Recombinant Human Intestinal Alkaline Phosphatase Expression And Regulation Of TNF-? And IL-6 Release From Human Leukocytes

Leukocytes play an important and understated role in mediating human inflammatory diseases.The ability of leukocytes to migrate into disease sites and secrete pro-inflammatory cytokines,such as TNF-? and IL-6,is of particular interest for developing therapeutic approaches to treat inflammatory pathologies.LPS is a toxic component of the outer membrane of Gram-negative bacteria.It can enter the blood circulation and bind to TLR4 receptors to induce inflammatory cells and vascular endothelial cells to release the key inflammatory factor TNF-?,and activate the inflammatory cascade.The use of human intestinal alkaline phosp hatase(IAP)has already been validated as a novel treatment for endotoxin lipopolysaccharide(LPS)-induced inflammatory diseases,which is mediated through IAP' s ability to detoxify LPS.It is unclear whether IAP has a role in treating inflammatory diseases that are not dependent on LPS(that is,not through TLR4 receptors).To investigate the effect of IAP on the release of human leukocyte inflammatory factors TNF-? and IL-6 and the non-LPS-dependent signaling pathway of IAP.Firstly,high-purity recombinant human intestinal alkaline phosphatase(rec IAP)was prepared using the CHO expression system.Secondly,we use freshly extracted human leukocytes to study effects of a recombinant human intestinal alkaline phosphatase(rec IAP)on secretion of TNF-? and IL-6 by the leukocytes in absence of LPS.Finally,physiological substrates of alkaline phosphatase and their dephosphorylated products at neutral p H including ATP,ADP,AMP and adenosine were employed to investigate their effects on secretion of TNF-? by the leukocyte.Using human freshly extracted leukocytes to study the relevant functions of rec IAP is close to the actual pharmaceutical application of rec IAP in humans and is of great significance.The main findings are as follows:(1)Expression of rec IAP in CHOIn order to obtain high-purity rec IAP,CHO cells were used for expression,and 5 stable CHO strains expressing rec IAP were successfully screened.Based on the basal expression level and the results of feed culture,a CHO cell line with the highest expression level was selected as a seed cell line to expand the culture.After collecting the cell culture solution,it was concentrated and purified.Finally,rec IAP with a molecular weight of about 70 k Da,a purity of 92.45%,and an enzyme activity of 140.125 U / m L was obtained.(2)Research on the activity of rec IAP on human leukocyte TNF-? and IL-6Using Limulus reagent to detect the effect of rec IAP on LPS activity,it was verified that rec IAP inactivation of LPS is dose-dependent and p H-dependent.LPS induces freshly extracted human leukocytes to secrete TNF-? in a dose-dependent manner.The results showed that whether LPS and rec IAP stimulated leukocytes after pretreatment,or LPS and rec IAP stimulated leukocytes at the same time,they significantly inhibited the secretion of TNF-? of leukocytes to a similar degree,indicating that the effect of rec IAP on inhibiting the secretion of TNF-? by leukocytes is not related to LPS.close.And in the presence and absence of LPS,rec IAP significantly inhibit ed the secretion of TNF-? and IL-6 by leukocytes(p <0.05),proving that rec IAP inhibits the secretion of TNF-? and IL-6 by human leukocytes independent of LPS.At physiological p H,rec IAP can dephosphorize ATP and gradually produce ADP,AMP and adenosine.In the absence of LPS,rec IAP can inhibit the secretion of leukocyte TNF-? by dephosphorylation of specific molecules(such as ATP,ADP and AMP),and inhibit the release of leukocyte TNF-? by producing free adenosine.In conclusion,this study found that rec IAP's non-LPS-dependent inhibition of human leukocyte TNF-? and IL-6 indicates that rec IAP can not only treat diseases related to LPS,but also hope to become a therapeutic candidate for diseases related to leukocyte TNF-? and IL-6 secretion Inject medicine,expanding the clinical application of rec IAP.It was further found that rec IAP dephosphorylates ATP to produce adenosine and inhibits the secretion of TNF-? from human leukocytes by adenosine in a non-LPS-dependent signaling pathway.In addition,the extracted bovine small intestine alkaline phosphatase(b IAP)is similar to rec IAP,inhibiting the secretion of human leukocytes TNF-? and IL-6 in the presence and absence of LPS(p <0.05),indicating that the humans established in the experiment The cell model has certain potential and can be used as a stable and repeatable activity assay for injectable IAP drugs.