Mechanism Of Cystathionine Gamma-lyase(cth) Induces Efferocytosis In Macrophages Via ERK1/2 Pathway To Modulate Intestinal Mucosa Repair

Objectives:(1)To investigate whether macrophage efferocytosis is involved in intestinal mucosal repair in the context of intestinal ischaemia-reperfusion injury;(2)To confirm whether macrophage efferocytosis plays a role in intestinal mucosal injury repair;(3)To identify the key molecules driving efferocytosis,and to demonstrate whether Cth is involved in intestinal mucosal injury repair by driving efferocytosis in an ERK1/2 pathway-dependent manner;(4)To observe the correlation between the level of efferocytosis and intestinal function recovery in clinical patients.Methods: In this project,we proposed to use Cth inhibitor,activator,silencing and overexpression,ERK1/2 inhibitor and activator to intervene in cells and mouse models,and q-PCR,western blot,flow cytometry,cell co-culture and bioinformatic analysis were applied to investigate whether efferocytosis is involved in intestinal injury repair and the specific molecular mechanisms involved from different dimensions such as molecular,cellular,clinical tissue and biochemical.It will provide new theories and rationale to explore the molecular targets of intestinal mucosal repair.Results:(1)Macrophage efferocytosis was present in the intestine of ischemia-reperfusion injury,and the level of efferocytosis gradually increased with the duration of repair.(2)Transcriptome sequencing analysis showed that a large number of efferocytosis markers such as Gas6 and Mfge8 were also altered when intestinal ischemia-reperfusion injury occurred,and the results of analysis and validation revealed that Cth was significantly overexpressed during injury repair.(3)When Cth expression was increased or suppressed,q-PCR and Western blot detected that its downstream ERK1/2 pathway and the level of efferocytosis were also increased or decreased.(4)When Cth expression was increased,the previously increased levels of efferocytosis and higher levels of repair factors were significantly reversed by the addition of ERK1/2 pathway inhibitors;when Cth expression was inhibited,the previously decreased levels of efferocytosis and lower levels of repair factors were significantly increased by the addition of ERK1/2 pathway activators.(5)The addition of Cytochalasin,the efferocytosis inhibitor,resulted in a significant decrease in the previously high level of repair factors when Cth expression was enhanced.(6)we found a significant positive correlation between Cth expression and efferocytosis markers at 48 h postoperatively(R = 0.65,P = 0.031),and Cth expression correlated positively with anti-inflammatory molecules(R=0.52,P=0.1).In addition,we also found a significant negative correlation between the level of Cth48 h postoperatively and the length of time at which patients resumed defecation(R =-0.65,P = 0.03)and bowel movements(R =-0.67,P = 0.023),indicating that high expression of Cth was positively correlated with the recovery of intestinal function in patients.Finally,we found that the expression level of Cth was lower in patients with oral dietary intolerance at 48 h postoperatively and in patients with infectious complications at 7 days postoperatively(p=0.031,p=0.018 respectively).Conclusion(s): Cth can activate the ERK1/2 signalling pathway,induce macrophage efferocytosis,and thus promote intestinal barrier repair.