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Objective To explore whether augmenter of liver regeneration (ALR) can serve as early diagnostic biomarker in the patients with acute kidney injury(AKI).Methods The inpatients with possibility to AKI in the central ICU of Second Affiliated Hospital of Chongqing Medical University from October 2014 to October 2015 were recruited and assigned to the AKI group and non-AKI group according to the KDIGO guidance.Blood and urine ALR and blood creatinine were detected at 0,6,12,24,48,72 h after entering the group.Results Among all cases,40 cases(62.5％) developed to AKI.Blood ALR and urine ALR at 6h after entering the group in the AKI group began to significantly increase compared with the non-AKI group(P＜0.05),the blood and urine ALR levels reached to the peak value at 12,24 h after entering the group;the blood creatinine level at 12 h after entering the group in the AKI group began to significantly increase compared with the non-AKI group(P＜0.05),blood creatinine level was still slowly and progressively elevated at 72 h after entering the group.Conclusion Serum and urine ALR levels are significantly increased in the early stage of AKI,which indicates that ALR may be a new type biomarker for diagnosing AKI.
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Augmenter of liver regeneration (ALR) is a thermostable cytokine that was originally identified to promote the growth of hepatocytes. This study was conducted to explore the expression and function of ALR in multiple myeloma (MM), a common hematologic malignancy. Real-time PCR and western blot analysis were performed to detect the expression of ALR in U266 human MM cells and healthy peripheral blood mononuclear cells (PBMCs). U266 MM cells were exposed to 20 or 40 μg/mL of recombinant ALR and tested for cell proliferation. Small interfering RNA-mediated silencing of ALR was done to investigate the role of ALR in cell proliferation, apoptosis, and cytokine production. Compared to PBMCs, U266 MM cells exhibited significantly higher levels of ALR at both the mRNA and protein levels. The addition of recombinant ALR protein significantly promoted the proliferation of U266 cells. In contrast, knockdown of ALR led to a significant decline in the viability and proliferation of U266 cells. Annexin-V/PI staining analysis demonstrated that ALR downregulation increased apoptosis in U266 MM cells, compared to control cells (20.1±1.1 vs 9.1±0.3%, P<0.05). Moreover, ALR depletion reduced the Bcl-2 mRNA level by 40% and raised the Bax mRNA level by 2-fold. Additionally, conditioned medium from ALR-depleted U266 cells had significantly lower concentrations of interleukin-6 than control cells (P<0.05). Taken together, ALR contributed to the proliferation and survival of U266 MM cells, and targeting ALR may have therapeutic potential in the treatment of MM.
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Humans , Apoptosis/drug effects , Cell Proliferation/drug effects , Multiple Myeloma/metabolism , Proteins/pharmacology , Blotting, Western , Cell Line, Tumor , Cytokines/biosynthesis , Down-Regulation , Flow Cytometry , Leukocytes, Mononuclear/metabolism , Multiple Myeloma/immunology , Proteins/physiology , Real-Time Polymerase Chain Reaction , Recombinant Proteins/pharmacology , RNA, Small Interfering/metabolismABSTRACT
Objective To construct recombinant adenovirus expressing augmenter of liver regeneration (ALR) gene and investigate its anti-apoptosis effect. Methods Recombinant shuttle vector pAdTrack-TO4-mALR and pAdTrack-TO4-hALR were constructed by recombinant DNA technology and recombinant adenovirus Ad-GFP-mALR and Ad-GFP-hALR were acquired by homologous recombination. After four cycles of amplification, high titers of the recombinant adenovirus Ad-GFP-mALR and Ad-GFP-hALR were obtained. The expression of green fluorescent protein was detected to evaluate the infection efficiency of Ad-GFPmALR and Ad-GFP-hALR, Western blotting was applied to assess the expression of ALR and Bcl-2/Bax protein, and CCK-8 assay was used to evaluate the proliferation activity of L02 cells when L02 cells were infected with either Ad-GFP-mALR or Ad-GFPhALR. Flow cytometry was utilized to detect the L02 cells' apoptosis rate induced by palmitic acid (PA). Results pAdTrack-TO4-mALR, pAdTrack-TO4-hALR, Ad-GFP-mALR and Ad-GFP-hALR were constructed successfully; the recombinant adenovirus Ad-GFP-mALR and Ad-GFP-hALR could infected L02 cells efficiently and expressed steadily in L02 cells. The L02 cells infected with either Ad-GFP-mALR or Ad-GFP-hALR had significantly increased proliferation activity compared with those uninfected and those infected with Ad-GFP. L02 cells with the infection of either Ad-GFP-mALR or Ad-GFP-hALR had significantly lower apoptosis rate, enhanced the Bcl-2 expression and decreased the Bax expression when L02 cells were treated with palmitic acid compared with those uninfected and those infected with Ad-GFP. Conclusion Recombinant adenovirus Ad-GFP-mALR and Ad-GFP-hALR here have been constructed successfully and the over expression of either mALR or hALR has the ability to promote the proliferation activity of L02 cells and the effect against L02 cell apoptosis induced by palmitic acid.
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Objective To investigate the expression and clinical significance of augmenter of liver regeneration (ALR) in patients with HBV related acute on chronic liver failure (ACLF) .Methods The serum and clinical data of patients with ACLF (ACLF group ,n=214) ,patients with mild chronic hepatitis B (mild chronic hepatitis B group ,n=196) were collected from outpatient and inpatient in the hospital ,and control group(n=200) people were the blood transfusion healthy blood donors .The level of ALR was measured by ELISA method .The correlation between ALR and MELD score of patients with ACLF were analyzed by linear regres-sion analysis .Unconditioned binary response logistic regression model was used to determine the correlation between ALR and mor-tality at 24 weeks of patients with ACLF .Results Serum ALR level was higher in ACLF group than in mild chronic hepatitis B group and control group(P<0 .05) .There were negative correlations between the serum ALR level and MELD score of patients with ACLF(r2 = -0 .249 ,F=13 .955 ,P<0 .01) .Serum ALR level of patients with ACLF was more significant in survival group than in dead group(P=0 .004) .Logistic regression analysis identified that high serum ALR level was related to the good prognosis (P=0 .012 ,OR=0 .807) .Conclusion The serum ALR level was significantly increased in patients with HBV related ACLF which played an important role in liver regeneration and improve the prognosis of patients .
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Objective To investigate the effects of recombinant human augmenter of liver regeneration (rhALR) on renal inflammation in acute kidney injury (AKI) induced by renal ischemia reperfusion (IR). Methods SD rats were randomly divided into sham-operated group,IR group,rhALR1 group (100 μg/kg) and rhALR2 group (200 μg/kg).Both renal pedicles of rats were identified and occluded with microvascular clamps for 60 min to induce acute kidney injury (AKI).Blood urea nitrogen and serum creatinine levels were evaluated using a Hitachi 747 automatic analyzer. For histological examination, sections were stained with HE. The activity of myeloperoxidase (MPO) was detected by spectrophotometer.Expression of TNF-α,ICAM-1,MCP-1 was determined by Western blotting. Results Blood urea nitrogen,serum creatinine levels and the injury of kidney were improved significantly in rhALR group as compared with IR group (all P＜ 0.05).They were improved more significantly in rhALR2 group as compared to in rhALR1 group (all P＜0.05).The protein levels of TNF-α,ICAM-1,MCP-1 and the activity of MPO in kidneys from the sham-operated rats were low,and increased significantly after renal ischemia reperfusion injury (all P＜0.05).After treated with rhALR,the expression of TNF-α,ICAM-1,MCP-1 and the activity of MPO were decreased significantly in kidneys as compared to those in IR group (all P＜0.05),which decreased more significantly in rhALR2 group than those in rhALR1 group (all P＜ 0.05). Conclusions nhALR can protect kidneys from ischemia reperfusion injury in rats.The mechanism may be associated with the inhibition of renal inflammatory cells infiltration and down-regulated expressions of YNF-α,ICAM-1 and MCP-1 in the kidney.
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Objective: To construct recombinant expression vector of human augmenter of liver regeneration (ALR) and study its protective effect on liver function. Methods: ALR cDNA was synthesized and inserted into expression vector pET28a+. The recombinant plasmid was tranformed into BL21 and the expression of ALR was induced by isopropyl-β-D-thiogalactoside(IPTG). MTT method was used for cell proliferation assay; the protective effect of recombinant product on liver function was observed in CCl4-induced acute toxic mouse model. Results: Recombinant expression plasmid of ALR was confirmed correct by restriction enzyme digestion and sequencing. The purified expression product had strong stimulatory effect on hepatocyte proliferation. Low and medium dosages of expression product decreased aminotransferase level in acute chemical injury mouse model. Conclusion: The recombinant expression vector of ALR has been correctly constructed and the expressed rALR can simulate hepatocyte regeneration.
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Objective To investigate the protective effects of recombination rat augmenter of liver regeneration (rrALR) on tubular cell injury and renal dysfunction in rats with acute renal failure (ARF)induced by gentamicin. Methods One hundred fifty female Wistar rots were randomly divided into 5 groups: group A (control), group B (gentamincin 140 mg/kg+ saline 100 μg/kg), group C (gentarnincin 140 mg/kg +blank plasmid 100 μg/kg), group Dl (gentaminein 140 mg/kg+rrALR 80 μg/kg), group D2 (gentamincin 140 mg/kg +rrALR 160 μg/kg). Rats were sacrificed at day 4, 8, 12, 16 and 21 after gentamicin first injection. Blood urea nitrogen (BUN), serum creatinine (Scr) and urine N-aeetyl-β-D-glucosaminidase (UNAG) were measured. The histopathologic changes were observed by periodic aeid-Schiff (PAS) staining. Protein expression of ALR and PCNA in renal tissue was detected using immtmohistochemistry and Western blot. Results Compared with control rots, the levels of BUN, Scr and UNAG were significantly increased in ARF rats at day 4, 8, 12 and 16 (P<0.05), however, the renal dysfunction and the renal histopathologieal injury were significantly improved in ARF rats simultaneously administered with rrALR (group D) compared with ARF rats (group B and C). The protein level of ALR, the number of PCNA positive tubular ceils and the proliferation index (PI) in group D were significantly higher than those of group B and C (P<0.05). Conclusion rrALR can promote the regeneration of tubular cells in nephrotoxie ARF rats and ameliorate renal dysfunction.
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Objective To study the relationship between CXXC motif and biologic activity of human augmenter of liver regeneration protein(hALRp).Methods The function of CXXC motif and its relationship with bioactivity were investigated between hALRp and hALRp-C65A groups by detecting the sulfhydryl oxidase activity;the interaction of target protein with GST-Na~+,K~+-ATPase fusion protein was examined by GST-Pulldown and MTT was used to study the ability of hALRp-C65A to promote HL-7702 hepatic cells proliferation in vitro.Results The turnover number(TN) of hALRp in sulfhydryl oxidase activity assay was 1.25?0.21,while TN was 0 of hALRp-C65A,there was significant difference between them(P
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Objective:To detect the existence of expression of human augmenter of liver regeneration(hALR) in HepG2 cells,and to observe proliferation of HepG2 cells after neutralizing hALR with anti-hALR McAb.Methods:the expression of hALR in HepG2 cells was observed immunocytochemistry and reverse transcriptive PCR(RT-PCR).Proliferation of HepG2 cells after neutralization was detected by ~3?H-TdR incorporation approach.Results:① hALR was expressed by HepG2 cells.② Anti-hALR McAb inhibited partially the autonomous growth of HepG2 cells.Conclusion:Anti-hALR McAb inhibit the autonomous growth of hepatoma cells partially;moreover,hALR maintain the autonomous growth of hepatoma cells in vitro through autocrine mechanism.
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Objective To Investigate the mechanism of augmenter of liver regeneration (ALR) in promoting proliferation of damaged hepatocyte. Methods The inhibitory effects of ALR on the expression of TGF-?_1 in hepatic stellate cell and Kupffer cell were studied by immunohistochemistry and Western blot. The effects of hepatic stellate cell (Ito cell) conditioned medium (ICCM+) and Kupffer cell conditioned medium (KCCM+) prepared by treatment of using augmenter of liver regeneration (ALR) on damaged hepatocytes proliferation were studied by MTT. The antagonistical role of ALR on TGF-?_1, which inhibited damaged hepatocyte proliferation was investigated by MTT determination. Results Immunoreactive positive signal of TGF-?_1 in hepatic stellate cell and Kupffer cell stimulated by ALR were decreased. Immunolabeling of TGF-?_1 in hepatic stellate cell stimulated by ALR was weakened. The proliferation of damaged hepatocytes was increased significantly by administration of ICCM and KCCM. ALR could reverse the inhibitory role of TGF-?_1 on the proliferation of damaged hepatocyte. Conclusion ALR can promote proliferation of injured hepatocyte indirectly by inhibiting expression of TGF-?_1 in hepatic stellate cell and Kupffer cell.
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Objective To investigate the influence of recombinant human augmenter of liver rege neration (hALR) on stromelysin 1 gene expression in experimental liver fibrosis. Methods Two kinds of rat model of experimental liver fibrosis induced by CCl 4 and albumin were established and different dosages of recombinant human augmenter of liver regeneration were given during the process of model making. Total RNA of liver tissues was extracted and stromelysin 1 gene expression levels were measured by reverse transcription polymerase chain reaction (RT PCR). Results In both rat models of experimental liver fibrosis, stromelysin 1 gene expression levels in hALR treating group were significantly higher than those of model groups in different periods of model forming. Stromelysin 1 gene expression levels in high dose hALR treated group were significantly higher than those of low dose hALR treated group.Conclusion Recombinant human augmenter of liver regeneration may have effects of promoting gene expression of stromelysin 1 in experimental liver fibrosis.
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Hepatic stellate cell (HSC-T) was cultured in medium containing different concentrations of recombinant human augmenter of liver regeneration (hALR),and cells were collected at different incubation period. Total RNA of HSC was isolated and collagenⅠ and Ⅲ gene expression levels were measured with reverse transcription polymerase chain reaction. The results showed that, collagen Ⅰ gene expression levels of HSC in high(0 2ng/L),middle (0 02ng/L) and low (0 002ng/L) concentrations of three hALR groups were much lower than those of control group after exposure to hALR at 8h, 24h, 48h and 72h. Collagen Ⅰ gene expression levels of HSC in high dose group were also significantly lower than those of middle and low dose groups . Collagen Ⅲ gene expression levels of HSC in middle and low dose hALR group were much lower than those of control group at 24h,48h and 72h, Collagen Ⅲ gene expression levels in high dose group were significantly lower than those of control group at 8h, 24h, 48h and 72h, and were also much lower than those of middle and low dose groups. It suggested that hALR could inhibit collagen Ⅰ and Ⅲ gene expression in hepatic stellate cells.
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To investigate the influence of recombinant human augmenter of liver regeneration (hALR) on stromelysin 1 gene expression in rats with fibrotic liver. CCl 4 or albumin induced liver fibrosis in rats was established, and different dosages of recombinant human augmenter of liver regeneration were given to rats with liver fibrosis.Liver specimens were obtained at different intervals of treatment , total RNA of liver tissues were isolated and stromelysin 1 gene expression was measured by reverse transcription polymerase chain reaction (RT PCR) . The results showed that in both rat models of experimental liver fibrosis , stromelysin 1 gene expression levels were significantly higher in hALR treated rats than those without the treatment at various intervals. Stromelysin 1 gene expression levels in high dose hALR treatment group were significantly higher than that in low dose hALR treatment group. It suggested that recombinant human augmenter of liver regeneration may enhance stromelysin 1 gene expression in rats with fibrotic liver.
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Objective:To prepare monoclonal antibody(McAb) against human augmenter of liver regeneration(hALR) by hybridoma technique with unpurified recombinant protein expressed by E.coli as screening antigen.Methods:BALB/c mice were immunized with hALR thioredoxin(hALR Thi) fusion protein twice.The splenocytes were fused with myeloma cells by polyethylene glycol and the hybridomas were selected in HAT medium.The positive hybridomas secreting anti hALR McAb were screened by means of ELISA,with the lysates of E.coli containing the recombinant plasmid pQE30 hALR induced by IPTG as screening antigen and containing plasmid pQE30 as controlling antigen.The reactivity of McAb secreted by the positive hybridoma to hALR expressed in eukaryocyte and native hALR in human serum was confirmed by means of ELISA and Western blot methods.Results:A positive hybridoma was screened successfully;the specific antigen antibody reaction was confirmed between the McAb prepared and hALR expressed in eukaryocyte and native hALR in human serum.Conclusion:Without any purification procedure,recombinant protein expressed by E.coli can be used for screening bybridomas in preparing of McAb with the expressed product of empty plasmid as control;anti hALR McAb can be used to the further study of hALR.
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Objective:Identification of the antigenic determinants of hALR.Methods:Theoretic antigenic determinants of hALR was predicted by using Hopp&Wodds method and and Goldkey software package.The four polypeptides according to the amino acid sequences of the predictive linear epitopes of hALR were synthesized and were used to analyze the antigenic determinants of hALR recognized by antibodies.Results:The polypeptides corresponding to residues 6～5,68～80 and 105～112 of hALR were its antigenic determinants.Conclusion:Prediction of the protein antigenic determinants by computer program and detection of them by competitive ELISA with synthesized polypeptides is a useful method of identification of the antigenic determinants.
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Objective:To construct recombinant expression vector of human augmenter of liver regeneration(ALR)and study its protective effect on liver function.Methods:ALR cDNA was synthesized and inserted into expression vector pET28a+.The recombinant plasmid was tranformed into BL21 and the expression of ALR was induced by isopropyl-?-D-thio- galactoside(IPTG).MTT method was used for cell proliferation assay;the protective effect of recombinant product on liver function was observed in CCl_4-induced acute toxic mouse model.Results:Recombinant expression plasmid of ALR was con- firmed correct by restriction enzyme digestion and sequencing.The purified expression product had strong stimulatory effect on hepatocyte proliferation.Low and medium dodges of expression product decreased aminotransferase level in acute chemical injury mouse model.Conclusion:The recombinant expression vector of ALR has been correctly constructed and the expressed rALR can simu- late hepatocyte regeneration.
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Objective To investigate the influence of recombinant human augmenter of liver regeneration (hALR) on gelatinase A(MMP 2) gene expression in experimental liver fibrosis. Methods Two kinds of animal model of rat experimental liver fibrosis induced by CCl 4 and albumin were established and different dosage of hALR (10,50 ?g?kg -1 ?d -1 )was given during the process of model making. Total RNA of liver tissues was isolated and MMP 2 gene expression levels were measured by reverse transcription polymerase chain reaction (RT PCR). Results In both rat models of experimental liver fibrosis, MMP 2 gene expression levels in two hALR preventing group were significantly lower than those of model group in different periods of model forming. MMP 2 gene expression levels in high dose of hALR treating group were significanfly lower than those of low dose hALR treating group. Conclusion hALR may have an effect on inhibiting gene expression of MMP 2 in experimental liver fibrosis.
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Augmenter of liver regeneration(ALR)is a novel hepatic stimulator. Owing to its peculiar characteristic of gene structure and many kinds of biological functions, it is thought to be an important hepatotrophic factor. This review summarized the advance in ALR research in recent years including characteristics of ALR gene, molecular structure, physiochemical characteristics, biological functions and roles in liver injury.
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AIM: To investigate the mechanisms of augmenter of liver regeneration (ALR) in promoting damaged hepatocyte proliferation.METHODS: The effects of Kupffer cell condition medium (KCCM+) stimulated by ALR on damaged hepatocyte proliferation were studied by MTT. The localization of ALR binding to Kupffer cell membrane and in intact rat liver was studied by immunohistochemistry. The IL-6 expression in Kupffer cells stimulated with ALR was observed by immunohistochemistry. RESULTS: The proliferation of damaged hepatocytes stimulated with KCCM+ was increased significantly. ALR immunostaining particles in plasm of hepatocyte were found in intact liver. The rough immunostaining particles of ALR were seen on the surface of Kupffer cell membrane. Immunostaining particles of IL-6 in Kupffer cells induced by ALR increased. CONCLUSION: ALR promotes proliferation of damaged hepatocytes indirectly by stimulating Kupffer cells.
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Objective:To construct a recombinant prokaryotic expression vector pQE30-hALRIP for further research.Method:PCR was used to amplify hALRIP codon region.The gene was inserted into pMD18-T vector and sequenced.Then the prokaryotic expression vector pQE30-hALRIP was constructed and identified. Results:Clonal recombinant vector pMD18- hALRIP and expression vector pQE30-hALRIP were constructed. DNA sequence analysis and restriction analysis showed both of the vectors were the same as the predicted results. Conclusion:Prokaryotic expression vector pQE30-hALRIP is successfully constructed.