| Background:Pneumonia is a common disease in pediatric pulmonary system, ranking first in the general hospital pediatric cases of single disease statistics. It has been one of three important global pediatric diseases by WHO.In the children with acute lower respiratory tract infections in China, the virus accounts for about one-third with upward trend with a maximum of respiratory syncytial virus (RSV). RSV pneumonia is more common in hot syndrome, with syndrome of obstruction of lung by phlegm heat is the most common and important kind of syndrome.A syndrome of multiple organ contains more complex linkages between indicators, it is difficult to use a single indicator or a simple sum of several indicators to explain the material basis of syndrome.How scientific and comprehensive exposition of TCM syndrome essence and content is an important issue in modern Chinese medicine needed to solve.Research has shown that, for the treatment of viral pneumonia in children,the lack of an ideal western medicine drug, the only American Academy of Pediatrics recommended as "Maybe you can use" drugs is ribavirin.Chinese medicine treatment has some advantages, and has been for a large number of clinical and experimental research institute confirmed.Nanjing University of Chinese Medicine Professor Wang Shouchuan led our group engaged in traditional Chinese medicine treatment of viral pneumonia in children for nearly 20 years.and developed an effective oral medicine named Jinxin oral liquid,which can open the inhibited lung-energy、clear away heat-evil and toxic factor,dissipate phlegm and relieve cough. Jinxin oral liquid in the role of RSV infection has been a lot of research.however, due to the complexity of active chemical ingredient in Jinxin oral liquid, metabolic network regulation effect of Jinxin oral liquid on RSV pneumonia is still unclear.Metabonomics is a newly developed subject in the mid twentieth Centurys, the science of biological endogenous metabolites type, quantity and variation, is an important component of systems biology technology. Because metabolome is the end of all organisms gene and protein functional activity, and thus be regarded as "the biochemical phenotype" of the functional status of the organism as a whole.Metabonomics can immediately and sensitively state the response and regulatory function of the organism in the stimulation of a variety of external factors,avoiding the previous single or a few indicators to study some physiological and pathological changes of the state.The methodological feature of metabolomics technology is mixture of integral, dynamic, comprehensive analysis in one of the characteristics, which consistent with the overall concept of the medicine. Therefore, this study uses metabolomics methods, combined with other biochemical detection, comprehensively characterize metabolomic changes in RSV pneumonia with syndrome of obstruction of lung by phlegm heat, and thus attempt to clarify the essence and mechanism of Jinxin oral liquid.Objective:Study on the plasmic and urinary metabolic characteristics of RSV pneumonia with syndrome of obstruction of lung by phlegm heat to explore the essence of syndrome. Study on the plasmic and pulmonary metabolic characteristics of RSV pneumonia in BALB/c mice to explore the intervention on RSV pneumonia of Jinxin oral liquid and the possible mechanism of metabolic regulation.Methords:Clinical experiment of metabolomics study about RSV pneumonia with syndrome of obstruction of lung by phlegm heat:Select 30 cases of RSV pneumonia with syndrome of obstruction of lung by phlegm heat in line with Western medicine diagnostic conditions to disease group,while 30 cases setting a healthy control group.Plasma and urine ere respectively collected as early as diagnosed and detected by ultra-performance liquid chromatography/linear ion trap orbitrap mass spectrometry.The target is to establish syndrome related metabolism spectrum after principal component analysis and orthogonal partial least squares discriminant analysis,and to analyze metabolic pathways and explore the essence of RSV pneumonia with syndrome of obstruction of lung by phlegm heat.Animal experiment of BALB/c mice RSV pneumonia intervention by Jinxin oral liquid:BALB/c mice were inoculated with RSV, and treated with Jinxin oral liquid,and set ribavirin as a positive control.After 6 days of the first inoculated with RSV, killed the mice and collected plasma and the lung tissue,to evaluate pulmonary lesions by Histological.Based on ultra-performance liquid chromatography/linear ion trap orbitrap mass spectrometry,metabolic changes in plasma and lung tissue of mice in each group were studied. Through principal component analysis and orthogonal partial least squares discriminant analysis, potential biomarkers and related metabolic pathways in plasma and lung tissue were screened.The target was to evaluate the efficacy of Jinxin oral liquid and the possible mechanism of metabolic regulation.Results:Clinical experiment of metabolomics study about RSV pneumonia with syndrome of obstruction of lung by phlegm heat:1. pattern recognition analysis of plasma metabolism showed that the integral value of principal component of majority of RSV pneumonia with syndrome of obstruction of lung by phlegm heat and normal children concentrated in a scatter plot of the oval(95% confidence interval).Although some separation tendency, but there are still overlap.OPLS-DA models were further constructed and the model parameters are R2Y= 0.907, Q2= 0.567(upper-phase plasma samples) and R2Y= 0.816, Q2=0.619 (lower-phase of plasma samples).Normal gruop and RSV pneumonia with syndrome of obstruction of lung by phlegm heat could be clearly separated along the first principal component t[1] axis direction,without cross and overlap indicating a clear difference about metabolites between the two groups.Twenty-one potential biomarkers were screened and identified one of the 20, wherein SM (18:2/24:0), SM (d18:2/24:1), SM (d18: 0/18:1), TG (16:0/18:1/22:6), TG (16:0/18:0/22:6), TG (16:0/20:4/22:5), TG (18:1/18:3/20:4), TG (16:0/20:3/22:4) were increased in plasma of RSV pneumonia with syndrome of obstruction of lung by phlegm heat,PC(18:0/18:2), PC(18:0/18:1), PC(16:0/20:3), PC(18:0/20:3), PC(16:0/22:5), PC(18:2/20:0), LysoPC(18:2), TG(14:0/16:0/18:2), TG(14:0/16:0/16:1), Bilirubin, Carnitine, Tryptophan significantly decreased compared with normal children.2. pattern recognition analysis of plasma metabolism showed that majority of RSV pneumonia with syndrome of obstruction of lung by phlegm heat and normal children concentrated in a scatter plot of the oval(95% confidence interval) after principal component analysis principal component analysis.OPLS-DA models were further constructed and the model parameters are R2Y=0.865, Q2=0.580(urine samples detected with aQ column) and R2Y=0.902, Q2=0.593(urine samples detected with HILIC column).Normal gruop and RSV pneumonia with syndrome of obstruction of lung by phlegm heat could be completely separated,indicating a clear difference between the two groups in terms of metabolites.Fifteen difference biomarkers were screened and identified 11 of them. Acetylcarnitine,3-Methylglutarylcarnitine, Kynurenine, Tryptophan, Acetylspermidine, Cytosine, 1-Pyrroline-4-hydroxy-2-carboxylate, Tryptamine significantly increased compared with normal children;Methyldopam N-Methylhydantoin in childreen with RSV pneumonia were showed a downward trend in the urine.Animal experiment of BALB/c mice RSV pneumonia intervention by Jinxin oral liquid:1. Pathological changes in lung tissue after RSV infection in BALB/c mice were mainly as interstitial pneumonia,alveolar wall thickening congestion,interstitial mononuclear macrophage and neutrophil infiltration, bronchial epithelial cells without obvious degeneration and necrosis,bronchial and alveolar exudate no obvious, pulmonary congestion and inflammation degree were relieved in Jinxin oral liquid group.Pathological scores showed the treatment group and the model group was statistically significant difference.2. OPLS-DA model based on plasma data between RSV pneumonia BALB/c mice and control mice showed completely separated of two groups after 6d of infection with RSV.The model parameters are R2Y= 0.917, Q2= 0.835 (upper-phase plasma samples) and R2Y= 0.967, Q2= 0.936 (lower-phase plasma sample) indicating successful model. RSV infection in mice caused a significant influence of metabolic networks in plasma.Twenty-five biomarkers were screened and identified. PC(18:1/22:6), PC(18:0/20:5), TG(18:1/18:2/18:2), TG(18:1/18:1/18:2) were increased significantly in RSV pneumonia BALB/c mice compared with control mice; PC(18:1/18:3), PC(18:0/22:6), PC(P-18:1/22:2), PC(O-18:0/22:6), PC(O-20:0/22:6), PC(16:0/20:4), PC(P-20:0/14:0), LysoPC(16:0), LysoPC(18:2), LysoPC(18:0), LysoPC(20:4), LysoPC(18:1), LysoPC(22:6), SM(d18:1/24:1), SM(dl8:0/18:1), SM(d18:2/24:1), PE(18:3/19:0), Phytosphingosine, TG(16:0/18:0/14:0), TG(16:0/16:0/18:0), Phytanic acid showed a downward trend.We evaluated the efficacy of oral Jinxin oral liquid based on the difference markers.lt showed that Jinxin oral liquid can callback partial biomarkers,and mainly through regulation of phosphatidylcholine metabolism pathway.3. OPLS-DA model based on lung tissue data between RSV pneumonia BALB/c mice and control mice showed completely separated of two groups after 6d of infection with RSV.The model parameters are R2Y=0.879, Q2=0.789 (upper-phase lung tissue samples) and R2Y=0.918, Q2=0.853 (lower-phase lung tissue sample),indicating successful model. RSV infection in mice caused a significant influence of metabolic networks in lung tissue.Twenty-five biomarkers were screened and identified.All of the bio biomarkers including PC(18:0/18:1),PC(18:0/18:2), PC(22:6/18:2),PC(18:2/20:4),PC(P-20:0/14:0),LysoPC(18:0),LysoPC(l 6:0),LysoPC(18:1),LysoP C(20:4),LysoPC(18:2),SM(40:1),SM(d18:1/20:0),Sphinganine,Phytosphingosine,TG(18:1/18:1/1 8:1),TG(18:1/16:0/20:1),TG(18:1/18:1/18:2),TG(18:1/18:2/18:2),TG(16:0/18:0/22:6),TG(18:1/1 8:0/20:1), Phytanic acid, Leucine, Phenylalanine, Choline, LysoPE(16:0) were decreased significantly in RSV pneumonia BALB/c mice compared with control mice.We evaluated the efficacy of oral Jinxin oral liquid based on the difference markers.lt showed that Jinxin oral liquid can callback partial biomarkers,and mainly through regulation of Phenylalanine, tyrosine and tryptophanbiosynthesis, Phenylalanine metabolism; Valine, leucine and isoleucine biosynthesis,Glycerophospholipid metabolism,Sphingolipid metabolism pathway.Conclusion:1. UPLC-LTQ/Orbitrap-MS is capable of identifying and explaining the metabolic modes of RSV pneumonia with syndrome of obstruction of lung by phlegm heat and normal healthy children.2. RSV pneumonia with syndrome of obstruction of lung by phlegm heat mainly marked with tryptophan and glycerophospholipid metabolism disorder.3. BALB/c mice infected with RSV characterized by Phenylalanine, tyrosine and tryptophanbiosynthesis, phenylalanine metabolism, Valine, leucine and isoleucine biosynthesis、 glycerophospholipid metabolismand sphingolipid metabolism disorders.4. Jinxin oral liquid can typically improve the lung lesions after the infection with RSV in BALB/c mice.5. Jinxin oral liquid is able to partially adjust metabolic disorders caused by RSV infection, mainly by regulating Phenylalanine, tyrosine and tryptophanbiosynthesis, Phenylalanine metabolism, Valine, leucine and isoleucine biosynthesis,Glycerophospholipid metabolism, Sphingolipid metabolism pathway. |
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