Role Of Nos2 In Liver Injury In Mice

Nitric oxide synthase as isozymes has three subtype,NOS1、NOS2、NOS3,also called nNOS,iNOS,eNOS.Nitric oxide synthase 2,work and produce lots of nitric oxide when the cell is stimulated and activated.Previous investigation indicated that Nos2 play a important role in chronic neurodegenerative diseases,inflammation,obstructive diseases,tumour,implantation,damage and resistance to parasitic infections.Although the effect of Nos2 has been investigated in Nos2 knockout young mice,it remained unclear in aged mice.It becomes more and more popular using the gene knockout technology to research gene function.Gene knockout technology has become the hotspot and frontier of biology,and has the profound influence on many biological areas.With the increasing of aged population,the number of the old people with hepatopathy suffering partial liver resection has obvious rising trend.Old people PH had higher mortality and morbidity.NO synthesized by NOS2 plays an important role in the liver regeneration after partial hepatectomy（PH）.During this research,we investigated the effect of liver regeneration after partial hepatectomy in the Nos2-knockout aged mice.2/3 liver resection was performed in 24-mouth-old Nos2-knockout and wild type C57BL/6 mice;liver tissue was collected after PH.The mRNA expression of the Nos2 gene in WT mice was examined by qRT-PCR;the mRNA expression of the immediate early genes Fos and Jun,cell cycle related genes Cyclin D1,Cyclin A2,Cyclin B1 in both types of mice was examined by qRT-PCR;the proteins expression of the proinflammatory cytokines TNF-α and IL-6,cell proliferation marker Ki67,cell proliferation related proteins JNK,NF-kB,STAT3,apoptosis related proteins CASPASE3,CASPASE9,BCL2 and BAX in both types of mice were examined by Western blot analysis.As compared to WT control,at the termination stage of liver regeneration,Nos2-knockout aged mice had lower survival ratio（P=0.039）and low liver index（from 120 hours to 192 hours,p=0.020,0.047 and 0.002 respectively）;The mRNA expression of immediate early gene Fos increased in the early phase of liver regeneration but decreased in the the middle and later periods;The mRNA expression of immediate early gene Jun is no difference in the early phase of liver regeneration,but decreased in the later phase;The mRNA expression of Cyclin D1,Cyclin A2,Cyclin B1 and the protein expression of proliferation related transcription factors NF-kB,STAT3,JNK is reduced and postponed;the proteins expression of apoptosis related genes Caspase3,Caspase9 and Bax increased;the protein expression of the inhibiting apoptosis gene Bcl2 nearly has no change.Nos2-knockout in aged mice made the liver regeneration slow down due to the increasing apoptosis and the decreasing proliferation.From the morphological and biochemical point of view,the rodent’s acute liver failure induced by CCl₄ is similar to human acute liver failure.NO synthesized by NOS2 plays an important role in acute hepatic failure.In this research,we studied the effect of CCl₄-induced acute liver failure in Nos2 knockout aged mice.The 24-mouth-old Nos2-knockout mice and wild C57BL/6 mice were treated with CCl₄ to induce acute hepatic failure;the serum and liver tissue were collected in different time.The result indicated that the content of serum ALT,AST in Nos2-knockout mice was higher than wild mice at most of time points.The expression of Nos2 gene in CCl₄ treatment’s wild mice was higher than normal WT mice,which reached the peak round 16 h and reduced subsequently.The mRNA expression of inflammatory factors Tnf-a、Ifn-γ and Emr1 in Nos2-knockout aged mice was higher at two time points;the mRNA expression of IL-6、Mcp-1 and Ccr2 in Nos2-knockout aged mice was higher at three time points.H.E.staining showed lipid droplets at the later stages in the Nos2-knockout aged mice were bigger and had more quantity than WT mice.Western blot analysis showed the protein expression of the liver function gene SOD2 and BCHE in Nos2-knockout mice was lower than wild mice.As compared with WT mice,proteins expression of inhibitory apoptosis gene Bcl2 was lower at two time points,but the protein expression of pro-apoptotic gene BAX was higher at three time points in Nos2 KO mice.Detecting the change of glutathione which has antioxidant and relieving liver poisoning function found that in Nos2 KO mice its concentration is lower than that in WT mice at later phase.Nos2 knockout exacerbated CCl₄-induced aged mice’s acute liver injure,which mainly is through the increased expression of the inflammatory factors and apoptosis related genes,decreased expression of liver function protein and reduced glutathione.