Zhao-Sheng Huang, Zong-Wei Wang, Ming-Ping Liu, Shi-Qing Zhong, Qiao Mei Li and Xiang-Lu Rong,  University of Traditional Chinese Medicine (TCM), College of Pharmacy, Guangzhou 510407, China
Zhao-Sheng Huang, male, born on 1953-09-02 in Jieyang City, Guangdong Province, graduated from Guangzhou University of TCM as a postgraduate in 1984, master of TCM pharmacy, now associate professor and director of the college, ha ving more than 20 papers and 6 books published.
*Supported by the Bureau of TCM Administration of Guangdong Provi nce, No.96033.
Correspondence to: Zhao-Sheng Huang, College of Pharmacy, Guang zhou University of TCM, 12 Jichang Road, Guangzhou 510407, Guangdong Province, China
Telephone: +86-20-86591233 Ext. 2425
Received: 1998-03-08

Subject headings: polydatin;injury,hepatocyte;CC1₄

Huang ZS, Wang ZW, Liu MP, Zhong SQ, Li QM, Rong XL. Protective effects of polydatin against CCl4-induced injury to primarily cultured rat hepatocytes. World J Gastroentero, 1999;5(1):41-44

Abstract
AIM: To investigate the protective effects of polydatin (PD) a gainst injury to primarily cultured rat hepatocytes induced by CCl₄.

METHODS: Rat hepatocytes were separated by methods of liver infusion in vivo and cultured medium (7.5×10⁵ cells/mL). Two mL or 0.2mL was added into 24-well or 96-well plates respectively. Twenty-four hours a fter cell preculture, PD at concentrations of 10^-7mol/L-10^-4mol/L was added into each plate. At 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 nation of GPT, GSH and MDA. At 48h, the survivability of rat hepatocytes was assayed by the MTT colormetric method.

RESULTS: After CCl₄ challenge, the release of GPT and the form ation of MDA in rat hepatocytes markedly increased and maintained at a high leve l in 48h, whereas PD with different concentrations could markedly inhibit this elevation with 10^-5mol/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 hepatocyte survivability from 91.0%±7.9% to 35.4%±3.8%. On the other hand, PD at 10^-7mol/L-10^-4mol/L could reverse this change and improve t he cell survival rates to 56.1%±5.2%, 65.8%±5.0%, 88.7%±6.8% and 75.2%±7.3%, respectively.

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

INTRODUCTION
Polygonum cuspidatum-Sieb. et Zucc. (Polygonaceae) is a traditional Chinese herbal drug, with bitter taste and cold nature. It mainly acts upon the liver, gallbladder and lung meridians. It is well known that -P. cuspidatum-has various activities such as promoting blood circulation, relieving swelling and pain, eliminating 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 and chrysophanol. Another important compound is resveratrol^［1］.
      Polydatin (PD), 3,4′,5 trihydroxystibene-3-β-mono-D-gluc osid e, also named piceid, is the glycoside of resveratrol^［1］. Some previous studies demonstrated that PD could lower the level of blood lipid, inhibit the p latelet aggregation, dilate blood vessels, protect cardiocytes, reduce cerebral ischemic damage and inhibit lipid peroxidation^［2-6］. However, the effects of PD on hepatocytes and its mechanisms have not been reported up to date. In this paper we report the details of protective effects of polydatin against inju ry to primarily cultured rat hepatocytes induced by CCl₄.

MATERIALS AND METHODS
Materials
Collagenase (type Ⅳ), 3-［4,5-dimethylthiazol-2-yl］-2,5-diphenyltetrazol ium bromide (MTT), dexamethasone, N-2-hydroxyethyl-piperazine-N ′-2 ′etha ne sulfonic 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 echnologies INC (Grand Island, NY). Fetal calf serum was obtained from Institute of Hemopathy, Chinese Academy of Medical Sciences (Tianjin). PD (Purity＞90%), which was isolated from the root and rhizome of P. cuspidatum^［7］, pr ovided by the Department of Chemistry, the First Military Medical University.

Animals
Wistar rats, male, 6 weeks old, weighing 160g-180g, were used for he patocyte isolation. 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 liver parenchymal cells of rat were isolated by the collagenase perfusion met hod following the procedure of Seglen and Koji^［8,9］. Simply, the portal vein of rat liver was exposed and cannulated with a teflon catheter. The liver w as perfused with Ca²⁺ free solution containing NaCl 142, KCl 6-7, HEPES 10, NaOH 5.5 (mmol/L), pH 7.4, at 37℃, with a flow rate of 40mL/min . Twelve minutes later, recirculation 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- penicillin and streptomycin, 10mmol/L insulin and 10mmol/L- dexamethasone. The content of hepatocytes was adjusted to 7.5×10⁸ cells/L with the above med ium. Cultured medium ²mL and 0.2mL were added into 24-well and 96-well plates respectively. The cells were incubated for 4h at 37℃ under 50mL/L CO₂ in air. Non-adherent hepatocytes 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 ontaining 10mmol/L-CCl₄ and various concentrations of PD. At 6, 12, 24 and 48h after CCl₄ intoxication, 0.1mL and 1mL culture so lution were removed from 96 well and 24 well plates respectively for determina tion.

Measurement of glutamic pyruvic transaminase (GPT)
The kits of GPT analysis, provided by the Shanghai Institute of Biological Products of Ministry of Health, were used to measure the activity of GPT in 0.1mL- culture 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 culture 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^［10］. At 48h after CCl₄ challenge, 20μL/well- MTT stock solution (5g/L) was added into each well of 96-well plates. The cells were continuously incubated for another 4h before 0.1mL/well dimethyl sulfoxid e was added to all wells and mixed thoroughly to dissolve the brown black cryst als. The plates were read on microplate reader, using a test wavelength of 570nm with a reference wavelength of 655nm.

Statistical analysis
The results were expressed as mean±SD and significant difference was a ssessed by Student′s t test.

RESULTS
Effects of PD on GPT activity in culture medium
The concentration of GPT in culture medium significantly increased after CCl₄ challenge, and maintained at a high level in 8h (Table 1). Fur thermore, a progressively elevated trend existed with time-dependence. PD could significantly inhibit the level of GPT in accordance with the doses used. Espec ially, PD -10μmol/L- had the strongest effects and the inhibiting rate wa s over 50%.

Table 1 Effect of PD on GPT activity in culture medium  (mean±SD, n=8)

^aP＜0.05, ^bP＜0.01, vs CCl₄ treated 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 in 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 10μmol/L PD showed a most signifi cant activity.

Table 2 Effects of PD on GSH content in culture supernatant  (mean±SD, n=8)

^aP＜0.05, ^bP＜0.01, 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 marked rise in time dependence 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 elevated when PD concentration was up to 10^-4-mol/L (Table 3).

Table 3 Effects of PD on MDA formation in rat hepatocytes  (mean±SD, n=8)

^aP＜0.05, ^bP＜0.01, 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 viability (91.0%±7.9%) and CCl₄ induced marked decrease of hepatoc ytes survivability (35.4%±3.8%, P＜0.01 vs normal group), whereas t he level of cell survivability could be significantly enhanced by PD at the conc entrations of 10^-7mol/L-10^-4-mol/L to 56.1%±5.2% (P＜0.05 , vs CCl₄-treated control group), 65.8%±5.0% (P＜0.05), 88.7%±6.8%(P＜0.001) and 75.2%±7.3% (P＜0.01) respectively. It reached a maximum value at 10^-5mol/L and slightly declined when the concentration of PD was up to 10_-4-mol/L (Figure 1).

Figure 1(PDF) 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 hepatitis and hepatocirrhosis. We have been trying to search for hepatoprotective compounds of P. cuspidatum. Our previous in vitro studies showed that emodin, another active compound, had a hepatoprotective effect^［11］. The present in vitro study also indicated that PD had a protective effect against CCl₄ induced injury to primarily cultured rat hepatocytes. Since the extractio n and isolation of PD are relatively simple and have a high content of 1.23% in the root of P. cuspidatum^［7］, we may take these advantages to furth er study its mechanisms of hepatoprotective effect and develop a new drug fromit. CCl₄ is a well known example of a chemical that produces free radical-mediated liver injury. It generates CCl₄ by the activation of liver cytochrome P-450, initiating lipid peroxidation of bio membranes^［12］. In the present e xperiment, it was found that CCl₄ induced both the increase of GPT in supernat ant and the elevation of MDA in rat hepatocytes. However, administration of 10^-7mol/L-10^-4mol/L PD could partly reduce GPT and MDA. Therefor e, there may be two possible mechanisms contributing to the hepatoprotective act ions of PD. One is that PD inhibits 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 anti oxidant substance. The decrease of GSH content may be due to increased GSH consumption as it participates in the detoxifica tion system for the metabolism of CCl₄, and results in an enhanced susceptibil ity of hepatocytes to CCl₄ toxicity^［13］. Our results showed that CCl₄ obviously decreased GSH content in the hepatocytes, but PD could partly revers e it. This suggested that the nature of PD protecting SH compounds (such as GSH ) from CCl₄ injury may be the third mechanism of its hepatoprotection.It is interesting that 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 10^-7-mol/L-10^-5-mol/L. Its mechanisms of action need to be further studied.

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