Study On Anti-inflammatory Effect Of ESP-B4 From Ephedra Sinica Stapf On Copd And Acute Lung Injury

In China,Ephedra has more than 5,000 years of medicinal history.Ephedra,acrid,little bitter,has the effect of "freeing lung and relieving asthma".Ephedra was firstly recorded in"Shen Nong Ben Cao Jing".Alkaloids have been considered to be the most important biologically active ingredients in Ephedra.However,our previous study founded that the Ephedra polysaccharides had the effects of anti-inflammation and immunity.ESP-B4,a purified and highest content in Ephedra polysaccharides,has the significantly anti-inflammatory effect on chronic obstructive pulmonary disease（COPD）and acute lung injury（acute respiratory distress syndrome）（ALI/ARDS）,laying the foundation for the study of new drugs for COPD and ALI/ARDS.The research content of this paper is as follows:1.Preparation and Identification of ESP-B4The method of water extraction and alcohol precipitation was used to extract the total Ephedra polysaccharides.ESP-B4 was obtained through anion ion resin column and DEAE-52 chromatographic column.In this paper,high-performance liquid chromatography and UV were used to identify ESP-B4;the method of PMP precolumn derivatization was used to detect the monosaccharide composition of ESP-B4;Methylation analysis was used to detect the connection sequence of sugar linkage.The HPLC chromatogram showed that it was a single symmetrical peak,indicating that the ESP-B4 is homogenous with high purity.There was no obvious absorption peaks at 260 nm and 280 nm,indicating that ESP-B4 did not contain nucleic acids and proteins.The monosaccharide composition analysis showed that ESP-B4 was consisted of Xyl,Ara,Glc,Rha,Man,Gal,GlcUA,GalUA with molar percentages of 1.9%,7.2%,1.8%,3.3%,1.7%,8.7%,2.7%,and 72.7.%.Methylation analysis showed that ESP-B4 was likely to have a linear 1,4 linked galacturonic acid as the backbone.2.Study on the anti-inflammatory effect of ESP-B4 on COPD ratsCOPD model was established by combining lipopolysaccharide（LPS）with cigarette smoke（CS）to study the anti-inflammatory effects of ESP-B4.Fifty healthy male SD rats,were randomly divided into 5 groups:control group,model group,ESP-B4 group（140 mg/kg）,ESP-B4 group（280 mg/kg）and dexamethasone group（5 mg/Kg/day）.During the experiment,the behavioral changes of animals were observed and the weight was recorded.After the experiment,the lung index,thymus index and spleen index of the rats were measured;the number of white blood cells,lymphocytes,neutrophils,macrophages and eosinophils in the whole blood of the rats were measured by a fully automatic three-class blood analyzer.The levels of TNF-α,IL-6,IL-8 and MMP-9 in the lung tissue were measured by Elisa.HE staining was used to observe the changes of lung tissue in rats.The expression of TGF-β/Smad2 pathway-related proteins was detected by immunohistochemistry.The urine samples were collected before and during the experiment,and the serum was collected after the experiment.The potential biomarkers and metabolism were determined by UPLC-Q-TOF/MS detection method.The results showed that ESP-B4 significantly reduced the number of leukocytes in the blood,decreased of the levels of TNF-α,IL-6,IL-8 and MMP-9,lowered the expression of TGF-β1,P-Smad2 and P-Smad3 proteins,raised the expression of Smad7.The result of HE staining showed that ESP-B4 significantly relieved the lung tissue phlogosis and bronchial mucus.The results showed that 47 biomarkers related COPD and 13 pathways were identified,namely Alpha-Linolenic acid metabolism,Vline,leucine and isoleucine biosynthesis,Arachidonic Acid Metabolism,Pentose and glucuronate interconversions,Retinol metabolism,Amino sugar and nucleotide sugar metabolism,Purine metabolism,Tryptophan metabolism,Starch and sucrose metabolism,Arginine and proline metabolism,Pyrimidine metabolism,Glutathione metabolism,Primary bile acid biosynthesis.The results showed that 28 biomarkers related ESP-B4 and 7 pathways were identified,and Alpha-Linolenic acid metabolism,Arachidonic Acid Metabolism had the stronger correlation.The level of alpha-linolenic acid involved in Alpha-Linolenic acid metabolism was decreased in model group,and ESP-B4 increased the level of alpha-linolenic acid.Thromboxane and Arachidonic acid were involved in Arachidonic Acid Metabolism,and the levels increased in model group,and decreased after the intervention of ESP-B4.It was concluded that ESP-B4 possibly played anti-inflammatory effects by promoting a-linolenic acid metabolism and inhibiting arachidonic acid metabolism.3.Study on anti-inflammatory effect of ESP-B4 on COPD cell modelThe inflammation model of A549 cells induced by LPS combined with CSE was established to study the anti-inflammatory effect of ESP-B4 on COPD cell model.Cells were grouped into control group,model group,ESP-B4（5 μg/ml）,ESP-B4（25 μg/ml）,ESP-B4（125 μg/ml）.The effects of ESP-B4,LPS,and CSE on cell viability were measured by MTT assay.The levels of TNF-α,IL-6,and IL-8 in cell supernatants were measured by Elisa method.The TGF-β/Smad2 pathway-related proteins were detected by Western blot.The results showed that ESP-B4 had no inhibitory effect on cell proliferation.LPS and CSE had a significant inhibitory effect on cell viability,and the inhibitory effect was enhanced with increasing concentration.ESP-B4 could significantly reduce TNF-α,IL-6,IL-8,and decrease the expression of TGF-β1,P-Smad2,P-Smad3 protein,raise Smad7 with a dose-dependent manner.4.Study on anti-inflammatory effect of ESP-B4 on mice with ALIALI model was established using Streptococcus pneumoniae and virus FM1 to study the anti-inflammatory effects of ESP-B4.In this study,Balb/c mice were infected intranasally with 2×107 CFU/mL Streptococcus pneumoniae and 1:640 virus FM1 saline solution.Animal behaviors,body weight,and coat color changes were recorded during the experiment.The lung index,thymus index,spleen index were measured.HE staining was used to observe the pathological changes of lung tissue and intestine.16s gene sequencing technic was used to detect the intestinal microbial species and abundance.In the experiment of Streptococcus pneumoniae infection,ESP-B4 improve the activity of mice,has the tendency to decrease lung index,thymus index and spleen index.HE staining showed that ESP-B4 reduced the degree of acute lung injury and intestine injury caused by S.pneumoniae infection.In addition,16S gene sequencing results showed that the intestinal flora of mice was mainly composed of Firmicutes,Bacteroidetes,Actinobacteria and Proteobacteri.Compared with control,the levels of abundance of Ruminococcaceae,Erysipelotrichaceae,Bifidobacteriaceae,Lactobacillaceae,Bacteroidales_S24-7_group,Coriobacteriaceae,Deferribacteraceae,Gastranaerophilales_NA,Clostridiales_vadinBB60_group decreased,and the levels of abundance of Anaeroplasmataceae、Enterobacteriaceae,Streptococcaceae,Prevotellaceae increased.ESP-B4 increased Bacteroidales_S24-7_group,Bifidobacteriaceae,Gastranaerophilales_NA,Coriobacteriaceae,and Lactobacillaceae,and decreased Anaeroplasmataceae,Clostridiales_vadinBB60_group,Clostridiales NA and Bacteroidales_NA.In the experiment of virus FM1 infection,ESP-B4 improve the activity of mice,decreased lung index.HE staining showed that ESP-B4 reduced the degree of acute lung injury and intestine injury caused by virus FM1 infection.Compared with control,the levels of abundance of Ruminococcaceae,Bifidobacteriaceae,Lactobacillaceae,Gastranaerophilales_NA,Clostridiales_vadinBB60_group,Clostridiaceae₁,Christensenellaceae decreased,and the levels of abundance of Enterobacteriaceae,Streptococcaceae,Prevotellaceae,Verrucomicrobiaceae,Enterococcaceae,Bacteroidales_NA,Defluviitaleaceae,Peptococcaceae and Moraxellaceae increased.ESP-B4 increased Gastranaerophilales_NA,Erysipelotrichaceae,Bifidobacteriaceae and decreased Verrucomicrobiaceae,Enterobacteriaceae,Deferribacteraceae,Moraxellaceae,Enterococcaceae,Peptococcaceae,Defluviitaleaceae,Pseudomonadaceae,Clostridiales_vadinBB60_group and Listeriaceae.In summary,this study found that ESP-B4 has anti-inflammatory effects on COPD and ALI/ARDS,and elucidates part of its anti-inflammatory molecular mechanisms from endogenous small molecules and intestinal immunity,respectively.On the basis of traditional theory,we further deepened understanding of the efficacy of traditional Chinese medicine ephedra,and discovered the new applications of ESP-B4 in COPD and ALI/ARDS.The findings of this paper lay the foundation for the research and development of new drugs that have definite efficacy,exact efficacy,low toxic and side effects,safety,and clinical application.