Effects And Mechanism Of Inhibiting Liver Kupffer Cells Function On Intestinal Mucosal Mechanical Barrier In Septic Rats

Objective:The classic"gut-liver axis"theory suggests that the intestinal barrier function is impaired,which causes the intestinal bacteria and endotoxin to shift into the liver,and activates the liver Kupffer cells,resulting in the release of a large number of inflammatory factors into the blood,which will further destroy the intestinal mucosal barrier function.It forms a vicious circle that exacerbates the systemic inflammatory response.The inflammatory response is closely related to the occurrence and development of sepsis.Injury of the intestinal mucosal barrier function has been considered to be the"initiation factor"for the development of multiple organ dysfunction syndrome in patients with sepsis.Our study is to investigate whether Kupffer cell function inhibited by GdCl₃ can alleviate intestinal inflammatory response and protect the intestinal mucosal mechanical barrier function.And further explore its mechanism,provide a theoretical basis for elucidating the mechanism of sepsis development,and lay a foundation for exploring new treatments for sepsis.Part 1:effects of gadolinium chloride inhibition of liver Kupffer cell function on intestinal mechanical barrier function following cecal ligation and puncture-induced sepsis in ratsMethods:In this study,the sepsis model was modeled by cecal ligation and perforation（CLP）.120 SD rats were randomly divided into 4 groups:Sham operation group（Sham group）,GdCl₃ pretreatment Sham operation group（Sham+GdCl₃ group）,CLP group,and GdCl₃pretreatment CLP group（CLP+GdCl₃ group）.Two pretreatment groups were given 5 mg/kg,10 mg/kg,20 mg/kg and 40mg/kg GdCl₃ respectively.GdCl₃ was injected into the tail vein 24hours and 48 hours before the establishment of the model.The sham operation group and CLP group were given the same amount of saline.Models were made after abdominal anesthesia.The rats were executed 12 hours after the establishment of the model.Blood was taken from the abdominal aorta and intestinal tissues were retained.The levels of TNF-α,IL-6 and IL-10were detected by ELISA.Western-blot was used to detect the levels of Occludin,ZO-1 in intestinal tissues.Hematoxylin-eosin staining（HE）was applied to observe the degree of damage in intestinal tissue.The tunnel was used to detect apoptosis of intestinal tissue and the apoptosis rate was calculated.Results:（1）5,10,20 mg/kg GdCl₃ inhibition of Kupffer cell function had no significant effects on serous and intestinal inflammatory factors（TNF-a,IL-6 and IL-1β）,intestinal permeability and injury（DAO,FD4,Chiu’s score）,intestinal tight junction protein（Occludin and ZO-1）and apoptosis in sham-group rats（P>0.05）,but increased dose to 40 mg/kg,it could promote the expression of TNF-a,IL-6 and IL-1βin serum of sham-group rats（P<0.05）.（2）5,10,20 mg/kg GdCl₃ inhibition of Kupffer cell function could reduce the expression of inflammatory factors in serum and intestine,reduce intestinal permeability and injury,promote the expression of tight junction protein in intestinal cells and reduce abnormal apoptosis of intestinal cells in septic rats（CLP-induced septic rats）（P<0.05）.However,when the dose increased to 40 mg/kg,GdCl₃ inhibition of Kupffer cell function could increased the expression of inflammatory factors,increased intestinal permeability and injury,decreased the expression of tight junction protein,and increased abnormal apoptosis of intestinal cells.（3）There was no significant difference in the protective effects of 10 mg/kg and 20 mg/kg GdCl3inhibition of Kupffer cell function on the expression of inflammatory factors in serum and intestine,intestinal permeability and injury,and intestinal barrier function in septic rats（P>0.05）.Preliminary Summary:（1）GdCl3 inhibition of Kupffer cell function had a protective effect on intestinal mucosal mechanical barrier function,which may be related to GdCl3 inhibiting the secretion of inflammatory factors in liver Kupffer cells.（2）This protective effect of GdCl3inhibition of Kupffer cell function on intestinal mucosal mechanical barrier function was related to the dose of GdCl₃.Part 2:protection mechanism of gadolinium chloride inhibition of liver Kupffer cell function on intestinal mechanical barrier function following CLP-induced sepsis in rats Methods:In this study,the sepsis model was also modeled by CLP.144 SD rats were randomly divided into 4 groups:Sham operation group（Sham group）,GdCl₃ pretreatment Sham operation group（Sham+GdCl₃ group）,CLP group,and GdCl₃ pretreatment CLP group（CLP+GdCl3 group）.20 mg/kg GdCl3 was injected into the tail vein 24 hours and 48hours before the establishment of the model.The sham operation group and CLP group were given the same amount of saline.Models were made after abdominal anesthesia.In each group,blood was taken from the abdominal aorta and intestinal tissue was collected at 6h,12h,and 24h.The levels of TNF-α,IL-6 and IL-1βwere detected by ELISA.Western-blot was used to detect the levels of Occludin,ZO-1,MLCK,NF-κB,and Caspase-3 in intestinal tissues.HE was applied to observe the degree of damage in intestinal tissue.The tunnel was used to detect apoptosis of intestinal tissue and the apoptosis rate was calculated.Results:（1）GdCl₃ inhibition of Kupffer cell function,at 6,12 and 24 hours,had no effect on serous and intestinal inflammatory factors（TNF-a,IL-6 and IL-1β）,intestinal permeability and injury（DAO,FD4,Chiu’s score）,intestinal tight junction（Occludin）,apoptosis of intestinal cells and Caspase-3 apoptotic protein in sham-group rats（P>0.05）.（2）GdCl3inhibition of Kupffer cell function,at 6h and 12h,could reduce the expression of inflammatory factors in serum and intestine of septic rats（P<0.05）.Howere,it only reduced the expression of TNF-a,IL-6 and IL-1βin intestinal tissues at 24 hours,but did not alleviate the inflammatory reaction（the the expression of TNF-α,IL-6 and IL-1β）in serum（P>0.05）.（3）GdCl3 inhibition of Kupffer cell function,at 6,12 and 24 hours,could decrease the expression of NF-κB protein of intestinal tissues in septic rats（P<0.05）.（4）GdCl₃ inhibition of Kupffer cell function,at 6,12 and 24 hours,could reduce intestinal permeability and damage,increase the expression of tight junction protein Occludin and ZO-1 in intestinal cells,and decrease the expression of MLCK protein of intestinal tissues in septic rats（P<0.05）.（5）GdCl3 inhibition of Kupffer cell function could decrease the abnormal apoptosis of intestinal tissue and inhibit the expression of Caspase-3（P<0.05）.Preliminary Summary:（1）GdCl3 inhibition of Kupffer cell function could protect intestinal mucosal barrier function by decreasing the intestinal inflammatory response due to down-regulating expression of NF-κB.（2）GdCl₃ inhibition of Kupffer cell function could protect intestinal mucosal barrier function by up-regulating the expression of Occludin and ZO-1 which due to being down-regulated the expression of MLCK by inhibiting expression of NF-κB.（3）GdCl3 inhibition of Kupffer cell function could protect intestinal mucosal barrier function by reducing the excessive apoptosis of intestinal tissue in septic rats,which was related to the inhibition of Caspase-3 expression.Conclusion:（1）GdCl₃ inhibition of Kupffer cell function had a protective effect on intestinal mucosal mechanical barrier function,which may be related to GdCl3 inhibiting the secretion of inflammatory factors in liver Kupffer cells,and also related to the dose of GdCl₃.（2）GdCl3 inhibition of Kupffer cell function could alleviate systemic and intestinal inflammatory responses and protect intestinal mucosal barrier function in septic rats,which may be related to NF-κB/MLCK and Caspase-3 pathway.