| Objective:In this experiment,the main active ingredient of Panax notoginseng(PNS),a local medicinal herb from Yunnan Province,was selected to observe the effects of PNS on the inflammatory lesions in the lungs of rats with COPD and its modulating effect on macrophage polarization,and to investigate whether PNS can intervene in the inflammatory lesions in the lungs of COPD by modulating macrophage polarization,and to provide a theoretical and experimental basis for the TCM treatment of target therapy for COPD,and also provide new ideas for the clinical treatment of the disease.Methods:Animal experiment:The COPD rat model was replicated by cigarette smoke combined with tracheal drip injection of lipopolysaccharide for a total of eight weeks.The success of the model replication was judged by observing the changes in behaviour,lung function,lung pathology and the concentration of inflammatory factors in the lung tissue homogenates of the rats.The model was successfully fumigated every other day to maintain the lesions,while Panax ginseng total saponin solution was given daily by gavage for a total of four weeks.The rats were anaesthetized with a small animal anaesthesia apparatus,and the lung function indexes were measured using the EMMS forceful lung function test system;HE and Masson staining were used to observe the inflammatory and collagen fibrous deposition lesions in the lungs of the rats;the expression of TNF-α,IL-1β,IL-6,IL-8 and other inflammatory factors and TGF-β1,IL-10 and other anti-inflammatory factors in the lung tissue homogenates of the rats were measured by ELISA.-The protein expression of M1 macrophage surface marker(CD11b~+CD86~+)and M2 macrophage surface marker(CD11b~+CD206~+)in rat lung tissue was detected by immunofluorescence;the protein expression of M1 macrophage surface marker(CD11b~+CD86~+)and M2 macrophage surface marker(CD11b~+CD206~+)in rat lung tissue was detected by flow cytometry;the protein expression of M1 macrophage surface marker(CD11b~+CD86~+)and M2 macrophage surface marker(CD11b~+CD206~+)in rat lung tissue was detected by flow cytometry.The mRNA expression of M1 macrophage surface markers(CD11b~+、CD86~+)and M2 macrophage surface markers(CD163~+、CD206~+)was detected by RT-PCR.The experimental data were statistically processed using SPSS 26.0 software,and the measurement data were expressed as mean±standard deviation((?)±s).The test method was one-way ANOVA or non-parametric ANOVA.p<0.05 indicated that the difference was statistically significant,and p>0.05 indicated that the difference was not statistically significant.Statistical plots were produced using Graph Pad Prism 9.0.Cell experiment part:Raw264.7 macrophages were used to observe and verify the modulation of macrophage polarization by PNS.The optimal dosing concentration was determined by MTS method,and the dosing group was given the optimal dosing concentration of Panax ginseng total saponin for intervention.ELISA was used to detect changes in the concentrations of IL-1β,TNF-α,IL-10 and TGF-β1 in the cell culture medium;RT-PCR was used to detect the expression of mRNA of M1-type macrophage surface markers(CD86~+、iNOS)and M2-type macrophage surface markers(CD163~+、CD206~+);Western Blot method The protein expression levels of M1 macrophage-related factors(TNF-α、iNOS)and M2 macrophage-related factors(TGF-β1、Arg-1、CD206)were detected.The experimental data were statistically processed using SPSS26.0 software,and the measurement data were expressed as mean±standard deviation((?)±s).The test method was one-way ANOVA or non-parametric ANOVA.p<0.05 indicated that the difference was statistically significant,and p>0.05 indicated that the difference was not statistically significant.Statistical plots were produced using Graph Pad Prism 9.0.Results:Animal experiment part.1.After tracheal drip injection of lipopolysaccharide combined with cigarette smoke modeling,rats in the model group showed slow weight gain,dry and burnt hair with shedding;frequent sneezing and nose scratching behaviors;HE pathological sections and Masson staining results showed a large amount of inflammatory factor infiltration and collagen fiber deposition in the lung tissue of rats in the model group;lung function test results showed that the lung ventilation The results of lung function tests showed that the lung ventilation function(FEV0.5,FVC,FEV0.5/FVC,MMEF)and lung volume function(RV,TLC,RV/TLC,FRC)of the rats in the model group were all impaired to varying degrees,and lung function was in a state of decline;the above indicators indicated that the model was successfully replicated.2.Compared with the blank group,the rats in the model group had dry,yellowish hair and were prone to shedding,scratching their noses,sneezing and other behaviours,and their body weight increased slowly.3.The results of lung function showed that compared with the blank group,the lung ventilation function(FEV0.5,FVC,FEV0.5/FVC,MMEF values were significantly lower(p<0.05)and lung volume function(RV,TLC,RV/TLC,FRC values were significantly higher(p<0.05))of the model rats were decreased.The lung ventilation function and lung volume function of the rats were significantly improved after the intervention with Panax notoginseng(p<0.05).4.The results showed that the alveolar structure of the rats in the blank group was clear and intact,the alveolar lumen was not enlarged,the bronchial epithelium was intact,the mucosal epithelial cells were neatly arranged,the cilia were not damaged and shed,there was no congestion and edema,and no obvious exudation was seen.In the model group,the lung interstitium was congested and edematous,and the alveolar wall was infiltrated by a large number of inflammatory cells such as macrophages,neutrophils and a small number of lymphocytes,and the cilia of the mucous membrane layer were inverted and shed.Some of the alveolar structures disappeared and fused to form large alveoli.The lung tissues of the rats in each administration group were less infiltrated than those in the model group,the congestion and edema were significantly reduced,and there was no obvious exudation.5.The results showed that the collagen fibre content in the lung tissues of rats in the model group increased significantly compared with that in the blank group,and the difference was statistically significant(p<0.05),and the collagen fibre content in the lung tissues of rats in each administration group showed a decreasing trend after the intervention of Panax ginseng total saponin,and the most significant was in the medium dose group of Panax ginseng total saponin and the positive drug group(p<0.05).6.The expression of M1-type macrophage secretory factors(pro-inflammatory factors)and M2-type macrophage secretory factors(anti-inflammatory factors)in the lung tissue homogenates of rats was detected by ELISA,and the results showed that compared with the blank group,the expression of pro-inflammatory factors TNF-α,IL-1β,IL-6,IL-8 and IL-12 in the lung tissue homogenates of rats in the model group was significantly higher(p<0.05),and the expression of anti-inflammatory factors TGF-β1,IL-8 and IL-12 in the model group was significantly higher(p<0.05).The expression levels of pro-inflammatory factors TNF-α,IL-1β,IL-6,IL-8 and IL-12 in lung tissue homogenates of rats were significantly lower(p<0.05)and the expression levels of anti-inflammatory factors TGF-β1and IL-10 were significantly higher(p<0.05)compared with those of the model group.7.Immunofluorescence detection of M1/M2 macrophage protein expression levels in rat lung tissues showed that compared with the blank group,the protein expression levels of M1macrophage surface markers(CD11b~+CD86~+)were significantly higher in the model group,and the difference was statistically significant(p<0.05),and the protein expression levels of M2 macrophage surface markers(CD11b~+CD206~+)were statistically significant(p<0.05).CD206~+)in the lung tissues of the rats administered with the drug were significantly lower compared to the model group,and the difference was statistically significant(p<0.05).CD206~+)was significantly increased and the difference was statistically significant(p<0.05).8.The expression of M1/M2 macrophage count levels in rat lung tissues was detected by flow cytometry,and the results showed that compared with the blank group,the cell count levels of M1 macrophages(CD11b~+CD86~+)in rat lung tissues of the model group were significantly higher,and the difference was statistically significant(p<0.05),and the cell count levels of M2 macrophages(CD11b~+CD206~+)were significantly lower,and the difference was statistically significant(p<0.05).Compared with the model group,the cell count levels of M1 macrophages(CD11b~+CD86~+)in the lung tissues of rats were significantly lower and the difference was statistically significant(p<0.05),while the cell count levels of M2 macrophages(CD11b~+CD206~+)were significantly higher and the difference was statistically significant(p<0.05).The difference was statistically significant(p<0.05).9.RT-PCR was used to detect the mRNA expression levels of M1/M2 macrophage surface markers,and the results showed that compared with the blank group,the mRNA expression levels of M1 macrophage surface markers(CD11b~+,CD86~+)were significantly higher in the lung tissues of rats in the model group,and the differences were statistically significant(p<0.05),and the mRNA expression levels of M2 macrophage surface markers(CD163~+,CD206~+)were statistically significant(p<0.05).Compared with the model group,the mRNA expression levels of M1 macrophage surface markers(CD11b~+,CD86~+)were significantly lower in the lung tissues of rats in each administration group,and the differences were statistically significant(p<0.05).The mRNA expression levels of M2 macrophages(CD163~+,CD206~+)were significantly increased and the difference was statistically significant(p<0.05).Cell experiment section.1.Normally cultured Raw264.7 were round or oval in shape under the microscope,growing in sheets and in good growth condition.After stimulating Raw264.7 with LPS for 24 hours and IL-4 for 48 hours respectively,the cell morphology changed significantly,with the cells showing long shuttle shape or irregular shape,the number of cells decreased,and there were cell debris floating in the cell culture medium.2.After reviewing the literature and basing on the previous experiments,7 PNS dosing concentrations were set at 0,100ug/ml,200ug/ml,300ug/ml,400ug/ml,500ug/ml and600ug/ml,respectively,and the optimal dosing concentration was determined by the MTS method:the cells were divided into 7 groups and 5 replicate wells were set up for each group.After incubation,the cells were incubated with MTS reagent for 3 hours and the absorbance was measured at 490 nm to determine the effect of different concentrations of PNS on cell activity.The results showed that a concentration of 200ug/ml of PNS had the least effect on cell activity and therefore 200ug/ml was used as the concentration for cell administration.3.Results of M1 macrophage experiments:3.1 Effect of PNS on the expression of inflammatory levels in M1-type macrophage cultures:after stimulation of Raw264.7 with LPS,the expression levels of inflammatory factors IL-1βand TNF-αwere significantly higher and the expression levels of anti-inflammatory factors IL-10 and TGF-β1 were significantly lower in the cultures of cells from the model group compared to cells from the blank group,and the differences were statistically significant(p<0.05).The expression levels of inflammatory factors IL-1βand TNF-α,and the expression levels of anti-inflammatory factors IL-10 and TGF-β1 were significantly increased in the cell supernatant after the intervention of Panax ginseng total saponin compared with the cells in the model group,and the difference was statistically significant(p<0.05).3.2 Effect of PNS on mRNA expression of M1-type macrophage surface markers:compared with the cells in the blank group,the mRNA expression levels of M1-type macrophage surface markers CD86~+and iNOS in the model group cells were significantly increased,and the differences were statistically significant(p<0.05);compared with the cells in the model group,after the intervention of total saponin of Panax ginseng,the mRNA expression levels of M1-type macrophage surface markers in the administered group cells were significantly increased,and the differences were statistically significant(p<0.05).The mRNA expression levels of the macrophage surface markers CD86~+and iNOS were significantly reduced,while the mRNA expression levels of the macrophage surface markers CD163~+and CD206~+were significantly increased in the M2 type cells after the administration of Panax ginseng total saponin intervention,and the differences were statistically significant(p<0.05).3.3 Effect of PNS on protein expression of M1-type macrophage-related factors:compared with cells in the blank group,the protein expression levels of M1-type macrophage-related factors TNF-αand iNOS were significantly increased in cells in the model group;compared with cells in the model group,the protein expression levels of M1-type macrophage-related factors TNF-αand iNOS in cells in the administration group were significantly.4.Results of experiments on M2 type macrophages:4.1 Effect of PNS on the expression of inflammatory levels in the culture medium of M2type macrophages:after stimulation of Raw264.7 with IL-4,the expression levels of the inflammatory factors TGF-β1 and IL-10 in the culture medium of cells in the model group were significantly higher and the expression levels of the inflammatory factors IL-1βand TNF-αwere significantly lower compared to the cells in the blank group,and the differences were statistically significant(p<0.05);compared with the cells in the model group,the expression levels of inflammatory factors IL-1βand TNF-αand the expression levels of anti-inflammatory factors TGF-β1 and IL-10 in the cell supernatant were significantly increased after the administration of Panax ginseng total saponin intervention,and the differences were statistically significant(p<0.05).4.2 Effect of PNS on mRNA expression of M2 macrophage surface markers:compared with the cells in the blank group,the mRNA expression level of the M2 macrophage surface marker CD206~+in the model group cells was significantly increased,and the difference was statistically significant(p<0.05);compared with the cells in the model group,after the intervention with Panax ginseng total saponin,the mRNA expression level of the M1macrophage surface marker CD86~+in the administered group cells was significantly increased,and the difference was statistically significant(p<0.05).The mRNA expression level of CD86~+,a marker of M1 macrophages,was significantly increased and that of CD206~+,a marker of M2 macrophages,was significantly decreased after the administration of Panax ginseng saponin,and the differences were statistically significant(p<0.05).4.3 Effect of PNS on protein expression of M2-type macrophage-related factors:compared with cells in the blank group,the protein expression levels of M2-type macrophage-related factors TGF-β1,Arg-1,CD206~+were significantly increased in cells in the model group;compared with cells in the model group,after the intervention with Panax ginseng total saponin,the protein expression levels of M2-type macrophage-related factors TGF-β1,Arg-1,CD206~+were significantly increased in cells in the administration group.The protein expression levels of M2 macrophage-related factors TGF-β1,Arg-1 and CD206~+were significantly reduced in the administered cells compared with the model group.Conclusion:1.Panax ginseng total saponin can improve lung function,reduce the level of inflammation and alleviate collagen deposition in the lung tissue of COPD rats.2.Panax ginseng total saponin reduces the expression of M1-type macrophages and the release of inflammatory factors in lung tissues of COPD rats,increases the expression of M2-type macrophages and the expression level of lung anti-inflammatory factors,and promotes the dynamic balance of M1/M2-type macrophages,thus reducing the level of inflammation in lung tissues and alleviating the lesion process of COPD. |
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