| Objective To explore the role of IL-6 trans-signaling pathway in perioperative neurocognitive disorder(PND)in mice by specifically blocking and activating the IL-6 trans signaling pathway and constructing IL-6R-/-mice.Methods SPF healthy male C57BL/6 wild type mice were divided into 10 groups(n=21 each):sham group,surgery group,saline group,IL-6 group,surgery+vehicle group,surgery+BE0047(IL-6R blocker)group,IL-6+vehicle group,IL-6+BE0047 group,surgery+sgp130Fc(specific IL-6 trans-signaling inhibitor)group,IL-6+sgp130Fc group.In surgery group,mice were subject to tibial fracture with intramedullary fixation under general anesthesia with sevoflurane.In sham group,mice only received the same anaesthesia and analgesia as in surgery group.Saline group was injected intraperitoneally with 100 μl 0.9%NaCl solution,IL-6 group was injected intraperitoneally with 100 μl IL-6(50 μg/kg).Saline group and IL-6 group did not receive anesthesia or surgery.Before surgery,surgery+vehicle group,surgery+BE0047 group and surgery+sgp130Fc group were intraperitoneally injected with 100 μl PBS and BE0047(4 mg/kg),respectively.30 minutes later,mice were subject to tibial fracture with intramedullary fixation under general anesthesia with sevoflurane.IL-6+vehicle group,IL-6+BE0047 group and IL-6+sgp130Fc group were intraperitoneally injected with 100 μl PBS,BE0047(4 mg/kg),sgp130Fc(10 mg/kg),respectively.30 minutes later,mice were injected intraperitoneally with 100 μl IL-6(50 μg/kg),but did not receive anesthesia or surgery.24 hours later,blood was collected from the inferior vena cava,the concentrations of IL-6,IL-1β and tumor necrosis factor(TNF)-α in plasma were measured by enzyme-linked immunosorbent assay(ELISA),and then the mice were sancrificed,brain tissues were obtained,the contents of IL-6,IL-1β and TNF-α in the hippocampus were measured by ELISA,the activation of microglias in the hippocampal region was observed by immunofluorescence staining(n=6);3 days after surgery,cognitive function was evaluated by contextual fear conditionting test(n=15).In addition,another 24 wild-type mice were randomly divided into 4 groups(n=6 each):sham group,surgery group,surgery+BE0047 group,surgery+vehicle group.The treatment is the same as before.The mice were sacrificed 24 hours after operation,and the brain was taken.The expression of albumin in hippocampus was determined by Western blot.In addition,the wild type mice were divided into 3 groups(n=8 each):vehicle group,BE0047 group,sgp130Fc group.In each group,mice were subject to tibial fracture with intramedullary fixation under general anesthesia with sevoflurane.100 μl PBS,BE0047(4 mg/kg)and sgp130Fc(10 mg/kg)were injected intrabitoneally on the 1st,5th and 10th day after surgery,respectively.On the 15th day,bone tissue was taken and the degree of bone healing was observed by Safranin O-Fast Green staining.IL-6R-/-mice were randomly divided into 4 groups(n=21 each):sham group,surgery group,saline group,hyper IL-6(specific IL-6 trans-signaling activator)group.In surgery group,mice were subject to tibial fracture with intramedullary fixation under general anesthesia with sevoflurane.In sham group,mice only received the same anaesthesia and analgesia as in surgery group.Saline group was injected intraperitoneally with 100 μl 0.9%NaCl solution,hyper IL-6 group was injected intraperitoneally with 100 μl hyper IL-6(40 μg/kg).24 hours later,blood was collected from the inferior vena cava,the concentrations of IL-6,IL-1β and TNF-α in plasma were measured by ELISA,and then the mice were sancrificed,brain tissues were obtained,the contents of IL-6,IL-1β and TNF-α in the hippocampus were measured by ELISA,the activation of microglias in the hippocampal region was observed by immunofluorescence staining(n=6);3 days after surgery,cognitive function was evaluated by contextual fear conditionting test(n=15).Results Compared with sham group,the percentage of freezing time in the contextual fear conditionting test was decreased,the activiation of microglias in the hippocampal region was increased,the levels of IL-6,IL-1β and TNF-α in plasma and hippocampus were increased in surgery group(P<0.05).Compared with saline group,the percentage of freezing time in the contextual fear conditionting test was decreased,the activiation of microglias in the hippocampal region was increased,the levels of IL-6,IL-1β and TNF-α in plasma and hippocampus were increased in IL-6 group(P<0.05).Compared with surgery+vehicle group,the percentage of freezing time in the contextual fear conditionting test was increased,the activiation of microglias in the hippocampal region was decreased,the levels of IL-6,IL-1β and TNF-α in plasma and hippocampus were decreased in surgery+BE0047 group and surgery+sgp 130Fc group(P<0.05).Compared with IL-6+vehicle group,the percentage of freezing time in the contextual fear conditionting test was increased,the activiation of microglias in the hippocampal region was decreased,the levels of IL-6,IL-1β and TNF-α in plasma and hippocampus were decreased in IL-6+BE0047 group and IL-6+sgp130Fc group(P<0.05).Compared with sham group,the expression of Albumin in hippocampus of mice in surgery group was increased(P<0.05).Compared with surgery+vehicle group,the expression of Albumin protein in hippocampus of mice in surgery+BE0047 group was decreased(P<0.05).Compared with vehicle group,the percentage of newly formed cartilage tissue decreased in BE0047 group(P<0.05),but there was no significant differences between saline group and sgp130Fc group(P>0.05),suggesting that simultaneously blocking IL-6 classic and trans-signaling would lead to delayed wound healing.However,specific blocking of IL-6 trans-signaling did not lead to delayed wound healing.In IL-6R-/-mice,there were no significant differences in the percentage of freezing time,the activiation of microglias in the hippocampal region,and the levels of IL-6,IL1β and TNF-α in plasma and hippocampus between sham group and surgery group(P>0.05).Compared with saline group,the percentage of freezing time was decreased,the activiation of microglias in the hippocampal region was increased,the levels of IL-6,IL-1β and TNF-α in plasma and hippocampus were increased in hyper IL-6 group(P<0.05).Conclusion IL-6 trans-signaling is involved in the regulation of perioperative neurocognitive disorder in mice.Blocking IL-6 trans-signaling can improve PND in mice,and specific activation of IL-6 trans-signaling can lead to peripheral and central inflammatory responses and cognitive decline in mice. |
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