| objective:Firstly,establishing endotoxin tolerance model in mice.Endotoxin tolerance in mice to be a large dose of lipopolysaccharide(LPS)again,researching the expression of TLR4/IRAK-4(Toll-like receptor4)signaling pathway related positive factor IRAK-4 and negative factor TRAF3 in spleen tissues of mice.Method:1.Feeding the experimental animals:A total of 84 ICR mice(age 35±2days,weight 25±5g),raised in the same environment(temperature,humidity,and the same food etc.),adaptive feeding one week by simulation their natural living conditions.2.Animal grouping: The 84 ICR mice using random number table,randomly assigned to the following three groups,endotoxin tolerance group,non-endotoxin tolerance group,the blank control group,28 mice per group.3.Establishment of animal model:(1)Advance a small dose of LPS: By intraperitoneal injection,in the endotoxin tolerance group by intraperitoneal injection of LPS0.5mg/kg(low-dose),the non-endotoxin tolerance group and the blank control group were to be an equal volume of normal saline(NS).(2)The second large dose of LPS: Intraperitoneal injection of a small dose of LPS 24 hours later,to be again in the endotoxin tolerance group by intraperitoneal injection of LPS5mg/kg(high dose),while the non-endotoxin tolerance group by intraperitoneal injection of LPS also be 5mg/kg,the blank control group still be the same volume of NS intraperitoneally.4.Taking Specimens and detecting index:(1)Inaccordance with the randomly selected 7 mice in each group before the second large dose of LPS injection(0 hours),injection after at 3 hours,6 hours and 24 hours,We use enucleation of eyeball to obtain mice peripheral blood and collect serum after centrifugation,then by enzyme-linked immunosorbent assay(ELISA)to detect different time points mice serum cytokine interleukin-10(IL-10)expression.(2)Mice were sacrificed after collecting blood,aseptically quickly removed intact mouse spleen tissue and placed in paraformaldehyde fixed save,then immunohistochemistry was used to detect each group mice at different time points spleen tissues TRAF3 and IRAK-4expression,while pathological changes of mice spleen tissues were also observed in the light microscope.Results:1.Changes in daily behavior of each group mice: The first injection of a small dose of LPS(0.5mg/kg)after,the mice of endotoxin tolerance group appeared different degrees of reduction activities and eating food,scattered hair and other symptoms.However,the mice of non-endotoxin tolerance group and blank control group had no significant change.The second injection of large doses of LPS(5mg/kg)after,the mice of endotoxin tolerance group and non-endotoxin tolerance group appeared obviously eat less,move less,stray,scattered hair and other symptoms,the more obvious than the small dose injection of LPS(Some mice of non-endotoxin tolerance group appeared diarrhea,cyanosis of the lips).The mice of blank control group still had no significant change.2.The expression of IL-10 in serum:(1)Before the second large dose of LPS injection(0 hours),mice serum IL-10 expression were compared among the three groups,IL-10 expression levels of the endotoxin tolerance group was higher than the non-endotoxin tolerance group and the blank control group,this shows mice cytokine synthesis inhibitory factor IL-10 expression already began to rise after injection of a small dose of LPS;The difference between the non-endotoxin tolerance group and the blank control group was not significant.(2)3 hours,6 hours and 24 hours after the second large dose of LPS injection,mice serum IL-10 expression were compared among the three groups,the endotoxin tolerance group compared with the blank control group and the non-endotoxin tolerance group,the difference was statistically significant(P<0.05),IL-10 expression in the endotoxin tolerance group is greater than the non-endotoxin tolerance group and the blank control group;The difference between the blank control group and the non-endotoxin tolerance group was statistically significant(P<0.05),IL-10 expression in the non-endotoxin tolerance group is greater than the blank control group;This shows that in mice in advance of low dose LPS injection,to be a large dose of LPS again,mice serum IL-10 expression increased.(3)mice serum IL-10 expression were compared in the three groups,0 hours compared to 3 hours,6hours,24 hours respectively in the endotoxin tolerance group and the non-endotoxin tolerance group,the differences was statistically significant,3h,6h,and 24 h were compared between each two,the difference was not statistically significant.The blank control group differences in the expressionof IL-10 at various times of the experiment were not statistically significant(P>0.05).3.Histopathological changes in mice spleen: endotoxin tolerance group and non-endotoxin tolerance group spleen tissues of mice have different degrees of pathological damage,manifested as spleen congestion,structural changes,red pulp and white pulp unclear boundaries,and this injury change endotoxin tolerance group was significantly lighter than the non-endotoxin tolerance group;the blank control group did not change the above.It also shows that the establishment of experimental endotoxin tolerance model is successful.4.The expression of IRAK-4 in the spleen tissues of mice:(1)the expression of IRAK-4 among the three groups was compared:(1).Before the second large dose of LPS injection(0 hours),the difference of expression of IRAK-4 among the three groups was not statistically significant.(2).3 hours,6hours after the second large dose of LPS injection,IRAK-4 expression levels of the non-endotoxin tolerance group was higher than the endotoxin tolerance group and the blank control group,and IRAK-4 expression levels of endotoxin tolerance group was higher than the blank control group.(3).24 hours after the second large dose of LPS injection,the non-endotoxin tolerance group compared with the endotoxin tolerance group and the blank control group,IRAK-4 expression levels of the non-endotoxin tolerance group was higher than the endotoxin tolerance group and the blank control group;the endotoxin tolerance group compared with the blank control group,the difference was not statistically significant(P>0.05).(2)The expression of IRAK-4 in the threegroups was compared: mice spleen IRAK-4 expression of endotoxin tolerance group and non-endotoxin tolerance group has been raised at the 3 hours,may be maintained until 6 hours,24 hours has declined.The difference of IRAK-4expression between the respective time points was not statistically significant in the blank control group.5.The expression of TRAF3 in the spleen tissues of mice:(1)the expression of TRAF3 among the three groups was compared:(1).Before the second large dose of LPS injection(0 hours),the difference of expression of TRAF3 among the three groups was not statistically significant.(2).3 hours,6 hours after the second large dose of LPS injection,TRAF3 expression levels of the endotoxin tolerance group was higher than the non-endotoxin tolerance group and the blank control group,and TRAF3 expression levels of non-endotoxin tolerance group was higher than the blank control group.(3).24 hours after the second large dose of LPS injection,the endotoxin tolerance group compared with the non-endotoxin tolerance group and the blank control group,TRAF3 expression levels of the endotoxin tolerance group was higher than the non-endotoxin tolerance group and the blank control group;The difference between the non-endotoxin tolerance group and the blank control group was not significant.(2)The expression of TRAF3 in the three groups was compared: mice spleen TRAF3 expression of endotoxin tolerance group and non-endotoxin tolerance group has been raised at the 3 hours,may be maintained until 6 hours,can still be maintained until 24 hours in endotoxin tolerance group,24 hours has declined in non-endotoxintolerance group,but still exists.Conclusions: 1.it can be established mice model of endotoxin tolerance by mean of intraperitonealinjection of 5mg/kg lipopolysaccharide after 24 hours of the intraperitoneal injection of 0.5mg/kg lipopolysaccharide.2.Mice in endotoxin tolerance,serum IL-10 expression increased and spleen tissue pathological damage reduction.3.Endotoxin tolerant mice spleen TLR4/IRAK-4 signaling pathway related positive factor IRAK-4 was down-regulated,while the expression of related negative factor TRAF3 was up-regulated,may be involved in the formation of endotoxin tolerance. |
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