Preparation of hepatic stimulator substance from neonatal porcine liver by enzymatic hydrolysis and characterization of the liver proteins by LC-MS/MS bottom-up approach.

Hepatic stimulator substance (HSS) is prepared by the freeze-thaw method on a large scale, but it is time-consuming and inefficient. It is necessary to find a better method to improve the efficiency and yield of HSS. In this study, HSS was prepared by enzymatic hydrolysis of neonatal porcine liver with trypsin, papain, dispase, and alcalase. Relatively, dispase was found to be the best enzyme, based on the results of the degree of hydrolysis and MTT assay. Box-Behnken design-response surface method was used to optimize the conditions, which were as follows: enzyme (dispase) concentration, 4164 U/g; substrate concentration, 7% (w/v); hydrolysis time, 4.3 hr; temperature, 45 °C; and pH, 6.5. The degree of hydrolysis was (54.99 ± 1.57)%. Shotgun proteomics coupled with Gene Ontology (GO) analysis based on the PANTHER classification system was used to screen proteins from neonatal porcine liver. The results profiled the proteins into biological processes, molecular functions, and cellular components, thus laying a foundation for further studies on components involved in and mechanisms of liver regeneration. Overall, the results suggest that enzymatic hydrolysis might be a promising method for industrial application.

Deficiency in augmenter of liver regeneration accelerates liver fibrosis by promoting migration of hepatic stellate cell.

BACKGROUND: Augmenter of liver regeneration (ALR) protects liver from various injuries, however, the association of ALR with liver fibrosis, particularly its effect on hepatic stellate cells (HSC), remains unclear. In this study, we investigated the impact of ALR on the activation of HSC, a pivotal event in occurrence of liver fibrosis. METHODS: Liver fibrosis was induced in vivo in mice with heterozygous ALR knockdown (ALR-KD) by administration of CCl4 or bile duct ligation. The effect of ALR-KD and ALR-overexpression on liver fibrosis was studied in mice and in HSC cells as well. RESULTS: Hepatic collagen deposition and expression of α-smooth muscle actin (α-SMA) were significantly increased in the ALR-KD mice compared to wild-type mice. In vitro, ALR-shRNA resulted in the activation of HSC cell line (LX-2). Furthermore, ALR-shRNA promoted LX-2 cell migration, accompanied by increased filamentous actin (F-actin) assembly. The ALR-KD-mediated increase in HSC migration was associated with mitochondrial fusion, resulting in mitochondria elongation and enhancing ATP production. In contrast, ALR transfection (ALR-Tx) decelerated HSC migration and inhibited F-actin assembly, concomitantly enhancing mitochondrial fission and reducing ATP synthesis. Mechanically, stimulation of HSC migration by ALR-shRNA was attributed to the increased mitochondrial Ca2+ influx in HSCs. Treatment of ALR-shRNA-cells with Ruthenium Red (RuR), a specific inhibitor of mitochondrial calcium uniporter (MCU), significantly suppressed mitochondrial Ca2+ influx, HSC migration, mitochondrial fusion and ATP production. ALR-KD-induced HSC migration was verified in vitro in primary mouse HSCs. CONCLUSION: Inhibition of ALR expression aggravates liver fibrosis, probably via promoting HSC migration and mitochondrial fusion.

Molecular mechanisms of hepatic stimulator substance gene knockout in promoting development and progression of nonalcoholic steatohepatitis / 中华传染病杂志

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.

Amelioration of nonalcoholic fatty liver disease by hepatic stimulator substance via preservation of carnitine palmitoyl transferase-1 activity.

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease and so far is supposed to be related with mitochondrial impairment. Hepatic stimulator substance (HSS) has been defined as a liver-protective factor promoting hepatocyte DNA synthesis and hepatic proliferation after liver intoxication. We previously reported that HSS ameliorated hepatocyte death, probably because of its preservation of mitochondria. This study aims to explore whether HSS could protect carnitine palmitoyl transferase-1 (CPT-1), an essential enzyme responsible for ß-oxidation of free fatty acids in mitochondria, from lipotoxicity, thus alleviating hepatic lipid deposition. To test this, the HSS gene was delivered into C57BL/6J mice and efficiently expressed in the liver. NASH mice were prepared with high-fat diet or methionine-choline-deficient diet. The results showed that hepatic inflammation and liver functions were alleviated in the HSS-transfected mice; meanwhile, the activity of CPT-1 was obviously protected. Moreover, oleic acid (OA) treatment resulted in remarkable lipid accumulation in HepG2 cells; this deposition was improved by HSS transfection. Simultaneously, the CPT-1 activity, which was impaired by OA treatment, was profoundly rescued in the HSS-expressing cells. CPT-1 activity was more severely impaired if the OA treatment was combined with S15176, a CPT-1 inhibitor. However, this impairment was effectively reduced by the HSS transfection, and the effect was enhanced by C75, a CPT-1 activator. Interestingly, if the cells were transfected with HSS-siRNA, the preservation of CPT-1 provided by HSS was again diminished. In conclusion, HSS reduces lipotoxicity to mitochondria most likely via preservation of CPT-1.

Deceleration of liver regeneration by knockdown of augmenter of liver regeneration gene is associated with impairment of mitochondrial DNA synthesis in mice.

Hepatic stimulator substance, also known as augmenter of liver regeneration (ALR), is a novel hepatic mitogen that stimulates liver regeneration after partial hepatectomy (PH). Recent work has indicated that a lack of ALR expression inhibited liver regeneration in rats, and the mechanism seems to be related to increased cell apoptosis. The mitochondria play an important role during liver regeneration. Adequate ATP supply, which is largely dependent on effective mitochondrial biogenesis, is essential for progress of liver regeneration. However, ALR gene expression during liver regeneration, particularly its function with mitochondrial DNA synthesis, remains poorly understood. In this study, ALR expression in hepatocytes of mice was suppressed with ALR short-hairpin RNA interference or ALR deletion (knockout, KO). The ALR-defective mice underwent PH, and the liver was allowed to regenerate for 1 wk. Analysis of liver growth and its correlation with mitochondrial biogenesis showed that both ALR mRNA and protein levels increased robustly in control mice with a maximum at days 3 and 4 post-PH. However, ALR knockdown inhibited hepatic DNA synthesis and decelerated liver regeneration after PH. Furthermore, both in the ALR-knockdown and ALR-KO mice, expression of mitochondrial transcription factor A and peroxisome proliferator-activated receptor-γ coactivator-1α were reduced, resulting in impaired mitochondrial biogenesis. In conclusion, ALR is apparently required to ensure appropriate liver regeneration following PH in mice, and deletion of the ALR gene may delay liver regeneration in part due to impaired mitochondrial biogenesis.

Downregulation of hepatic stimulator substance during the early phase of liver regeneration inhibits E-cadherin expression in mice.

Hepatic stimulatory substance (HSS), which encodes a sulfhydryl oxidase enzyme, promotes liver regeneration (LR) and maintains the viability of hepatocytes. Surprisingly, we found that the levels of the HSS mRNA and expressed protein were both strongly repressed at 12h after a 70% partial hepatectomy (PH) in mice. Understanding the mechanism and effect of this extraordinary suppression can provide a novel path for exploring the molecular function of HSS during LR. We observed that the EGF levels in the serum were negatively correlated with HSS expression in regenerating livers. Treating primary mouse hepatocytes or Hepa1-6 cells with EGF suppressed HSS mRNA expression. This suppression was transcriptional and was mediated by the effect of EGF on the phosphorylation of CCAAT/enhancer-binding protein ß (C/EBPß), which regulates HSS expression. We further showed that the enhanced phosphorylation of C/EBPß after PH promoted its interaction with the HSS promoter and repressed HSS expression at early time-points after PH. Interestingly, the knockdown of HSS caused a dramatic decrease in E-cadherin expression in hepatocytes. E-cadherin expression was also significantly suppressed at 12h after PH. Moreover, the pre-injection of HSS-expressing adenovirus vectors prevented E-cadherin suppression after PH. Treatment with C/EBPß siRNA reversed the EGF-mediated inhibition of HSS expression and led to enhanced E-cadherin expression and reduced cell migration. Our findings suggest that C/EBPß directly inhibits the HSS promoter after PH and that this inhibition can downregulate E-cadherin expression. These data provide novel insight into the potential role of HSS in hepatic structural reconstruction during LR.

Enhanced endoplasmic reticulum SERCA activity by overexpression of hepatic stimulator substance gene prevents hepatic cells from ER stress-induced apoptosis.

Although the potential pathogenesis of nonalcoholic fatty liver disease (NAFLD) is unclear, increasing evidence indicates that endoplasmic reticulum (ER) stress may link free fatty acids to NAFLD. Since we previously reported that hepatic stimulator substance (HSS) could protect the liver from steatosis, this study is aimed to investigate whether HSS protection could be related with its inhibition on ER stress. The HSS gene was stably transfected into BEL-7402 hepatoma cells and effectively expressed in ER. The palmitic acid (PA)-induced heptocyte lipotoxicity was reproduced in the HSS-transfected cells, and HSS alleviation of the ER stress and apoptosis were subsequently examined. The results showed that PA treatment led to a heavy accumulation of fatty acids within the cells and a remarkable increase in reactive oxygen species (ROS). However, in the HSS-expressing cells, production of ROS was inhibited and ER stress-related marker glucose-regulated protein 78 (GRP-78), sterol regulatory element-binding protein (SREBP), anti-phospho-PRK-1ike ER kinase (p-PERK), anti-phospho-eukaryotic initiation factor 2α (p-eIF2α), and anti-C/EBP homologous protein (CHOP) were downregulated compared with the wild-type or mutant HSS-transfected cells. Furthermore, PA treatment severely impaired the activity of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), leading to imbalanced calcium homeostasis during ER stress, which could be rescued in the HSS-trasfected cells. The protection provided by HSS to the SERCA is identical to that observed with N-acetyl-l-cysteine (NAC) and sodium dimercaptopropane sulfonate (Na-DMPS), which are two typical free radical scavengers. As a consequence, the rate of ER stress-mediated apoptosis in the HSS-expressing cells was significantly reduced. In conclusion, the protective effect of HSS against ER stress may be associated with the removal of ROS to restore the activity of the SERCA.

THE EFFECT OF RECOMBINANT HUMAN HEPATIC STIMULATOR SUBSTANCE ON GROWTH OF LIVER OVAL CELLS / 解剖学报

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.

Protection of hepatic stimulator substance against chemotherapeutic liver damage induced by cyclophosphamide in mice inoculated with S_(180) cells / 中国临床药理学与治疗学

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.

Effects of human fetal hepatic stimulator substance on D-galac-tosamine-induced liver necrosis in rats / 第三军医大学学报

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%.

EFFECT OF HEPATIC STIMULATOR SUBSTANCE ON CELLS GROWTH IN VITRO / 第二军医大学学报

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.

THE ESTABLISHMENT AND PRELIMINARY USE OF D-GALACTOSAMINE CYTOTO- XICITY MODEL OF PRIMARILY CULTURED HEPATOCYTES / 解放军医学杂志

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.

INCREASED CELLULAR PROLIFERATION AND PROTECTION OF RECOMBINED HUMAN HEPATIC STIMULATOR SUBSTANCE / 解剖学报

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. [