ABSTRACT
Objective To explore the molecular mechanisms of hepatic stimulator substance (HSS) gene knockout in promoting the development and progression of nonalcoholic steatohepatitis (NASH).Methods NASH model mice (n=20) with HSS wild-type (HSS+/+) or HSS gene knockout (HSS-/-) were constructed using modified choline-deficient diet (CD-diet),untreated C57BL6-HSS-/-and C57BL6-HSS+/+ mice (n=20) were considered as control.Ten mice of each group were killed at month 1 and 2,respectively.The levels of triglyceride (TG) and total cholesterol (TC) in liver were measured using ELISA method.Histopathology and collagen deposition in liver tissue were observed using HE staining and Masson staining,respectively.Lipid content in liver tissue was observed and calculated using oil red O staining.The levels of mRNA and proteins of peroxisome proliferators activated receptor gama coactivator 1 alpha (PGC-1α),mitochondrial transcription factor A (TFAM),transcription factor-E2 related factor α (Nrf2),[-loop,dynamin-related protein 1 (Drp1),mitochondrial fission 1 protein (Fis1),mitofusins 1 (Mfn1),autophagy related gene 3 (Atg3) in liver tissue were detected using Real-time PCR and Western blot,respectively.Content of malonaldehyde (MDA),cyclooxygenase Ⅳ (COX Ⅳ) and adenosine tirphosphate (ATP) were measured using kits,and the activity of respiratory chain complex Ⅴ and cytochrome C oxidase in liver tissue were measured using spectrophotometry.the comparison between groups was done by t test.Results The levels of HSS mRNA and protein in mice-HSS-/-were 0.154± 0.04 and 0.08± 0.01,respectively,which were both significantly lower than those in mice-HSS+/+ (0.952 ± 0.08 and 1.362±-0.130,respectively),and t he differences had statistical significance (t =10.244 and 10.375,respectively,both P＜0.05).One month and 2 months after NASH modeled,TC contents in mice-HSS-/ were (248.6±21.5) μmol/g and (217.4±18.0) μmol/g,respectively,which were both remarkably higher than those in mice-HSS+/+ [(153.5 ± 11.2) μmol/g and (140.8 ±7.5) μmol/g,respectively],and the differences had statistical significance (t=15.270 and 10.524,respectively,both P＜0.05).The results form HE staining,oil red O staining and Masson staining indicated that fat deposition,collage deposition and inflammation in liver tissues of mice-HSS-/-were severer than those in mice-HSS+/+.One month after NASH modeled,protein levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/ were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=10.705,24.072,9.892 and 17.540,respectively,all P＜ 0.05).Two months after NASH modeled,protein levels of Drp1,Fis1,Mfn1and Atg3 in liver tissues of mice-HSS-/ were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=125.378,15.926,34.330 and 13.437,respectively,all P＜0.05).One month after NASH modeled,mRNA levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/-were all significantly decreased compared with those in mice-HSS+/+,the differences had statistical significance (t=36.337,40.825,33.508 and 28.104,respectively,all P＜0.05).Two months after NASH modeled,mRNA levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/-were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=35.210,42.375,27.753 and 20.560,respectively,all P＜0.05).The protein levels of PGC-1α,TFAM,Nrf2 and D-loop in liver of C57BL6-HSS-/-group were lower than those in liver of C57BL6-HSS+/+ group,and the differences had statistical significance (one month:t=20.548,31.036,19.445 and 10.974,respectively;two months:t=9.887,13.330,22.375 and 18.903,respectively,all P＜0.05).The mRNA levels of PGC-1α,TFAM,Nrf2 and D-loop in liver of C57BL6-HSS-/-group were all lower than those in C57BL6-HSS+/+ group,and the differences had statistical significance (one month:t=9.087,12.582,21.451 and 7.774,respectively;two months:t=23.758,17.924,9.924 and 15.209,respectively,all P＜0.05).One month and 2 months after NASH modeled,the levels of ATP mRNA in liver of C57BL6-HSS / group were both significantly lower than those in C57BL6-HSS+/+,and the differences had statistical significance 0=43.775 and 28.375,respectively,both P＜0.05);the levels of COXⅣ mRNA in liver of C57BL6-HSS / group were 0.142 ± 0.06 and 0.068± 0.001,respectively,which were both significantly lower than those in C57BL6-HSS+/+ group (0.255± 0.08 and 0.172 ±0.06,respectively),and the differences had statistical significance (t=28.337 and 19.782,respectively,both P＜0.05);the levels of MDA mRNA in liver of C57BL6-HSS-/-group were 0.973 ±0.112 and 1.253±0.054,respectively,which were both significantly lower than those in C57BL6-HSS+/+ group (0.366±0.02 and 0.872±0.05,respectively),and the differences had statistical significance (t=8.357 and 6.582,respectively,both P＜0.05).Conclusion Deletion of HSS accelerates NASH progression via inhibiting mitochondrial fusion,which leads to dysfunction of mitochondrial respiratory chain and inhibition of fatty acid oxidation.
ABSTRACT
Objective Hepatic stimulator substance(HSS)is newly defined as a growth mitogen to hepatocytes.This sutdy is aiming to investigate the effect of recombinant human hepatic stimulator substance(rhHSS) on growth of liver oval cells(OVCs). Methods OVCs were prepared from intoxicated rats with 3'-methyl-dimethylaminoazobenzene.After confirmation of OVC morphologically and histochemically,the cell cultures of OVC were exposed to various dosages of rhHSS for 12 and 24?h,respectively.The cellular proliferation was analyzed by MTT and flow cytometry. Results Administration of rhHSS(160-400 mg/L)inhibited the proliferation of OVC,as indicated by MTT and cell cycle analysis,the effect appeared non-dose dependent pattern.The peak of inhibition occurred at 240?mg/L.After incubation with 240?mg/L of HSS for 12 and 24?h,the percentages of S-phase were reduced 47.8% and 35.8% to those of the untreated cells.respectively.Conclusion rhHSS exhibits the inhibitory effect on growth of OVC.
ABSTRACT
AIM: To study the protective effect of hepatic stimulator substance(HSS) on chemotherapeutic liver damage induced by cyclophosphamide and its possible mechanism.METHODS: The serum biochemical parameters,oxidative parameters and anti-oxidative parameters of liver tissue of male Kunming mice inoculated with S_(180) cells were measured.The pathology of the liver was observed with microscope.The weight of S_(180) sarcom was weighted.RESULTS: The elevation of MDA content of liver tissue and the less of GSH content and CAT,GSH-PX,GST,and SOD activities induced by CP were restored remarkably by HSS.The ALT and LDH activities of serum in HSS group which was compared with control group and CP group were not statistically distinctive.In the pathologic examination,spotty and focal necroses of hepatocytes in the liver of CP group were found with a great deal of inflammatory cells infiltration in the necrotic and portal areas,while the damage in the liver of HSS group was ameliorated obviously;The weight of S_(180) sarcom in CP group and HSS group was not statistically distinctive.CONCLUSION: The effect of HSS on CP-induced chemotherapeutic liver damage maybe due to anti-oxidative activity.
ABSTRACT
The effects of human fetal hepatic stimulator substance (h-HSS) and liver cytosol (h-FLC) on the survival rate,the intensity of liver damage,and liver regeneration in rats with liver necrosis induced with D-galactosamine were observed.The physico-chemical properties of h-HSS were preliminarily determined.It was found that both h-HSS and H-FLC could markedly increase the.survival rate of the rats with toxic liver necrosis,significantly decrease the serum level of ornithine carbamoyl transferase and mitochondria aspartic trans-aminase,improve the hepaplastin activity,and elevate the incorporation rate of 3H-thymidine with liver DNA.h-HSS was stable after heated to 95℃ for 30 minutes,and sensitive to trypsin treatment.It demonstrated one major band at 15 000 daltons and 3 minor ones at 24 000,34 000,and 40 000 daltons respectively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The relative content of the major band was 47.57%.
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The effects of hepatic stimulator substance (HSS) extracted from wealing rats on 4 kinds of cell lines are reported.In LW13 normal liver cell line, HSS significantly increased the incorporation of 3H-TdR into liver cells, but it had no effect oo K562 erythrokukcmia cell line and primary culture of rabbit epidermal cells, the latter can be stimulated by keratinocyte growth factor at a dose of 38?g/ml. Study of response of SMMC-7721 human hepatoma cell line to HSS revealed a dose-dependent decrease in 3H-TdR incorporation.
ABSTRACT
A cytotoxicity model of primary cultured hepatocytes with D-galactosamine (D-Gal) has been established. The ALT value of culture medium was used as the marker of cell injury. Using this model, the in vitro antihepatotoxic effects of hepatic stimulator substance (HSS), isolated from regenerating adult rat liver, as well as Potenlin and 15-amino acid solution were observed. The results showed that although HSS could stimulate DNA synthesis in serum-free culture of adult rat hepatocytes, it had no effect to reduce ALT value in vitro. However the latter could be reduced by Potenlin or 15-amino acid solution in high concentrations. The described model may be useful for preliminary screening of antihepatotoxic activity of drugs.
ABSTRACT
Objective Based on the result that a gene coding for human HSS had been identified and prokaryotically expressed, this study is aiming to investigate the proliferative and protective effect of recombinant HSS on hepatoma cells. Methods hHSS protein was produced in BL\|21 strain of E.Coli containing pET\|42a vector and purified with His\5Tag affinity chromatography. A various dosages of 80\|400??g/L were administrated into cell culture. The cell proliferation was analyzed by MTT, cell\|count and flow cytometry methods. Furthermore, cellular growth signaling as indexed by phosphorylation of mitogen\|activated protein kinase (MAPK) was determined with Western blot. In addition to cell growth, protection of hHSS on the cells against H\-2O\-2 was also observed. Results It was showed that recombinaht HSS of 400??g/L was able to promote DNA synthesis by 21^5% as compared to non\|treated cells. After 24?h of HSS action, the cell division measured by MTT method as well as cell\|count was significantly enhanced. Meantime, it was indicated that the phosphorylation of MAPK Thr202/Tyr204 was increased in 79^0% as compared with that of the non\|treated cells. Pretreatment of the cells with hHSS for 12?h prior to H 2O 2 injury preserved the survival of them.Conclusion It is postulated that hHSS is an active protein to stimulate liver proliferation. [