AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombin...AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intravenously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals.RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the most effective group after four times of injection of recombinant pcDNA3-ALR plasmid. The indexes of PCNA significantly increased in the recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The level of serum AST and ALT remarkably reduced in recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The results showed that the effect of 200 μg/kg recombinant pcDNA3-ALR plasmid in the rats with acute liver injury was stronger than that of 50μg/kg pcDNA3-ALR DNA.The effect of intravenous injection of recombinant pcDNA3ALR plasmid was better. After the rats with acute hepatic failure were treated with recombinant pcDNA3-ALR plasmid,the survival rate (40%) significantly increased in treatment groups compared to control group (15%, P＜0.01).CONCLUSION: The ALR gene may play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing level ofAST and ALT in CCl4-intoxicated rats.展开更多
OBJECTIVE To investigate the compatibility of the prescription Rhodiolarosea,Cordycepsmilitaris,and Rhubarb root.METHODS The DIO mice accompanying with insulin resistance,hypercholesterolemia and obesity were induced ...OBJECTIVE To investigate the compatibility of the prescription Rhodiolarosea,Cordycepsmilitaris,and Rhubarb root.METHODS The DIO mice accompanying with insulin resistance,hypercholesterolemia and obesity were induced by high fat diet(HFD)in C57BL/6mice.For evaluating the insulin sensitivity in vivo,the values of serum insulin,HOMA-IR,and the glucose infuse rate(GIR)in hyperinsulinemiceuglycemic clamp test were determined;the time-dependent blood glucose changes were measured after insulin loading in insulin tolerance test(ITT),or after glucose loading in glucose tolerance test(GTT),respectively.Recombinant human protein tyrosine phosphatase 1B(PTP1B)was overexpressed by hGST-PTP1BBL21 E.coli and purified by GST affinity chromatography.The activities of PTP1 Band glutamine:fructose 6-phosphate amidotransferase(GFAT)were measured by the pNPP and GDH method,respectively.The activity ofα-glucosidase was determined with the substrates of p-nitrophenyl-alpha-D-glucopyranoside(pNPG).The IC50 value was calculated using the software GraphPad Prism 5.RESULTS 1st,the standard for the raw materials and for the extraction process of Rhodiolarosea,Cordycepsmilitaris,and Rhubarbroot are formulated according to the inhibition on PTP1 B,α-glucosidase,and GFAT in vitro,and the improvement on impaired glucose tolerance(IGT),hypercholesterolemia,and obesity in vivo,respectively.2nd,the composition was determined by the synergistic effects of Rhodiolarosea,Cordycepsmilitaris,and Rhubarb root on insulin resistance in DIO mice.3rd,in accordance with the IC50 values from the L9(34)and L8(27)orthogonal experiments targeting on PTP1 B and α-glucosidase,respectively,the best rate of Rhodiolarosea∶Cordycepsmilitaris∶Rhubarbroot was selected.4th,the effective dose for the treatment of metabolic syndrome was assessed based on the effects on IGT,hypercholesterolemia,obesity,insulin resistance,respectively,in DIO mice.CONCLUSION The prescription compatibility of Rhodiolarosea,Cordycepsmilitaris,and Rhubarbroot with the rate of 20∶1∶1was formed to treat the metabolic syndrome,such as IGT,hypercholesterolemia,and obesity,with the effective dose of 200 mg·kg-1 by the major mechanism of the improvement on insulin sensitivity.Monarch drug,Rhodiola,can clear away the lung-heat,tonify Qi,resolve stasis and nourish the heart.Adjuvant drug,Cordycepsmilitaris,can tonify the lung Qi and the kidney essence,strong waist and knee,compatibility with Rhodiola to enhance the function of invigorating lung and kidney.Assistant drug,Rhubarb,can clear heat,detoxify,remove blood stasis.These three herbal are compatible to show the effects of tonifying Qi,nourishing essence,cleaning heat,reducing phlegm and resolving masses for the treatment of metabolic syndrome.展开更多
基金Supported by the Natural Science Foundation of Hebei Province, No. 302489
文摘AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intravenously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals.RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the most effective group after four times of injection of recombinant pcDNA3-ALR plasmid. The indexes of PCNA significantly increased in the recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The level of serum AST and ALT remarkably reduced in recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The results showed that the effect of 200 μg/kg recombinant pcDNA3-ALR plasmid in the rats with acute liver injury was stronger than that of 50μg/kg pcDNA3-ALR DNA.The effect of intravenous injection of recombinant pcDNA3ALR plasmid was better. After the rats with acute hepatic failure were treated with recombinant pcDNA3-ALR plasmid,the survival rate (40%) significantly increased in treatment groups compared to control group (15%, P＜0.01).CONCLUSION: The ALR gene may play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing level ofAST and ALT in CCl4-intoxicated rats.
基金The project supported by National Major Special Project on New Drug Innovation of China(2009ZX09103-4322012ZX09103-101-063+1 种基金2012ZX09301002-004)We also appreciate the support of Novo Nordisk Union Diabetes Research Talent Fund
文摘OBJECTIVE To investigate the compatibility of the prescription Rhodiolarosea,Cordycepsmilitaris,and Rhubarb root.METHODS The DIO mice accompanying with insulin resistance,hypercholesterolemia and obesity were induced by high fat diet(HFD)in C57BL/6mice.For evaluating the insulin sensitivity in vivo,the values of serum insulin,HOMA-IR,and the glucose infuse rate(GIR)in hyperinsulinemiceuglycemic clamp test were determined;the time-dependent blood glucose changes were measured after insulin loading in insulin tolerance test(ITT),or after glucose loading in glucose tolerance test(GTT),respectively.Recombinant human protein tyrosine phosphatase 1B(PTP1B)was overexpressed by hGST-PTP1BBL21 E.coli and purified by GST affinity chromatography.The activities of PTP1 Band glutamine:fructose 6-phosphate amidotransferase(GFAT)were measured by the pNPP and GDH method,respectively.The activity ofα-glucosidase was determined with the substrates of p-nitrophenyl-alpha-D-glucopyranoside(pNPG).The IC50 value was calculated using the software GraphPad Prism 5.RESULTS 1st,the standard for the raw materials and for the extraction process of Rhodiolarosea,Cordycepsmilitaris,and Rhubarbroot are formulated according to the inhibition on PTP1 B,α-glucosidase,and GFAT in vitro,and the improvement on impaired glucose tolerance(IGT),hypercholesterolemia,and obesity in vivo,respectively.2nd,the composition was determined by the synergistic effects of Rhodiolarosea,Cordycepsmilitaris,and Rhubarb root on insulin resistance in DIO mice.3rd,in accordance with the IC50 values from the L9(34)and L8(27)orthogonal experiments targeting on PTP1 B and α-glucosidase,respectively,the best rate of Rhodiolarosea∶Cordycepsmilitaris∶Rhubarbroot was selected.4th,the effective dose for the treatment of metabolic syndrome was assessed based on the effects on IGT,hypercholesterolemia,obesity,insulin resistance,respectively,in DIO mice.CONCLUSION The prescription compatibility of Rhodiolarosea,Cordycepsmilitaris,and Rhubarbroot with the rate of 20∶1∶1was formed to treat the metabolic syndrome,such as IGT,hypercholesterolemia,and obesity,with the effective dose of 200 mg·kg-1 by the major mechanism of the improvement on insulin sensitivity.Monarch drug,Rhodiola,can clear away the lung-heat,tonify Qi,resolve stasis and nourish the heart.Adjuvant drug,Cordycepsmilitaris,can tonify the lung Qi and the kidney essence,strong waist and knee,compatibility with Rhodiola to enhance the function of invigorating lung and kidney.Assistant drug,Rhubarb,can clear heat,detoxify,remove blood stasis.These three herbal are compatible to show the effects of tonifying Qi,nourishing essence,cleaning heat,reducing phlegm and resolving masses for the treatment of metabolic syndrome.
