Effects of TJN-101, a lignan compound isolated from Schisandra fruits, on liver fibrosis and on liver regeneration after partial hepatectomy in rats with chronic liver injury induced by CCl4

TJN-101 ((+)-(6S,7S,R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy -6,7-dimethyl-10,11-methylenedioxy-6-dibenzo[a,c]cyclooctenol) is one of the lignan compounds isolated from Schisandra fruits. 1) Effect of TJN-101 on liver fibrosis was investigated in rats which were injected with CCl4 (1 ml/kg) subcutaneously twice a week for 12 weeks. TJN-101 was given orally at the dose of 10 or 30 mg/kg/day for 6 or 3 weeks beginning on the 6th or 9th week after the start of CCl4-intoxication, respectively. The elevations of serum transaminase activities and the increase of liver 4-hydroxyproline content were observed depending on the period of CCl4-intoxication. These changes were marked on the 9th and 12th weeks after. In the histopathological study, the degenerative fatty change on the 6th week after and the formation of pseudolobule caused by fibrosis proliferation on the 9th or 12th week after were mainly observed. When rats were treated with TJN-101, the abnormalities in biochemical parameters and the fibrosis proliferation caused by CCl4-intoxication were improved. 2) Chronic liver injury was induced by the treatment with CCl4 (1 ml/kg) subcutaneously twice a week for 10 weeks to investigate the effect of TJN-101 on liver regeneration after partial hepatectomy. TJN-101, which was given orally at the dose of 10, 30 or 100 mg/kg/day for 6 days from the 1st day after partial hepatectomy, dose-dependently increased the liver regeneration rate and improved the serum BSP retention rate. These results suggest that TJN-101 suppresses the fibrosis proliferation and accelerates both the liver regeneration and the recovery of liver function after partial hepatectomy in chronic liver injury.     

Effect of Gomisin A (TJN-101) on liver regeneration.

We studied the effect of TJN-101, a lignan component of Schisandra fruits (Schisandrae fructus), on liver regeneration after partial hepatectomy. TJN-101 was given orally to male Wistar rats 30 min before partial hepatectomy. The mitotic index and the level of DNA synthesis increased after partial hepatectomy and their increase was significantly enhanced by TJN-101. Ornithine decarboxylase (ODC) activity increased in the early stages of liver regeneration and it was also significantly enhanced by TJN-101. Besides, TJN-101 enhanced the increase in hepatic putrescine. These results suggest that TJN-101 stimulates liver regeneration after partial hepatectomy by enhancing ODC activity, which is an important biochemical event in the early stages of liver regeneration.     

Effect of gomisin A (TJN-101), a lignan compound isolated from Schisandra fruits, on liver function in rats

TJN-101 [+)-(6S, 7S, R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy- 6,7-dimethyl-10,11-methylenedioxy-6-dibenzo [a, c] cyclooctenol) is one of the lignan compounds isolated from Schisandra fruits. When TJN-101 was administered orally at the doses of 3-100 mg/kg/day for 4 days, bile secretion, hepatic excretion of dye or hepatic hemodynamics 24 hr after the last dose was investigated in comparison with the phenobarbital (100 mg/kg/day)-treated group. Bile flow was dose-dependently increased; in contrast, biliary concentration of bile acids was decreased in TJN-101 (30 and 100 mg/kg/day)-treated groups. Similar changes were also observed in the phenobarbital-treated group. These results suggested that the enhancement of bile secretion caused by TJN-101 or phenobarbital was due to an increase of a bile acid-independent fraction. In the bromosulfophthalein (BSP) clearance test for liver function, both TJN-101 (30 and 100 mg/kg/day) and phenobarbital accelerated the disappearance from the blood and biliary excretion of BSP. Hepatic hemodynamics was examined by the hydrogen clearance method and measurement of liver wet and dry weight. Liver blood flow tended to increase in the TJN-101 (10-100 mg/kg/day) or phenobarbital-treated group. On the other hand, TJN-101 (3-100 mg/kg/day) or phenobarbital hardly altered the water content of the liver. These results suggested that the liver enlargement caused by both compounds was not accompanied with hepatic edema and that the enhancement of bile secretion or hepatic excretion of BSP might be related to an increase of liver blood flow.     

The effect of hepatic stimulator substance (HSS) on liver regeneration arrest induced by 5-HT2 receptor blockade

BACKGROUND: The restorative effect of hepatic stimulator substance (HSS) against hepatic regeneration arrest induced by 5-HT2 receptor blockade was investigated. MATERIALS AND METHODS: Male Wistar rats were subjected to 60-70% partial hepatectomy and to 5-HT2 receptor blockade at 16 h after partial hepatectomy by ketanserin administration (6 mg/kg bodyweight intraperitoneally; group I). HSS at the dose of 100 mg protein/kg bodyweight was administered at 10 or 17 h after partial hepatectomy in ketanserin-treated rats (groups II and III). The mitotic index in hematoxylin-eosin-stained liver sections, immunochemical detection of PCNA and Ki 67 nuclear antigens and the rate of [3H]-thymidine incorporation into hepatic DNA were used as indices of liver regeneration. RESULTS: Liver regeneration, as evaluated by [3H]-thymidine incorporation into hepatic DNA, mitotic index, PCNA and Ki67 nuclear antigens, peaked at 40 h in groups I, II and III of rats and no significant differences were observed between the studied groups. CONCLUSION: HSS administration is not capable of reversing the liver regeneration arrest induced by 5-HT2 receptor blockade.     

Acceleration of liver regeneration by malotilate in partially hepatectomized rats

The effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on liver regeneration was studied by using partially hepatectomized rats. Malotilate administration (100 mg/kg/day, p.o.) facilitated the weight gain of the liver after partial hepatectomy. Protein, RNA and DNA contents of the regenerating liver correlated well with the weight gain. The weight gain, RNA and DNA contents, and mitotic index were significantly suppressed in the alloxan-diabetic rats 24 hr after partial hepatectomy. However, malotilate administration significantly improved the delayed recovery of RNA content. Other parameters were not significantly improved by malotilate, but tended to increase to a level comparable to those of partially hepatectomized control rats. These results show that malotilate accelerates cell proliferation, resulting in facilitated liver regeneration in rats (as well as in alloxan-diabetic rats).     

Activation of mitochondrial functions by malotilate in relation to accelerated liver regeneration in partially hepatectomized rats

Malotilate was orally administered to rats at a dose of 100 mg/kg 2 hr after partial hepatectomy. Mitochondrial state 3 respiration of the liver increased significantly from 23.4 nmol/min/mg protein in the control rats to 29.3 nmol/min/mg protein in the rats administered with malotilate at 3 hr after the hepatectomy (1 hr after the administration). The administration also resulted in higher tendencies in the respiratory control ratio after 3, 6 or 20 hr (1, 4 or 18 hr after the administration) than in the control rats. Although partial hepatectomy made hepatic ATP concentration remarkably low, it gradually increased (from 1.53 mumol/g liver after 3 hr) to a level of 2.03 mumol/g liver after 20 hr in the rats administered with malotilate. No increase in ATP concentration was observed in the control rats. Correspondingly, the adenylate energy charge also showed higher tendencies in the malotilate administered rats. From these result, it is supposed that malotilate activates the mitochondrial functions which results in the increases of ATP concentration and adenylate energy charge. These changes in energy metabolism can be associated with accelerated regeneration of the liver by malotilate.     

Reparative regeneration of the liver as a model for the preclinical study of the hepatotropism of anti-alcohol agents

It has been established that administration of teturam (200 mg/kg) per os once a day for 14 days retards reparative regeneration of the liver in rats following partial hepatectomy and exerts an additional damaging action on the population of regenerating hepatocytes. Administration of potassium orotate (100 mg/kg) per os once a day for 14 days speeds up, on the contrary, organ regeneration. The combined use of the drugs potentiates paradoxically the negative hepatotropic effect of teturam. It is concluded that experimental reparative regeneration of the rat liver can be applied to the assessment of hepatotropism of the antialcoholic drugs.     

Effects of cianidanol on chronic liver injury induced by carbon tetrachloride and on liver regeneration after partial hepatectomy in rats

Effects of cianidanol on chronic liver injury induced by prolonged administration of carbon tetrachloride (CCl4) and on liver regeneration after partial hepatectomy of normal liver and CCl4 chronically injured liver were investigated by the measurement of plasma and liver biochemical parameters. Cianidanol increased the total plasma protein and 14C-Leu incorporation into plasma protein, while it reduced the contents of liver cholesterol and triglycerides. In rats with chronically injured liver or regenerating liver after partial hepatectomy of chronically injured liver, cianidanol improved the retention rate of BSP and the content of liver sugar. In rats with chronically injured liver, plasma GPT and GOT activities were reduced with the administration of cianidanol. Cianidanol had no effect on the regeneration rate after partial hepatectomy of normal liver, but it increased the regeneration rate after partial hepatectomy of chronically injured liver. These results suggest that cianidanol has the effect of improving the function of liver cells damaged by CCl4 treatment and of promoting the recovery of cell function to a normal level.     

In vivo glycyrrhizin accelerates liver regeneration and rapidly lowers serum transaminase activities in 70% partially hepatectomized rats

The in vivo effects of glycyrrhizin on restoration of liver mass and recovery of liver function were compared with those of epidermal growth factor (EGF), ibuprofen and dexamethasone in 70% partially hepatectomized rats. Hepatic regenerative activity was assessed based on the ratio of liver weight to 100 g body weight, and 5-bromo-2'-deoxyuridine (BrdU) incorporation into hepatocyte DNA in the remnant liver. Glycyrrhizin (50 mg/kg/day, i.p.)- or EGF (1.0 microg/kg/day, i.p.)-treated rats showed an approx. 1.4-fold increase in liver weight/100 g body weight ratio over saline-treated control rats on days 2 and 3 after 70% partial hepatectomy. BrdU labeling index in the remnant regenerating liver was significantly higher in glycyrrhizin- or EGF-treated rats when compared with saline-treated control rats on days 0.5 and 1. Ibuprofen (100 mg/kg/day, i.p.) and dexamethasone (0.1 mg/kg/day, i.p.) did not significantly increase either liver weight/100 g body weight ratio or BrdU labeling index. Serum activity of liver-related transaminases, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), elevated rapidly on day 1 and decreased to near pre-operative levels on day 5 after 70% partial hepatectomy in saline-treated control rats. Injection of glycyrrhizin or EGF significantly decreased the elevated serum ALT and AST activities on days 2 and 3 after hepatectomy when compared with saline-treated control rats. The transaminase-lowering effects of glycyrrhizin or EGF were smaller than those of ibuprofen and dexamethasone. These results demonstrate that injection of glycyrrhizin or EGF significantly enhances regeneration of liver mass and function, as well as recovery from the liver damage induced by surgical resection.     

Dimethylnitrosamine demethylase activity of rat liver microsomes after partial hepatectomy

It is known that partial hepatectomy increases the hepatocarcinogenicity of dimethylnitrosamine (DMN). To investigate why this procedure increases the hepatocarcinogenicity of DMN, the activity of liver DMN demethylase was determined on Wistar male rats at different time intervals after partial hepatectomy. The ld₅₀ for DMN administered 1 day after partial hepatectomy was also compared to that for the control animals. The enzyme activity reached its lowest point (47 per cent) 1 day after operation, and at 3 days had recovered to about 90 per cent of the control values. The ld₅₀ for partially hepatectomized rats 44 hr after i.p. injection of DMN was 114 mg/kg body weight as compared to 82 mg/kg for control animals. The reduction rather than increase of enzyme activity after hepatectomy shows that increased DMN carcinogenicity after hepatectomy is not caused by a compensatory increase of demethylase activity associated with liver regeneration.     

The effect of different doses of dimethylbiguanide (DMB) on liver blood flow, blood glucose and plasma immunoreactive insulin in anaesthetized rats

The effect of different doses of dimethylbiguanide (DMB) administered by the i.v. and i.d. route on liver blood flow, blood glucose and plasma immunoreactive insulin was investigated in anaesthetized rats. Liver blood flow was measured by thermocouples implanted into the liver utilising Grayson's principle of “internal calorimetry”. In addition blood glucose and immunoreactive insulin in the plasma was measured at different time intervals following the different doses of DMB. Intraarterial BP was monitored during the whole experiment. Following 100 mg/kg DMB i.v. a significant increase in liver blood flow, blood glucose, plasma immunoreactive insulin and BP was observed. 50 mg/kg DMB i.v. did not affect any of the parameters measured, while 150 mg/kg DMB was already a toxic dose. After i.d. administration no changes were seen following a dose of 175 mg/kg DMB and 1500 mg/kg DMB was already toxic. 500 mg/kg, however, showed a similar significant increase in liver blood flow, blood glucose, plasma immunoreactive insulin and BP was observed. 50 mg/kg effects observed could be suppressed by a simultaneous administration of 1 mg/kg propranolol i.v. In addition a decrease in liver blood flow occurred which is explained by a direct effect of the β-adrenergic blocking agent propranolol on the liver vasculature. Therefore a selective β-adrenergic effect of the biguanides seems a likely explanation for the increased liver blood flow following DMB administration.Administering 1.6 g/kg glucose i.d. an increase in liver blood flow, blood glucose and plasma immunoreactive insulin was observed. This influence on blood glucose and plasma immunoreactive insulin was inhibited by simultaneous administration of 1.6 g/kg glucose and 500 mg/kg DMB, while liver blood flow showed the same increase when glucose alone was given. After changing the experimental design in giving the 500 mg/kg DMB i.d. and 30 min later 1.6 g/kg glucose a much higher increase in liver blood flow occurred, which might reflect an additive effect.     

The role of hepatic stimulator substance (HSS) on liver regeneration arrest induced by cadmium

BACKGROUND: The mechanism of cadmium-induced liver regeneration arrest in relation to hepatic stimulator substance (HSS) biological activity was investigated. MATERIALS AND METHODS: In Wistar rats subjected to 65 - 70% partial hepatectomy, saline, cadmium, cadmium and HSS were administered. The rats were also subjected to 30 - 34% partial hepatectomy. Mitotic index, immunochemistry for PCNA, 3[H]-thymidine incorporation into DNA and thymidine kinase activity were used as indices of liver regeneration. HSS biological activity was evaluated in all groups of rats using a bioassay. Results: Liver regeneration and HSS activity were arrested by cadmium during the first 24 h after partial hepatectomy. Both in normal and in cadmium-treated rats, the HSS activity was increased and liver regeneration coincided. HSS activity was stable in 30 - 34% hepatectomized rats. HSS administration was able to restore liver regeneration arrest induced by cadmium. Conclusion: The biological activity of HSS increased at the time of G1/S transition of hepatocytes in the cell cycle and no increase was observed with asynchronous G1/S transition (30 - 34% partial hepatectomy). The suppression of HSS biological activity by cadmium seems to represent an important factor for liver regeneration arrest induced by the metal and HSS administration is able to restore liver regeneration.     

Effects of cyclopiazonic acid on the ultrastructure of rat liver

Groups of Sprague-Dawley rats were dosed per os for 4 consecutive days with 0.0, 0.2, 2.0 or 4.0 mg cyclopiazonic acid (CPA)/kg body weight/day, and killed on the fifth day. Sections of liver were prepared for electron microscopic examination. Dilatation of the rough endoplasmic reticulum was observed in all hepatocytes examined from the 2 highest dose groups, and in about 25% of liver cells from the 0.2 mg CPA/kg/day group. Vesiculation of the rough endoplasmic reticulum also occurred in these groups, an increasing amount of vesiculation being observed with increasing dosage. Control sections exhibited neither of these characteristics. No proliferation of smooth endoplasmic reticulum, or blockage of bile canaliculi was observed in any group. Lysing cells were present only in the 4.0 mg CPA/kg/day group; mitochondria in the 2.0 and 4.0 mg CPA/kg/day dose groups were swollen. Nuclei were ultrastructurally normal in all groups. The primary cellular effect of CPA was on the endoplasmic reticulum, even at relatively low doses. Possible interactions of CPA with other toxins likely to be produced by the same fungus, such as aflatoxin, are considered.     

Effect of calcium channel blockers and trifluoperazine on rat liver regeneration

The activity of hepatic thymidylate synthetase and thymidine kinase at 24 h after 70% partial hepatectomy of rats was suppressed significantly compared with that in the control group by the administration of calcium channel blockers (verapamil, diltiazem and nifedipine) 8 h after partial hepatectomy. The decrease of thymidylate synthetase and thymidine kinase activities was accompanied by a reduction of DNA content in 24 h regenerating liver. Trifluoperazine showed an effect similar to that of the calcium channel blockers on DNA synthesis during liver regeneration. These results suggest that calcium entry into the hepatic cell is an essential event in liver regeneration.     

Determination of gomisin A (TJN-101) and its metabolite in rat serum by gas chromatography-mass spectrometry]

Gomisin A (TJN-101) is one of the lignan components isolated from Schisandra Fruits. A high sensitive and precise method for the determination of TJN-101 and its major metabolite (Met. B) in the rat serum was developed by selected ion monitoring (SIM) with gas chromatography-mass spectrometry (GC/MS) using a fused silica capillary column (SPB-1, Supelco). A 100 microliter serum sample was used for the solid phase extraction. The calibration curves of TJN-101 and Met.B both showed a good linearity between 2.0 and 2000.0 ng/ml. The analytical precision (intra-assay, C.V. less than 4.7%), recoveries (98.4 +/- 10.1%), and detection limit (2 ng/ml) of TJN-101 indicated that this system was suited for the determination of TJN-101 in biological fluid. In case of Met.B, the same results as TJN-101, were obtained. After oral administration of TJN-101 at a dose of 10 mg/kg to male rats, the average values of the maximal serum concentration of TJN-101 and Met.B were 1446.1 +/- 131.8 and 317.4 +/- 18.5 ng/ml, respectively. The serum concentrations of these substances could be monitored sufficiently for 8 h after dosing.     

Effect of tritoqualine on liver regeneration after partial hepatectomy in rats

Effect of tritoqualine (TRQ) on liver regeneration after partial hepatectomy in normal rats and chronically injured rats treated with carbon tetrachloride (CCl4) for 12 weeks were investigated by the measurement of serum and liver biochemical parameters concerning the hepatic function. The results are as follows: 1) In normal rats, the liver regeneration rate after partial hepatectomy was increased dose-dependently with the administration of TRQ for 7 days after the operation. TRQ improved BSP retention rate which was decreased after partial hepatectomy. In addition, protein synthetic activity in the liver microsomes prepared from TRQ-administered rats was higher than that prepared from control rats, and the contents of serum total protein, serum albumin and liver protein were also higher in TRQ-administered rats. 2) In the rats treated with CCl4 for 12 weeks, the liver regeneration rate after partial hepatectomy was increased dose-dependently with the administration of TRQ for 6 days. TRQ also improved the contents of serum total protein, serum albumin and liver protein. Though the amount of collagen in the liver chronically injured by CCl4 increased more than twice compared with that in the normal liver, the amount of collagen in the regenerating liver of CCl4-treated rats whose liver regeneration was accelerated by TRQ was not different from that in the normal liver. These results suggest that TRQ has the effect of improving the various hepatic functions through the activation of the protein synthesis in hepatocytes.     

Differential effects of hepatic microsomal enzyme inducing agents on liver blood flow

Male and female rats were injected i.p. for 5 days with phenobarbitone (80 mg/kg/day), antipyrine (80 mg/kg/day), phenytoin (65 mg/kg/day) or chlordiazepoxide (40 mg/kg/day). Seven days after start of treatment, radioactive microspheres were used to determine liver blood flow in some animals from treatment groups. Other animals were used to measure liver microsomal protein, cytochrome c reductase and cytochrome P450. Pentobarbitone sleeping time was also determined. In males, all the drugs significantly increased cytochrome P450 content and liver weight/100 g body weight (bw) relative to salinetreated controls. Also, pentobarbitone sleeping time was significantly decreased by all 4 drugs. However, only phenobarbitone changed liver blood flow; liver weight was increased by 23 per cent and this was paralleled by a 32 per cent increase in liver blood flow/100 g bw. Female rats receiving saline had lower liver cytochrome P450 contents and longer pentobarbitone sleeping times than the control males. In addition, the drugs were less potent in the females; all significantly reduced sleeping time but liver weight/100 g bw and cytochrome P450 content were only significantly increased by phenobarbitone and phenytoin. After phenytoin, antipyrine and chlordiazepoxide, liver blood flows/100 g bw were within 4 per cent of the control value whereas with phenobarbitone there was a 9 per cent increase which accompanied an 11 per cent increase in liver weight/100 g bw. The dose-effect relations of phenobarbitone were determined in male rats using doses of 5, 10 and 80 mg/kg/day and some animals were given amylobarbitone (80 mg/kg/day) in order to see if it also changed liver blood flow. Phenobarbitone gave dose-dependent effects on the biochemical parameters, liver weight; 100 g bw and liver blood flow/100 g bw. Amylobarbitone was much less potent than phenobarbitone but did cause parallel changes in liver blood flow and liver weight/100 g bw. It is concluded that, of all the hepatic microsomal enzyme inducing agents which have been studied, only-barbiturates increase both liver blood flow and liver weight.     

Effects of chlorinated diphenyl ethers on the mixed-function oxidases and ultrastructure of rat and trout liver

The effects of chlorinated diphenyl ethers (CDEs) on hepatic mixed-function oxidase (MFO) activities and ultrastructure were examined in the rat and trout after ip administration of 100 mg/kg/day of 4-chlorodiphenyl ether (4-CDE), 2,4-dichlorodiphenyl ether (2,4-diCDE), 2,4,4'-trichlorodiphenyl ether (2,4,4'-triCDE), or 2,4,5,4'-tetrachlorodiphenyl ether (2,4,5,4'-tetraCDE) for 3 days. The 4-CDE, 2,4-diCDE, and 2,4,4'-triCDE pretreatments did not alter significantly the MFO activities of trout liver; however, 2,4,5,4'-tetraCDE pretreatment increased significantly the hepatic cytochrome b5 content and 7-ethoxycoumarin O-deethylase activity of trout liver. No histological changes were observed in the hepatocytes of trout treated with any of the CDEs. Pretreatment of rats with 4-CDE and 2,4-diCDE also did not alter significantly the MFO activities of the liver. In contrast, pretreatment of rats with 2,4,4'-triCDE and 2,4,5,4'-tetraCDE increased significantly hepatic MFO activities and also caused a proliferation of smooth endoplasmic reticulum in the liver. 2,4,4'-triCDE and 2,4,5,4'-tetraCDE appear to be a phenobarbital-type inducer and a mixed-type inducer of rat liver MFO activity, respectively.     

Correlation of liver growth and function during liver regeneration and hepatocarcinogenesis.

We have previously studied some parameters of rat liver activity and compared the kinetics of cell proliferation (normal growth or after partial hepatectomy) with some specific hepatic enzymes. The mutually exclusive relationship between division and tissue function, their specific circadian rhythm as well as the "chalone effect" have been used to characterize the normal homeostatic regulatory mechanism in the liver. The same parameters have been recently determined during chemical carcinogenesis. Adult rats, fed long term with diethylnitrosamine (DENA, 10 mg/kg/day) develop liver carcinoma after 90 days of carcinogen administration. The results show that the relationship between the above parameters is progressively disturbed during the second month of treatment. A minimum of 4 weeks of continuous DENA feeding is found to be necessary for the induction of liver cancers. Giving the carcinogen for a second month decreases the delay before death with cancer. Protracting the treatment after the second month has no further effect either on survival or on cancer induction. The mechanism of carcinogenesis is explained by postulating that preneoplastic lesions evolution would closely depend on the homeostatic control disturbances.     

匹格列酮抑制小鼠肝切除术后肝脏再生

目的:探讨匹格列酮在小鼠肝切除术后肝脏再生中的作用。方法:对C57BL/6J小鼠实施2/3肝切除,建立小鼠肝再生模型。实验组小鼠按体重给予匹格列酮20mg.kg-1.d-1口服,对照组给予安慰剂口服,在术后不同时间点收集小鼠残余肝脏和血清,计算肝脏体重比;监测术后肝功能和血糖变化;H&E染色观察肝脏形态学变化,免疫组化染色观察肝细胞增殖情况。结果:匹格列酮20mg.kg-1.d-1对小鼠术后肝功能和血糖无明显影响。与对照组相比,匹格列酮组小鼠术后肝脏生长缓慢,肝细胞增殖受到抑制(P<0.05)。结论:匹格列酮抑制小鼠肝切除术后肝脏再生。