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Effets de Polydatin sur l'activit¨¦ de GPT dans le milieu de culture, Polydatin s

From: micheal derrida
Category: ÉÌÒµÐÅÏ¢
Date: 9/15/2003
Time: 9:25:50 PM
Remote Name: 218.95.200.222

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Effets de Polydatin sur l'activit¨¦ de GPT dans le milieu de culture, Polydatin s uperbe comme m¨¦decine d'herbe pour des affections h¨¦patiques chroniques de grou pe de MDidea?Polydatin 90%95%98%99%HPLC(Origine:Polygonum cusPldatum Sleb.et Zucc .)

Amanuensis&Calligraphe de trace de symbole: Michael Derrida tous les int¨¦resser, transfert juste un email simplement et nous seraient heure ux davantage que l'aide d'ust de j.

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Polydatin 90%95%98%99%HPLC(Origine:Polygonum cusPldatum Sleb.et Zucc.) ¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó¡ó

Donn¨¦es De base:

[Nomm¨¦ Chimique]:3,4¡ä,5trihydroxystibene-3-¦Â-mono-D-glucoside [nom commun]:Piceid Normal [Formule mol¨¦culaire et poids mol¨¦culaire]:C20H22O8;M.W.:390 [nom Botanique]:Polygonum cusPldatum Sleb.et Zucc. [Synoms Botanique]:le cuspidaturn de Cuspldatum P.E.);Polygonurn de cuspidatur n(Polygonum d'urne de polygone d'mauvaise herbe, connu sous le nom de racines de Hu-Chang et de Ko-jo-kon;the de cuspidaturn Sieb de Polygonurn. et pouce de multi florurn de Zucc.;Polygonurn. Fonction De base: les aides stimulent les nerfs dans tout le corps, effet est en particulier exp ¨¦riment¨¦es dans le secteur g¨¦nital du corps, augmentent la production sexuelle d'hormone accrue par ex¨¦cution sexuelle.

Effets de Polydatin sur l'activit¨¦ de GPT dans le milieu de culture, Polydatin superbe comme m¨¦decine d'herbe pour des affections h¨¦patiques chroniques de gr oupe de MDidea?

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Effets protecteurs de polydatin contre des dommages de CCl4-induced ¨¤ principal ementcultured rat hepatocytes

Abstract AIM Pour ¨¦tudier les effets protecteurs du polydatin (palladium) a dommages de gainst aux hepatocytes principalement cultiv¨¦s de rat induits par CCl 4.

METHODS Des hepatocytes de rat ont ¨¦t¨¦ s¨¦par¨¦s in vivo par des m¨¦thodes d' infusion de foie et de milieu cultiv¨¦ (7.5¡Á105 cells/mL). 2 mL or 0.2mL was add ed into 24-well or 96-well plates respectively. Twenty-four hours a fter cell pre culture, PD at concentrations of 10-7mol/L-10-4mol/L was added into each plate. A t the same time injury to hepatocytes was induced by adding 10mmol/L-CCl4. Then, 0.1mL or 1mL-culture solut ion was removed from the 96-well or 24-well plates at 6h, 12h, 2 4h and 48h after CCl14 intoxication respectively for the determi natio n of GPT, GSH and MDA. At 48h, the survivability of rat hepatocytes was assayed b y the MTT colormetric method.

RESULTS After CCl4 challenge, the release of GPT and the form ation of MDA in r at hepatocytes markedly increased and maintained at a high leve l in 48h, whereas PD with different concentrations could markedly inhibit this elevation with 10-5 mol/L PD having the strongest effects and inhibi ting rate was over 50%. PD could also improve the decreased content of GSH cause d by CCl4 in accordance with the doses used. CCl4 evidently decreased the he patocyte survivability from 91.0%¡À7 .9% to 35.4%¡À3.8%. On the other hand, PD at 10-7mol/L-10-4mol/L could reverse th is change and improve t he cell survival rates to 56.1%¡À5.2%, 65.8%¡À5.0%, 88.7% ¡À6.8% and 75.2% ¡À7.3%, respectively.

CONCLUSION PD at 10-7mol/L-10-4mol/L could protect primarily cultured rat hepa tocytes against CCl4 induced injury.

INTRODUCTION Polygonum cuspidatum-Sieb. et Zucc. (Polygonaceae) is a traditional Chinese herb al drug, with bitter taste and cold nature. It mainly acts upon the liver, gallbl adder and lung meridians. It is well known that -P. cuspidatum-has various activi ties such as promoting blood circulation, relieving swelling and pain, eliminatin g phlegm, alleviating cough, clearing away heat, and removing dampness and toxin. The drug has been widely used for cardiovascular and liver diseases. Its active compounds mainly consist of free anthraquinones which include emodin , physcion a nd chrysophanol. Another important compound is resveratrol£Û1£Ý. Polydatin (PD), 3,4¡ä,5ª²trihydroxystibene-3-¦Â-mono-D-gluc osid e, also nam ed piceid, is the glycoside of resveratrol£Û1£Ý. Some previous studies demonstrat ed that PD could lower the level of blood lipid, inhibit the p latelet aggregatio n, dilate blood vessels, protect cardiocytes, reduce cerebral ischemic damage and inhibit lipid peroxidation£Û2-6£Ý. However, the effect s of PD on hepatocytes an d its mechanisms have not been reported up to date. In this paper we report the d etails of protective effects of polydatin against inju ry to primarily cultured r at hepatocytes induced by CCl4.

MATERIALS AND METHODS

Materials Collagenase (type ¢ô), 3-£Û4,5-dimethylthiazol-2-yl£Ý-2,5-diphenyltetrazol ium b romide (MTT), dexamethasone, N-2-hydroxyethyl-piperazine-Nª«¡ä-2 ¡äetha ne sulfon ic acid (HEPES), insulin, penicillin and streptomycin were purchased fr om Sigma Chemical Corp (St. Louis, USA). RPMI 1640 was a product of Gibco Life T echnologi es INC (Grand Island, NY). Fetal calf serum was obtained from Institute of Hemop athy, Chinese Academy of Medical Sciences (Tianjin). PD (Purity£¾90%), which was isolated from the root and rhizome of P. cuspidatum£Û7£Ý, pr ovided by the Depart ment of Chemistry, the First Military Medical University.

Animals Wistar rats, male, 6 weeks old, weighing 160g-180g, were used for he patocyte is olation. They were provided by Laboratory Animal Center, Guangzhou Un iversity of TCM.

Isolation and culture of rat hepatocytes Rats were anesthetized with sodium pentobarbital (50mg/kg, i.p.). Then t he live r parenchymal cells of rat were isolated by the collagenase perfusion met hod fol lowing the procedure of Seglen and Koji£Û8,9£Ý. Simply, the portal vein of rat li ver was exposed and cannulated with a teflon catheter. The liver w as perfused wi th Ca2+ free solution containing NaCl 142, KCl 6-7, HEPES 10, NaOH 5.5 (mmol/L), pH 7.4, at 37¡æ, with a flow rate of 40mL/min . Twelve minutes later, recirculati on started with collagenase solution composed of NaCl 67, KCl 6-7, CaCl2¡¤2H2O5, HEPES 100, NaOH 66, collagenase 0.2g/L, pH 7.6. Isolated cells were cultured in RPMI 1640 containing 100mL/L-fetal calf serum, 10mmol/L-HEPES, 100kU/L- penicilli n and s treptomycin, 10mmol/L insulin and 10mmol/L- dexamethasone. The content of hepatocytes was adjusted to 7.5¡Á108 cells/L with the above med ium. Cultured me dium 2mL and 0.2mL were added into 24-well and 96-well plates respectively. The c ells were incubated for 4h at 37¡æ under 50mL/L CO2 in air. Non-adherent hepatocy tes were eliminated by replac ing the medium, and adherent hepatocytes continued to be incubated, and the medium was changed every 24h.

CCl4-induced hepatocytes injury After pre-culture for 24h, the hepatocytes were exposed to fresh medium c ontain ing 10mmol/L-CCl4 and various concentrations of PD. At 6, 12, 24 and 48h after C Cl4 intoxication, 0.1mL and 1mL culture so lution were removed from 96ª²well and 24ª²well plates respectively for determina tion.

Measurement of glutamic pyruvic transaminase (GPT) The kits of GPT analysis, provided by the Shanghai Institute of Biological Produ cts of Ministry of Health, were used to measure the activity of GPT in 0.1mL- cul ture medium.

Determination of reduced glutathione (GSH) and malondialdehyde (MDA) Utilizing the kits of GSH analysis and the kits of MDA analysis, all pu rchased from Nanjing Jiancheng Bio-engineering Institute, the content of GSH in 1mL cult ure medium and the level of MDA in 0.1mL culture medium we re measured.

Cell survivability assay The survivability of rat hepatocytes was assayed by the MTT colormetric method £Û 10£Ý. At 48h after CCl4 challenge, 20¦ÌL/well- MTT stock solution (5g/L) was add ed into each well of 96-well plates. The cells were continuously incubated for an other 4h before 0.1mL/well dimethyl sulfoxid e was added to all wells and mixed t horoughly to dissolve the brownª²black cryst als. The plates were read on micropl ate reader, using a test wavelength of 570nm with a reference wavelength of 655nm .

Statistical analysis The results were expressed as x-¡Às and significant difference was a ssessed by Student¡äs t test.

RESULTS

Effects of PD on GPT activity in culture medium The concentration of GPT in culture medium significantly increased after CCl4 ch allenge, and maintained at a high level in 8h (Table 1). Fur thermore, a progress ively elevated trend existed with time-dependence. PD could significantly inhibi t the level of GPT in accordance with the doses used. Espec ially, PD -10¦Ìmol/L- had the strongest effects and the inhibiting rate wa s over 50%. Table 1 Effect of PD on GPT activity in culture medium (x-¡Às,n=8)

Group c/(mol/L) GPT(U) 6h 12h 24h 48h Normal 13.5¡À2.5b 13.8¡À3.1b 13.7¡À5.6b 14.1¡À3.3b Control 72.3¡À14.1 79.7¡À10.3 85.4¡À9.2 88.3¡À19.6 PD 10-7 60.3¡À17.1a 62.0¡À15.6a 68.8¡À17.5a 71.4¡À20.5a PD 10-6 55.0¡À10.3a 58.3¡À16.7a 64.1¡À13.6a 69.1¡À19.2a PD 10-5 30.6¡À10.6b 38.3¡À5.5b 42.5¡À7.0b 45.0¡À7.6b PD 10-4 42.1¡À7.8a 47.5¡À9.8a 56.8¡À11.3a 59.2¡À10.7a

aP£¼0.05, bP£¼0.01, vs CCl4ª²treated control group.

Effects of PD on GSH content in culture medium (Table 2). The content of GSH in culture medium decreased obviously as compared with that i n normal hepatocytes after 6h incubation with CCl4 (Table 2). On the other hand, PD of various concentrations could improve GSH in a dose dependence manner, and 1 0¦Ìmol/Lªª PD showed a most signifi cant activity. Table 2 Effects of PD on GSH content in culture supernatant (x-¡Às, n=8)

Group c/(mol/L) GSH (ng/L) after CCl4 challenge 6h 12h 24h 48h Normal 9.8¡À0.8b 10.1¡À0.8b 10.4¡À0.7b 10.6¡À1.2b Control 4.2¡À0.6 4.1¡À0.7 4.1¡À0.3 3.8¡À0.6 PD 10-7 5.0¡À0.3 5.4¡À0.5 5.6¡À0.9 6.1¡À1.0a PD 10-6 5.3¡À0.8 5.6¡À0.9 6.4¡À0.6a 6.8¡À1.1a PD 10-5 8.4¡À1.2b 5.9¡À1.3a 7.7¡À0.8a 9.0¡À1.2b PD 10-4 6.7¡À0.4a 6.1¡À1.0a 6.8¡À0.7a 7.6¡À0.9a

aP£¼0.05, bP£¼0.01, vs CCl4 treated control group.

Effects of PD on MDA formation in rat hepatocytes CCl4 challenge obviously elevated the MDA formation in rat hepatocytes, with a m arked rise in timeª²dependence manner, whereas MDA formation of rat hepatocyte s decreased significantly at various concentrations of PD as compared with that in CCl4 control group, and it reached minimum value at 10-5-mol/L and s lightly elev ated when PD concentration was up to 10-4-mol/L (Table 3). Table 3 Effects of PD on MDA formation in rat hepatocytes (x-¡Às, n=8)

Group c/(mol/L) GSH (ng/L) after CCl4 challenge 6h 12h 24h 48h Normal 4.0¡À0.4b 4.5¡À0.6b 4.8¡À0.4b 4.6¡À0.7b Control 15.5¡À1.8 16.0¡À2.7 17.5¡À2.1 19.0¡À2.4 PD 10-7 13.1¡À2.0 13.8¡À3.3 13.0¡À4.3a 14.5¡À1.8a PD 10-6 11.4¡À1.7a 12.0¡À1.8a 12.1¡À3.1a 12.5¡À2.0a PD 10-5 6.5¡À1.2b 6.7¡À1.2b 7.5¡À2.3b 8.2¡À2.7b PD 10-4 8.7¡À3.5b 8.9¡À2.8b 9.8¡À2.6b 10.3¡À3.0b

aP£¼0.05, bP£¼0.01, vs CCl4 treated control group.

Effects of PD on cell survivability in primary culture rat hepatocytes The results of MTT assay showed that normal hepatocytes had high level of cell v iability (91.0%¡À7.9%) and CCl4 induced marked decrease of hepatoc ytes survivabi lity (35.4%¡À3.8%, P£¼0.01 vs normal group), whereas t he level of cell survivabi lity could be significantly enhanced by PD at the conc entrations of 10-7mol/L-10 -4-mol/L to 56.1%¡À5.2% (P£¼0.05 , vs CCl4-treated control group), 65.8%¡À5.0% (P £¼0.05), 88.7%¡À6 .8%(P£¼0.001) and 75.2%¡À7.3% (P£¼0.01) respectively. It reache d a maximum value at 10-5mol/L and slightly declined when the concentration of PD was up to 10-4-mol/L (Figure 1).ª¤ Figure 1 Effects of PD on cell survivability in prim ary culture rat hepatocytes . 1. CCl4-treated control group; 2. normal hepatocytes; 3. PD 10-7-mol/L; 4. PD 10 -6-mol/L; 5. PD 10-5-mol/L; 6. PD 10-4-mol/L.

DISCUSSION P. cuspidatum has been used to treat some chronic liver diseases such as hepatit is and hepatocirrhosis. We have been trying to search for hepatoprotective compou nds of P. cuspidatum. Our previous in vitro studies showed that emodin, another a ctive compound, had a hepatoprotective effect£Û11£Ý. The present in vitro study a lso indicated that PD had a protective effect against CCl4 induced injury to prim arily cultured rat hepatocytes. Since the extractio n and isolation of PD are rel atively simple and have a high content of 1.23% in the root of ª«P. cuspidatum£Û 7£Ý, we may take these advantages to furth er study its mechanisms of hepatoprote ctive effect and develop a new drug from i t. CCl4 is a wellª²known example of a chemical that produces free radical-med iated liver injury. It generates CCl4 by the activation of liver cytochrome P-450 , initiating lipid peroxidation of bioª²membranes£Û12£Ý. In the present e xperime nt, it was found that CCl4 induced both the increase of GPT in supernat ant and t he elevation of MDA in rat hepatocytes. However, administration of 10-7mol/L-10-4 mol/L PD could partly reduce GPT and MDA. Therefor e, there may be two possible m echanisms contributing to the hepatoprotective act ions of PD. One is that PD inh ibits further production of lipid peroxidation in rat hepatocytes, and the other is that it inhibits the destructive action of lip id peroxidation on liver cells. GSH is an important endogenous antiª²oxidant substance. The decrease of GSH c ontent may be due to increased GSH consumption as it participates in the detoxifi ca tion system for the metabolism of CCl4, and results in an enhanced susceptibil ity of hepatocytes to CCl4 toxicity£Û13£Ý. Our results showed that CCl4 obviousl y decreased GSH content in the hepatocytes, but PD could partly revers e it. This suggested that the nature of PD protectingª²SH compounds (such as GSH ) from CCl 4 injury may be the third mechanism of its hepatoprotection.It is interesting tha t PD of 10-5-mol/L was more effective than that of 10-4-mol/L, at the same time, the hepatoprotective action of PD was i n dose dependence at concentrations of 1 0-7-mol/L-10-5-mol/L. Its mechanisms of action need to be further studied.

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Amanuensis&Calligraphe de trace de symbole: Michael Derrida

==================================================== Amanuensis&Calligraphe de trace de symbole: Michael Derrida

tous les int¨¦resser, transfert juste un email simplement et nous seraient heure ux davantage que l'aide d'ust de j.

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