Effects Of Different Concentrations Of Putrescine On Proliferation Of Peripheral Blood Mononuclear Cells,proliferation And Apoptosis Of CD₄⁺ Lymphocytes,CD₁₄⁺ Cells And Secretion Of Related Cytokines

Patients with severe burns,trauma,crush injuries,severe diabetic feet,and severe infections throughout the body,whether trauma or disease-induced large-area skin or soft tissue infection and necrosis,have severe systemic inflammatory reactions in critical conditions.The systemic inflammatory response syndrome that occurs at this time is not only critically ill and difficult to treat,but also the patient’s condition progresses rapidly.In the absence of clinical active intervention,most patients will quickly develop DIC,MODS,or even death.The previous research of the research team found that:wound necrotic tissue produced putrescine,cadaverine and other polyamine decomposition products under the action of protease and decarboxylation;in clinically severely infected diabetic patients,there is a large amount of putrescine in the wound necrotic tissue,which is absorbed into the bloodand even lead to the damage of liver and kidney function and cell withering in the body at a higher concentration.The systemic inflammatory response caused by putrescine has a high degree of fitto the inflammatory response curve caused by necrotic tissue homogenate.In addition,putrescine isdetected in the urine of diabetic patients with severe infections in the body,and putrescine is excreted inthe urine;high concentrations of putrescine have obvious toxic effects on liver and kidney cells,fibroblasts,vascular endothelial cells,etc.,and can promote cell apoptosis.In the clinic,we also found that the immune function of chronically invasive patients was obviously suppressed,which was prominently manifested in the decline in the number of T cells,but the reason is not clear.Under normal circumstances,the integrity of immune cell function plays an important role in wound repair and the hot immune cells related to wound repair include various T lymphocytes,macrophages and so on.Peripheral blood monocyte cells are the population of mononuclear cells in peripheral blood,including lymphocytes(T cells,B cells and NK cells),monocytes and DC cells.Mononuclear cells in peripheral blood chemoattract to peripheral tissues and differentiate into macrophages(CD14+).Macrophages(CD14+)play an important immune defense function in local tissues byrecognizing,phagocytosing pathogens and presenting antigen signals to CD4+lymphocytes,causing the secretion of corresponding cytokines,causing proinflammatory responses,and clearing the invasion of pathogenic microorganisms.CD4+ T lymphocytes,accounting for about 65%of T cells,are an important component of adaptive immunity in the body and play a key role in cellular immunity and immune regulation:receiving antigens presented by macrophages,differentiating into differenthelper T lymphocytes and secreting different inflammatory factors,causing adaptive immunity.The subpopulation of T cells in the body can reflect the level of immunity of the body,and CD4+ Lymphocyte proliferation and differentiation disorder plays an important role in the immunosuppression in the late stage of sepsis caused by severe infection and other reasons.CD4+ lymphocytes and macrophages(CD14+ cells)play important rolesin adaptive immunity and innate immunity,respectively.However,in the presence of a large number of necrotic tissue in chronic wounds,whether the main damage,putrescine,damage the function of immune cells,what is the characteristic of such damage,which is the goal we want to study at present.As a result,we intend to start with the main immune cells in the process of wound repair as PBMCs、CD4+ lymphocytes and CD14+ cell to understand the effect of putrescine on the function of different types of immune cells,and then to understand the effect of putrescine on wound repair.Objective:To study the proliferation,apoptosis,and cytokines(IL-2,IL-10,IL-12)of different concentrations of putrescine on peripheral blood monocytes,CD4+ lymphocytes,CD14+ cells,the effect of secretion.To explore the effects of putrescine at different concentrations on the immune defense of wounds and related immune cells in wound repair,and then understand the effects of putrescine on wound repair.Methods:1、Using the CCK-8 proliferation experiment method,we studied the induction of PBMC and PBMC into monocytes(CD14+ cells)when putrescine concentrations were 0,0.5,1.0,2.5,5.0,10,25,50,100,250,500,and 1000μg/ml,CD4+lymphocytes,and CD14+ cells proliferation after 24 hours of culture(CCK-8 method).Each group had 3 samples,and the experiment was repeated 3 times.2、Using flow cytometry to detect the apoptosis of CD4+ lymphocytes and CD14+ cells cultured in the above putrescine concentration range after 24 h.Each group had3 samples,and the experiment was repeated 3 times.3、Using ELISA to detect the concentration of cytokine IL-2 in the supernatant of CD4+lymphocytes cultured in the above putrescine concentration range after 24.Each group had 3 samples,and the experiment was repeated 3 times.4、Using ELISA to detect the concentration of cytokine IL-2 in the supernatant of CD14+cell cultured in the above putrescine concentration range after 24.Each group had 3 samples,and the experiment was repeated 3 times.Statistical methods:The data was analyzed using SPSS 24.0 software,and the data was subjected to one-way analysis of variance.When the data did not obey the normal distribution or the variance between groups was uneven,rank sum test was used,that is,Kruskal-Wallis test,and the one-way ANOVA test was used for pairwise comparison.The LSD method was used for comparison.When the normal distribution was not followed or the variance between groups was uneven,the Dunnett method was used.P<0.05 indicated that the difference was statistically significant compared with the controlgroup.The two groups of data were compared using person product moment correlation analysis,and Spearman correlation analysis was used when the data did not follow the normal distribution.P<0.05 was considered statistically significant.Results:1、The OD of PBMCs CCK-8 in the control group was 0.116±0.003,and the OD value of PBMCs was 0.118±0.000,after the putrescine concentration of 0.5,1.0,2.5,5.0,10,25,50,100,250,500 and 1000μg/ml.0.120±0.001,0.115±0.001,0.118±0.002,0.121 ±0.001,0.116±0.003,0.110±0.001,0.110±0.001,0.113±0.001,0.124±0.002,0.114±0.001,no difference between groups(F=25.999,P>0.05).2、The OD values of putrescine treatment at 0.5,1.0,2.5,5,10,25,50,100,250,500,and 1000μg/ml concentration(0.381 ±0.022,0.390±0.028,0.400± 0.015,0.403±0.001,0.407±0.008,0.382±0.013,0.366±0.018,0.372±0.014,0.347±0.018,0.320±0.018,0.286±0.022)were compared with the control group(F=17.306),the difference is statistically significant(P values are<0.05).3、The OD value of CD4+lymphocytes CCK-8 in the control group was 0.060±0.009,and the absorbance value of CD4+lymphocytes in the positive control group was 0.083±0.001.Compared with the control group,the OD values of CD4+lymphocyte putrescine treated with 0.5,2.5,5,10,25,50,100μg/ml putrescine(0.089±0.003,0.077±0.003,0.074±0.006,0.098±0.014,0.084±0.011,0.099±0.012,0.096±0.012)were higher(F=15.225).Compared with the control group,the OD values(0.052±0.002,0.051±0.004,0.046±0.001)treated with putrescine at concentrations of 1.0,250,500,and 1000μg/ml were not statistically significant(P value>0.05).4、The control group CD14+cells CCK-8 had an OD value of 0.355±0.008.The OD values of putrescine treated with 0.5,1.0,2.5,5.0,10,25 and 100μg/ml of putrescine in the CD±4+cells putrescine group(0.384±0.010,0.407±0.006,0.409±0.001,0.401±0.005,0.412±0.001,0.383±0.006,0.383±0.002)were increased compared with the control group(F=109.933,P=0.000),and the differences were statistically significant(P values were all<0.05).And when the putrescine concentration is 10μg/ml,the absorbance value of CCK-8 reaches the peak;the OD value(0.327±0.004,0.291 ±0.011)treated with putrescine at 500 and 1000μg/ml concentration is lower than the control,The difference is statistically significant(all P values<0.05),and the absorbance value decreases with increasing concentration.The OD values of putrescine-treated groups with 50 and 250 ug/ml concentrations(0.353±0.006,0.359±0.004)were not significantly different from those of the control group(P>0.05).5、The apoptotic rate of CD4+ lymphocytes control group was 76.76±1.24%.The apoptotic rate of 50,100,250,500,1000μg/ml putrescine group(85.7±0.7%,87.05± 1.65%,87.05±0.15%,87.75±0.05%,87.95±0.25%)was higher than that of control group(F=40.165),and the difference was statistically significant(P values were all<0.05).The apoptotic rates in the putrescine groups of 5,0.5,1.0,2.5,5.0,10,and 25μg/ml(77.7 ± 0.1%,81.35 ± 0.05%,82.05± 0.95%,82.95± 0.35%,83.15±0.15%,84.75±2.45%)were not significantly different from those in the control group(all P values>0.05).6、The apoptosis rate of the CD14+ cells control group was 47.910±0.01%,and the apoptosis rate of the putrescine group of 0.5,1.0,2.5,250,500μg/mL(54.10±0.08%,49.29±0.05%,49.39±0.1%,48.46±0.45%,54.90±0.55%)was higher than the control group(F=2096.84),the difference was statistically significant(all P values<0.05);5.0,10,25,50,100,1000μg/mL putrescine group The rate of apoptosis(45.50±0.1%,44.25±0.42%,34.50±0.22%,31.22±1.11%,38.59±0.09%,32.76±0.37%)was lower than that of the control group,and the differences were statistically significant(all P values<0.05).7、The content of IL-2 control group in CD4+ lymphocytes supernatant was 2.62±0.02pg/ml,0.5,1.0,2.5,5.0,10,25,50,100,250,500,1000μg/ml putrescine group IL-2(2.24±0.01,2.30±0.01,2.20±0.01,1.99±0.02,1.78±0.02,1.76±0.01,1.88±0.02,2.16±0.02,2.01 ±0.02,2.10±0.01,2.53±0.05)pg/ml compared with the control group,the difference was statistically significant(P<0.05).When the putrescine concentration was lower than 25μg/ml,As the putrescine concentration increases,the IL-2 concentration in the supernatant gradually decreases;when the putrescine concentration is higher than 25μg/ml,As the concentration of putrescine increased,the concentration of IL-2 in the supernatant gradually increased.8、The content of the IL-2 control group in the CD 14+cells supernatant was 3.01 ±0.020 pg/ml,and the IL-2 of 1.0,2.5,5.0,10,25,50,100,250,500,1000μg/ml putrescine group(2.61 ±0.010,2.17±0.020,1.81±0.025,2.07±0.030,2.04±0.010,2.33±0.006,2.30±0.030,2.10±0.010,1.92±0.015,2.17±0.030,1.95±0.020pg/ml)compared with the control group,the content was reduced,and the difference was statistically significant(P<0.05);The content of IL-10 control group in CD4+ cells supernatant was 41.24±0.549pg/ml,0.5,1.0,2.5,5.0,10,25,50,100,250,500,1000μg/ml putrescine group IL-10(46.42±0.967,48.80±0.122,54.38±0.613,57.96±0.070,62.33±0.892,68.50±0.761,71.98±1.543,75.21 ±0.967,77.90±1.318,82.43±0.428,84.51 ±0.930 pg/ml)compared with the control group,the content was increased(F=570.54),the difference was statistically significant(P<0.05);The content of IL-12 in CD14+cells supernatant was 40.95 ± 1.178pg/ml,0.5,1.0,2.5,5.0,10,25,50,100,250,500,1000μg/ml putrescine group IL-12(44.99±0.560,50.28± 1.017,54.72± 1.222,60.65±0.980,62.25±0.463,66.09± 1.560,70.83±0.071,72.67± 1.000,78.96± 1.000,80.21±1.025,84.74±0.373 pg/ml)compared with the control group(F=439.90),the difference was statistically significant(P<0.05).With the increase of putrescine concentration,the concentration of IL-10 and IL-12 in the supernatant of CD14+cells increased.9、Correlation analysis between each group of data curvesThere is a negative correlation between the effect of putrescine on the proliferation of CD14+ cells and the content of IL-10 and IL-12 in the supernatant under the action of putrescine(r=-0.610,P=0.035,r=-0.599,P=0.039);There is a positive correlation between IL-10 and IL-12 in the supernatant of different concentrations of putrescine on CD14+cells(r=0.994,P=0.000).Conclusion:1、Low concentration of putrescine can significantly promote the proliferation of different types of immune cells,while high concentration of putrescine can significantly promote the apoptosis of different types of immune cells,especially for T lymphocytes(CD4+).2、High concentration of putrescine can promote the secretion of IL-2 of CD4+lymphocytes,IL-2,IL-10 and IL-12 of macrophages(CD14+),but it has no significant effect on the classification of macrophages to M1 and M2.3、Putrescine is significantly more sensitive to the proliferation of induced mononuclear macrophages(CD14+)than PBMC,while the sensitivity of peripheral blood mononuclear cells(PBMC)to putrescine proliferation is very low.4、Putrescine is significantly more sensitive to the proliferation of induced mononuclear macrophages(CD14+)than PBMC,while the sensitivity of peripheral blood mononuclear cells to putrescine proliferation is very low.Different concentrations of putrescine can have different effects on different immune cells.Basically,low concentrations of putrescine play a positive role in promoting,while high concentrations of putrescine have a negative effect.Locallyhigh concentrations of putrescine will inhibit normal immune function,which in turn will affect wound repair.