| For a long time,acute exacerbation of chronic obstructive pulmonary disease(AECOPD)caused by multi-drug resistant bacteria infection is the main cause of death in clinical patients,and due to the abuse of antibiotics and the decrease of drug sensitivity of glucocorticoids,the clinical efficacy of conventional western medicine in the treatment of AECOPD patients needs to be improved.The animal model of acute exacerbation of chronic obstructive pulmonary disease caused by multidrug-resistant bacteria infection is not yet mature,so it is of great significance to establish a controllable animal model of AECOPD and develop new traditional Chinese medicine and combination of traditional Chinese and Western medicine for the treatment of AECOPD.Objective:To establish rat chronic obstructive pulmonary disease(COPD)models by passive cigarette smoking plus intratracheal instillation of cigarette tar,and an animal model of acute exacerbation of chronic obstructive pulmonary disease(AECOPD)caused by multi-drug resistant Pseudomonas aeruginosa infection was established by intratracheal instillation of multi-drug resistant Pseudomonas aeruginosa on the basis of the COPD rat model.To study the pulmonary function,blood gas analysis,pathological changes and related inflammatory changes in AECOPD rat model.The therapeutic effect and mechanism of Shufeng Jiedu capsule on acute exacerbation of chronic obstructive pulmonary disease caused by multi-drug resistant Pseudomonas aeruginosa were further studied.Methods:1 The establishment of chronic obstructive pulmonary disease animal modelFirst,the animal model of chronic obstructive pulmonary disease(COPD)was established.During the establishment of the animal model of COPD,two strains of rats(SD,Wistar)with three different body weights(50-70 g,130-150 g,180-200 g)were used for the study.The rat COPD models by passive cigarette smoking plus intratracheal instillation of cigarette tar was established.Lung function,blood gas analysis was tested to identify whether the rat COPD model was successful.On the basis of the animal model of chronic obstructive pulmonary disease,the animal model of acute exacerbation of chronic obstructive pulmonary disease was established by intratracheal instillation of multi-drug resistant Pseudomonas aeruginosa.After intratracheal instillation of multi-drug resistant Pseudomonas aeruginosa,the modeling method was investigated by lung function,gas and blood analysis,lung colony count,pathological examination of lung tissue and detection of drug resistance gene content in lung tissue.2 Pathological and immunological studies on the acute exacerbation model of chronic obstructive pulmonary disease in Wistar rats infected with multidrug-resistant Pseudomonas aeruginosa.Animal models were established under the fumigation conditions and infection conditions screened in experimental method 1.The rats were subjected to cigarette fumigation for one hour every day for the first six days of the seven-day cycle,twice a day,with an interval of more than one hour.On the seventh day,50 μl of tar was instilled into the trachea.When the lung function of the rats decreased significantly,isoflurane was used for mild anesthesia,and 3 McBurney’s concentration of multi-drug resistant Pseudomonas aeruginosa solution was injected into the trachea for infection,200 μL/rat.Lung function test,blood analysis and he staining were performed on the 5 th day after infection,and the contents of inflammatory cytokines and bacterial infection markers in lung tissue were determined by ELISA.3 Study on the therapeutic effect of Shufeng Jiedu Capsule on AECOPD Wistar rat model induced by multi-drug resistant Pseudomonas aeruginosa infectionThe modeling method was the same as that in Experiment 2.Seventy Wistar rats(130150 g)were randomly divided into normal control group,COPD model group,AECOPD model group,drug-resistant levofloxacin group,sensitive amikacin group,Shufeng Jiedu capsule high dose group,and Shufeng Jiedu capsule low dose group.In addition to the normal control group,the remaining rats were subjected to fumigation modeling according to Experimental Method 2.The lung function and the oxygen saturation detection are performed before infection and on the fifth day after infection,blood is taken from the abdominal aorta after the detection is completed,the blood routine detection is performed,the pathological change of the lung tissue is detected by HE staining of the lung tissue,the expression level of a multi-drug resistant pseudomonas aeruginosa drug resistance gene in the lung tissue is detected by adopting a RT-PCR method,and the contents of inflammatory cytokines and bacterial markers in the lung tissue are determined by multi-factor analysis and ELISA method,Through the above method,we studied the therapeutic effect of Shufeng Jiedu Capsule on Wistar rat model with acute attack of chronic obstructive pulmonary disease caused by multi-resistant Pseudomonas aeruginosa infection.4 Study on the mechanism of Shufeng Jiedu Capsule on AECOPD Wistar rat model caused by multi-drug resistant Pseudomonas aeruginosa infectionThe rats in experiment 3 were taken for blood collection from the abdominal aorta and the proportion of T lymphocyte subsets was analyzed by flow cytometry.After the samples were taken,Masson staining was performed to mark the collagen content in the lung tissue and quantitatively detect the collagen deposition in the lung tissue.The goblet cells were stained by AP-PAS staining method and the mucus deposition in the lung tissue was quantitatively detected.The collagen contents of COL-1 and COL-3 in the lung tissue were detected by ELISA.EGFR and MUC5AC protein markers were stained by immunofluorescence in the lung for quantitative detection.Western Blot was used to quantitate EGFR,P-EGFR and MUC5AC mucin secretion pathway-related proteins in the lung tissue and TGF-β1,Smad2/3 and P-Smad2/3 pulmonary fibrosis pathway-related proteins in the lung tissue.The mechanism of Shufeng Jiedu Capsule in the treatment of AECOPD was evaluated.Results:1 The establishment of chronic obstructive pulmonary disease animal modelWe evaluated the indicators related to the establishment of rat models with three kinds of body weights(50-70 g,130-150 g,and 180-200 g)and two strains of rats(SD,Wistar).We finally selected the Wistar rats with 130-150 g as the model rats from the aspects of general signs of rats,modeling time,model stability,and so on.Finally,we determined that the concentration of infection of multi-resistant Pseudomonas aeruginosa solution to Wistar rats with chronic obstructive pulmonary disease was 3 McF(6×108cfu/ml).The establishment method of COPD rat model is as follows:Selection of test methods:According to the clinical etiology of COPD(cigarette smoke and air pollution,etc.):passive cigarette rats were selected for modeling.Selection of cigarettes:Huangguoshu(10 mg of tar,0.9 mg of nicotine and 12 mg of carbon monoxide in smoke)with relatively high content of tar and carbon monoxide and relatively cheap price was used as the cigarette for this experiment.Smoking time and number of cigarettes:Smoking time:The rats were smoked for one hour each time and twice a day to achieve the optimal state of all aspects(poor state and no death).Number of cigarettes:130-150 g Wistar rats Smoked 0.5 cigarettes per rat on averageSmokey machine setting parameters:Fixed parameters of smoking machine:According to the experiment,the fixed parameters of smoking machine are as follows:smoke discharge flow rate of 1.8L/min,cigarette lighting time and residual smoke discharge time of 10s.The smoke machine needs to adjust parameters:the number of cigarettes can be determined according to the number of rats,the operation time of the smoke exhaust pump can be determined according to one hour of each smoke inhalation,and the oxygen addition flow rate can be determined according to the operation time of the pump.as follows:#12 Determination of condition of tar gas pipe instillation:The relevant experimental results showed that according to the relevant preliminary experiments,the tar concentration was determined to be non-diluted,and the rats trachea were injected with 50 μL tar infusion each time,with the cycle of once every seven days,and the rats were in good condition.In the establishment of COPD animal model,two rat strains and three body weight specifications were screened in this experiment.With the development of smoking,the rats showed general signs such as malaise,hair explosion,yellow hair,decreased activity,cough,increased water intake and decreased food intake,and the body weight increased slowly,which was significantly different from the normal control group(P<0.01);Pulmonary function,respiratory peak flow rate and tidal volume in COPD group were significantly decreased(P<0.01),and blood oxygen saturation was also significantly decreased(P<0.01),which reached the standard of COPD model.2 Pathological and immunological studies on the acute exacerbation model of chronic obstructive pulmonary disease in Wistar rats infected with multidrug-resistant Pseudomonas aeruginosa.The specific test method is as follows:General conditions showed that after infection,the rats had tachypnea,curled up in a corner of the cage,and barely moved for 24 hours,accompanied by an increase in body temperature and serious hair explosion,which gradually recovered over time.The pathological results showed that at 24h after infection,the lung tissue of mice in the AECOPD model control group was pathologically examined for heart failure cells,infiltration of congestive neutrophils in the alveolar cavity and the presence of macrophages.The inflammatory cell infiltration gradually decreased over time at 72h and 120h after infection.Pulmonary function results showed that pulmonary function was further limited after infection,but gradually recovered over time,with a high degree of consistency with clinical AECOPD patients;The respiratory frequency curve,relaxation time curve,and other related parameters showed that the lung function of the AECOPD group was in an acute decline state(P<0.01)compared with the COPD model group at 24-120 h after infection with drug-resistant Pseudomonas aeruginosa,and it was in the recovery period at 168h,with significant recovery in related parameters.Oxygen saturation results showed that:Oxygen saturation was in a stable state of acute decline from 48 h to 120 h,significantly different from that in the COPD model group(P<0.01),and it recovered at 168h;The results of colony count in lung tissue showed that the viable count in lung tissue of rats reached the maximum at 24h and then gradually decreased,but the decrease in colony count within five days was relatively gentle,and it decreased rapidly on the seventh day,but inflammation persisted.The results showed that rats recovered quickly.However,five days after infection with the resistant bacteria,the model of acute exacerbation of chronic obstructive pulmonary disease induced by multi-resistant Pseudomonas aeruginosa infection in Wistar rats was stable,and the relevant results showed that the rats were in the acute exacerbation of chronic obstructive pulmonary disease within 120h after infection with the resistant Pseudomonas aeruginosa.3 Study on the therapeutic effect of Shufeng Jiedu Capsule on AECOPD Wistar rat model induced by multi-drug resistant Pseudomonas aeruginosa infectionThe results of lung function showed that the relaxation time,tidal volume and expiratory volume in the high-dose group were significantly increased after giving Shufeng Jiedu capsule,which was significantly different from that in the AECOPD model group(P<0.01;P<0.05);Both the high-dose group and the low-dose group can significantly reduce the respiratory rate,which is significantly different from that of rats in AECOPD model group(P<0.01;P<0.05);The high-dose group and low-dose group of Shujiedu capsule can significantly increase the peak expiratory flow rate and the peak inspiratory flow rate,which is significantly different from that of rats in AECOPD model group(P<0.01;P<0.05),which proves that Shufeng Jiedu capsule can improve the lung function of AECOPD rats.The results of blood oxygen saturation showed that the high-dose group and low-dose group of Shufeng Jiedu capsule could significantly increase the blood oxygen saturation of rats,which was significantly different from that of rats in AECOPD model group(P<0.01),indicating that Shufeng Jiedu capsule could improve the blood oxygen saturation of rats with AECOPD.In addition,the two dosage groups of Shufeng Jiedu Capsule have improved the pathological changes such as cilia breakage and shedding,inflammatory cell infiltration,etc.,and the two dosage groups can significantly reduce the number of colonies in lung homogenate,which is significantly different from that of rats in AECOPD model group(P<0.01).The detection results of drug-resistant genes show that the high and low dosage groups of Shufeng Jiedu Capsule can significantly reduce the expression of two drug-resistant genes mexA and mexB in rat lung tissue,indicating that Shufeng Jiedu Capsule can reduce the colonization of drug-resistant bacteria in lung tissue.The results of bacterial markers,inflammatory factors and chemokines in lung tissue showed that CRP,bacterial marker of high and low dose groups of Shufeng Jiedu Capsule,was significantly reduced,and there was significant difference as compared with the control group of AECOPD model(P<0.01).The chemokine CXCL1 was significantly decreased in the high-dose group of Shufeng Jiedu Capsule,which was significantly different from that in the control group of AECOPD model(P<0.01).Besides,chemokine 12(CXCL12)was significantly reduced in the Shufeng Jiedu Capsule low-dose group,which was significantly different from that in the AECOPD model control group(P<0.05).The levels of IL-1β,TNF-αand IL-6 in the lung tissue of rats in the Shufeng Jiedu Capsule group were significantly lower than those in the AECOPD model control group(P<0.01).In addition,IL-10 in the lung tissue of rats in the Shufeng Jiedu Capsule high-dose group was significantly reduced,and compared with the control group of AECOPD model,there was a significant difference(P<0.01).The blood routine results showed that the number of lymphocytes in peripheral blood of rats in the high-dose group of Shufeng Jiedu capsule increased significantly,which was significantly different from that of AECOPD group(P<0.01).The number of neutrophils in peripheral blood of rats in the high-dose group of Shujiedu capsule decreased significantly,which was significantly different from that of AECOPD group(P<0.05).The white blood cell count in peripheral blood of rats in the low-dose group of Shufeng Jiedu capsule decreased significantly,which was significantly different from that of AECOPD group(P<0.05),indicating that Shufeng Jiedu capsule can inhibit the inflammatory reaction of the body.After 4 days of treatment with Shufeng Jiedu Capsule,both dosage groups of Shufeng Jiedu Capsule showed a certain therapeutic effect.Within its scope,it can reduce bacterial colonization in lung,improve pathological changes of lung tissue,reduce the level of inflammatory cells in peripheral blood,improve lung function,increase oxygen saturation,etc.The above results show that Shufeng Jiedu Capsule has obvious therapeutic effect on Wistar rats with acute exacerbation of chronic obstructive pulmonary disease caused by multi-drug resistant Pseudomonas aeruginosa infection.4 Study on the mechanism of Shufeng Jiedu Capsule on AECOPD Wistar rat model caused by multi-drug resistant Pseudomonas aeruginosa infectionThe results of flow cytometry showed that the proportion of CD4+ T lymphocytes in peripheral blood of rats in the low-dose Shufeng Jiedu capsule group was significantly higher than that in the AECOPD model control group(P<0.05).The proportion of CD8+T lymphocytes decreased significantly,which was significantly different from that of AECOPD model control group(P<0.05).The ratio of CD4+T lymphocytes to CD8+T lymphocytes increased significantly,which was significantly different from that of AECOPD model control group(P<0.05),indicating that dispelling wind and detoxifying can improve the immune level of the body.Masson staining and Elisa detection of collagen showed that Shufeng Jiedu capsule could reduce collagen deposition in lung tissue,thus alleviating pulmonary fibrosis in rats.AP-PAS staining showed that Shufeng Jiedu capsule could reduce proliferation and metaplasia of goblet cells and mucus secretion.Fluorescence and WB results showed that EGFR phosphorylation decreased significantly after treatment with Shufeng Jiedu capsule,which indicated that EGFR activation was blocked and downstream MUC5AC mucin content decreased significantly.The content of TGF-β protein decreased obviously,and the phosphorylation level of Smad2/3 decreased,indicating that the activation of TGF-β1/Smad2/3 pathway was blocked.After 4 days of treatment with Shufeng Jiedu Capsule,both dosage groups of Shufeng Jiedu Capsule showed a certain therapeutic effect.Within its scope,it can reduce and enhance the body’s immunity,reduce collagen deposition,reduce mucus secretion,improve lung tissue pathological changes,reduce the level of inflammatory cells in peripheral blood and so on,thus improving lung function and increasing blood oxygen saturation,thus playing a therapeutic role in AECOPD,and its mechanism of action may be to reduce mucus secretion,reduce collagen deposition by inhibiting EGFR-β/Smad2/3 pathway,improve the body’s immunity and reduce the body’s inflammatory response,etc.Conclusion:In this experiment,we explored the relevant parameters such as three body weights of two animal strains,smoking condition,tar tracheal instillation condition,and the infection rate of multi-resistant Pseudomonas aeruginosa.Through the lung function,blood oxygen saturation,lung lesions and the changes in the number of pulmonary colonies after infection,we determined the relevant parameter conditions for the establishment of AECOPD model.Under the relevant conditions,the AECOPD model could be reproduced,proving that the AECOPD model was stable.Shufeng Jiedu Capsule has obvious therapeutic effect on acute attack Wistar rat model of chronic obstructive pulmonary disease caused by multi-drug resistant Pseudomonas aeruginosa infection,and it exerts the therapeutic effect by reducing mucus secretion and pulmonary fibrosis.Innovation:1 The animal model of COPD was optimized.The tar injection through the trachea was used instead of LPS injection through the trachea,which is similar the morbidity conditions of clinical COPD patients and made it more in line with the clinical practice.Moreover,the model was stable and had high repeatability.2 At present,there are reports on AECOPD in China and abroad and analysis on clinical medication,but no reports on the pharmacodynamic mechanism of AECOPD in vivo.After the establishment and stability of this model,large-scale drug screening can be performed,which has guiding significance for clinical medication. |
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