| Inflammation is the basic pathological process of most human diseases,and it is particularly important to control the development of inflammation.The main inflammatory cells in the human body are leukocytes in the blood.Leukocytes mainly include neutrophils.Under inflammatory stimulation,neutrophils are activated by inflammatory mediators after leaving the blood.Their accumulation and migration,phagocytosis,and excessive release of ROS will aggravate the inflammatory response,causing extensive damage to the surrounding normal tissues,while the timely apoptosis of inflammatory cells will reduce the inflammatory response.Therefore,the control of neutrophils and mononuclear cells migration,neutrophils phagocytosis and apoptosis,and ROS release can regulate the inflammatory response of the body,thus playing an important role in the prevention and treatment of related inflammatory diseases.Intestinal alkaline phosphatase(IAP)is only used to treat inflammatory diseases caused by LPS by inactivating LPS.It is unclear whether IAP has a therapeutic effect on non-LPS-dependent inflammatory diseases(without TLR4 receptors).In order to study the action of IAP on the migration of neutrophils,the phagocytosis and apoptosis of neutrophils,and the release of ROS and the non-LPS-dependent signal conduction pathway of IAP,this study first used CHO expression system to prepare high-purity recombinant human intestinal alkaline phosphatase(recIAP).Secondly,the model of inflammatory cells was established by using freshly extracted neutrophils,and the effect of recIAP on the migration,phagocytosis,apoptosis and release of ROS under the conditions of the presence and non-existence of LPS was studied.Finally,phosphomolybdic acid method was used to explore the mechanism of recIAP affecting human neutrophil migration,apoptosis,phagocytosis and ROS release in non-LPS dependent signal transduction pathways.In order to obtain high purity recIAP,CHO-S cells were used for expression,and five cho stable strains of expression recIAP were successfully screened.Based on the results of basic expression and flow culture,a highest expression CHO-recIAP cell line was selected as a seed cell line,expanded culture and received fermentation fluid,after separation and purification,the molecular weight of recIAP was obtained at about 60 k Da,purity was 95.51%,and enzyme activity was 137.28 U/m L.This study used freshly extracted human neutrophils to establish an inflammatory cell model,and studied the effects of recIAP on the migration,phagocytosis and apoptosis of neutrophils under the conditions of the presence and non-existence of LPS.The results of the migration test showed that recIAP inhibited the migration of human neutrophils,whether or not LPS existed.The results of the phagocytosis test showed that recIAP inhibited the phagocytosis of human neutrophils,whether or not LPS existed.In addition,the results of apoptosis test showed that recIAP can reduce the inhibition of LPS on human neutrophil apoptosis.The results of the release ROS test showed that recIAP inhibited the release of human neutrophil ROS,whether or not LPS existed.In this study,the phosphomolybdic acid method was used to detect the dephosphorization effects of recIAP and purine signal transduction pathways CD39 and CD73 on ATP,ADP,and AMP.The test results showed that recIAP removes ATP,ADP,AMP free phosphate and purine signal transduction pathway CD39 and CD73 remove ATP,ADP,AMP free phosphate content was similar.It showed that recIAP and the dephosphorization of the purine signal transduction pathway CD39 and CD73 had a distribution cross.The above results proved that in the LPS-dependent transduction pathway,recIAP inhibited the migration of neutrophils,phagocytosis and the release of ROS through the mechanism of inactivating LPS,and weakened the neutrality of LPSinhibition of granulocyte apoptosis.In the LPS-independent transduction pathway,recIAP inhibited the migration of neutrophils phagocytosis,and the release of ROS through the mechanism of dephosphorylation of ATP,ADP,and AMP.It showed that recIAP can not only treat LPS-related diseases,but also is expected to become a candidate medicine for the treatment of non-LPS-dependent inflammatory diseases,expanding the scope of clinical application of recIAP. |
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