| Purpose:In this study,we selected those key genes of macrophages in burns releated through bioinformatics technology.Then we monitored changes in macrophage inflammatory markers at different time points of burn model mice after injury,and found out the subsequent variations in key cytokines at different time points during inflammatory outbreak,so as to monitor and intervene the process of inflammatory waterfall.Method:Our research methods have two main parts.1.Screen out trauma-related high-expressed differential genes in macrophages by bioinformatics methods;the macrophage expression gene library was obtained by single-cell gene sequencing;the GSE19743 data of GEO gene database were used for bioinformatics analysis.Differential gene expression data in different periods after injury was analyzed based on the Affymetrix human genome U133 Plus 2.0(GPL570)array platform.2.In vivo experiments to verify the expression of macrophage inflammatory markers in different phases of trauma.First,experimental mice were divided into three groups: pseudo-burn group,15% body surface burn group,and 30% body surface burn group.Then mice spleens were taken for RT-q PCR at different time points after burn to detect m RNA transcription level of key genes CCL3,CCL4,MMP9,CD86,which were obtained in Part 1.Flow cytometry to detect the expression level of corresponding inflammatory markers in macrophages.ELISA to measure the expression of serum inflammatory factors IL-6 and TNF-α.At last,show the changes of inflammatory cell infiltration in different injury stage by HE staining.Results:1.Filter out differentially expressed genes in macrophages related to burns by bioinformatics.We selected CCL3、CCL4、MMP9、CD86 as four trending obvious genes based on |log FC|>1.5,p <0.01.Among them,the gene expression up-regulated by macrophages during burns is MMP9,while down-regulated genes are CCL3,CCL4 and CD86.2.1 Verify the m RNA levels of CCL3,CCL4,MMP9,and CD86 in spleen of15% and 30% burn models by RT-q PCR.The m RNA expression level of CD86 increased rapidly at 6h after injury and dropped rapidly to the lowest point at 2d after injury and the trend has statistically different(P < 0.01).Meanwhile,the m RNA expression level of CCL3 was gradually increased to the peak at 2d after injury and then gradually decreased,and this trend has statistically different(P<0.05).In 30%body surface burn model,the m RNA expression level of CD86 increased immediately after injury and then gradually decreased(P<0.01).The m RNA expression level of MMP9 increased significantly at 6h after injury and then decreased to the lowest point at 2d,then back to gradually increased(P <0.01).The m RNA expression level of CCL3 gradually increased to the peak at 1d after injury and then gradually decreased(P<0.05).The m RNA expression level of CCL4 showed fluctuating changes in both scald models,and its trend has no statistically difference.2.2 Verify proportion of spleen cells expressing CCL3,MMP9,and CD86 by Flow cytometry.In 15% body surface burn model,the proportion of spleen cells expressing CCL3 and MMP9 increased from 0h to 6h after injury and then decreased,and gradually increased after reaching the lowest value at 1 to 2 days.This trend is statistically significant(P < 0.01);The proportion of spleen cells expressing CD86 increased rapidly at 6h after injury and then rapidly decreased to the lowest value at1 d,then increased again after reaching the highest value at 2d.This trend has statistics significance(P<0.01).Among them,the proportion of CD86 expressed in macrophages increased to the highest value at 2d after injury and then gradually decreased;while the proportion of MMP9 expressed has an opposite trend,which increased rapidly to the apex at 0h after injury and then gradually decreased to the lowest point during 1-2d,then increased again.Both trends of these two kinds of cells have statistics significance(P<0.01).In 30% body surface burn model,the proportion of spleen cells expressing CD86 and MMP9 increased immediately after injury,then decreased to the lowest point at 1d,and then gradually increased.This trend is statistically significant(P<0.05).The proportion of CCL3 expression gradually increased to the maximum value at 7d;this trend has statistics significance(P<0.05).The changing of CD86 and MMP9 expressed proportion in macrophages has the same trend with the proportion of the same factor expressed in spleen cells,which increased immediately after injury then decreased to the lowest point at 1d and then gradually increased,while only the changing of CD86 expression proportion is statistically significant(P<0.01);the proportion of CCL3 expression increased slightly till 1d after injury,then suddenly dropped to the lowest on the second day,after that the value gradually increased;this trend has no statistics significance(P>0.05).Comparing the 15% and 30% burn groups,the trend of CCL3 expressed proportion in all spleen cells and macrophages is statistically significant(P<0.01).There were differences between the groups as well.Moreover,the proportion of macrophages in spleen cells increased significantly during aggravation of the damage and reached the highest point on the 2nd day.This trend has differences between groups(P<0.01).2.3 Elisa verified expression levels of inflammatory factors IL-6 and TNF-α in peripheral serum.In 15% body surface burn model,IL-6 expression increased rapidly to the highest point at 6h after injury,and then gradually decreased to the lowest point on the 3rd day,then increased again on Day 5.This trend is statistically significant(P<0.01);Meanwhile,TNF-α expression level gradually increased to the highest point on the 7th day after injury,while the trend has no statistics significance(P>0.05).In 30% body surface burn model,the expression of IL-6 and TNF-αreached the highest point at 6h after injury,and then the volatility decreased and reached the lowest point on the 7th day.This trend is statistically significant(P <0.05);The expression of IL-6 was highest at 6h after injury and lowest at 24h/1d,and this trend has differences between groups(P<0.05).2.4 Changes in spleen length.In 15% body surface burn model,the length of mice spleen kept shortest 0h～2d after injury and then gradually increased to the length before injury on the 7th day.This trend is statistically different(P<0.01).The change of spleen length in the 30% body surface burn model has similar trend,but has no statistics significance.2.5 HE staining.In 15% body surface burn model,0h and 6h after burn skin edema was severe and normal structure of skin and subcutaneous tissue disappeared;2d～7d after burn,subcutaneous edema gradually reduced,and inflammatory cell infiltration gradually reduced;0h and 6h after burn,spleen had obvious lymphocyte infiltration in red and white pulp;On 2d and 3d,the volume of red and white pulp significantly reduced,and lymphocyte infiltration decreased;Recover to pre-injury level on Day 7.In 30% body surface burn model,the trend of skin and spleen changes is consistent with the trend in 15% body surface burn model;30% burn model showed more severe edema and inflammatory cell infiltration at the same time point compared with 15% burn model.Conclusion:In the first part,we use bioinformatics technology to screen out those burn-related significant difference genes in macrophages.They are CCL3,CCL4,MMP9,and CD86.The high expression of MMP9 and the low expression of CCL3,CCL4,and CD86 may become important observation indicators in the burn process.In the second part,the proportion of macrophages in spleen cells reached its maximum at 2d after burns,and increased with severity of the injury increased during acute phase of burns.The expression ratio of chemokine CCL3 in macrophages reached the lowest point 2d after injury and then gradually increased,and this trend became more pronounced with the severity of the damage increasing.The expression ratio of CD86 in macrophages reached the highest point at 2d after injury,and then gradually decreased to the lowest at 7d;this trend became more pronounced as the degree of injury increased.The expression ratio of metalloproteinase MMP9 in macrophages reached the minimum at 1d after injury and then fluctuated up,and in15% body surface burn models,a significant peak of expression was observed at 0h after injury.IL-6 and TNF-α were expressed at the highest level at 6h which is correlated with the degree of injury positively.1～2d after burns is the acute inflammatory edema period.High expression of CD86 and low expression of CCL3 and MMP9 in macrophages appear during this period,which are closely related to the severity of the injury.Monitoring and regulating these sensitive indicators may have some contributions to assess severity of burns and reduce inflammatory blow to the body from burns. |
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