Expression And Purification Of Extracellular Histones And Investigation On The Mechanism Of Their Cytotoxicities Related Mechanism Research

Extracellular histones,also named circulating histones,are toxic proteins released by injured,pressured or dead cells.They can be released to the lumen for many reasons and exhibit strong cytotoxicities to the cells,thus play pivotal roles in the pathogenesis and progression of many diseases such as sepsis,acute lung injuries and rheumatoid arthritis.However,due to the lack of appropriate histone standard,the extracellular histone levels in the patients' plasma can not be quantified.Therefore,classification of the disease stage and the accordingly drug dose usage cannot be calculated based on the levels of circulating histones in patients.In the present study,we expressed denatured histone H3 and H4 monomers in E.coli.After purification with affinity column,the refolded H3 and H4 monomers as well as H3/H4 complex could be obtained by either gradient dilution or dialysis.By comparing the stability of different forms of histones,we found that the H3/H4 complex,existing as(H3/H4)2 tetramer,was the most stable form of recombinant histones.Treatment of HUVECs with 50 ?g/m L recombinant H3/H4 complex could induce about 80% cell death,consistent with the result treated with 200 ?g/m L calf thymus histones.Such cytotoxicity could be inhibited by human serum albumin in a dose dependent manner(0.625 mg/m L-10 mg/m L),suggesting a native-like structure of the recombinant H3/H4 complex,as well as certain interactions between histones and albumin through a yet to be determined manner.Therefore,with antibodies of higher sensitivity,the recombinant H3/H4 complex obtained in the present study could be used as the standard for histone quantification and provide a valuable tool for histone level-based disease classification as well as the mechanism study of histone-induced cytotoxicities,Based on these,we have treated HUVECs with 10 ?g/m L recombinant histone H3/H4 complex for various periods of time,and found that,in contrast to the cell pyrotosis induced by high dose histones,low dose histone could continously induce endothelial cells death.Since caspase3 was activated after 12-hr treatment,such cell death might be apoptosis.In addition,results from Path Scan Stress and Apoptosis Signaling Antibody Array Kit suggested that proteins like AKT could also be related to histones' epithelial cytotoxicities.Evidences from cell morphology changes and the viability also suggested that 10 ?g/m L of H3/H4 is more relavent to the actual level of extracellular histones in patients with sepsis and other infectious diseases.Our findings could provide some information for further investigation on the underlying mechanisms of histone-induced cytotoxicities in clinical infectious diseases.