Effect And Mechanism Of Inhibiting Liver Kupffer Cells Function On Intestinal Dendritic Cells In Septic Rats

Objective:By inhibiting the function of Kupffer cells in liver,monitoring the inflammation and the changes of intestinal dendritic cells in sepsis rats,and further exploring its mechanism.To establish a theoretical foundation for exploring the mechanism of Liver-Intestine interaction in the occurrence and development of sepsis and proposing new strategies for the treatment of sepsis.PART ONE:Effects of inhibiting Kupffer cell function in liver with different doses of gadolinium chloride on inflammatory response and dendritic cells in intestinal lamina propria of sepsis rats.Methods:Sixty SD rats were randomly divided into four groups:sham operation group,gadolinium chloride pretreatment sham operation group,sepsis group and gadolinium chloride pretreatment sepsis group,with 6rats in each group.The GdCl₃ pretreatment group was injected with 5 mg/kg,10 mg/kg,20 mg/kg and 40mg/kg via tail vein 48 hours and 24 hours before operation respectively,and the sham operation group was injected with the same amount of saline.The sepsis model was duplicated by cecal ligation and puncture?CLP?.The rats were sacrificed 24 hours after operation.The expression of inflammatory factors TNF-?,IL-6 and IL-10 in peripheral serum and intestinal tissue of rats in each group was detected by ELISA.Histopathological evaluation of intestinal injury in rats.Dendritic cells in lamina propria?LPDCs?of intestinal mucosa of rats were obtained by digestion with collagenase IV.The CD103 antibody positive rate of LPDCs in rats of each group was detected by flow cytometry and the number of LPDCs was calculated.The expression of major histocompatibility complex?MHC?II and costimulatory molecule CD86 on LPDCs of rats in each group was detected by flow cytometry.Results:?1?In serum and intestinal tissues,the expression of pro-inflammatory factors TNF-?and IL-6 in 5mg/kg,10 mg/kg,20 mg/kg GdCl₃ inhibition group was significantly lower than that in sepsis group?P<0.05?,while the expression of anti-inflammatory factor IL-10 was significantly increased.The expression of pro-inflammatory factors TNF-?and IL-6 in 40 mg/kg GdCl₃ inhibition group was significantly higher than that in other groups.?2?According to the intestinal histopathological evaluation,the intestinal inflammation injury in sepsis group was significantly worse than that in sham operation group.GdCl₃ of 5 mg/kg,10 mg/kg and 20 mg/kg could significantly reduce the inflammatory injury of intestinal tissue,and the injury of intestinal tissue in 40 mg/kg GdCl₃ group was more serious than that in sepsis group?P<0.05?.?3?The number of LPDCs decreased significantly 24 hours after CLP?P<0.05?.The number of LPDCs increased significantly in 5 mg/kg,10 mg/kg and 20 mg/kg groups?P<0.05?.There was no significant difference in the number of LPDCs between 40 mg/kg group and CLP group.?4?Compared with sham group,the expressions of MHC-II and CD86 in CLP group were significantly increased?P<0.05?,while the expressions of MHC-II and CD86 in 20 mg/kg GdCl₃ pretreatment group were significantly lower than those in sepsis group?P<0.05?.PART TWO:Temporal changes in the effects of inhibiting Kupffer cell function on inflammatory response and intestinal dendritic cells in sepsis rats.Methods:Seventy-two SD rats were randomly divided into four groups:sham group,GdCl₃ pretreatment sham group,sepsis group and GdCl₃ pretreatment sepsis group,with 18 rats in each group.In GdCl₃pretreatment group,20 mg/kg of GdCl₃ was injected into tail vein 48 hours and 24 hours before operation,respectively.In control group,rats were injected with the same dose of saline.Sepsis model was duplicated by cecal ligation and puncture?CLP?.Six rats were sacrificed at 6,12 and 24 hours after operation in each group.The expression of inflammatory factors TNF-?,IL-6 and IL-10 in peripheral serum and intestinal tissue of rats in each group was detected by ELISA.Histopathological evaluation of intestinal injury in rats.Dendritic cells in lamina propria?LPDCs?of intestinal mucosa of rats were obtained by digestion with collagenase IV.The CD103 antibody positive rate of LPDCs in rats of each group was detected by flow cytometry and the number of LPDCs was calculated.The expression of major histocompatibility complex?MHC?II and costimulatory molecule CD86 on LPDCs of rats in each group was detected by flow cytometry.Detection of apoptotic rate of LPDCs with flow cytometry.Detection the expression of autophagy-related protein?LC3-II?of LPDCs in rats by Western Blot.Results:?1?In serum and intestinal tissues,the expression of TNF-a,IL-6 and IL-10 increased significantly at 6 h after CLP compared with sham operation group?P<0.05?,and reached the highest level at 24 h.After GdCl₃ inhibiting the function of Kupffer cells in the liver,the expression of pro-inflammatory factors TNF-alpha and IL-6 was significantly down-regulated?P<0.05?and anti-inflammatory factor IL-10 was significantly up-regulated?P<0.05?.?2?In the evaluation of intestinal inflammation injury,compared with Sham group,the intestinal inflammation injury was significantly aggravated in sepsis group at 6 h,12 h and24 h,and GdCl₃ inhibited the function of Kupffer cells in liver significantly reduced intestinal tissue injury in sepsis rats at various time points.?3?The number of LPDCs increased significantly at 6 h after CLP,reached the maximum at 12 h?P<0.05?,and decreased significantly at 24 h.GdCl₃ inhibited the function of Kupffer cells in the liver and significantly increased the number of LPDCs in septic rats at various time points.?4?GdCl₃ significantly inhibited the expression of MHC-II and CD86 on LPDCs of septic rats at various time points?P<0.05?.?5?GdCl₃ inhibited the function of Kupffer cells in liver,significantly reduced the expression of LPDCs autophagy-related proteins at different time points in sepsis rats,and decreased the apoptotic rate?P<0.05?.Conclusions:?1?GdCl₃ can inhibit the function of Kupffer cells in the liver of sepsis rats in a dose-dependent manner.GdCl₃ at 20 mg/kg inhibited the function of Kupffer cells in the liver of sepsis rats,and did not aggravate the excessive inflammation and intestinal tissue damage in sepsis rats.?2?GdCl₃ inhibits the function of Kupffer cells in the liver of sepsis rats and can alleviate the inflammatory reaction and intestinal tissue damage in sepsis rats.The mechanism may be related to the inhibition of the expression of pro-inflammatory factors TNF-a and IL-6 in sepsis rats.?3?GdCl₃ inhibits the function of Kupffer cells in the liver of sepsis rats,reduces the ability of LPDCs to present antigen and activate T cells,and maintains the stability of LPDCs by promoting the migration of peripheral DCs to intestinal tract and lowering the levels of autophagy and apoptosis.The mechanism may be related to the promotion of the expression of anti-inflammatory factor IL-10.