| Viral pneumonia is an acute respiratory infection caused by viral infection.In recent years,the incidence of viral pneumonia is increasing gradually,which brings great harm to the society.Maxingshigan Decoction is formulated with the legislation of cooling and promoting lung,clearing heat and relieving asthma.It has definite curative effect on viral pneumonia,but its mechanism is not clear.Previous studies have found that viral pneumonia can lead to intestinal flora disorder,increased expression of energy metabolites and related proteins,and accompanied by changes in a variety of inflammatory factors.Glycolysis plays an important role in the occurrence of inflammation and affects the release of immunomodulatory factors.Maxingshigan Decoction can regulate intestinal flora and inflammatory response.This study aims to investigate the effects of intestinal flora on glycolysis and inflammation in mice with viral pneumonia through animal experiments,and study the mechanism of treating viral pneumonia with Maxingshigan Decoction based on "intestinal flora-glycolysis".Experiment 1:Effect of Ma Xingshi Gan decoction on glycolysis and inflammatory response of lung tissue of viral pneumoniaObjective:To explore the relationship between the glycolysis process of viral pneumonia and inflammation,and the intervention effect of Ma Xingshigan Decoction on it.Method:Using SPF-level 6-8 weeks old BALB/c male mice as the research object,under mild isofluorane anesthesia,the nasal cavity was inoculated with viral solution containing 1 unit LD50 per 50 μL to prepare viral pneumonia model mice,intervened with Ma Xingshi Gan Decoction,and ose with ose and glycolycolytic inhibitor 2 deoxyglucose as positive control drugs.There was a blank control group(N),pneumonia group(P),oseltamivir group(OT),Ma Xingshi Gan Decoction Treatment Group(MT),Glycolycolytic Inhibitor Group(DGT).Record mouse weight,calculate lung index and lung index inhibition rate,observe lung histopathology with HE staining,target the metabolism group to detect the level of glycogenic acid intestinal contents of lung tissue,biochemically detect the activity of key enzyme hexylase and lactate dehydrogenase in mouse lung tissue glysis,The expressions of Ml-type macrophage markers F4/80 and CD80 and M2-type macrophage markers F4/80 and CD206 in lung tissue were detected by IF,and the contents of TNF-α and IL-10 in lung tissue were detected by ELISA.Results1.Weight changeOn the 7th day after molding,group P lost weight compared with Group N(P<0.01);compared with Group P,group OT and MT group gained weight significantly(P<0.01),and DGT group gained weight(P<0.05).2.Pulmonary index and lung index inhibition rateCompared with group N,the lung index in group P increased(P<0.01).Compared with group P,the lung index in OT group and MT group decreased significantly(P<0.01),the lung index in the DGT group decreased(P<0.05),the lung index suppression rate in the MT group was 29%,the lung index suppression rate in the OT group was 36.5%,and the lung index inhibition rate in the DGT group was 16.1%.3.Pulmonary histopathologyGroup N mice had a regular alveolar morphology and clear structure,and no inflammatory cell infiltration and bleeding.Compared with Group N,the cell membrane of lung tissue in Group P mice was destroyed,the alveolar wall was thickened and the tissue structure disappeared,and a large number of inflammatory cells could be infiltrated in the alveolar cavity.The pathological damage of the lung tissue of mice in each treatment group was improved to varying degrees.Among them,the alveolar morphological structure of the OT group and MT decoction group was complete,there were fewer inflammatory cells infiltration in the alveolar,the pulmonary histopathological damage was significantly improved,the alveolar morphological structure of the DGT group was relatively complete,the alveolar wall was thick,and there were more inflammatory cells infiltration in the alveolar cavity.4.Detection of glucose metabolism levels of lung tissue and intestinal contents in the targeted metabolism group(1)Inter-group pulmonary tissue glucose metabolism levelCompared with Group N,the content of fructose 6 phosphoric acid in Group P increased significantly(P<0.01),the content of glucose 6 phosphoric acid and xanthin mononucleotide increased significantly(P<0.01),the content of oxalic acid,thioammonium pyrophosphoric acid,lactic acid and cyclic adenosine increased(P<0.05),and the content of adenosine triphosphate decreased(P<0.05).Compared with group P,the phosphoric acid content of nicotinamide adenine dinucleotide in OT group increased significantly(P<0.001),guanosine content increased significantly(P<0.01),adenosine content(P<0.05),and yanhusolic acid and cisoteric acid decreased(P<0.05).Compared with Group P,the content of flavin single nucleotide and oxalic acid in MT group decreased significantly(P<0.01),fructose 6 phosphoric acid and glucose 6 phosphate decreased(P<0.05),and the content of nicotinamide adenine dinucleotide phosphate and adenosine triphosphate increased(P<0.05).Compared with Group P,the content of yanhusolic acid,succinic acid,citric acid,lactic acid and ciscephalic acid in the DGT group decreased(P<0.05).(2)Changes in intestinal contents between groupsCompared with group N,the content of α-ketoglutaric acid and pyruvate in group P was significantly higher than that in the normal group.Compared with group P,the content ofα-ketoglutaric acid decreased in OT and DGT groups.The content of α-ketoglutaric acid in the MT group increased,and the content of lactic acid and oxaloacetic acid decreased significantly.(3)Levels of important metabolites of lung tissue and intestinal contents15 important metabolites between the lung tissue group and the intestinal content group:fructose 6 phosphoric acid,oxalic acid,pyruvate,isocitric acid,glucose 6 phosphoric acid,nicotinamide adenine dinucleotide phosphate phosphate,proxoric acid,triphosphate or5.Activity of lung tissue glycolysis key enzymes hexose kinase and lactate dehydrogenaseCompared with group N,the activity of hexose kinase and lactate dehydrogenase in group P was enhanced(P<0.01).Compared with group P group,hexose kinase and lactate dehydrogenase in group OT,MT group and DGT group decreased(P<0.01).6.M1 macrophage markers and M2 macrophage markersCompared with group N,the expressions of M1-type macrophage markers F4/80 and CD80 in lung tissue of group P were significantly increased.After the intervention of oseltamivir,Maxinshigan Decoction and glycolytic inhibitor,the expressions of Ml-type macrophage markers F4/80 and CD80 in lung tissue of mice in OT,MT and DGT groups were decreased.Compared with group N,the expressions of M2-type macrophage markers F4/80 and CD206 in lung tissue of group P were significantly decreased.After the intervention of oseltamivir,Mayxingshigan Decoction and glycolytic inhibitor,the expressions of M2-type macrophage markers F4/80 and CD206 in lung tissue of mice in OT,MT and DGT groups were increased.7.Levels of serum inflammatory factors and anti-inflammatory factors in lung tissueCompared with Group N,the level of TNF-α of pulmonary tissue inflammatory factor in Group P increased significantly(P<0.01),and the level of anti-inflammatory factor IL-10 decreased significantly(P<0.01);compared with Group P,the level of TNF-α of pulmonary tissue inflammation factor in OT,MT and DGT groups decreased significantly(P<0.01);and the level of anti-inflammatory factor IL-10 increased significantly increased(P<0.01).Conclusions1.Changes of glycolytic metabolites in lung tissue and intestinal contents of viral pneumonia were observed,the content of glycolytic metabolites in lung tissue was increased,the activity of key glycolytic enzymes was enhanced,the activation of M1-type macrophages driven by glycolysis was increased,the level of pro-inflammatory factor TNF-α was increased,and the level of anti-inflammatory factor IL-10 was decreased,the glycolytic metabolism in lung tissue of viral pneumonia was active,and the inflammatory response was obvious.2.Maxingshigan Decoction can inhibit lung tissue inflammation and reduce lung injury caused by viral pneumonia by inhibiting the glycolysis process and the activation of M1 macrophages driven by glycolysis.Experiment 2:Effect of MaxingShigan Decoction on intestinal flora and glycolysis of viral pneumoniaObjective:To explore the effect of intestinal flora of viral pneumonia mice on glycolysis and inflammation,and the intervention effect of Ma Xing Ganshi Decoction on it.Methods:SPF-level 6-8 weeks old BALB/c male mice were used as the research object.Under mild isoflurane anesthesia,viral liquid containing 1 unit LD50 per 50 μL was inoculated in the nasal cavity to prepare viral pneumonia model mice.Ma Xingshigan decoction was used for intervention,and oseltami Agent 2 deoxyglucose is used as a positive control drug.Set the blank control group(N),pneumonia group(P),oseltamivir group(OT),Ma Xingshi Gan decoction treatment group(MT),and glycolysis inhibitor group(DGT).16S rRNA amplifier sequencing detects intestinal flora,biochemically detects the levels of lactic acid,pyruvate and succinic acid in serum and lung tissue of mice,and detects the content of IL-6,IL-1β and LPS in serum and lung tissue with ELISA.Western blot detected the expression changes of glycolysis key enzyme pyruvate kinase PKM2 in lung tissue,regulatory glycolysis key factor hypoxia induction factor HIF-1α,succinic acid specific protein G-coupled protein GPR91.Results1.Pulmonary histopathologyGroup N mice had a regular alveolar morphology and clear structure,and no inflammatory cell infiltration and bleeding.Compared with Group N,the cell membrane of lung tissue in Group P mice was destroyed,the alveolar wall was thickened and the tissue structure disappeared,and a large number of inflammatory cells could be infiltrated in the alveolar cavity.The pathological damage of the lung tissue of mice in each treatment group was improved to varying degrees.Among them,the alveolar morphological structure of the OT group and MT decoction group was complete,there were fewer inflammatory cells infiltration in the alveolar,the pulmonary histopathological damage was significantly improved,the alveolar morphological structure of the DGT group was relatively complete,the alveolar wall was thick,and there were more inflammatory cells infiltration in the alveolar cavity.2.Intestinal flora(1)The results of flora diversity analysis show thatCompared with group N,there was no significant difference in the Chao1 index of group P flora(P>0.05),the Shannon index of flora diversity increased significantly(P<0.01);there was no significant difference in the Chaol index of group MT group richness(P>0.05),and the diversity Shannon index increased significantly(P<0.05);the Chao1 index of the richness of the OT group and the DGT group decreased significantly(P<0.01),and there was no significant difference in the diversity Shannon index.Compared with group P,there was no significant difference between the Chao1 index and the diversity Shannon index of the MT group(P>0.05);the Chao1 index of the richness of the OT group decreased(P<0.05)and the diversity Shannon index decreased(P<0.05).The Chao1 index of the richness of bacteria in the DGT group decreased(P<0.01),and the diversity Shannon index was different(P<0.01).(2)Differential flora between groupsLactobacillus family of thick-walled bacteria was detected as the dominant strain in group N.The rumenococcus family of the thick-walled bacteria in the 10 main families of Group P is the dominant strain,and Prevorella is the dominant genus.Compared with Group P,the MT group takes Lactobacillus and Clostridium as the dominant bacteria,and Lactobacillus as the dominant bacteria.The OT group and the DGT group took the paraplesiella genus as the dominant strain.3.Serum and lung tissue inflammatory factor levelsCompared with group N,the content of lung tissue and serum IL-1β and IL-6 in group P increased significantly(P<0.01).Compared with group P,the content of lung tissue and serum IL-1β and IL-6 in OT group,MT group and DGT group decreased significantly(P<0.01).4.Content of pyruvate,lactic acid and succinic acid in serum and lung tissueCompared with Group N,the content of serum and lung tissue succinic acid,pyruvate and lactic acid in Group P increased(P<0.01);compared with Group P,the content of serum and lung tissue succinic acid,pyruvate and lactic acid in the OT group,MT group and DGT group decreased(P<0.01).5.Levels of LPS in serum and lung tissueCompared with Group N,the content of lung tissue and serum LPS in Group P increased significantly(P<0.01);compared with Group P,the content of lung tissue and serum LPS in OT,MT and DGT groups decreased significantly(P<0.01).6.Changes in protein expression in PKM2,HIF-1α and GPR91 in lung tissueCompared with group N,the expression of PKM2,HIF-la and GPR91 proteins in group P increased(P<0.01);compared with group P,the expression of PKM2,HIF-1α and GPR91 proteins in pulmonary tissues in OT,MT and DGT groups decreased(P<0.01).7.Correlation analysis of succinic acid,LPS and intestinal floraCompared with Group P,succinic acid is negatively correlated with the genus Rikenella,Candidatus_Arthromitus and Lactobacillus,and positively correlated with the genus Butyricicoccus,Buchnera,Parabacteroides,Allobaculum,Prevotellaceae_Prevotella,Akkermansia and Paraprevotella.LPS is negatively associated with the genus Rikenella,Roseburia,Candidatus_Arthromitus and Lactobacillus;it is associated with the genus Butyricicoccus,Buchnera,Parabacteroides,Sutterella,Prevotellaceae Prevotella.Compared with the MT group,succinic acid was positively correlated with Parabacteroides and Oscillospira,and negatively correlated with Rikenella,Clostridium,Candidatus_Arthromitus and Lactobacillus.LPS is positively correlated with Butyricicoccus,Gemmiger,and negatively related to Mycoplana,Kaistobacter,Neisseria,Candidatus_Arthromitus and Lactobacillus.Conclusions1.Viral pneumonia can cause structural and dysfunction of the intestinal flora.The molecule LPS and the metabolite succinic acid of the intestinal flora can affect the glysis process of lung tissue through circulation,thus exacerbating the inflammatory response.2.Ma Xingshigan Decoction can regulate the structure and function of the intestinal flora,reduce the level of molecule LPS and metabolite succinic acid in the circulation of the intestinal flora structure,improve the glysis of lung tissue,and thus reduce inflammatory reactions. |
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