| Objective:By conducting a small dose of endotoxin stimulation, establishing the model of endotoxin tolerance rat, research the expression of serum inflammatory factor TREM-1 (triggering receptor expressed on myeloid cells-1) and calcium binding protein S100A8, which is the positive factor of the S100A8/TLR4(Toll-like receptor 4) pathway.Method:1.Grouping of the experimental animals:the 96 ICR rats divided into saline-stimulation group,non-pre-endotoxin-stimulation group and pre-endotoxin-stimulation group,according to the random number table.The 96 rats were randomly assigned to the three groups.Each group has 32 rats.2.Adaptive feeding of the experimental animals:After the grouping of the experimental rats,gave the rats one week of adaptive feeding by simulating the natural live conditions of them.3.Establishing the experimental rats models:(1) by using the method of intraperitoneal injection of lipopolysaccharide (LPS), the pre-endotoxin-stimulation group rats were injected of 0.5μg/g LPS, both the non-pre-endotoxin-stimulation group and the saline-stimulation group rats were injected of an equal volume of saline.(2) 24 hours later,the pre-endotoxin-stimulation group and non-pre-endotoxin-stimulation group rats were injected of 5μg/g LPS,while the saline-stimulation group rats were injected of an equal volume of saline also.4. Testing index:(1) Before the second injection (Ohour),3hours, 10hours and 24hours after injection, using the mothod of removal the eyes of the rats to get the peripheral blood,and then isolated the serum. After that,using the enzyme-linked immunosorbent assay to detect the expression of TREM-1 in each group at every timing. (2) After taking blood,killed the rats immediately and got the whole liver tissue samples under aseptic, and placed them into 4% paraformalde-hyde fixative to save and embedded in pafaffin. Then detected the pathological changes of the liver tissue under an optical microscope and used immunohistochemistry method to detect the expression of S100A8 in the liver tissue.Results:1.The daily behavior changes of the rats:after the small dose of LPS (0.5μg/g) stimulation, all of 32 rats in the pre-endotoxin-stimulation group had those behaviors in different degrees,such as eating less,lacking of exercise,outliers and hair scattered.Those performances occured about one hour after injection,and went on to 12 hours.The rats in the other two groups(non-pre-endotoxin-stimulation group and saline-stimulation group) which were given equal volume of saline had no such performances.After the high dose of LPS (5μg/g) stimulation, the rats of pre-endotoxin-stimulation group and non-pre-endotoxin-stimulation group behaved such as eating less,lacking of exercise,outliers and hair scattered.Those performances occured about 3 hours after the injection,and reached the peak about 12hours and continued until the end of this observation (24 hours after injection of large doses of LPS). Further more,those performances were more obvious than that in the small-dose LPS stimulation group,two rats of the non-pre-endotoxin-stimulation group also had convulsions performance.The rats of saline-stimulation group had no behavior changes also.2.TREM-1 expression:(1) Before high-dose of LPS injection (0h), the difference between the pre-endotoxin-stimulation group and non-pre-endotoxin-stimulation group was statistically significant (P<0.01),and the difference between the pre-endotoxin-stimulation group and the saline-stimulation group was statistically significant (P<0.05).It means that a small dose of LPS (0.5μg/g) can also cause the realse of the inflammatory factor;The difference between the non-pre-endotoxin-stimulation group and the saline-stimulation group was not statistically significant (P>0.5).(2) 3 hours,10 hours and 24 hours after high-dose of LPS injection, comparison of the pre-endotoxin-stimulation group with the non-pre-endotoxin-stimulation group and the saline-stimulation group, the differences was statistically significant (P<0.01). The difference between the non-pre-endotoxin-stimulation group and the saline-stimulation group was statistically significant (P<0.01). And the expression of TREM-1 in non-pre-endotoxin-stimulation group was higher than that in the pre-endotoxin-stimulation group, the pre-endotoxin-stimulation group was higher than the saline-stimulation group, which indicated that the establishing of the endotoxin tolerance model is successful. (3)Before the high-dose of LPS injection (0h), 3hours,10hours,and 24hours after injection, we compared the expression of TREM-1 within the groups.Both the expression of the pre-endotoxin-stimulation group and the non-pre-endotoxin-stimulation group at each timing were statistically significant, while the expression of the saline-stimulation group at each timing was not statistically significant.3.The pathological changes in the liver:the pathological injury of the pre-endotoxin-stimulation group was significantly lower than the non-pre-endotoxin-stimulation group at every timing,which also indicated that the establishing of the endotoxin tolerance model is successful.4.The expression of S100A8:(1) Before the high-dose of LPS injection (0h),comparison the expression of S100A8 of the pre-endotoxin-stimulation group with the non-pre-endotoxin-stimulation group and the saline-stimulation group, the differences was statistically significant (P<0.05). It means that a small dose of LPS (0.5μg/g) can activate S100A8/TLR4 pathway,resulting in the release of S100A8.The difference between the non-pre-endotoxin-stimulation group and the saline-stimulation group was not statistically significant.(2) 10 hours after high-dose of LPS injection,pairwise comparisons between the three groups, the differences were statistically significant, indicating that endotoxin-tolerance model was successfully established. (3)3hours and 24hours after high-dose LPS injection, compared the pre-endotoxin-stimulation group with the non-pre-endotoxin-stimulation group, the difference was not statistically significant; but when the pre-endotoxin-stimulation group,and non-pre-endotoxin-stimulation group compared with the saline-stimulation group, the difference was statistically significant.(4) Before the high-dose of LPS injection (0h), 3hours,10hours,and 24hours after injection, we compared the expression of S100A8 within the groups. The pre-endotoxin-stimulation group had statistically significant differences in the expression of Oh and 10h.The differences in non-pre-endotoxin-stimulation group at the timing of 10h and 24h were not statistically significant.And the expression of the saline-stimulation group at each timing was not statistically significant.Conclusions:Ⅰ.It is a viable way to establish the endotoxin tolerance model by pre-injection of a small-dose of LPS (0.5μg/g) and then give a high-dose of LPS (5μg/g) 24hours later.2.Ralative to the non-pre-endotoxin-stimulation group,the rats behavior changes of the pre-endotoxin stimulation group were lighter.we also found that the pathological injury of liver and the release of inflammatory factor were significantly reduced. We can see that,S100A8,which is the positive factor of the S100A8/TLR4 pathway,may play an important role in the endotoxin tolerance. |
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