| Objective:On the basis of previous research in the laboratory,this study used the trend of polarization of M1 and M2 influencing the IPF process as a clue,systematically reviewed the historical origins and clinical applications of ascending and descending,floating and sinkin resistance,and analyzed the effects of Ephedra and Schisandrae on M1/M2 polarization.We attempted to elucidate the scientific significance of IPF treatment guided by the ascending and descending,floating and sinking from the perspective of the" AM/TGF-?1/Smad" signaling pathway.Method:(1)Literature research:The ancient books and literature related to the theory of lifting and sinking and sinking and sinking were systematically reviewed,combing the historical origins,theoretical research and clinical research of lifting and sinking drug resistance theory.searching CNKI,VIP database,Wanfang database,Pubmed and Chinese biomedical literature.The databases for the past decade have summarized the role of macrophage polarization in pulmonary fibrosis,and combing the regulation of TGF-?1/Smad signaling pathway on different diseases.(2)In the in-vivo study,120 SPF Wistar male rats were randomly divided into sham group,model group.Ephedra group,Schisandrae group and Dex group.In addition to sham group,bleomycin was used to obtain IPF of rats.The rats in the sham group were instilled with nonnal saline.The lung tissues were removed on the 7th,14th,and 28th day after modeling.The histopathological morphology of the lungs were observed by HE staining.The expressions of CD163 and iNOS in the lung tissue were observed by immunohistochemical staining.(3)In thein-vitro study,15 SPF Wistar male rats were fed intragastrically with Ephedra and Schisandrae decoction for 7 days.After 7 days,their blood was taken to obtain drug-containing serum;30 SPF Wistar male rats were obtained,AMs were extracted by alveolar lavage and cultured for 48 hours with drug-containing serum.MTT assay was used to detect the effect of drug-containing serum on cell viability.The effects of Ephedra and Schisandrae on polarizations of M1 and M2 were detected by flow cytometry.The ELISA assay was used to detect the expressions of TNF-a and TGF-?1 in cell culture medium.The effect of Ephedra and Schisandrae containing serum on the expression of NF-?B,Smad3,Smad4 and Smad7 in cells were detected by WestenBlot method.The expressions of NF-?B,Smad3,Smad4 and Smad7 mRNA in the cells were detected by RT-PCR.Results:(1)In the in-vivo study,? in the bleomycin-induced pulmonary fibrosis model,The respiratory rate of rats in the model group was significantly higher than that in the sham group(P<0.01).Both Ephedra and Schisandraecould reduce the respiratory frequency of IPF rats,which was significantly different from that of the model group(P<0.05).Among them.Ephedra decreased significantly at 7 days after modeling compared with Schisandrae group,Schisandrae had amore significant effect than Ephedra at 28 days after modeling.HE staining showed that the alveolar structure of rats in the sham group was relatively intact,and no fusion or inflammatory infiltration occurred and no fiber formation was observed.At 7 days after modeling,a large number of inflammatory cell infiltrates occurred in the alveolar cavity of model rats,some alveolar cavities were fused and damaged,and some alveolar interstitials were thickened.However,there was no obvious fiber bundle formation.The alveolar structures of rats in the Ephedra and Schisandrae groups had different degree of alveolar fusion and damage,and there was a small amount of inflammatory cell infiltration in the alveoli,but the extent and scope were lighter than that of the model group.On the 14th day after modeling,there were some fiber bundles around the bronchus in the model group,alveolar damage and formation of bullae,alveolar damage in the Ephedra and Schisandrae groups to some extent,a small amount of fiber bundles,but lighter than the model group.At 28 days,a large number of fiber bundles appeared in the lungs of the model group,and even around the bronchus,the alveolar structure was disordered,the alveolar wall was damaged,and pulmonary bulla appeared,but the inflammatory infiltration was relieved when compared with 7 days;In the Ephedra and Schisandrae group,the lung tissues of rats showed different degrees of alveolar wall damage,interval thickening,and fiber bundle formation,but the degree was lighter than that of the model group.Compared with the pathological morphology of the model group rats,Ephedra and Schisandraecould improve the pathological morphology of pulmonary fibrosis in rats,the difference was statistically significant(P<0.05).?In immunohistochemical staining,the expression of iNOS in the model group was significantly increased compared with the sham group(P<0.01).Compared with the model group,both Ephedra and Schisandrae inhibited the expression of iNOS.the difference was significant(P<0.01);and the inhibitory effect of Ephedra was significantly stronger than Schisandrae,there was a significant difference between the two groups at 14d and 28d after modeling(P<0.01).Besides,both Ephedra and Schisandrae groups could significantly inhibited the expression of CD 163,which had a significantly differencecompared with the model group(P<0.01).Moreover,the inhibitory effect of Schisandrae was stronger than that of Ephedra,there was a significant difference between the two groups at 7d and 28d after modeling(P<0.01).(2)In in-vitro studies,?In the experiment of cell viability of Ephedra and Schizandrae containing serum,when the final concentration of Ephedra and Schizandrae containing serum was 10%,the growth state of cells was stable than other concentrations.?In vitro M1/M2 polarization of rats' alveolar macrophages induced by Ephedra and Schizandrae containing serum,the number of M1 macrophages in the LPS+IFN-y group was significantly increased,compared with the blank group,there was a significant difference(P<0.01).The number of M2 macrophages in the IL-4 group increased significantly compared with the blank group(P<0.01);the proportion of M2 macrophages in the Ephedra group was significantly lower than that of the IL-4 group,which has a significant difference(P<0.01),and M1 macrophages were significantly lower,compared with LPS+IFN-y group,there was a significant difference(P<0.01).The number of M2 macrophages in Schizandrae group was significantly reduced,compared with the IL-4 group,it has a significant difference(P<0.01);the number of M1 macrophages in Schizandrae group decreased,and compared with the LPS+IFN-y group,it has a significant difference(P<0.01).?Ephedra and Schizandrae containing serum affect the release of TNF-a and TGF-?1 from M1 and M2.The levels of TNF-a and TGF-?1 in LPS+IFN-? and IL-4 groups showed an upward trend,compared with the blank group.there were a significant difference(P<0.01).The contents of TNF-a and TGF-?1 in the Ephedra and Schizandrae groups were all significantly decreased.Compared with the LPS+IFN-y group and the IL-4 group,the results have significantly difference(P<0.01),but they were all higher than those in the blank group,and there was a significantly difference compared with the blank group(P<0.01).? In the experiments of Ephedra and Schizandrae containing serum affecting NF-?B,Smad3,Smad4,and Smad7 in M1 and M2,the Ephedra and Schizandrae groups all inhibited the expressions of NF-?B.Smad3,and Smad4,and there were a statistical difference or significant difference(P<0.05 or P<0.01)compared with the blank group.At the same time,the expression of Smad7 was upregulated,and there was a significant difference compared with the blank group(P<0.01).? In the effects of Ephedra and Schizandrae containing serum on the expressions of NF-?B.Smad3,Smad4 and Smad7 mRNA in M1 and M2,both Ephedra and Schisandrae inhibited the expressions of NF-?B,Smad3 and Smad4mRNA,compared with the control group.There were statistically significant differences or significant differences(P<0.05 or P<0.01).At the same time,the Ephedra and Schizandrae groups promoted the expression of Smad7mRNA,which have a significantly difference compared with blank group(P<0.01).Conclusion:In this study,the "AM/TGF-?1/Smad" signaling pathway was used as a clue to explore the differences in the mechanism of AM polarization between Ephedra and Schisandrae from both in-vivo and in-vitro experiments.The in-vivo experiments showed that there was damage of alveolar structure and proliferation of fiber bundles in the process of pulmonary fibrosis.In the stage of alveolitis,AM was an important regulatory cell of IPF,and it was also a cellular target of this signaling pathway.Ephedra and Schisandrae inhibited the expression of iNOS in the alveolitis stage to suppress the polarization of M1,decreased the secretion of inflammatory factors,and Ephedra was more effective than Schisandrae,thus inhibited the development of alveolitis.In the fibrosis stage,Ephedra and Schisandrae can inhibit the expression of CD 163.and Schisandrae inhibiting effect was more significant,and inhibit the hyperpolarization of M2,reduced the formation of fiber bundles and inhibited the process of pulmonary fibrosis.This was consistent with the results of previous studies that Ephedra is more effective in the early stage of alveolitis,and Schisandrae has a more significant effect in the stage of advanced pulmonary fibrosis.Then the mechanism of action of this difference was explored from the perspective of"AM/TGF-?1/Smad".The in-vitro experiments showed that Ephedra and Schisandrae could inhibit the polarization of AM to Ml and M2 subtypes,and Ephedra inhibited the polarization of AM to M1 subtype was stronger than that of Schisandrae,the inhibitory effect of Schisandrae on the polarization of AM to M2 subtype was stronger than that of Ephedra.Through in-depth research,we found that in the inhibition of M1 subtype.Ephedra and Schisandrae may be activated byinhibiting TNF-a secretion,reducing TNF-?stimulation of NF-?B and then activating NF-?B.At the same time,the two drugs could inhibit the expression of NF-?B mRNA and then inhibited NF-?B production,thus blocking the role of NF-?B and STAT signaling pathway to inhibit the M1 polarized signaling pathway,inhibited AM polarization to the M1 subtype and inhibited the inflammatory response.In this process,Ephedra had a strong inhibitory effect.In the inhibition of M2 subtype.TGF-?1 is the initiator of the signaling pathway,and Smad protein is the key receptor in the signal transduction process.Ephedra and Schisandra may be by surpressingthe secretion of TGF-?1,it inhibited the activation of T?R I receptor and surpressed the expressions of Smad3 and Smad4 mRNA to inhibit the expressions of Smad3 and Smad4.The inhibition of Smad3 and Smad4 expressions reduceed Smad3 binding to T?RI type receptors to dimers,inhibited dimer phosphorylation,and later it binded to Smad4 to oligomers,and blocked the TGF-?1 signaling pathway.At the same time,Ephedra and Schisandrae promoted the expression of Smad7,Smad7 and Smad3 could competitively binded T?RI receptors to inhibit the phosphorylation of Smad3,which in turn interfered the binding of Smad3 and Smad4,thus exerted a negative regulatory effect,and inhibited TGF-?1 signal transduction into cells.And Schisandrae's inhibition was more pronounced in this process.As a promoter of TGF-?1/Smad signaling pathway,inhibition of TGF-?1 secretion reduced the production of TGF-?1 signal,and Smad3 and Smad4 were suppressed,which were the key receptors of this pathway,Smad7 protein which had negative regulation was promoted toexpressed at the same time,so the TGF-?1 conduction pathway was blocked that its signal could not be transferred into the nuclear transcription in time,thereby polarization of AM to M2 subtype was suppressed.In this study,the "AM/TGF-?1/Smad"signal pathway was used as the guide,and Ephedra and Schisandrae were taken as the representative drugs.Through investigating the tendency of traditional Chinese medicines with typical ascending and descending,floating and sinkin characteristics to AM polarization,we initially obtained the rise in IPF.The drug may inhibit the polarization of AM to the M1 subtype at the alveolitis stage,and the Schisandrae may inhibit AM polarization to the M2 subtype at the fibrotic stage.This study also provides a deeper explanation from the perspective of analytical biology on the results of the previous study that Ephedra in the early stage of alveolitis efficacy is more significant,Schisandraeacted more effectivein the late stage of pulmonary fibrosis.And provided beneficial exploration of the molecular biology of ascending and descending,floating and sinkin resistance. |
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