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Suppression of Rat Stellate Cell Activation and Liver Fibrosis by a Japanese Herbal Medicine, Inchinko-to (TJ135)

February 2004
Comparative Hepatology 3 Suppl 1:S11

February 2004
3 Suppl 1:S11

DOI:10.1186/1476-5926-2-S1-S11

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Comparative Hepatology

Open Access

Proceedings

Suppression of Rat Stellate Cell Activation and Liver Fibrosis by a

Japanese Herbal Medicine, Inchinko-to (TJ135)

Yukihiro Imanishi*1, Naoto Maeda1, Hiroko Matsui1, Tokuko Takashima1,

Shuichi Seki1, Tetsuo Arakawa2 and Norifumi Kawada1

Address: 1Department of Hepatology, Graduate School of Medicine, Osaka City University, Asahimachi, Abeno, Osaka 545-8585, Japan and

2Department Gastroenterology, Graduate School of Medicine, Osaka City University, Asahimachi, Abeno, Osaka 545-8585, Japan

Email: Yukihiro Imanishi* - yukihiro@med.osaka-cu.ac.jp; Naoto Maeda - kyojin@msic.med.osaka-cu.ac.jp; Hiroko Matsui - hiroko-

mat@med.osaka-cu.ac.jp; Tokuko Takashima - yukihiro@med.osaka-cu.ac.jp; ShuichiSeki-yukihiro@med.osaka-cu.ac.jp;

Tetsuo Arakawa - yukihiro@med.osaka-cu.ac.jp; Norifumi Kawada - kawadanori@med.osaka-cu.ac.jp

* Corresponding author

Introduction

Herbal medicine has been recognized as one of useful

treatments for chronic liver diseases. Inchinko-to (TJ135)

consists of Artemisia Capillaris Spike, Gardenia Fruit and

Rhubarb Rhizome. Artemisia Capillaris Spike and Gardenia

Fruit promote bile secretion [1,2]. Rhubarb Rhizome is used

for constipation. Rhubarb Rhizome contains anthraqui-

none derivatives, such as aloe emodin and emodin. TJ135

is used for cholestasis, primary biliary cirrhosis [3] and

hepatitis C [4] in Japan. Although therapeutic benefit of

TJ135 was recently reported on acute liver injury induced

by Fas-induced apoptosis of hepatocytes [5], its effect on

liver fibrosis has never been studied so far. In the present

study, we investigated the effect of TJ135 and its compo-

nent on the activation of rat hepatic stellate cells in pri-

mary culture [6]. In vivo effect of TJ135 on an experimental

liver fibrosis caused by thioacetamide (TAA) administra-

tion was also studied.

Methods

Isolated rat HSCs were cultured on plastic dishes in

DMEM in the presence or absence of 10% FBS for 2 days

and were successively exposed to TJ135 or its component

for the following 2 days. DNA synthesis was evaluated by

BrdU uptake. Effect of TJ135 and its component on the

expression of smooth muscle alpha-actin and PDGFR beta

was evaluated by western blot. Effect of these agents on

the phosphorylations of MAPK, Akt, c-raf-1 and PDGFR

beta was estimated using phospho-specific antibodies.

In vivo experimental liver fibrosis was induced in rats by

injecting TAA. TJ135 was administered every day orally.

After 3 weeks, rats were sacrificed. Formalin-fixed liver sec-

tions were stained with sirius red.

Results

HSCs exposed to TJ135 dose-dependently maintained

quiescent phenotype in culture. TJ135 dose-dependently

inhibited serum-stimulated DNA synthesis of HSCs with-

out affecting the expressions of smooth muscle alpha-

actin and PDGFR beta. In addition, TJ135 dose-depend-

ently inhibited DNA synthesis of HSCs under PDGF stim-

ulation (20 ng/ml). Analysis of the signal transduction

under PDGF stimulation indicated that TJ135 inhibited

expressions of phospho-tyrosine at 170 kDa, phospho-

Raf, phospho-MAPK and phospho-Akt without affecting

their total protein level.

HSCs exposed to Rhubarb Rhizome, a TJ135 component,

dose-dependently maintained quiescent morphology

with dendritic processes. Rhubarb Rhizome dose-depend-

ently inhibited DNA synthesis of HSCs, but had negligible

effect on the expressions of smooth muscle alpha-actin

and PDGFR beta. Further analysis revealed that emodin, a

component of Rhubarb Rhizome, dose-dependently inhib-

ited DNA synthesis of HSCs.

In vivo, TJ135 administration suppressed the development

of liver fibrosis.

from 11th International Symposium on the Cells of the Hepatic Sinusoid and their Relation to Other Cells

Tucson, Arizona, USA, 25–29 August, 2002

Published: 14 January 2004

Comparative Hepatology 2004, 3(Suppl 1):S11

<supplement> <title> <p>11th International Symposium on the Cells of the Hepatic Sinusoid and their Relation to Other Cells</p> </title> </supplement>

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Discussion

In the present study, TJ 135 was found to suppress the pro-

liferation of HSC [7] by hampering the action of PDGF-

BB. Emodin contained in Rhubarb Rhizome may be a major

player inhibiting HSC proliferation and morphological

transformation into myofibroblasts. In vivo, TJ135 was

found to suppress the development of liver fibrosis. Thus,

TJ135 might have a clinical potential suppressing liver

fibrogenesis.

References

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prescription "Inchinko-to" on intrahepatic cholestasis

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Komatsu Y, Fukuda K, Ito T, Terasawa K: Genipin, a metabolite

derived from herbal medicine Inchinko-to, and suppression

of Fas-induced lethal liver apoptosis in mice. Gastroenterology

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6. Liu YZ, Luo GA, Shen ZW: Inhibiting effect of emodin on

smooth muscle cells proliferation. Acta Biophys Sinica 1998,

14:241-244.

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Shugan-Huayu powder (SHP) has been administered to outpatients with chronic liver disease without clear anti-fibrosis mechanism. To investigate the anti-fibrotic effects of SHP on liver fibrosis in a rat model and in hepatic stellate cells (HSCs) in vitro, rats were gavaged with CCl4 at 1.0 g/kg body weight twice a week for 8 weeks to induce liver fibrosis and the rats were randomly assigned to one of the three groups: -CCl4 alone, low-dose SHP and high-dose SHP. SHP was given by gavages 5 times a week for 8 weeks. Serum, livers and HSCs were assayed for serology, pathology, western blot, zymography and quantitative RT-PCR. Hepatic function improved as decreased serum aspartate aminotransferase and alanine aminotransferase, and collagen deposition and active HSCs were significantly reduced in CCl4-induced liver by SHP treatment. The expression of matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta1 (TGF-beta1) mRNA in fibrotic liver showed significant downregulation after SHP treatment. In vitro, inhibition of alpha-smooth muscle actin (alpha-SMA) expression and MMP-2 secretion of active HSCs were also noticed by SHP treatment. SHP has an antifibrotic effect on CCl4-induced liver fibrosis in rats. Anti-fibrotic mechanisms were probably inhibiting activation of HSCs and decreased expression of MMP-2 and TGF-beta1.

Adjuvant therapy in biliary atresia: hopelessly optimistic or potential for change?

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PEDIATR SURG INT

Mark Davenport

Given that the aetiology of biliary atresia (BA) is complex and that there is a multiplicity of possible pathogenic mechanisms then it is perhaps not surprising that the evidence for effect of a number of different agents is contradictory. Post-operative cholangitis for instance is common, bacterial in origin and various antibiotic regimens have been tested (although none in a randomized trial) but continuation beyond the early post-operative period does not appear to offer any greater protection. There is an inflammatory reaction in about 25–35% of cases of BA illustrated by abnormal expression of class II antigen and upregulation of ICAM, VCAM and E-selectin with an infiltrate of immune-activated T cells (predominantly CD4 + Th1 and Th17) and NK cells and a systemic surge in inflammatory cytokines (e.g. TNF-α, IL-2, IL-12). This has potential as a therapeutic target and is the main hypothesis behind the rationale use of steroids. The first report of steroids was published in 1985 by Karrer and Lilly as “blast” therapy to treat recalcitrant cholangitis, followed by a multiplicity of small-scale uncontrolled studies suggesting benefit. To date there has been one randomized placebo-controlled study with a low-dose (prednisolone 2 mg/kg/day) regimen (2007); one with a high-dose (IV prednisolone 4 mg/kg/day regimen) (2014); two prospective high-dose open-label studies (2013); a prospective comparison of low- and high-dose regimen and a large (380 infants) retrospective comparison. The most recent meta-analysis (2016) identified a significant difference in clearance of jaundice at 6 months (OR 1.59, 95% CI 1.03–2.45, P = 0.04), in patients treated with high-dose steroids, particularly if < 70 days at surgery. Ursodeoxycholic acid (UDCA) may increase choleresis or change the ratio of endogenous bile acids to a less hydrophobic and, therefore, less toxic millieu. UDCA may protect cholangiocyte membranes against damage and perhaps reduce the tendency to fibrogenesis. Biochemical benefit has been shown in a single crossover trial in older BA children who had cleared their jaundice. Other potential adjuvant therapies include immunoglobulin therapy, anti-viral agents and Chinese herbs although real evidence of benefit is lacking.

Treatment strategy for successful hepatic resection of icteric liver

Article

Mar 2018

Background: The treatment strategy for jaundiced patients with hilar cholangiocarcinoma (HC) is not well established. In this study, we evaluate the feasibility of our perioperative protocol for jaundiced patients with HC. Methods: Twenty patients with HC who underwent hepatic resection at our institute were enrolled, and patients were divided into icteric(n=6) and normal(n=14) group. As a perioperative protocol, Oral administration of Inchinkoto(ICKT), steroid and nafamostat mesilate were introduced. The evaluation of functional future remnant liver(FRL) by asiaroscintigraphy, and postoperative outcomes were retrospectively compared. Results: Indocyanine green dye retention rate at 15 minutes was higher, and LHL15 values was lower in icteric group. However, in the functional evaluation of FRL, which was the sum of GSA uptake of the future FRL, there was no significant difference of LHL15 values of the remnant liver functional reserve between the two groups. As results, according to the difference of liver function, serum AST level was not different between two groups. The number of patients with postoperative morbidity in the two groups was comparable. Conclusions: Even in HC patients with icteric liver, accurate assessment of liver functional reserve and effective perioperative treatment may attribute to successful hepatectomy and favorable post-operative outcomes. J. Med. Invest. 65:37-42, February, 2018.

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Spinal cord injury (SCI) often results in a cavitary lesion, contained within the dura, which involves only a portion of the cord. Injectable biopolymers are an attractive treatment option for SCI to re‐establish cell migratory pathways within the lesion while minimizing the collateral damage attendant to an open surgical procedure. In this study we evaluate a thermoresponsive soluble collagen gel incorporating genipin, an amine reactive covalent cross‐linker with low cytotoxicity and fluorogenic attributes. Unlike previous studies, genipin is being investigated as an in situ covalent cross‐linker that will continue to act on the gel after injection. Physical characterization studies show that the addition of genipin provides control over the mechanical and degradative behavior of the gel, to meet design specifications of an injectable material for neural tissues. Additionally, an improved in situ assay to predict the extent of cross‐linking reaction is investigated. Encapsulation of mesenchymal stem cells (MSCs) in collagen–genipin gels show the gels support cell viability and proliferation, and thus serve as a cell delivery vehicle. Neural stem cells are found to be more sensitive to genipin, with respect to toxicity, as compared to MSCs. From our studies, 0.25‐0.5 mM is an appropriate genipin concentration to use for an in situ forming scaffold capable of delivering cells and therapeutic agents.

Effect of Kampo treatment on chronic viral hepatitis on the basis of traditional diagnosis

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Full-text available

Jan 1997

rights: 本文データは和漢医薬学会の許諾に基づき複製したものである

Combined use of ursodeoxycholic acid and Inchin‐ko‐to in jaundiced patients with primary biliary cirrhosis

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Jan 1990

Four compounds of anthraquinone in Rheum officinale on Helicobacter pylori inhibition

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OBJECTIVE: To study the effect of four anthraquinone extracts in Rheum officinale on Helicobacter pylori inhibition. METHODS: Four compounds of anthraquinone in Rheum officinale were extracted with gradient alkaline extraction. The susceptibility of Helicobacter pylori inhibition was studied with the dilution method in test tube and the paper diffusion. RESULTS: The minimum inhibitory concentration and Helicobacter pylori inhibited annulus of anthraquinone extracts in Rheum officinale were emodin 0.40 μg · ml-1 (30 mm), rhein 0.60 μg · ml-1 (28 mm), chrysophanol 0.78 μg · ml-1 (24 mm) and aloe-emodin 0.85 μg · ml-1 (23 mm), respectively. Control sample- Rheum officinale extract was 17.24 μg · ml-1 (15 mm). CONCLUSION: Four compounds of anthraquinone in Rheum officinale were effective in Helicobacter pylori inhibition.

Effects of the Chinese prescription 'Inchinko-to' on intrahepatic cholestasis induced by the cholestatic factor

Article

Nov 1985

The effects of "Inchinko-to", a Chinese prescription frequently applied to hepatobiliary diseases, on bile flow were investigated using the erythritol clearance methods. As a result, it was found that when Inchinko-to was administered into the duodenum of normal rats, bile flow was enhanced. Moreover, it seemed to affect the bile formation mainly in the bile canaliculi but not the distal fluid transfer. Furthermore, Inchinko-to significantly blocked the reduction of bile flow induced by the cholestatic factor.

Pharmacologic studies of Gardeniae Fructus. I. Effects of geniposide and genipin on the biliary excretion, the gastric juice secretion and the gastric contraction, and other pharmacologic actions (Japanese)

Article

Mar 1974

Pharmacological studies were made on geniposide, genipin, and gardenia fruit extract on the first general screening test and on their effect on digestive organs, especially on biliary excretion, gastric juice secretion, and gastric contraction. Geniposide is the main component of fruits of gradenia (Gardenia jasminoides ELLIS) and genipin is its aglucone. Gardenia fruit is an important crude drug which is used in prescriptions of Chinese medicine, Shishishi-to ( ?? ?? ?? ?? ), Shishikankyo-to ( ?? ?? ?? ?? ?? ), Inchinko-to ( ?? 〓 ?? ?? ), etc., and may be expected to show sedative, anti-inflammatory, choleretic, antipyretic, and diuretic effects. In the first general screening test, geniposide and genipin inhibited the writhing behavior in mice induced by acetic acid, and genipin showed a weak anti-acetylcholine action and a weak antihistamine action on the isolated mouse. ileum and isolated guinea pig ileum, respectively. Genipin increased biliary excretion by either intravenous, intraduodenal, or oral administration in rats. The choleretic activity of genipin was almost equal to that of sodium dehydrocholate. Genipin inhibited the gastric secretion in the pylorusligated rat, the potency being 1/10 to 1/5 of atropine sulfate. Geniposide and genipin inhibited both the spontaneous contraction and the pilocarpine-induced contraction of the rat stomach. Such an inhibition, however, was transient and geniposide was less potent.

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Mar 2000
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We showed previously that a Kampo (Chinese/Japanese herbal) medicine, Inchin-ko-to (ICKT), inhibits hepatocyte apoptosis induced by transforming growth factor beta1 in vitro. The present study investigated whether ICKT or its ingredients inhibit Fas-mediated liver apoptosis in vivo. Acute liver injury was induced by an intravenous injection of anti-Fas antibody, Jo2. The effects of ICKT and its ingredients on lethality, histology, apoptotic index, serum transaminase levels, caspase activation, mitochondrial membrane potential (Deltapsi(m)), and mitochondrial permeability transition (MPT) were analyzed. Apoptosis in mouse hepatocytes in vitro was also evaluated. Pretreatment with ICKT rescued 75% of Jo2-treated mice and markedly suppressed liver apoptosis/injury. Genipin, an intestinal bacterial metabolite of geniposide that is a major ingredient of ICKT, was found to be an active principle of ICKT. Genipin also suppressed in vitro Fas-mediated apoptosis in primary-cultured murine hepatocytes. Activation of caspase 3 and 8 in the liver homogenate and rapid reduction of triangle uppsi(m) of hepatocytes isolated from Jo2-treated mice were inhibited by genipin preadministration. The resistance to Ca(2+)-induced MPT was enhanced in liver mitochondria of genipin-treated mice. These results suggest that the antiapoptotic activity of genipin via the interference with MPT is a possible mechanism for therapeutic effects of ICKT.

Inhibiting effect of emodin on smooth muscle cells proliferation

YZ Liu
GA Luo
ZW Shen

Inhibiting effect of emodin on smooth muscle cells proliferation

Jan 1998
241-244

Y Z Liu
G A Luo
Z W Shen

Liu YZ, Luo GA, Shen ZW: Inhibiting effect of emodin on smooth muscle cells proliferation. Acta Biophys Sinica 1998, 14:241-244.

Suppression of Rat Stellate Cell Activation and Liver Fibrosis by a Japanese Herbal Medicine, Inchinko-to (TJ135)

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