| Background:Pseudomonas aeruginosa is a kind of gram-negative,non fermentative bacillus,which is a common opportunistic pathogen with inherent resistance to a variety of antibiotics.At present,the treatment of Pseudomonas aeruginosa includes β-lactams,fluoroquinolones and aminoglycosides.These drugs reduce the sensitivity to antibiotics,have high toxicity and low eradication rate.Therefore,it is necessary to find solutions to supplement and replace the existing drugs.Tanreqing injection is a classic Chinese medicine compound.Since it was listed in China in 2003,it has been widely used in clinic for its remarkable and safe clinical efficacy.Previous in vitro experiments showed that Tanreqing could kill the strain by inhibiting DNA replication,and could destroy the complete biofilm of the strain,showing a better killing effect than antibiotics.Objective:To evaluate the therapeutic effect of Tanreqing on acute pulmonary infection of Pseudomonas aeruginosa in rats by observing the survival rate,bacterial count,histopathological injury,inflammatory state and oxidative stress of lung tissue Objective to explore the mechanism of JNK1/p38 α/MAPK pathway of Tanreqing in the treatment of pulmonary infection caused by Pseudomonas aeruginosa,and to provide support for the treatment of human pulmonary infection caused by Pseudomonas aeruginosa.Methods:The first part:the model of acute Pseudomonas aeruginosa pulmonary infection was established by endotracheal injection method,which was divided into control group,model group,levofloxacin group,Tanreqing group and combined drug group.Rats in Levofloxacin group,Tanreqing group and combined drug group were injected intraperitoneally at 5 p.m.every day.The control group and model group were injected with equal amount of saline for seven days.The general situation and survival condition of rats were observed daily after infection.On the first,third,fifth and seventh day after infection,randomly selected each group of rats,and the lung tissue was collected under anesthesia and aseptic conditions for bacterial count and alveolar lavage.The lung gross and micro pathological conditions were evaluated by naked eye and he staining microscope;cell classification was carried out by cell counting plate and gemsa staining The levels of cytokines such as IL-6,IL-10 and tnf-α were measured by ELISA method,and the blood was collected on the 7th day for routine detection.The second part:extract the lung tissue of rats,and detect the expression of pjnkl/pmapk8 and pp38 α/pmapk14 in lung tissue by immunohistochemistry,Western blotting method was used to measure the levels of pjnkl/pmapk8 and pp38 α/pmapk14 in the lung tissue of each group,jnkl/mapk8 and p38 α/mapk14 by qPCR The mRNA level was used to investigate the molecular biological mechanism of Tanreqing in the treatment of pulmonary infection of Pseudomonas aeruginosa.Results:The first part:(1)survival rate:the mortality rate of rats in model group was 25%within 7 days,all of them died within 48 hours after bacterial inoculation;compared with model group,the survival rate of rats in LF group was significantly increased on the second day after infection(P<0.05,log rank test);the survival rate of LF group was similar to that of TRQ group,and there was no statistical difference(P>0.05).(2)Bacterial count:on the first day after infection,the CFU value of lung tissue in model group and Tanreqing group was significantly higher than that of the other groups,while that of levofloxacin group and the combined drug group was not significantly different(P>0.05);on the third day,the CFU value of lung tissue in each treatment group was significantly lower than that of model group(P<0.01);on the 7th day,compared with other groups,the pulmonary fine of the model group was significantly lower than that of the model group(P<0.01);on the 7th day,compared with other groups,the pulmonary CFU value of the model group was significantly lower than that of the The bacteria were still not completely removed(P<0.01).(3)The number of BAL cells in model group and levofloxacin group was not significant(P>0.05)in the first to 7th days after infection,while the total BAL cells in Tanreqing group and combined drug group were significantly lower than those in model group and levofloxacin group(P<0.05);the number of neutrophils in model group and levofloxacin group was not significantly different from that of model group and levofloxacin group on day 1-3 The results showed that the neutrophil level of levofloxacin group,Tanreqing group and combined drug group decreased significantly(P<0.05).(4)Blood routine:on the 7th day after infection,the number and percentage of neutrophils in model group were higher than that of other groups,and the number and percentage of neutrophils and lymphocytes in model group were lower than that of other groups.There was no significant difference in the number and percentage of neutrophil and lymphocyte between control group and each treatment group(P>0.05).(5)General pathology of lung:①macropathology.On the first day after the model,the model group showed large area of pulmonary consolidation and pulmonary congestion,while the area of pulmonary consolidation and pulmonary congestion in each treatment group was smaller than that of the model group.On the third to fifth day,the pulmonary congestion disappeared in Tanreqing group and the combined drug group,and the model group developed into lung abscess and lung adhesion.On the 7th day,the lung tissue of Tanreqing group and the combined drug group tended to recover,and some lung tissue abscess remained in the model group and levofloxacin group.②LIMP.LIPM scores of Tanreqing group and combined drug group were significantly lower than those in model group at all four time points.③Macroscopic lung score.At different time points,the macro lung score of all treatment groups was lower(score 1-3),while the model group had higher macro lung score(score 4).(6)He staining and micro pathology:on the 7th day after infection,the microscopic pathological score of lung tissue in control group was grade Ⅰ,that of 80%model group was grade Ⅳ,30%levofloxacin group,40%Tanreqing group and 100%combined drug group were grade Ⅱ or Ⅰ,while 70%levofloxacin group and 40%tanerqing group were rated as grade Ⅲ.(7)ELISA and cytokines:① IL-6 level.Compared with the model group and levofloxacin group,the average level of IL-6 water in Tanreqing group and the combined drug group was lower at all time points(P<0.05).② TNF-αlevel.Compared with the model group,the TNF-αlevels of Tanreqing group and the combined drug group were lower at each time point.Compared with levofloxacin group,the TNF-α level of Tanreqing group was lower on the 5th to 7th days,and that of the combined drug group was lower on the 3rd to 7th days.③ IL-10 level.The levels of IL-10 in Levofloxacin group,Tanreqing group and combined drug group were significantly lower than those in model group on the 3rd to 7th days.On the first to 7th days,the level of IL-10 in the combined drug group was significantly lower than that of levofloxacin group,and similar significant differences were observed in Tanreqing group on the 7th day.(8)Oxidative stress response level:compared with model group and levofloxacin group,SOD and GSH-PX activity in Tanreqing group and combined drug group increased significantly on the 3rd to 7th day after infection,and MDA level decreased significantly on the 5th to 7th day after infection.The second part:(1)immunohistochemistry:① expression of pJNK1.On the 7th day after infection,compared with the blank group,the expression of pJNK1 in the model group(P<0.01)and levofloxacin group(P<0.01)increased;compared with the model group,the expression of pJNK1 in the combination group decreased(P<0.05).② pp38α was positive.Compared with the blank group,the expression of pp38α in model group(P<0.001),levofloxacin group(P<0.01)and Tanreqing group(P<0.05)was increased;compared with the model group,the expression of pp38αin Tanreqing group(P<0.01)and combination group(P<0.01)was decreased.(2)Western blotting:① pJNK1 level.Compared with the blank group,the protein level of pJNK1 in model group and levofloxacin group increased,but there was no statistical significance(P>0.05);compared with the model group,the protein kinase level of pJNK1 in Tanreqing group(P<0.05)and combination group(P<0.05)decreased.②pp38αlevel.Compared with the blank group,the level of pp38 α protein kinase increased in the model group(P<0.01);compared with the model group,the level of pp38a protein kinase decreased in the Tanreqing group(P<0.01)and the combination group(P<0.01).(3)rt-qPCR:compared with the blank group,the expression of JNK1 mRNA(P>0.05)and p38α mRNA(P>0.05)in the model group increased,but there was no statistical difference;compared with the model group,the expression of JNK1 mRNA(P<0.05)and p38α mRNA(P>0.05)in the combination group decreased.Conclusion:Tanreqing injection can improve the survival rate of rats with acute pulmonary infection caused by Pseudomonas aeruginosa,improve the severity of acute lung injury and inflammatory cell infiltration,inhibit the high secretion of inflammatory cytokines(TNF-α,IL-6,IL-10)and reactive oxygen species(MDA),and promote the activities of antioxidant enzymes(SOD and GSH-Px).These events may be related to the significant bactericidal effect of Tanreqing.Tanreqing combined with levofloxacin has synergistic bactericidal effect on Pseudomonas aeruginosa in model rats,thus improving the therapeutic effect.After infection with Pseudomonas aeruginosa,the JNK1/p38/MAPK pathway was activated,resulting in a significant increase in phosphorylated JNK1/MAPK8 and p38α/MAPK14 protein kinase levels.After treatment with Tanreqing or combination therapy,the JNK1/p38/MAPK pathway activated by bacterial infection was inhibited,resulting in a significant decrease in phosphorylated JNK1/MAPK8 and p38α/MAPK14 protein kinase levels and mRNA expression levels Tanreqing or combination therapy can inhibit inflammation and oxidative stress by regulating JNK1/p38α/MAPK pathway and reducing the phosphorylation and mRNA expression of JNK1/MAPK8 and p38α/MAPK14 protein kinase. |
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