A Study Of Modulation Of Augmenter Of Liver Regeneration On Cell Proliferation And Its Relationship Between Na～+, K～+-ATPase

Augmenter of liver regeneration (ALR) is a cytosine which can specifically protect liver function and stimulate hepatocyte cell proliferation. It's very possible to become a new genetic medicine. Previous research elucidated that ALR could interact with Na⁺, K⁺-ATPase directly in vitro and in vivo. However, the relationship about ALR and Na⁺, K⁺-ATPase in promoting hepatocyte cell proliferation has not been understand now. This article focused on the relationship of ALR and Na⁺, K⁺-ATPase in modulating cell proliferation. The conclusions were made as follows:In cell level, the effects of recombinant ALR protein on promoting cell proliferation were analyzed by MTT, [³H]-TdR and FACS. ALR could accelerate the DNA synthesized rate, change the cell cycle, and promote the hepatocyte proliferation in concentrate dependent effect. The work concentration was originated at 50μg/L, and the fittest concentration ranged from 100μg/L to 200μg/L. Anti-ALR mono-cloned antibody could block the effect of ALR on hepatocyte cell proliferation. As to other cells in this study, ALR would not induce cell proliferation. The proliferation caused by ALR had been obstructed by low Na⁺, K⁺-ATPase activity in HepG2 cell. When the Na⁺, K⁺-ATPase activity was partially inhibited, the proliferation was inhibited also, if the Na⁺, K⁺-ATPase activity was completely inhibited, ALR could not induced the proliferation.Both the measurement of Na⁺, K⁺-ATPase activity and the protein immunoblot (western blot) identified that ALR stimulated the activation of Na⁺, K⁺-ATPase on HepG2 cells in concentration-time dependent effect. The half maximum stimulation concentration was 42±11μg/L, the maximum Na⁺, K⁺-ATPase activity was obtained at 5 min after ALR stimulation. The mechanism of Na⁺, K⁺-ATPase activation caused by ALR was fit for a Michaelis-Menten equation model on HepG2 cell. ALR affected the turnover rate of Na⁺, K⁺-ATPase, the V_max increased from 0.84±0.11 μmol·mg^-1·min^-1 to 1.68±0.07μmol·mg^-1·min^-1, the K_m decreased from 23.54 mmol/L±0.12 to 20.86±0.13 mmol/L. Phosphorylation of serine or threonine were the key factor of Na⁺, K⁺-ATPase activation. Anti-ALR mono-cloned antibody could block the effect of ALR on Na⁺, K⁺-ATPase activation.Fluorescence probe analysis showed that mitochondria membrane potential and intracellular [Ca²⁺] were increased after the treatment of ALR on HepG2 cell, while the intracellular ROS level was decreased. ALR would influence the down-regulation of MMP and intracellular [Ca²⁺] which were caused by the partly...