround bead shape, and the number of necrótica cells increased in a concentration-dependent way.

2.5 Efecto de 2.5 Cistanche deserticola on H2O2-induced proliferación de conejo renal tubular epitelial células

Experimental Estudio en Cistanche deserticola

El supervivencia tasa de conejo renal tubular epitelial células bajo diferentes concentraciones (2, 20, 200 mg! L-1) de agua y alcohol extractos de Cistanche deserticola mostródono no significativo diferencia comparado a células cultivadas bajo normales condiciones sin intervención. As mostrado en Figura 3, comparado con el modelo grupo, el alta-concentración grupos de alcohol y agua extractos de Cistanche deserticola significativamente reducido el inhibidor efecto de H2O2 on la proliferación de conejo renal tubular epitelial células (P<0 01).

Fig. 2 AO/EB tinción de conejo RTEC tratado por diferentes concentraciones de H2O2 ( 100 × )

Nota: Comparado con el modelo grupo, * P<0 05,＊＊P ＜ 0. 01

Fig. 3  Efecto de C． deserticola on la inhibición de H2O2 － inducida conejo RTEC proliferación

2.6  Efecto de 2.6 Cistanche deserticola on H2O2 inducida daño a conejo renal tubular epitelial células

Los resultados de el impacto en el liberación de LDH en células son mostrados en Figura 4. El alto, medio, y bajo concentración grupos de etanol extracto y agua extracto de Cistanche deserticola resulted en significativamente menor LDH liberación en conejo renal tubular epitelial células después H2O2 tratamiento than in el modelo grupo (P<0 01), the results are concentration-dependent, with higher concentrations leading to lower cell LDH release.

Nota: Comparado con el modelo grupo, * P<0 05,＊＊P ＜ 0. 01

Fig. 4   Efecto of C． deserticola on H2O2 － inducida LDH liberación de conejo RTECs

3 Discusión

Cistanche deserticola, known as "desert ginseng", has extremely high medicinal value and is a famous Chinese tonic medicine. The "Shennong Materia Medica Classic" [7] describes top-grade medicine as: "Shangyao... mainly nourishes the life to respond to the heavens, non-toxic, and should be taken frequently without harming people..." Cistanche deserticola is listed as a top-grade medicine, with a sweet taste and a mild temperature. The "List of Famous Doctors" [8] points out that Cistanche deserticola is "sour, salty, and non-toxic". Afterward, the records of the nature, taste, and meridian of Cistanche deserticola in various dynasties of Chinese herbal medicine are consistent. Its nature is slightly warm, and its taste is sweet, sour, and salty, which belongs to the kidney and large intestine meridians. Traditionally used for kidney yang deficiency, waist and knee soreness, and muscle and bone weakness, its pharmacological effects and mechanisms have always been a research focus. Modern pharmacological studies have shown that Cistanche deserticola can resist aging, improve learning and memory abilities, and regulate immune functions. It is widely used in clinical prescriptions of traditional Chinese medicine, traditional Chinese patent medicines, and simple preparations and health care products.

Cistanche suplemento cerca yo

Oxidative stress is the key inducement of some major diseases, which is due to the imbalance between the formation and elimination of reactive oxygen species in the body, resulting in a certain degree of oxidative damage to histiocytes. Therefore, in-depth research on the mechanism of the stress response can find a reasonable way to effectively prevent, improve or even eliminate irritability diseases. Oxidative stress can lead to various cell and protein peroxidation in the body [9], and H2O2 is chosen as one of the exogenous substances for damage in research. It has a strong effect on inducing lipid peroxidation, causing chain lipid peroxidation reactions, and leading to cell apoptosis and dysfunction. At present, the research on the nephrotoxicity of this exogenous substance and its antagonistic effect mainly relies on establishing animal models, and there are relatively few literature reports on establishing in vitro models for related research. Compared with animal models of nephrotoxicity, the in vitro nephrotoxic cell model has advantages such as a clear target of the action, a short medication cycle, and low dosage.

Chino hierba cistanche

Study the in vitro nephrotoxic cell model induced by exogenous substances through changes in cell morphology, inhibitory effects of exogenous substances on cell proliferation, and changes in cell LDH release. Different concentrations of H2 O2 have a significant impact on the morphological changes of cells. As the concentration of exogenous substances increases, it is found that the number of cell deformations increases. MTT assay is a commonly used method for detecting the number of living cells. In experiments where different concentrations of Cistanche deserticola protect rabbit renal tubular epithelial cells from H2O2 damage, different concentrations of Cistanche deserticola significantly reduced the inhibitory rate of H2O2 on rabbit renal tubular epithelial cells, and it is concentration dependent. When the cell membrane is damaged, intracellular LDH will be released into the culture medium, The LDH content in the cell culture medium is an important indicator for detecting cell death. This study found that the LDH level in the cell culture medium of the model group with H2O2-induced damage to rabbit renal tubular epithelial cells significantly increased, while the LDH level in the experimental group treated with different concentrations of Cistanche deserticola significantly decreased in a concentration-dependent manner. El investigación resultados sugieren que establecer un modelo de H2O2-induced daño a conejo renal tubular epitelial células y preliminarmente explorando el protector efecto de Cistanche deserticola extract on renal tubular epitelial células, not only lays the foundation for research on cell damage caused by oxidative stress but also proporcions basic data for the development and utilization of Cistanche deserticola.

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