| Objective:In this study,U937 cells were induced by IL-4 to construct M2 macropha-ges and an asthma mouse model was established to observe the effects of Pingchuan granule on CFD,M2 macrophages,autophagy-related molecules and airway remodeling in asthma mice,and to explore the regulatory mechani-sm of Pingchuan granule on CFD-mediated M2 polarization of autophagymacrophages and airway remodeling.Method:Experiment I: wild type 239 t cells were transfected with LV3 CFD(gene interference lentivirus),and the lentivirus was titered by the well by well dilution method and CFD sh RNA was constructed.U937 cells were induced to differentiate into macrophages by PMA,and the macrophages were divided into four groups as follows: blank group,model group,antiasthmatic particle group,and CFD sh RNA group.The blank group was cultured normally,and both the model group and antiasthmatic particle group were induced with IL-4to construct M2 macrophage model,while the antiasthmatic particle group and CFD sh RNA group were cultured with antiasthmatic particle containing serum and lv3-cfd(gene interference lentivirus)solution,respectively.Seventy two hours after the intervention,the protein and m RNA levels of CFD in each group were determined by Western blot analysis;Group 5(CFD sh RNA + CFD group)was added,while the other 4 groups received macrophage extraction and culture,cell grouping and intervention methods in the same experiment,and group 5(CFD sh RNA + CFD group)received the same intervention method as the CFD sh RNA group with the additional addition of CFD recombinant proteins for intervention.Seventy two hours after cell intervention in each group,the protein and m RNA expression levels of Arg-1,FIZZ1,Ym1 and the percentage of cd206 positive cells in each group were determined by Western blot,RT q PCR as well as flow cytometry,respectively;The expression levels of Beclin1,LC3 Ⅱ / LC3 Ⅰ ratio,p62 protein as well as related proteins in FB / C3 b / c5 b signaling pathway in each group were detected by Western blot;Macrophage extraction and culture methods the same experiment was performed and divided into four groups: model group,antiasthmatic particle group,rapamycin group,and antiasthmatic particle + rapamycin group.All four groups were induced with IL-4 to construct M2 macrophage model,and the model group was cultured normally,while the antiasthmatic particle group and rapamycin group were given antiasthmatic particle containing serum and rapamycin(agonist of autophagy)intervention,respectively.The antiasthmatic granule + rapamycin group was then co intervened with antiasthmatic granule containing serum and rapamycin(autophagy agonist).The expression levels of CFD,Arg-1,FIZZ1,Ym1 and the percentage of cd206 positive cells in each group were determined by Western blot and RT q PCR;Subsequently,rapamycin(an autophagy agonist)was changed to 3-m A(an autophagy inhibitor)for the other 3 groups except the model group,and the expression levels of CFD,Arg-1,FIZZ1,Ym1 and the percentage of cd206 positive cells in each group were determined by Western blot and RT q PCR;Experiment 2: Fifty C57 BL / 6 mice were randomly divided into five groups,consisting of blank,model,antiasthmatic pellet,CFD sh RNA,and antiasthmatic pellet + CFD sh RNA groups,with 10 mice in each group.Mice in all groups(model group,antiasthmatic particle group,CFD sh RNA group and antiasthmatic particle + CFD sh RNA group),except the blank group,were subjected to intraperitoneal injection of ova sensitizing liquid combined with aerosolized challenge to establish the asthma model.The comparison between the antiasthmatic particle group and the antiasthmatic particle + CFD sh RNA group received intragastric treatment(31.2 g / kg)with 1 ml of an aqueous solution of antiasthmatic particles from day 15 to day 20;The blank group was gavaged with 1 ml distilled water from the 15 th to 20 th day;The CFD sh RNA group was given an airway instillation of CFD sh RNA Lentivirus on day 20 p.i.The general status of mice in each group,the total number of inflammatory cells,the numbers of neutrophils and eosinophils as well as the number of eosinophils in BALF were observed;MCH challenge test was applied to detect the airway hyperresponsiveness of asthmatic mice in each group;H & E staining,PAS staining,and Masson staining were applied to evaluate the histopathological changes in the lungs of asthmatic mice in each group;Serum levels of IL-4,IL-5,and IL-13 as well as ova Ig E in BALF were measured by ELISA;IHC was performed on all groups α-SMA protein expression levels;The protein and m RNA expressions of Arg-1,FIZZ1,YM-1and CFD in each group were determined by Western blot and RT q PCR;The expression levels of Beclin1,LC3 Ⅱ / LC3 Ⅰ ratio,p62 protein as well as related proteins in FB / C3 b / c5 b signaling pathway in each group were detected by Western blot.Result:1.Compared with the blank group,the levels of CFD protein and m RNA in the model group increased significantly,all of which were statistically significant(P < 0.05).Compared with the model group,the levels of CFD protein and m RNA significantly decreased between the antiasthmatic particle group and the CFD sh RNA group,all of which were statistically significant(P< 0.05).In which the expression of CFD in the antiasthmatic particle group was comparable to that in the CFD sh RNA group.2.Western blot and RT q PCR results showed that compared with the blank group,the expression levels of Arg-1,FIZZ1 and Ym1 protein and m RNA in the model group were all significantly increased.Meanwhile,the results of flow cytometry showed that compared with the blank group,the percentage of cd206 positive cells in the model group was significantly increased,Arg-1,FIZZ1,Ym1 protein and m RNA and the percentage of cd206 positive cells were significantly decreased between the antiasthmatic particle group and the CFD sh RNA group,all of which were statistically significant(P< 0.05);CFD significantly reversed the reduction in Arg-1,FIZZ1,Ym1 protein and m RNA expression levels and the percentage of cd206 positive cells due to CFD inhibition.However,there were no significant changes in the expression levels of Arg-1,FIZZ1 and Ym1 and the percentage of cd206 positive cells in the CFD sh RNA + CFD group compared with the model group,none of which were significant(P > 0.05);The ratio of Beclin1 to LC3 Ⅱ /LC3 Ⅰ in the model group was significantly increased compared with the blank group,and the expression level of p62 protein was significantly decreased,all differences were statistically significant(P < 0.05).Compared with the model group,the ratio of Beclin1 to LC3 Ⅱ / LC3 Ⅰ was significantly decreased and the expression level of p62 protein was significantly increased in the CFD sh RNA group compared with the antiasthmatic particle group(P < 0.05).However,when CFD recombinant proteins were added to the stable U937 cells with CFD sh RNA,the expression levels of autophagy marker proteins did not change significantly compared with the model group,and none of the differences were statistically significant(P > 0.05).3.The expression levels of Arg-1,FIZZ1 and Ym1 and the percentage of cd206 positive cells in the rapamycin group were significantly increased compared with those in the model group,all of which were statistically significant(P < 0.05).However,the expression levels of Arg-1,FIZZ1 and Ym1 and the percentage of cd206 positive cells in the antiasthmatic particle +rapamycin group were significantly higher than those in the antiasthmatic particle group(P < 0.05).Compared with the model group,there was no significant change in the expression level of CFD in the rapamycin group,and none of the differences were statistically significant(P > 0.05).The expression levels of CFD in the antiasthmatic granulate + rapamycin group were significantly decreased compared with those in the rapamycin group(all P < 0.05);Western blot and RT q PCR results showed that the expression levels of Arg-1,FIZZ1,Ym1 and the percentage of cd206 positive cells were significantly decreased in the 3-m A group and the antiasthmatic particle +3-m A group compared with the model group,all of which were statistically significant(P < 0.05).Among them,the expression levels of Arg-1,FIZZ1 and Ym1 and the percentage of cd206 positive cells in the + 3-m A group of antiasthmatic particles were significantly lower than those in the 3-m A and antiasthmatic particles groups(P < 0.05).Compared with the model group,there was no significant change in the level of CFD expression in the 3-m A group,and none of the differences were statistically significant(P > 0.05).The expression levels of CFD in the antiasthmatic particle + 3-m A group were significantly decreased compared with those in the 3-m A group,and all the differences were statistically significant(P < 0.05).4.Western blot showed that compared with the blank group,the expression of FB in the model group was significantly decreased,while the shear molecule FB of FBS α The expression of FBS was significantly increased and all the differences were statistically significant(P < 0.05),shear molecules FB β Expression was slightly reduced and the difference was not statistically significant(P > 0.05).FB and its splicing molecules FB in CFD sh RNA group with antiasthmatic particles group compared to model group β The expression level of FBS was significantly increased by the shear molecule FB α Their expressions were significantly decreased and all differences were considered statistically significant(P < 0.05).FB,FB)after addition of CFD recombinant proteins to stable U937 cells with CFD sh RNAα And FB β The expression levels of the proteins did not change significantly compared with those of the model group,and none of the differences were statistically significant(P > 0.05);In addition,compared with the blank group,the expression of C3 b and c5 b in the model group was significantly increased,and the differences were all statistically significant(P < 0.05).Compared with the model group,the expression levels of C3 b and c5 b were significantly decreased in the antiasthmatic particle group with the CFD sh RNA group,and the differences were all statistically significant(P <0.05).When the addition of CFD recombinant proteins to U937 cells stably transfected with CFD sh RNA,the expression levels of C3 b and c5 b proteins did not change significantly compared with those of the model group,and none of the differences were statistically significant(P > 0.05).5.During the application of the MCH challenge test,mice in each group showed mouth opening breathing,slower breathing and deepening of the amplitude as the MCH concentration increased,and the Penh value increased significantly compared with the basal value as the drug concentration increased,especially when the MCH concentration reached 6.25g/l,the Asthma Mouse Model in the model group exhibited obvious airway hyperresponsiveness.Intergroup comparison results showed that compared with the blank group,the Penh value of the model group was significantly higher,and the difference was statistically significant(P < 0.05).However,both pharmacological interventions could significantly improve the airway hyperresponsiveness of asthmatic mice,and both the antiasthmatic particle group and the CFD sh RNA group had significantly lower Penh values compared with the model group,with significant differences(P < 0.05);Among them,compared with the CFD sh RNA group,the Penh value of the antiasthmatic particle + CFD sh RNA group was significantly lower,and the difference was statistically significant(P < 0.05);Compared with the blank group,the numbers of total inflammatory cells,lymphocytes,neutrophils,eosinophils,and macrophages in the BALF of the model group were all significantly increased,and the differences were all statistically significant(P < 0.05);Compared with the model group,the numbers of total inflammatory cells,neutrophils and eosinophils were significantly decreased in both the antiasthmatic particle group and the CFD sh RNA group,which were statistically significant(P < 0.05),while the numbers of lymphocytes and macrophages were not significantly different(P > 0.05).The number of total inflammatory cells,neutrophils and eosinophils were significantly decreased in the antiasthmatic particle + CFD sh RNA group compared with the CFD sh RNA group(P < 0.05),while the number of lymphocytes and macrophages were not significantly different.6.ELISA results: compared with the blank group,the contents of IL-4,IL-5,IL-13 and ova Ig E in the serum of mice in the model group were significantly increased,all differences were statistically significant(P <0.05).Compared with the model group,the levels of IL-4,IL-5,IL-13 and ova Ig E in the serum of mice in the antiasthmatic particle group and CFD sh RNA group were significantly decreased,all of which were statistically significant(P < 0.05).The levels of IL-4,IL-5,IL-13 and ova Ig E in the serum of mice in the antiasthmatic particle + CFD sh RNA group were significantly decreased compared with those in the CFD sh RNA group(P < 0.05).7.The results of HE staining showed that: there were no obvious pathological changes in the lung tissues of mice in the blank group.However,a large number of inflammatory cells were observed in the bronchial and vascular periphery of mice in the model group,and the local area of alveoli contained a large number of inflammatory cells;The lumen was markedly narrow.In which,the airway inflammation scores of mice in the model group were significantly increased compared with the blank group,and the differences were statistically significant(P < 0.05).However,both pharmacological interventions could significantly improve the infiltration of inflammatory cells in the airways of asthmatic mice,in which the airway inflammatory scores of mice in the antiasthmatic particle group and the CFD sh RNA group were significantly reduced compared with those of the model group,all with significant differences(P < 0.05).The airway inflammation scores of mice in the antiasthmatic particle + CFD sh RNA group were significantly reduced compared with those in the CFD sh RNA group(P <0.05).8.The results of PAS staining showed that very few goblet cells could be observed in the trachea and bronchi of mice in the blank group,and there was no more obvious mucus secretion in the airways.The airways of mice in the model group showed more goblet cell hyperplasia and formed a large number of mucus plugs,in which the PAS score was significantly increased compared with the blank group,and the difference was statistically significant(P <0.05).However,pharmacological intervention both significantly improved goblet cell hyperplasia,and PAS scores of mice were significantly reduced in both antiasthmatic particle group and CFD sh RNA group compared with the model group,with all differences statistically significant(P < 0.05).Compared with the CFD sh RNA group,the PAS scores of mice in the antiasthmatic particle + CFD sh RNA group were significantly lower,all differences were statistically significant(P < 0.05).9.Masson staining results showed that very little collagen staining was visible around the airway in the blank group mice,while a large amount of collagen staining was visible around the airway in the model group mice.Compared with the blank group,the stained area of collagen in the model mice increased significantly,all differences were statistically significant(P <0.05).Compared with the model group,the area of collagen staining around the airways of mice in both the antiasthmatic particle group and the CFD sh RNA group were significantly decreased,and the differences were statistically significant(P < 0.05).Compared with the CFD sh RNA group,the area of collagen staining around the airways of mice in the antiasthmatic granulate + CFD sh RNA group was significantly decreased,and all differences were statistically significant(P < 0.05).10.The results of immunohistochemical staining showed that very few airways were visible in the mice of the blank group α-SMA protein was positively stained,while a large number were found around the airways in the model mice α-SMA protein positive staining.Compared with the blank group,the mice in the model group α-The mean optical density values of SMA proteins were significantly increased and all differences were statistically significant(P < 0.05).Compared with the model group,the airways of mice in the antiasthmatic particle group and the CFD sh RNA groupα-The mean optical density values of SMA proteins were all significantly lower and all differences were statistically significant(P < 0.05).Airways of mice in the antiasthmatic pellet + CFD sh RNA group compared to the CFD sh RNA group α-The mean optical density values of SMA proteins were significantly lower and all differences were statistically significant(P <0.05).11.Flow cytometry results showed that compared with the blank group,the percentages of mice in the model group that were positive for both CD86 and cd206 were significantly increased,while the ratios of CD86 to cd206 were significantly decreased and all differences were statistically significant(P < 0.05).Compared with the model group,the percentages of CD86-and cd206 positive cells were significantly decreased in both the antiasthmatic particle group and the CFD sh RNA group,while the ratio of CD86 to cd206 was significantly increased,all differences were statistically significant(P <0.05).Compared with the CFD sh RNA group,the percentages of both CD86-and cd206 positive cells in the mice of antiasthmatic particle + CFD sh RNA group were significantly decreased,while the ratios of CD86 to cd206 were significantly increased,all differences were statistically significant(P <0.05);Western blot and RT q PCR assays showed that compared with the blank group,the expression of Arg-1,FIZZ1,YM-1 and CFD protein and m RNA in the lung tissues of mice in the model group were all significantly increased,and the differences were statistically significant(P < 0.05).Compared with the model group,the protein and m RNA expressions of Arg-1,FIZZ1,YM-1and CFD in the lung tissues of mice in both the antiasthmatic particle group and the CFD sh RNA group were significantly decreased,with significant differences(P < 0.05).Compared with the CFD sh RNA group,the expression of Arg-1,FIZZ1,YM-1 and CFD protein and m RNA in the lung tissues of mice in the antiasthmatic particle + CFD sh RNA group were significantly decreased(P < 0.05);The results of Western blot showed that the ratio of Beclin1 to LC3 Ⅱ /LC3 Ⅰ in the model group was significantly increased compared with that in the blank group,and the expression level of p62 protein was significantly decreased,all of which were statistically significant(P < 0.05).Compared with the model group,the ratio of Beclin1 to LC3 Ⅱ / LC3 Ⅰ was significantly decreased and the expression level of p62 protein was significantly increased in the CFD sh RNA group compared with the antiasthmatic particle group(P < 0.05).However,the expression levels of autophagy marker proteins did not change significantly when CFD recombinant proteins were added to the stable U937 cells with CFD sh RNA compared with the model cells,and none of the differences were statistically significant(P > 0.05);Compared with the blank group,the expression of FB,a shear molecule of FBS,was significantly decreased in the model group α The expression of FBS was significantly increased,all of which were statistically significant(P<0.05),shear molecules FB β.Expression was slightly reduced and the difference was not statistically significant(P> 0.05).FB and its splicing molecules FB in CFD sh RNA group with antiasthmatic particles group compared to model group β.The expression level of FBS was significantly increased by the shear molecule FB α.Their expression was significantly reduced and all differences were statistically significant(P<0.05).FB,FB)after addition of CFD recombinant proteins to stable U937 cells with CFD sh RNA α And FB β.The expression levels of the proteins did not change significantly compared with those of the model group,and none of the differences were statistically significant(P>0.05).In addition,compared with the blank group,the expression of C3 b and c5 b in the model group was significantly increased,and the differences were all statistically significant(P < 0.05).Compared with the model group,the expression levels of C3 b and c5 b were significantly decreased in the antiasthmatic particle group with the CFD sh RNA group,and the differences were all statistically significant(P <0.05).When the addition of CFD recombinant proteins to U937 cells stably transfected with CFD sh RNA,the expression levels of C3 b and c5 b proteins did not change significantly compared with those of the model group,and none of the differences were statistically significant(P > 0.05).Conclusion:1.Pingchuan granule can reduce FB by downregulatingα 、The expression levels of C3 b and c5 b proteins further decrease the expression of CFD proteins;Also reduced the expression levels of M2 macrophage marker proteins Arg-1,FIZZ1,YM-1 and m RNA;2.Pingchuan granule could decrease the expression levels of autophagy related molecules Beclin1 and LC3 Ⅱ / LC3 Ⅰ and increase the expression levels of p62 protein.3.Pingchuan granule attenuate the pathological changes of airway hyperresponsiveness,airway inflammatory cell infiltration,thickening of smooth muscle layer,mucus secretion increase,goblet cell hyperplasia,and collagen deposition in the airways of asthmatic mice.4.Pingchuan granule reduce the levels of IL-4,IL-5,IL-13,and ova Ig E in the serum and BALF of asthmatic mice;Can be downregulated α-SMA protein expression;Can reduce the expression levels of M2 macrophage marker proteins Arg-1,FIZZ1,YM-1 and m RNA,as well as autophagy related molecules Beclin1 and LC3 Ⅱ / LC3 Ⅰ,and increase the expression levels of p62 protein in lung tissue of asthmatic mice;Can also reduce FB α 、Expression levels of C3 b and c5 b proteins.5.Pingchuan granule inhibit FB / C3 b / c5 b signaling pathway activation mainly through the expression of CFD,which inhibits the autophagy level of macrophages,and then inhibits the M2 polarization of macrophages and inhibits asthmatic airway inflammation and airway remodeling,especially airway remodeling. |
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