Based On The Theory Of "lung And Large Intestine Are Exterior And Interior", The Mechanism Of Action Of Shegan Zhike Capsule On MRSA-infected Pneumonia Was Explore

Purpose:Abuse of antibiotics leading to multidrug resistance and extensive drug resistance of bacteria,which is a major scientific problem urgently needs to be solved in clinical practice.Shegan Zhike capsule is a component of traditional Chinese medicine preparation funded by the 13th Five-Year Major New Drug Development Project of the Ministry of Science and Technology of the People’s Republic of China.On the basis of confirming that Shegan Zhike capsule has the effects of antitussive,expectorant,anti-inflammatory and immune enhancement,the research group used the Shegan Zhike capsule in vitro antibacterial model to verify that it has obvious inhibitory effect on methicillin-resistant Staphylococcus aureus(MSRA)and can enhance the sensitivity of antibiotics.Based on the traditional Chinese medicine theory of’lung and large intestine’,focusing on the efficacy of traditional Chinese medicine Shegan’reducing real fire and promoting large intestine’,this study aims to explore the effect and mechanism of Shegan Zhike capsule on MRSA pneumonia in rats by means of intestinal flora and metabolomics technology,and lay the foundation for solving the problem of clinical multi-drug resistant bacteria infection.Material and method:1.Theoretical and clinical studies:(1)Based on the development and application analysis of the theory of’lung and large intestine’in ancient books of traditional Chinese medicine:using the method of literature research,through the big data analysis platform of traditional Chinese medicine-’think tank of traditional Chinese medicine’to search and consult,looking for the records of the theory of’lung and large intestine’in ancient books,combing the development process of’lung and large intestine’theory,and the application of’lung and large intestine’theory in lung diseases is statistically analyzed.(2)Modern clinical study of traditional Chinese medicine in the treatment of severe pneumonia:By consulting and collating modern literature,the clinical status of traditional Chinese medicine in the treatment of severe pneumonia was analyzed.The advantages and disadvantages of traditional Chinese medicine in the treatment of severe pneumonia were summarized from the etiology and pathogenesis,syndrome differentiation and treatment of severe pneumonia.2.Using 16Sr DNA to analyze the effect of Shegan component preparation on intestinal flora of MRSA pneumonia rats:Sixty SPF SD rats,half male and half female,were randomly divided into 6 groups(10rats/group):negative control group,model control group,low,medium and high dose group of Shegan Zhike capsule,Suhuang Zhike capsule group.Except for the negative control group,each group was given intraperitoneal injection of pentobarbital sodium(30-60 mg/kg,2 m L/kg)for anesthesia,intranasally infected with multidrug-resistant Staphylococcus aureus(1×10~9CFU/m L,0.5 m L/mouse)for 5 days.During the modeling period,the drug was intragastrically administered once a day for 14 days.The negative control group and the model control group were given the same volume of pure water.Urine and feces were collected one day before the last administration and stored at-80℃.One hour after the last administration,abdominal aorta blood was collected from all survived rats after anesthesia,and the blood was centrifuged and stored at-80℃.Anatomy and take all rats left middle lobe with 10%formaldehyde fixed,HE staining for pathological sections;the right lung tissue was stored at-80℃.The ileocecal contents and colon tissues of rats in each group were taken for labeling.According to the results of blood routine test and lung tissue pathology,the negative control group,model control group,low and medium group of Shegan Zhike capsule,Suhuang Zhike capsule group were selected.The feces of six rats in each group were subjected to 16Sr DNA sequencing.The 16Sr DNA sequencing technology was used to analyze the change trend of intestinal flora of rats with MRSA pneumonia treated by Shegan Zhike capsule.Through the changes of intestinal flora diversity and richness in rats in each group,the main flora affecting the treatment of MRSA pneumonia rats by Shegan Zhike Capsule was searched,so as to speculate the possible mechanism of Shegan Zhike Capsule.3.Using metabolomics to analyze the effect of Shegan component preparation on metabolites of MRSA pneumonia rats:The lung tissue,abdominal aorta serum and feces of 6 rats with the same 16Sr DNA intestinal flora were taken for non-targeted metabolomics detection.Through the changes of metabolites in different tissues of MRSA pneumonia rats,the core metabolites of Shegan Zhike capsule in the treatment of MRSA pneumonia rats were found,and the possible mechanism of Shegan Zhike capsule was speculated.Finally,through the correlation analysis between these core differential metabolites and the main intestinal flora,it is further speculated that Shegan Zhike capsule may affect the changes of intestinal flora by regulating metabolites to treat MRSA pneumonia.Results:1.The theory of’lung and large intestine’runs through the whole development process of traditional Chinese medicine.The germination of the theory in the period of’Huangdi Neijing’to the formal proposal and promotion in the Sui and Tang Dynasties,the continuous improvement of the theory in the Song,Jin and Yuan Dynasties,until the theory of’lung and large intestine’in the Ming and Qing Dynasties has matured.The theory of’lung and large intestine’is mainly reflected in the following aspects in the diagnosis and treatment of lung diseases:(1)etiology and pathogenesis:dryness and heat evil,as common pathogenic factors of lung and intestine,lead to lung diseases and intestine,intestinal diseases and lung.From the perspective of pathogenesis,no matter the physiological function of the solid lung itself is obstructed,or the conduction function of the large intestine is out of duty,it will cause lung lesions.(2)Therapeutic principles:clearing away heat and detoxifying,purging and purging the bowels are the main therapeutic principles of’treating intestines with lung disease’and’treating both lung and intestines’clinically.’If the bowels are not real,the viscera cannot be real.’Lung and large intestine interact in pathology.2.Pneumonia caused by multidrug-resistant bacteria often leads to severe pneumonia due to the lack of effective antibiotics,which increases the difficulty of treatment for severe pneumonia.’Phlegm,heat,blood stasis and deficiency’are the main pathogenesis characteristics.The theories of’lung and large intestine’,’cultivating earth and generating gold’and’blood stasis and heat’are widely used in the treatment of severe pneumonia by traditional Chinese medicine.The addition of traditional Chinese medicine can not only reduce the inflammatory response of severe pneumonia,improve the clinical symptoms of patients,shorten the duration of mechanical ventilation(or ICU retention time),but also reduce the occurrence of bacterial resistance and improve the prognosis of patients.3.By analyzing the intestinal flora of MRSA pneumonia rats with 16Sr DNA,there was no significant difference in the diversity and richness of the flora between the model group and the negative control group.Compared with the model group,the Proteobacteria,Lachnospiraceae＿UCG-006,Rhizobiales,Anaerovoracaceae,Desulfovibrio＿fairfieldensis were significantly down-regulated in the low-dose Shegan Zhike capsule group;compared with the model group,the middle dose of Shegan Zhike capsule significantly up-regulated Bacteroidota,Bacteroidia,Bacteroidales,Muribaculaceae,and down-regulated Ligilactobacillus,Lactobacillus＿murinus,RF39,Ruminococcus＿sp＿N15.MGS-5.According to the 16S sequencing data,the bacterial community function was predicted based on the KEGG database.There were two metabolic pathways between the model group and the control group.There were 38 metabolic pathways with significant differences between the model group and the low dose group of Shegan Zhike capsule,and 4 metabolic pathways with significant differences between the model group and the middle dose group of Shegan Zhike capsule.4.Non-targeted metabolomics analysis of MRSA pneumonia rat serum by LC-MS:There were 16 significant metabolic differences between the negative control group and the model group,including 7 up-regulated metabolites and 9 down-regulated metabolites.Through KEGG enrichment of these metabolites,14 possible metabolic pathways were obtained.There were 33 significant metabolic differences between the model group and the low-dose group,including 24 up-regulated metabolites and 9 down-regulated metabolites.After KEGG enrichment,10 possible metabolic pathways were obtained.There were only three metabolites produced in the model group and the middle dose group of Shegan component preparation,which may not be detected in the middle dose group of Shegan component preparation.5.LC-MS was used to perform non-target metabolomics analysis on the lung tissue of MRSA pneumonia rats.There were 12 significant metabolic differences between the negative control group and the model group,of which 2 were up-regulated and 7 were down-regulated.Through the enrichment analysis of KEGG,we found that there were 12 metabolic pathways that might be affected by the negative control group and the model group.There were 8different metabolites in the model group and the low dose group of Shegan component preparation,up-regulated by 22 and down-regulated by 6.There were 9 different metabolites in the model group and the middle dose group of Shegan component preparations,including 3up-regulated and 6 down-regulated.KEGG enrichment analysis was performed on the two groups of differential metabolites.There were 15 metabolic pathways in the model group and the low dose group of the trunk component preparation,and 15 metabolic pathways in the middle dose group of the trunk component preparation,including 14 common metabolic pathways.6.Non-targeted metabolomics analysis of MRSA pneumonia rat feces by LC-MS:there were58 significant metabolic differences between negative control group and model group,including 24 up-regulated and 34 down-regulated metabolites.Through KEGG enrichment analysis,we found nine metabolic pathways that might be affected.There were 47 significant metabolic differences between the model group and the low-dose group,including 18up-regulated metabolites and 29 down-regulated metabolites.After KEGG enrichment,there were 11 main metabolic pathways affecting the low-dose group.There were 46 significant metabolic differences between model group and middle dose group,including 17 up-regulated metabolites and 29 down-regulated metabolites.After enrichment by KEGG,there were 11main metabolic pathways in low dose group.7.Correlation analysis between intestinal flora and differential metabolites in MRSA pneumonia rats:comparing between low dose group and model group,We found that Xanthosine,Phenylacetylglycine in serum metabolites,Methyl palmitate and Thromboxane B2 in feces were all significantly correlated with Proteobacteria in flora;Anaerovoracaceae was significantly associated with(+/-)11-HETE,Xanthosine in serum metabolites and Tyrosol in feces.In the comparison between the medium dose group and the model group,we found that Ligilactobacillus,Lactobacillus＿murinus and metabolites of feces such as Thromboxane B2,Tyrosol,and 17(S)-Hp DHA were significantly correlated;Ruminococcus＿sp＿N15.MGS-5 was significantly correlated with Methyl palmitate in feces;r F39 was significantly associated with Ricinoleic acid methyl ester,Adenosine and Tyrosol in feces.Conclusion:1.MRSA pneumonia can lead to change in intestinal flora in rats,it consistents with the theory of traditional Chinese medicine’lung and large intestine’;shegan Zhike capsule may maintain the balance of the enteral ecological environment by regulating the proteobacteria to exert an inhibitory effect on the inflammation of MRSA pneumonia rats.2.MRSA pneumonia can cause changes in serum,lung tissue and fecal metabolites in rats.Shegan Zhike capsule may inhibit MRSA pneumonia rats through amino acid metabolism pathway,purine metabolism pathway and arachidonic acid metabolism pathway.3.Shegan Zhike capsule may regulate the change of intestinal proteobacteria by purine metabolism and arachidonic acid metabolism,so as to achieve the therapeutic effect of MRSA pneumonia,which is consistent with the traditional effect of Shegan’reducing real fire and promoting large intestine’,it may be one of the mechanisms of Shegan Zhike capsule in the treatment of MRSA pneumonia.