Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.

Profile of hepatocyte apoptosis and bile lakes before and after bile duct decompression in severe obstructive jaundice patients

BACKGROUND:Excessive hepatocyte apoptosis and bile lakes in severe obstructive jaundice might impair liver functions.Although decompression of the bile duct has been reported to improve liver functions in animal studies,the mechanism of obstruction differs from that in humans.This study aimed to determine the profiles of hepatocyte apoptosis and bile lakes following bile duct decompression in patients with severe obstructive jaundice in the clinical setting.METHODS:We conducted a 'before and after study' on severe obstructive jaundice patients as a model of inhibition of the excessive process by bile duct decompression.Specimens of liver biopsies were taken before and after decompression of the bile duct and then stained by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling(TUNEL) to identify hepatocyte apoptosis and by hematoxilin-eosin(HE) to identify bile lakes.All measurements were independently done by 2 observers.RESULTS:Twenty-one severe obstructive jaundice patients were included.In all patients,excessive hepatocyte apoptosis and bile lakes were apparent.After decompression,the hepatocyte apoptosis index decreased from 53.1(SD 105) to 11.7(SD 13.6)(P<0.05),and the bile lakes from 23.6(SD 14.8) to 10.9(SD 6.9)(P<0.05).CONCLUSION:Bile duct decompression improves hepatocyte apoptosis and bile lakes in cases of severe obstructive jaundice,similar to the findings in animal studies.

Glycyrrhizin attenuates HMGB1-induced hepatocyte apoptosis by inhibiting the p38-dependent mitochondrial pathway

AIM： To examine how High-mobility group box I （HMGB1） regulates hepatocyte apoptosis and, furthermore, to determine whether glycyrrhizin （GL）, a known HMGB1 inhibitor, prevents HMGBl-induced hepatocyte apoptosis.

Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice

AIM: To investigate the role of caspase-12 and its downstream targets in carbon tetrachloride (CCl4)-induced hepatocyte apoptosis.

Differential effects on apoptosis induction in hepatocyte lines by stable expression of hepatitis B virus X protein

AIM： Hepatitis B virus protein X （HBx） has been shown to be weakly oncogenic in vitro. The transforming activities of HBx have been linked with the inhibition of several functions of the tumor suppressor p53. We have studied whether HBx may have different effects on p53 depending on the cell type. METHODS： We used the human hepatoma cell line HepG2 and the immortalized murine hepatocyte line AML12 and analyzed stably transfected clones which expressed physiological amounts of HBx. P53 was induced by UV irradiation.RESULTS： The p53 induction by UV irradiation was unaffected by stable expression of HBx. However, the expression of the cyclin kinase inhibitor p21^waf/cip/sdi which gets activated by p53 was affected in the HBx transformed cell line AML12-HBx9, but not in HepG2. In AML-HBx9 cells, p21^waf/ciP/sdi -protein expression and p21^waf/dip/sdi transcription were deregulated. Furthermore, the process of apoptosis was affected in opposite ways in the two cell lines investigated. While stable expression of HBx enhanced apoptosis induced by UV irradiation in HepG2-cells, apoptosis was decreased in HBx transformed AML12-HBx9. P53 repressed transcription from the HBV enhancer I, when expressed from expression vectors or after induction of endogenous p53 by UV irradiation. Repression by endogenous p53 was partially reversible by stably expressed HBx in both cell lines.CONCLUSION： Stable expression of HBx leads to deregulation of apoptosis induced by UV irradiation depending on the cell line used. In an immortalized hepatocyte line HBx acted anti-apoptotic whereas expression in a carcinoma derived hepatocyte line HBx enhanced apoptosis.

Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes

BACKGROUND Calpain-2 is a Ca^2+-dependent cysteine protease,and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.AIM To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum(ER)stress-related apoptosis in rat hepatocyte BRL-3A cells.METHODS BRL-3A cells were treated with varying doses of dithiothreitol(DTT),and their viability and apoptosis were quantified by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry.The expression of ER stress-and apoptosis-related proteins was detected by Western blot analysis.The protease activity of calpain-2 was determined using a fluorescent substrate,Nsuccinyl-Leu-Leu-Val-Tyr-AMC.Intracellular Ca^2+content,and ER and calpain-2 co-localization were characterized by fluorescent microscopy.The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified.RESULTS DTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis.DTT treatment significantly upregulated 78 kDa glucose-regulated protein,activating transcription factor 4,C/EBP-homologous protein expression by>2-fold,and enhanced PRKR-like ER kinase phosphorylation,caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence.DTT treatment also significantly increased intracellular Ca^2+content,calpain-2 expression,and activity by>2-fold in BRL-3A cells.Furthermore,immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2.Moreover,calpain-2 silencing to decrease calpain-2 expression by 85%significantly mitigated DTT-enhanced calpain-2 expression,caspase-12 cleavage,and apoptosis in BRL-3A cells.CONCLUSION The data indicated that Ca^2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.

HEPATOCYTE GROWTH FACTOR PROTECTS AGAINST APOPTOSIS INDUCED BY ADVANCED GLYCATION END PRODUCTS IN ENDOTHELIAL CELLS

Objective To investigate the effects of hepatocyte growth factor(HGF)on vascular endothelial cells apoptosis induced by advanced glycation end products(AGEs)and its possible mechanism. Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and intervened by different concentrations of AGEs and HGF.The cell inhibitory rates of each group with different culture time(12, 24, 48, and 72 hours)were measured by methyl thiazolyl tetrazolium(MTT)assay. The early stage apoptosis was detected by flow cytometry with Annexin V-FITC/PI double staining, morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the expression of apoptosis-associated genes Bax and Bcl-2 were determined by Western blotting.The activity of caspase-3 was detected by enzyme-linked immunosorbent assay (ELISA).Results Morphological observation indicated that high concentration of AGEs induced characteristic apoptotic changes in HUVECs.Within a certain concentration range, HUVECs apoptosis inducing rates by AGEs were in both dose- and time-dependent manners.HGF significantly inhibited the apoptosis of HUVECs induced by AGEs (P< 0.05).AGEs significantly promoted expression of Bax protein, but not Bcl-2.Whereas HGF significantly promoted the expression of Bcl-2(P<0.01)and decreased the activity of caspase-3(P<0.05)without affecting Bax level.Conclusions AGEs can induce the apoptosis of endothelial cells in vitro.HGF may effectively attenuate AGEs-induced endothelial cells apoptosis through upregulating Bcl-2 gene expression and inhibiting caspase-3 activation.

IGFBPrP1 induces liver fibrosis by inducing hepatic stellate cell activation and hepatocyte apoptosis via Smad2/3 signaling

AIM: To investigate the role and mechanism of insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) in the development of liver fibrosis.

Serotonin receptor agonist quipazine promotes proliferation and apoptosis of human hepatocyte strain of L-02 strain

BACKGROUND: Liver disease is commonly seen in the clinic and its pathological characteristic is combined hepatocellular death and apoptosis. Promoting hepatocyte regeneration is one of the main methods of treating liver disease. Serotonin (5-HT) is an important compound which participates in various life process, and 95% of it is carried by platelets in the blood. A recent finding showed that platelet-derived serotonin is the key factor in liver regeneration, which fails without serotonin. This study aimed to investigate the effects of quipazine, a selective 5-HT receptor agonist, on proliferation and apoptosis in the human hepatocyte strain L-02. METHODS: L-02 cells were cultured in medium with 5-HT and quipazine, and samples were collected at 24, 48, and 72 hours. The methyl thiazolyl tetrazolium (MTT) method was used to test viability, flow cytometry to assess the cell cycle, the Annexin-V/PI method to evaluate apoptosis, and immunohistochemistry to detect proliferating cell nuclear antigen (PCNA). RESULTS: Compared with the control group, the viability of L-02 cells was improved in the 10, 50, and 250 mu g/ml quipazine groups (P<0.05); the percentage of S-phase and PCNA-positive cells were increased in the 2, 10, 50, and 250 mu g/ml quipazine groups (P>0.05); and no difference in the percentage of apoptotic cells was found between the 50 mu g/ml quipazine and control groups (P>0.05). CONCLUSION: Quipazine improves proliferation of a human hepatocyte strain in vitro, and this is not based on the inhibition of apoptosis. (Hepatobiliary Pancreat Dis Int 2009; 8: 278-281)

Studies of hepatocytes apoptosis induced by TNF-α and its mechanism

Objective: To investigate the apoptosis effect of TNF-α on HepG2 cells and its mechanism in vitro. Methods: HepG2 cells were treated with TNF-α (400 U/mL). HepG2 cells treated with TNF-α for 24 h and apoptosis was proved by DNA fragments on gel electrophoresis. Fluorescent quantitative real-time PCR was used to detect Fas and FasL expression. HepG2 cells treated by TNF-α were co-cultivated with normal HepG2 cells. Apoptosis of HepG2 cells was determined by the method of FACS. Results: After 24 h TNF-α treatment, DNA was collapsed into fragments to form DNA ladder in gel elec- trophoresis; FasL expression increases and induces HepG2 cells apoptosis. By FACS, 98.4% of the co-cultivated cells were apoptosis, but 16.5% cells in control group were apoptosis. Conclusion: TNF-α can induce apoptosis of HepG2 cells in vitro. FasL expression on TNF-α pre-treated HepG2 cells increased and these cells can lead normal HepG2 cells to apoptosis. This may attribute to the cure of virus hepatitis and hepatoma.

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.

Effect of naphthalene acetic acid on proliferation and apoptosis of mice hepatocytes

Objective To investigate the effect of naphthalene acetic acid(NAA) on proliferation and apoptosis of mice hepatocytes.Methods Fifty mice were randomly divided into five groups: positive control,normal control,low,middle and high dose of NAA groups.Mice in low,middle and high dose of naphthalene acetic acid groups were fed with 200 mg/kg,1 000 mg/kg and 5 000 mg/kg NAA animal food,respectively.Mice in positive control,normal control groups were fed with usual animal food.After 4 weeks,mice were sacrificed and liver was separated.Three days before scarification,mice in positive control group were given 20 mg/kg ascyclophosphami daily by intraperitoneal injection.The proliferative activity of hepatocytes was detected by MTT assay.The expressions of PCNA protein and Caspase-3 were measured by immunohistochemistry method.Results The proliferative activities of mice hepatocytes in high dose group and normal control group were 0.794±0.019 and 1.055±0.019, respectively.The expressions of PCNA protein and Caspase-3 in high dose group were 11.2% and 37.9%,and those in the normal group were 33.1% and 6.3%, respectively.All differences were significant(P<0.01).Conclusion High dose of NAA acid could significantly inhibit proliferation and increase apoptosis of hepatocytes in mice.

Family with sequence similarity 134 member B-mediated reticulophagy ameliorates hepatocyte apoptosis induced by dithiothreitol

BACKGROUND Endoplasmic reticulum(ER)stress-related hepatocyte apoptosis is responsible for multiple hepatic diseases.Previous studies have revealed that endoplasmic reticulophagy(ER-phagy)promotes the selective clearance of damaged ER fragments during ER stress,playing a crucial role in maintaining ER homeostasis and inhibiting apoptosis.Family with sequence similarity 134 member B(FAM134B)is a receptor involved in ER-phagy that can form a complex with calnexin(CNX)and microtubule-associated protein 1 light chain 3(LC3).The complex can mediate the selective isolation of ER fragments to attenuate hepatocyte apoptosis.However,the precise regulatory mechanisms remain unclear.AIM To elucidate the effect of FAM134B-mediated ER-phagy on ER stress-induced apoptosis in buffalo rat liver 3A(BRL-3A)rat hepatocytes and the potential regulatory mechanisms.METHODS ER stress-related hepatocyte apoptosis was induced using dithiothreitol(DTT).Proteins related to ER stress and autophagy were measured with western blotting.Protein complex interactions with FAM134B were isolated by co-immunoprecipitation.ER-phagy was evaluated in immunofluorescence experiments.Cell cycle distribution and apoptosis were measured by flow cytometry.Mitochondrial Ca^(2+) levels were evaluated by the co-localization of intracellular Ca^(2+)-tracker and Mitotracker.The small interfering RNA against FAM134B was used to knockdown FAM134B in BRL-3A cells.RESULTS ER stress-related and autophagy-related proteins in BRL-3A cells were elevated by both short and long-term DTT treatment.Furthermore,co-immunoprecipitation confirmed an interaction between FAM134B,CNX,FAM134B,and LC3 in BRL-3A cells.Immunofluorescence assays revealed that autolysosomes significantly decreased following short-term DTT treatment,but increased after long-term treatment.Mitochondrial Ca2+levels and apoptotic rates were dramatically elevated,and more cells were arrested in the G1 stage after short-term DTT treatment;however,these decreased 48 h later.Moreover,FAM134B downregulation accelerated mitochondrial apoptotic pathway activation and aggravated hepatocyte apoptosis under ER stress.CONCLUSION FAM134B-mediated ER-phagy attenuates hepatocyte apoptosis by suppressing the mitochondrial apoptotic pathway.Our findings provide new evidence highlighting the importance of FAM134Bmediated ER-phagy in attenuating hepatocyte apoptosis.

Mesenchymal Stem Cells Alleviate Acute Liver Failure through Regulating Hepatocyte Apoptosis and Macrophage Polarization

Background and Aims:Acute liver failure(ALF)is a life-threatening clinical problem with limited treatment options.Administration of human umbilical cord mesenchymal stem cells(hUC-MSCs)may be a promising approach for ALF.This study aimed to explore the role of hUC-MSCs in the treat-ment of ALF and the underlying mechanisms.Methods:A mouse model of ALF was induced by lipopolysaccharide and d-galactosamine administration.The therapeutic effects of hUC-MSCs were evaluated by assessing serum enzyme activity,histological appearance,and cell apoptosis in liver tissues.The apoptosis rate was analyzed in AML12 cells.The levels of inflammatory cytokines and the phenotype of RAW264.7 cells co-cultured with hUC-MSCs were detected.The C-Jun N-terminal kinase/nuclear factor-kappa B signal-ing pathway was studied.Results:The hUC-MSCstreatment decreased the levels of serum alanine aminotransferase and aspartate aminotransferase,reduced pathological damage,alleviated hepatocyte apoptosis,and reduced mortality in vivo.The hUC-MSCs co-culture reduced the apoptosis rate of AML12 cells in vitro.Moreover,lipopolysaccharide-stimulated RAW264.7 cells had higher levels of tumor necrosis factor-α,interleukin-6,and interleukin-1β and showed more CD86-positive cells,whereas the hUC-MSCs co-culture reduced the levels of the three inflammatory cytokines and increased the ratio of CD206-positive cells.The hUC-MSCs treatment inhibited the activation of phosphorylated(p)-C-Jun N-terminal kinase and p-nuclear factor-kappa B not only in liver tissues but also in AML12and RAW264.7 cells co-cultured with hUC-MSCs.Conclusions:hUC-MSCs could alleviate ALF by regulating hepatocyte apoptosis and macrophage polarization,thus hUC-MSC-based cell therapy may be an alternative option for patients with ALF.

Glycyrrhizic acid inhibits apoptosis and fibrosis in carbontetrachloride-induced rat liver injury

AIM:To investigate anti-apoptotic effects of glycyrrhizic acid(GA) against fibrosis in carbon tetrachloride(CCl4)-induced liver injury and its contributing factors.METHODS:Liver fibrosis was induced by administration of CCl4 for 8 wk.Pathological changes in the liver of rats were examined by hematoxylin-eosin staining.Collagen fibers were detected by Sirius red staining.Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of cleaved caspase-3,Bax,α-SMA,connective tissue growth factor(CTGF),matrix metalloproteinase(MMP) 2 and MMP9 proteins were evaluated by western blot analysis,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were estimated by real-time PCR.RESULTS:Treatment with GA significantly improved the pathological changes in the liver and markedly decreased the positive area of Sirius red compared with rats in the CCl4-treated group.TUNEL assay showed that GA significantly reduced the number of TUNEL-positive cells compared with the CCl4-treated group.The expression levels of cleaved caspase-3,Bax,α-SMA,CTGF,MMP2 and MMP9 proteins,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were also significantly reduced by GA compared with the CCl4-treated group(P < 0.05).CONCLUSION:GA treatment can ameliorate CCl4-induced liver fibrosis by inhibiting hepatocyte apoptosis and hepatic stellate cell activation.

The bcl-2 mRNA Expression in GCDC-induced Obstructive Jaundice in Rats and Its Implication in Hepatocellular Apoptosis

The modulatory role of bcl 2 gene in hepatocellular apoptosis of rats with glycochenodeoxycholate (GCDC) induced obstructive jaundice was investigated. The hepatocytes in normal rats and those with bile duct ligation for 7 days, 14 days and 21 days were isolated and obtained by in situ collagenase perfusion and primary culture. The expression of bcl 2 mRNA in the hepatocytes was detected by RT PCR. Primary culture was performed on the hepatocytes from normal rats and those with bile duct ligation for 14 days. 100 μmol/L GCDC was added to the hepatocytes for incubation for 24 h. The hepatocellular apoptotic ratio was measured by using FCM and hepatocellular apoptosis detected in situ by using TUNEL technique. Results showed that the expression of bcl 2 mRNA was not detectable in the hepatocytes of normal rats by RT PCR technique, while detectable in the hepatocytes of those with bile duct ligation (BDL) for 7, 14 and 21 days. Hepatocellular apoptosis in the BDL group was obviously decreased as compared with normal control group after addition of 100 μmol/L GCDC to the cells for 24 h. It was concluded that the hepatocytes in the BDL rats expressed bcl 2. During obstructive jaundice, expression of bcl 2 from the hepatocytes can inhibit the bile salt induced hepatocellular apoptosis.

Inhibitory effects of grape procyanidins on free radical-induced cell damage in rat hepatocytes in vitro

AIM： To study the protective effect of grape procyanidins on oxidative injury induced by ethanol and carbon tetrachloride in rat hepatocytes. METHODS： Normal rat hepatocytes as well as cells damaged by ethanol or carbon tetrachloride were incubated with different doses of grape procyanidins for 24 h. Cell proliferation, apoptosis and TNFα mRNA expression were subsequently determined using MTT assay, cell death ELISA and in situ hybridization. RESULTS： Proliferative levels of the control cells from ethanol and CCh injury groups significantly decreased while apoptosis and TNFα mRNA expression significantly increased compared to the normal control and grape procyanidins co-treatment groups （0.455 ± 0.051 vs 0.318 ±0.045, P 〈 0.05）. In comparison with the normal control, 50 and 100 mg/L grape procyanidins significantly stimulated cell growth, with a better effect observed with 100 mg/L grape procyanidins. CONCLUSION： Grape procyanidins inhibit the hepatocyte damage induced by ethanol and carbon tetrachloride, and stimulate normal hepatocyte proliferation.

Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells

AIM:To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells (HSC) in vitro. METHODS: The study in vitro was carried out in the culture of HSC lines. Various concentrations of Yigan Decoction were added and incubated. Cell proliferation was detected with MTT colorimetric assay. Cell apoptosis was detected by electron microscopy, flow cytometry and TUNEL. RESULTS: The proliferation of HSC was inhibited by Yigan Decoction, which depending on dose and time significantly. The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L(-1)) were 21.62% and 40.54% respectively, significantly lower than that of normal control group(P【0.01). The HSC proliferation rates of groups at the end concentrations 36, 18 and 9(g.L(-1)) were 54.05%, 45.95% and 51.35% respectively, lower than that of control group (P【0.05). When the end concentration was 4.5 g.L(-1), the proliferation rate was 83.78%, which appeared no significant differences compared with control group. At the same concentrations of 18 g.L(-1), the inhibitory effects of Yigan Decoction at 24 h, 48 h and 72 h time point were observed, the effects were time-dependent, and reached a peak at 72 h. Meanwhile, it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose-dependent and time-dependent. The apoptosis index(AI) was detected by TUNEL. After Yigan Decoction had been incubated for 48 h at the end concentration of 18 g.L(-1), the AI (14.5+/-3.1)% was significantly higher than that of control group (4.3+/-1.3)% (P【0.01). When visualized under transmission electron microscopy, some apoptotic stellate cells were found, i.e. dilated endoplasmic reticulum, irregular nuclei, chromatin condensation and heterochromatin ranked along inside of nuclear membrane. By flow cytometry detection, after HSC was treated with Yigan Decoction at different concentrations of 36, 18 and 9(g.L(-1)) for 48 h, AI (%) were 13.3+/-3.2, 10.7+/-2.7 and 10.1+/-2.5 respectively, which were significantly higher than that of control group(4.1+/-1.9) (P【0.01). At the same concentration of 18 g. L(-1) for 24h, 48 h and 72 h, AI (%) were 9.3+/-1.8,10.7+/-2.7 and 14.6+/-4.3 respectively, which were significantly higher than that of control group (P【0.01). CONCLUSION: Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently, which may be related to its mechanism of antifibrosis.