| BackgroundAt present,the number of global surgical operations has reached 313 million per year,but at least 4.3 million of them have died within 30 days after surgery,accounting for 7.7 % of global deaths.It has become the third leading cause of death after ischemic heart disease and stroke.The reason is that postoperative infection is the main cause of postoperative death.The occurrence of postoperative infection not only prolongs the hospitalization days of patients,increases the global medical and health burden,but also leads to the inhibition of immune function,which increases the susceptibility of postoperative sepsis and affects the prognosis of the disease.Immune dysfunction is the core evolution mechanism of sepsis,sepsis is defined as life-threatening multiple organ dysfunction caused by the body ’s response to infection.Studies have shown that monocyte / macrophage phagocytosis dysfunction is an important feature of postoperative immune dysfunction,which can significantly reduce the host ’s ability to clear pathogens,thereby increasing the incidence of postoperative infection and postoperative sepsis,and monocyte / macrophage polarization is the key factor affecting monocyte / macrophage phagocytosis.Studies have reported that there is an inseparable relationship between monocyte /macrophage polarization and phagocytosis.Previous studies on the mechanism of surgical stress reducing the body ’s immune function and increasing the susceptibility to sepsis mainly focused on neutrophils,natural killer cells,and T lymphocytes.There are few studies on the molecular mechanism of monocyte / macrophage changes after surgical trauma increasing the susceptibility to postoperative sepsis.Kallikrein-related peptidase 8(KLK8),also known as Neuropsin,is a secreted serine protease with important biological effects such as tissue remodeling and cell function regulation.KLK8 can hydrolyze substrate proteins such as cell membrane surface proteins and extracellular matrix.Previous studies have shown that KLK8 is involved in the body’s innate and adaptive immune response process.KLK8 can induce the activation of PI3 K / Akt / m TOR and Notch signaling pathways,which play an important role in monocytes / macrophages.Therefore,this study mainly focused on KLK8 to explore the mechanism of monocytes / macrophages in surgical trauma increasing sepsis susceptibility,and expected to provide a new target for the prevention and treatment of postoperative immunosuppressive disorders.MethodIn this study,we first established a mouse surgical stress model to observe the effect of surgical trauma on the phagocytic function of peritoneal macrophages,the intraperitoneal infection model was established by intraperitoneal injection of E.coli to observe whether surgical trauma would increase the susceptibility to sepsis.Then,peripheral blood samples from 20 patients after radical resection of colorectal cancer were collected for flow analysis to observe the changes of polarization and phagocytic function of monocytes / macrophages after surgical trauma,Lung,liver and kidney tissue samples after surgical stress model of mice were collected for quantitative PCR to observe the changes of M1 and M2 indexes,and the changes of phagocytic function were observed by flow analysis after macrophage M1 and M2 phenotype induction on RAW264.7 cell line.Then,in order to explore the mechanism of macrophage M2 polarization after operation,KLK8 was found by genetic microarray data of macrophage polarization in NCBI database and verified by quantitative PCR,so the follow-up study focused on KLK8 and macrophage polarization mechanism.The effects of KLK8 on monocyte / macrophage polarization and phagocytosis after surgical stress were further verified by quantitative PCR,ELISA and flow cytometry at the cytological level and animal level.The potential substrates of KLK8 were screened by protein spectrum detection and co-immunoprecipitation,and CD44 was found.Finally,CD44 agonists were used to observe how KLK8 interacted with CD44 to affect monocyte macrophage polarization and phagocytosis after surgery in vitro cytological experiments and in vivo animal experiments.Results1.The results of flow cytometry analysis of peritoneal macrophages after surgical stress model in mice showed that the phagocytic function of peritoneal macrophages decreased significantly on the first day and the third day after operation,compared with that before operation.The model of surgical trauma abdominal infection found,compared with the control group,surgical stress significantly increased the mortality and abdominal bacterial load of mice after intraperitoneal injection of E.coil.2.The results of flow cytometry analysis of peripheral blood clinical samples showed that compared with preoperative,the phagocytic ability of peripheral blood monocytes / macrophages in patients after radical resection of colorectal cancer began to decrease immediately after surgery,and decreased significantly on the first and third days after surgery.The monocyte / macrophage M1 polarization marker CD86 began to increase immediately after surgery,peaked at 1 day after surgery,and returned to normal levels at 3 days after surgery,while the monocyte / macrophage M2 polarization marker CD206 gradually increased with the increase of postoperative time.The results of M1 and M2 polarization monitored by quantitative PCR in lung,liver and kidney tissues of mice after surgical stress were consistent with those of peripheral blood mononuclear / macrophage polarization.Quantitative PCR results showed that compared with the control group,the phagocytic function of macrophages induced by LPS was enhanced after M1 phenotype,while the phagocytic function of macrophages induced by IL4 was decreased after M2 phenotype.3.The biological information of four groups of genetic microarray data related to macrophage polarization in NCBI gene chip library was analyzed.The results showed that 31 genes were increased in macrophage M2 polarization compared with M0.Subsequently,IL4-induced M2 macrophages quantitative PCR found that the m RNA expression of KLK8,BATF3 and SMAP2 genes in M2 macrophages was significantly up-regulated compared with M0.By using si RNA to knock down the expression of KLK8,BATF3 and SMAP2 in RAW264.7 cells,quantitative PCR results showed that only KLK8 knockdown could block IL4-induced macrophage M2 polarization changes,so the subsequent focus was on the mechanism of KLK8 in macrophage M2 polarization.4.ELISA showed that the expression of KLK8 in plasma of patients undergoing radical resection of colorectal cancer increased significantly after three days of surgical stress.After successful overexpression of KLK8 in RAW264.7 cells,the results of flow cytometry showed that the phagocytic function of macrophage RAW264.7 decreased significantly after overexpression of KLK8.Flow cytometry analysis of KLK8 knockout mice showed that the phagocytic function of KLK8 KO mice was significantly enhanced after surgical stress.At the same time,compared with the control group,KLK8 KO significantly reduced the abdominal bacterial load after laparotomy in mice,and the survival rate was significantly increased.5.The interaction between KLK8 and CD44 was obtained by proteomics and co-immunoprecipitation.In vitro cytological experiments and in vivo animal experiments using CD44 agonist A6 showed that A6 could significantly reverse the M2 polarization and phagocytosis of macrophages caused by overexpression of KLK8 and high expression of KLK8 after surgical trauma stimulation.Conclusion1.Surgical trauma stress stimulation can cause monocyte / macrophage polarization to M2 and inhibit monocyte / macrophage phagocytosis,which significantly increases the susceptibility to postoperative infection and sepsis.2.KLK8 mediates the molecular mechanism of increased susceptibility to postoperative infection and sepsis by cleaving CD44,and KLK8 / CD44 is expected to be a new target for the prevention and treatment of postoperative infection and sepsis. |
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