Heat stress induced hepatocyte apoptosis in largemouth bass Micropterus salmoides via IRE1α/TRAF2/ASK1/JNK pathway

Heat stress(HS)has been shown to adversely affect fish livers and can lead to extensive apoptosis.To investigate the relationship between endoplasmic reticulum(ER)stress and HS-induced apoptosis in fish livers,we isolated and cultured primary hepatocytes of largemouth bass,Micropterus salmoides by trypsin method,then established an in-vitro model of liver cells under HS(35℃).The contents of lactic dehydrogenase(LDH)and hydrogen peroxide(H2O2)were determined to evaluate the effects of HS on hepatocyte injury and oxidative stress.RT-qPCR was performed to discover the key genes in unfolded protein response(UPR)pathways involved at different HS duration.ERS inhibitor 4-PBA and IRE1αinhibitor 4μ8C were used to further investigate the effects of HS on IRE1αapoptosis pathway in hepatocytes.Results show that HS led to significant increases in the release of LDH,the content of H2O2,and the expressions of oxidative protein folding genes(ero1αand pdi)under HS,suggesting severe hepatocyte injury and oxidative stress happened in heat-stressed largemouth bass hepatocytes.The continuous activation of IRE1αpathway genes(grp78,grp94,atf6,perk,eif2a,atf4,chop,ire1α,traf2,ask1,jnk1,and jnk2)indicated that HS led significantly to ER stress.In particular,the mRNA expression levels of ER stress-related genes(grp78,grp94,atf6,perk,ire1α,chop,jnk1,and jnk2)in the high temperature(HT)+4-PBA group and the HT+4μ8C group were significantly down-regulated under HS.After 4μ8C treatment,the expression levels of apoptosis-related genes(caspase-2,caspase-3,caspase-6,caspase-7,caspase-8,caspase-9,and caspase-10)and LDH content were significantly decreased,whereas the cell survival rate was significantly increased when given 4-PBA or 4μ8C treatment.These findings demonstrate that HS could induce liver apoptosis of largemouth bass through the IRE1αpathway,which may act as a key switch mediating liver apoptosis of largemouth bass under HS.

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.

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.

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.

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.

Effect of hepatocyte apoptosis induced by TNF-α on acute severe hepatitis in mouse models

AIM To study the effect of hepatocyteapoptosis and necrosis induced by TNF-α on thepathogenesis of acute severe hepatitis(ASH).METHODS The model of ASH was prepared inD-galactosamine(GAIN)sensitized BALB/c miceby injection of either endotoxin(ET)or tumornecrosis factor-α(TNF-α).Morphologicalchanges of apoptotic hepatocytes were studiedby both light and electron microscope and in siteend labeling method(ISEL).Molecular biologicalchanges of DNA ladder were observed byelectrophoresis of extract from liver tissues.Biochemical changes were measured by alanineaminotransferase(ALT),asparticaminotransferase(AST)and TNF-α.The relationbetween apoptosis and necrosis was evaluatedsimultaneously.RESULTS The sequence of hepatocyteapoptosis,necrosis,and final death from ASHwas observed both in GAIN/ET and GAIN/TNF-agroup.Apoptosis was prominent at 3.5 h and 5 hafter injection of inducer,while necrosis becamedominant at 9 h after challenge.The appearanceof apoptosis was earlier in GAIN/TNF-α groupthan that in GAIN/ ET group.Pretreatment ofmice with antiTNF IgG1 may completely preventthe liver injury induced by GalN/ET.CONCLUSION TNF-α can cause liver damageby inducing hepatic apoptosis and necrosis inmice with endotoxemia.

Glycyrrhizic acid attenuates CCl_4-induced hepatocyte apoptosis in rats via a p53-mediated pathway

AIM: To investigate the effect of glycyrrhizic acid (GA) on carbon tetrachloride (CCl4)-induced hepatocyte apo-ptosis in rats via a p53-dependent mitochondrial path-way. METHODS: Forty-five male Sprague-Dawley rats were randomly and equally divided into three groups, the control group, the CCl4 group, and the GA treatment group. To induce liver fibrosis in this model, rats were given a subcutaneous injection of a 40% solution of CCl4 in olive oil at a dose of 0.3 mL/100 g body weight biweekly for 8 wk, while controls received the same isovolumetric dose of olive oil by hypodermic injection, with an initial double-dose injection. In the GA group,rats were also treated with a 40% solution of CCl4 plus 0.2% GA solution in double distilled water by the intraperitoneal injection of 3 mL per rat three times a week from the first week following previously published methods, with modifications. Controls were given the same isovolumetric dose of double distilled water. Liver function parameters, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were de-termined. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen fibers were evaluated by Sirius red staining. Hepatocyte apoptosis was investigated using the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assay. The expression levels of p53 and apoptosis-related proteins were evaluated by immunohistochemistry or Western blotting analysis. RESULTS: After 8 wk of treatment, GA significantly re-duced serum activity of ALT (from 526.7 ± 57.2 to 342 ± 44.8, P<0.05) and AST (from 640 ± 33.7 to 462.8 ± 30.6, P<0.05), attenuated the changes in liver his-topathology and reduced the staging score (from 3.53 ± 0.74 to 3.00 ± 0.76, P<0.05) in CCl4 -treated rats. GA markedly reduced the positive area of Sirius red and the ratio of the hepatic fibrotic region (from 7.87% ± 0.66% to 3.68% ± 0.32%, P<0.05) compared with the CCl4 group. GA also decreased the expression level of cleaved caspase-3 compared to the CCl4 group. TU-NEL assay indicated that GA significantly diminished the number of TUNEL-positive cells compared with the CCl4 group (P<0.05). GA treatment clearly decreased the level of p53 (P<0.05) detected by immunohis-tochemistry and Western blotting analysis. Compared with the CCl4 group, we also found that GA reduced the Bax/Bcl-2 ratio (P<0.05), the expression of cleaved caspase-3 (P<0.05), cleaved caspase-9 (P<0.05), and inhibited cytochrome C and second mitochondria-derived activator of caspases (Smac) release from mito-chondria to cytoplasm, i.e. , GA reduced the expressionlevel of Smac, which inhibited c-IAP1 activity (P<0.05), ultimately inhibiting the activity of caspase-3, according to Western blotting analysis. As a result, GA suppressed activation of the caspase cascades and prevented he-patocyte apoptosis.

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.

Inhibitory effect of oxymatrine on hepatocyte apoptosis via TLR4/PI3K/Akt/GSK-3β signaling pathway

To evaluate the effect of oxymatrine (OMT) on hepatocyte apoptosis in rats with lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute liver failure (ALF).METHODSLPS/D-GalN was used to establish a model of ALF in rats. To evaluate the effect of OMT, we assessed apoptosis by transmission electron microscopy, and the pathological changes in the liver by light microscopy with hematoxylin and eosin staining. An automated biochemical analyzer was used to measure serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Enzyme-linked immunosorbent assay was used to determine the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Western blotting was used to detect protein levels in liver tissues. Streptavidin peroxidase immunohistochemistry was used to observe expression of Toll-like receptor (TLR)4, active caspase-3, Bax and Bcl-2.RESULTSAll rats in the normal control and OMT-pretreated groups survived. The mortality rate in the model group was 30%. OMT preconditioning down-regulated apoptosis of hepatocytes and ameliorated pathological changes in liver tissue. The levels of AST, ALT, TNF-α and IL-1β in the model group increased significantly, and were significantly reduced by OMT pretreatment. OMT pretreatment down-regulated expression of TLR4 and active caspase-3 and the Bax/Bcl-2 ratio, and up-regulated expression of P-AktSer473 (Akt phosphorylated at serine 473) and P-GSK3βSer9 (glycogen synthase kinase 3β phosphorylated at serine 9) induced by LPS/D-GalN.CONCLUSIONOMT inhibits hepatocyte apoptosis by suppressing the TLR4/PI3K/Akt/GSK-3β signaling pathway, which suggests that OMT is an effective candidate for ameliorating acute liver failure.

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)

Extraction and purification of TGFβ and its effect on the induction of apoptosis of hepatocytes

AIM To extract and purify the transforming growth factor β (TGF β), and to demonstrate its biological activity in vivo and induction of apoptosis of hepatocytes in vitro.METHODS TGF β was isolated from fresh bovine platelets by acid/ethanol extraction method and purified with ion exchange and gel chromatography. The extracted TGF β was injected subcutaneously to mice, and its biological activity in vivo was observed 72 hfs post-injection by HE staining. The morphological changes were observed by HE staining and the occurrence of apoptosis was detected by TUNEL method after the human normal hepatic cell line QZG was treated with 8 μg@L 1 TGFβ for 12 hrs in vitro.RESULTS The molecular mass 25 ku TGF β protein was successfully extracted. It was able to induce localized granulation tissue formation in vivo. TGF β-treated hepatocytes showed obvious apoptotic morphological changes, including the pyknosis and dense-stained nuclei and cytoplasm, the fragmentary, annular or crescent nuclei, and the "bubbling" cytoplasm. Moreover, its apoptotic rate was significantly higher than that of the control group (P＜0.05).CONCLUSION Biological active TGF β protein is extracted and purified successfully from bovine platelets, and it is able to induce the apoptosis of hepatocytes.

DNA Damage,Apoptosis and C-myc,C-fos,and C-jun Overexpression Induced by Selenium in Rat Hepatocytes

Objective To study the effects of selenium on DNA damage, apoptosis and c-myc, c-fos, and c-jun expression in rat hepatocytes. Methods Sodium selenite at the doses of 5, 10, and 20 μmol/kg was given to rats by i.p. and there were 5 male SD rats in each group. Hepatocellular DNA damage was detected by single cell gel electrophoresis （or comet assay）. Hepatocellular apoptosis was determined by TUNEL （TdT-mediated dUTP nick end labelling） and flow cytometry. C-myc, c-fos, and c-jun expression in rat bepatocytes were assayed by Northern dot hybridization. C-myc, c-fos, and c-jun protein were detected by immunohistochemical method. Results At the doses of 5, 10, and 20μmol/kg, DNA damage was induced by sodium selenite in rat hepatocytes and the rates of comet cells were 34.40%, 74.80%, and 91.40% respectively. Results also showed an obvious dose-response relationship between the rates of comet cells and the doses of sodium selenite （r=0.9501, P〈0.01）. Sodium selenite at the doses of 5, 10, and 20μmol/kg caused c-myc, c-fos, and c-jun overexpression obviously. The positive brown-yellow signal for proteins of c-myc, c-fos, and c-jun was mainly located in the cytoplasm of bepatocytes with immunohistocbemical method. TUNEL-positive cells were detected in selenium-treated rat livers. Apoptotic rates （%） of selenium-treated liver cells at the doses of 5, 10, and 20 μmol/kg were （3.72±1.76）, （5.82±1.42）, and （11.76±1.87） respectively, being much higher than those in the control. Besides an obvious dose-response relationship between apoptotic rates and the doses of sodium selenite （r=0.9897, P〈0.01）, these results displayed a close relationship between DNA damage rates and apoptotic rates, and the relative coefficient was 0.9021, P〈0.01. Conclusion Selenium at 5-20μmol/kg can induce DNA damage, apoptosis, and overexpression of c-myc, c-fos, and c-jun in rat hepatocytes.

Effects of Cadmium on Telomerase Activity,Expressions of TERT,C-myc and P53,and Apoptosis of Rat Hepatocytes

This study investigated the effect of cadmium on the telomerase activity,the expression of TERT,c-myc and p53 and the apoptosis of rat hepatocytes.The rats were administrated 5,10 and 20 μmol/kg cadmium chloride intraperitoneally and sacrificed 48 h after the initial treatment.The telomerase activity of the rat hepatocytes was measured by the telomeric repeat amplification protocol (TRAP),and apoptosis was detected by flow cytometry.The mRNA expressions of TERT,c-myc and p53 were measured by reverse transcription-polymerase chain reaction (RT-PCR).C-myc and P53 proteins were determined by immunochemistry.The results showed that cadmium chloride increased the hepatocellular telomerase activity in a dose-dependant manner and induced the apoptosis of hepatocytes significantly.The value of relative coefficient between the telomerase activity and the apoptosis rate was 0.9398.RT-PCR revealed that specific bands corresponding to the TERT mRNA,c-myc mRNA,and p53 mRNA were displayed at 185,342 and 538 bp respectively.Cadmium chloride could substantially increase the mRNA expressions of TERT,c-myc and p53 in rat hepatocytes,as compared with control.Moreover,cadmium chloride at the doses of 5,10 and 20 μmol/kg could increase the content of P53 protein in rat hepatocytes obviously,but only that at the doses of 10 and 20 μmol/kg substantially promoted the c-myc protein level in rat hepatocytes.Our study herein suggested that cadmium may contribute to the carcinogenesis by activating telomerase,and overexpressing the mRNAs of TERT,c-myc and p53,and causing apoptosis of normal cells.

Anti-oxidative role of quercetin derived from Allium cepa on aldehyde oxidase(OX-LDL)and hepatocytes apoptosis in streptozotocininduced diabetic rat

Objective:To study the role of Quercetin in streptozotocin-induced diabetes in rals.Methods:Wistar male rat(n=40) were allocated into three groups,control group(n=10) and Quercetin(QR) group received 15 mg/kg(IP) QR.(n=10),and diabetic group that received 55 mg/kg(IP)streptozotocin(STZ)(n=20) which was subdivided to two groups of 10;STZ group and treatment group.Treatment group received 55mg/kg(IP) STZ plus 15 nig/kg QR,daily lor 4 weeks,respectively:however,the control group just received an equal volume of distilled water dailv(IP).Diabetes was induced by a single(IP) injection of streptozotocin(55 mg/kg).Animal-were kept in standard condition.Twenty-eight days after inducing diabetic,5 mL blood were collected for TAC,MPA and Ox-LDL levels and liver tissues of rat in whole groups were removed then prepared for apoptosis analysis by Tunel method.Results:Apoptotic cells significantly decreased in group that has received 15 mg/kg(IP) Quercetin(P<0.05) in comparison to experimental groups(P<0.05).Conclusions:Since in our study 15 nig/kg(IP) Quercetin have significantly Preventive ellecl on liver cells damages by reducing number of apoptotic cells in Liver,so it seems that using it can be effective for treatment in diabetic rat.

DNA damage,apoptosis and cell cycle changes induced by fluoride in rat oral mucosal cells and hepatocytes

AIM： To study the effect of fluoride on oxidative stress, DNA damage and apoptosis as well as cell cycle of rat oral mucosal cells and hepatocytes.METHODS： Ten male SD rats weighing 80N120 g were randomly divided into control group and fluoride group, 5 animals each group. The animals in fluoride group had free access to deionized water containing 150 mg/L sodium fluoride （NaF）. The animals in control group were given distilled water. Four weeks later, the animals were killed. Reactive oxygen species （ROS） in oral mucosa and liver were measured by Fenton reaction, lipid peroxidation product, malondialdehyde （MDA）, was detected by thiobarbituric acid （TBA） reaction, reduced glutathione （GSH） was assayed by dithionitrobenzoic acid （DTNB） reaction. DNA damage in oral mucosal cells and hepatocytes was determined by single cell gel （SCG） electrophoresis or comet assay. Apoptosis and cell cycle in oral mucosal cells and hepatocytes were detected by flow cytometry.RESULTS： The contents of ROS and MDA in oral mucosa and liver tissue of fluoride group were significantly higher than those of control group （P〈 0.01）, but the level of GSH was markedly decreased （P〈 0.01）. The contents of ROS, MDA and GSH were （134.73 ＋ 12.63） U/mg protein, （1.48 ＋ 0.13） mmol/mg protein and （76.38 ~ 6.71） mmol/ mg protein in oral mucosa respectively, and （143.45＋ 11.76） U/mg protein, （1.44：1：0.12） mmol/mg protein and （78.83±7.72） mmol/mg protein in liver tissue respectively. The DNA damage rate in fluoride group was 50.20% in oral mucosal cells and 44.80% in hepatocytes, higher than those in the control group （P 〈0.01）. The apop- tosis rate in oral mucosal cells was （13.63 ＋ 1.81） % in fluoride group, and （t2.76＋ 1.67） % in hepatocytes, higher than those in control group. Excess fluoride could differently lower the number of oral mucosal cells and hepatocytes at G0/G1 and S G2/M phases （P〈 0.05）.CONCLUSION： Excess fluoride can induce oxidative stress and DNA damage and lead to apoptosis and cell cycle change in rat oral mucosal cells and hepatocytes.

Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes

AIM： To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells.METHODS： HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 μg/mL G418 and named HL-7702/HBV-encoded X protein （HBx） cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively.RESULTS： RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semiquantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than thosein control.CONCLUSION： HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner.