| Objectives: To establish endotoxin tolerance model. Toresearch the expression of the positive factor Myd88and the negativefactor Tollip in the TLR4/Myd88pathway in the liver tissue of baby ratwith endotoxin tolerance.Methods:1.Grouping. The96ICR baby rats(40±2days old) were randomly divided into3groups （namelyexperimental group， experimental control group, and blank controlgroup）. Each group had32ICR baby rats.2.Establishing endotoxintolerance model.(1) the experimental group was induced byintraperitoneal injection of0.5mg/kg lipopolysaccharide. Theexperimental control group and blank control group were induced byintraperitoneal injection of saline solution of the same volume.(2)24hours after the first injection, experimental group and experimentalcontrol group were induced by intraperitoneal injection of5mg/kglipopolysaccharide. And blank control group was induced byintraperitoneal injection of saline solution of the same volume.3.Testingindex.(1)8rats were selected randomly at each time point(0h,3h,8h,24h after the second injection). we collected the rat blood by eyeballremoval method, and separated the serum, then detected the amount ofhigh mobility group protein B-1（HMGB-1) in the serum of each baby ratat each time point by enzyme-linked immunoassay.(2) We took the livertissue of baby rat under aseptic operation after collecting blood, andpreserved rat liver tissue within4%paraformaldehyde fixed liquid, thendetected the amount of Myd88and Tollip in the liver tissue of each babyrat at each time point by paraffin section and immune histochemicalmethod,and observe the pathological change.Results:1The changes of baby rat. All the baby rats ate less, drunk less, moved less, becamelethargic and hair messy after both small dose and large dose oflipopolysaccharide stimulation. Those symptoms were more obvious afterlarge dose lipopolysaccharide stimulation. There was no significantchange after saline solution stimulation.2.Expression of HMGB-1.(1)We compared the HMGB-1expression quantity between each group atthe time points of0h,3h after the second injection. There was nostatistically difference between each group (P>0.05, P0h=0.686,P3h=0.356).(2) We compared the HMGB-1expression quantity betweeneach group at the time points of8h,24h after the second injection. TheHMGB-1expression quantity of experimental group significantlydecreased compared with experimental control group. The difference wasstatistically significant (P<0.05). It meant that endotoxin tolerance modelestablishment successes. The HMGB-1expression quantity of bothexperimental group and experimental control group significantlyincreased compared with blank control group. The difference wasstatistically significant (P<0.05).(3) HMGB-1in the serum of baby ratbegan to express between3h and8h after the second injection, did notreturn to the original basic level at the time point of24h after the secondinjection in both experimental group and experimental control group.3.The liver tissue pathological injury of baby rat were milder in theexperimental group than that of the experimental control group,it furthermeant that endotoxin tolerance model establishment successes.4.Expression of Myd88.(1) We compared the Myd88expression quantitybetween each group at the time point of0h after the second injection.There was no statistically difference between each group (P>0.05，P0h=0.956).(2) We compared the Myd88expression quantity betweeneach group at the time points of3h,8h after the second injection, theMyd88expression quantity of experimental control group significantly increased compared with both experimental group and blank controlgroup. The difference was statistically significant (P<0.05). Theexpression quantity of experimental group significantly increasedcompared with blank control group. The difference was statisticallysignificant (P<0.05).(3) We compared the Myd88expression quantitybetween each group at the time point of24h after the second injection.The Myd88expression quantity of experimental control groupsignificantly increased compared with both experimental group and blankcontrol group. The difference was statistically significant (P<0.05). Therewas no statistically difference between experimental group and blankcontrol group (P>0.05, P24h=0.08).(4) Myd88in the liver tissue of babyrat began to express before3h of the second injection, arrived the highestlevel at the time point of8h after the second injection, still existed at thetime point of24h after the second injection in experimental control group.5. Expression of Tollip.(1) We compared the Tollip expression quantitybetween each group at the time point of0h after the second injection.There was no statistically difference between each group (P>0.05,P0h=0.98).(2) We compared the Tollip expression quantity between eachgroup at the time points of3h,8h after the second injection. The Tollipexpression quantity of experimental group significantly increasedcompared with both experimental control group and blank control group.The difference was statistically significant (P<0.05). The Tollipexpression quantity of experimental control group significantly increasedcompared with blank control group. The difference was statisticallysignificant (P<0.05).(3) We compared the Tollip expression quantitybetween each group at the time point of24h after the second injection.The Tollip expression quantity of experimental group slightly decreasedcompared with experimental control group. The difference wasstatistically significant (P<0.05).The Tollip expression quantity of both experimental group and experimental control group significantlyincreased compared with blank control group. The difference wasstatistically significant (P<0.05).(4) Tollip in the liver tissue of baby ratbegan to express before3h of the second injection, arrived the highestlevel at the time point of8h after the second injection, still existed at thetime point of24h after the second injection in both experimental groupand experimental control group.6.There was positive correlation betweenexpression of Myd88and HMGB-1at8h,24h after the LPS injection（r8h=0.912，P8h<0.05；r24h=0.907，P24h<0.05）;There was negativecorrelation between expression of Tollip and HMGB-1at8h,24h after theLPS injection（r8h=－0.950，P8h<0.05；r24h=－0.829，P24h<0.05）.7.Therewas positive correlation between expression of Myd88and Tollip at3h,8h,24h after the LPS injection（r3h=0.960，P3h<0.05；r8h=0.844，P8h<0.05；r24h=0.895，P24h<0.05）. Conclusions:1.It is feasible to establishendotoxin tolerance model of baby rat by mean of intraperitonealinjection of5mg/kg lipopolysaccharide after24hours of theintraperitoneal injection of0.5mg/kg lipopolysaccharide.2.The HMGB-1expression quantity decreased in the serum and the damage of liver easedafter endotoxin tolerance.3.It is probably one of the mechanisms ofendotoxin tolerance that the positive factor Myd88expression quantitydecreased and the negative factor Tollip expression quantity increased inthe TLR4/Myd88pathway. |
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