Based On The "bacteria-gut-brain Axis" To Explore The Mechanism Of Yinlai Decoction In The Treatment Of Gastrointestinal Heat Accumulation Complicated With Pneumonia

Children's gastrointestinal tract is weak,and high-calorie food is easy to form gastrointestinal heat retention syndromes(GHRS),which is related to the incidence of pneumonia.Yinlai Decoction is a prescription for treating GHRS combine with pneumonia,but its mechanism is not clear yet."Microbiota-gut-brain axis" reflects the interaction between the gut microbes and the brain-gut axis.The gut microbes can affect the brain-gut axis through a variety of ways,causing immune and inflammatory reactions.Preliminary studies have found that pneumonia combined with GHRS can cause gut microbes disorders and inflammatory factors changes,Yinlai Decoction has a regulatory effect on gut microbes and brain-gut axis.This study intends to investigate the effects on short-chain fatty acids and 5-HT by the gut microbes of rats with GHRS combine with pneumonia,and to study how the gut microbes affects the immune and inflammatory response through the brain-gut axis in this rat model based on the "Microbiota-gut-brain axis",thereby elucidating the pathogenesis of Pneumonia combined with GHRS,and the treatment mechanism of Yinlai Decoction for the disease.Experiment 1:Effect of Yinlai Decoction on "Microbiota-gut-brain axis" Related Indexes in Rat Model of Pneumonia combined with GHRSObjective:To explore the changes of gut microbes,short-chain fatty acids,5-HT,and inflammatory factors in rat model of Pneumonia combined with GHRS,and the intervention effect of Yinlai Decoction on it.Methods:Male SPF-grade SD rats(4-week-old)were used,GHRS model was established by gavage with high-calorie feed and milk,pneumonia model was established by LPS aerosol inhalation,and the pneumonia combined with GHRS model was established by combining the two.Yinlai Decoction was used as intervention,and prednisone acetate was used as a positive control.Grouped as Normal group(N),Pneumonia group(P),GHRS group(S),Shiji combined pneumonia group(SP),Shiji combined pneumonia with the treatment of Yinlai Decoction group(SPT1),Shiji combined pneumonia with the treatment of prednisone acetate group(SPT2).Body weight,rectal temperature,food intake,the pathology tissue of lung and colon were observed;the organ index was calculated,the content of IFN-?,IL-2,IL-4,IL-6,IL-10,TNF-? in serum was detected by multi-factor tests,TGF-? and 5-HT content in serum were detected by ELISA,gut microbes was detected by 16S rDNA amplicon sequencing,the levels of short-chain fatty acids in serum and intestinal contents were detected by targeting the metabolome.Results1.General situation(1)Physical signs of Rats:Rats in the SP group showed reduced activity and irritability;they liked to curl up and pile up;their fur was not compliant and showed vertical hair or tresses;their stools were soft and yellowish in color,sticky and stinky,and irregular.After the treatment with Yinlai Decoction,rats in the SPT1 group were more calm and less irritable,curled up and piled up;their fur gradually became smooth and shiny,and their stools basically returned to normal.(2)Body weight:Compared with group N and P,the body weight of SP group rats was reduced to varying degrees(P<0.05).After the treatment with Yinlai Decoction,the body weight of rats in the SPT1 group increased to varying degrees.(3)Food intake:Compared with group N,the rats of group SP had a lower food intake throughout the experiment.After the treatment with Yinlai Decoction,the rats' food intake of group SPT1 changed more steadily during the experiment compared with group SP,and their food intake increased on the 5th day.(4)Rectal temperature:The rectal temperature of rats in group SP increased significantly(P<0.01)compared with group N.After the treatment with Yinlai Decoction,the rectal temperature of the rats in group SPT1 decreased to different degrees compared with the SP group on the 4th and 5th day.2.Organ IndexCompared with the group N and P,the liver,spleen and thymus indexes of rats in group SP all decreased to varying degrees(P<0.05).While the spleen index and thymus index of rats in group SPT1 had a higher tendency,and the liver index increased(P<0.05).3.Histopathology of lung and colon(1)Lung histopathology:Compared with group N,the alveolar structure of the rats of group SP was not clear,the alveolar wall was obviously thickened and fractured and fused,accompanied by a large number of inflammatory cell infiltration.Compared with the SP group,the alveolar structure of the rats in group SPT1 and SPT2 became clear,the alveolar wall became thinner,the rupture and fusion of the alveolar wall was reduced,and the infiltration degree of inflammatory cell was significantly reduced.(2)Colonic histopathology:Compared with group N,the epithelial cell structure of the colon tissue of the rats of group SP was clear,the goblet cells were irregularly arranged and were increased in number,and a small amount of inflammatory cell infiltration was seen.Compared with group SP,the epithelial cell structure of the colon tissue of the rats of group SPT1 was clear,the number of goblet cells was reduced,so as the infiltration of inflammatory cells.4.The level of inflammatory factors in serumCompared with group N,group SP's levels of pro-inflammatory factors IFN-?,IL-2,IL-6,and TNF-? in serum increased significantly(P<0.01);the group SP's level of anti-inflammatory factors IL-4 and IL-10 in serum increased significantly(P<0.01),while TGF-? increased(P<0.05).After the treatment with Yinlai Decoction,the levels of serum pro-inflammatory factors IFN-y,IL-2,IL-6,and TNF-? in group SPT1 were significantly reduced(P<0.01);the level of anti-inflammatory factor IL-10 in serum was significantly increased(P<0.01),and TGF-? increased too(P<0.05).5.The level of 5-HTCompared with group N,the 5-HT level in serum of group SP were significantly reduced(P<0.01).After the treatment with Yinlai Decoction,the 5-HT level in serum of group SPT1 increased(P<0.05).6.Gut microbes(1)The results of OTU clustering and species annotation showed that the number of OTU in the gut microbes of group SP was significantly lower than that of group N.(2)The results of the a diversity analysis of the gut microbes showed that compared with group N,the Shannon,Simpson,Ace,Chaol coefficients all reduced to varying degrees in group SP(P<0.05 or P<0.01).After the treatment with Yinlai Decoction,the Shannon coefficient of group SPT1 increased,while the Simpson,Ace,and Chaol coefficients decreased to varying degrees.(3)Species composition at each taxonomic level:The abundance of Actinobacteria increased and the abundance of Bacteroides decreased in the gut microbes of rats in group SP.Opposite change showed in group SPT1:the abundance of Bacteroides increased while the abundance of Actinobacteria decreased.From the perspective of all classification levels,the abundance of Coriobacteriia,Coriobacteriales and Peptostreptococcaceae increased,and the abundance of Lactobacillus and Lactobacillu.reuter decreased in the gut microbes of rats in group SP;the abundances of Bacteroidetes,Bacteroidia,Bacteroidiales,Bacteroidaceae and Bacteroides all increased,while the abundances of Coriobacteriia,Coriobacteriales,Peptostreptococcaceae decreased in group SPT1.(4)Prediction of gut microbes' function:The functions of gut microbes in rats of group SP are differences from those of group N in carbohydrate transport and metabolism,amino acid transport and metabolism,membrane transport,transcription,signal transduction and metabolism,cell cycle control,cell division and chromosome division,defense mechanism.The functions of gut microbes in rats of group SPT1 are differences from those of group N in metabolism(especially carbohydrate transportation and metabolism),signal transduction mechanism and defense mechanism,cardiovascular disease and endocrine system.7.Short-chain fatty acid level:Compared with group N,the levels of acetic acid,propionic acid,butyric acid and total short-chain fatty acid in the feces of rats in group SP were significantly reduced(P<0.01),and the levels of acetic acid,butyric acid and total short-chain fatty acid in serum showed an upward trend.After the treatment with Yinlai Decoction,the fecal level of acetic acid,propionic acid,and butyric acid of rats in group SPT1 all had an increasing trend,and the serum level of acetic acid,propionic acid,butyric acid and total short-chain fatty acid had a decreasing trend.Conclusions:GHRS combine with pneumonia can change the structure and function of gut microbes of rats,resulting in abnormal secretion of short-chain fatty acids,decreased levels of acetic acid,propionic acid,and butyric acid,and decreased serum 5-HT levels.Serum inflammatory factor levels are elevated,which aggravates the body's inflammatory response.Yinlai Decoction can improve these changes.Experiment 2:The Effect of Gut Microbes on "Brain-gut Axis" Related Indexes in Rat Model of GHRS Combine with Pneumonia.Objective:To explore the effect of the gut microbes of rat model of GHRS combine with pneumonia on short-chain fatty acids,5-HT and inflammatory factors,and whether Yinlai Decoction exerts a therapeutic effect through the gut microbes.Methods:Male SPF-grade SD rats(4-week-old)were used,the rat model of GHRS combine with pneumonia were established and Yinlai Decoction was used as an intervention.Mixed antibiotics were administered to prepare Antibiotic rats.Grouped as fecal supply group 1(providing normal fecal bacteria,G1),fecal supply group 2(providing GHRS combine with pneumonia fecal bacteria,G2),fecal supply group 3(providing fecal bacteria after Yinlai Decoction treatment,G3),fecal microbe transplantation group 1(FMT1),fecal microbe transplantation group 2(FMT2),fecal microbe transplantation group 3(FMT3),Normal group(N),Shiji combined pneumonia group(SP),Shiji combined pneumonia with the treatment of Yinlai Decoction group(SPT),of which FMT1 and FMT2 groups are antibiotic rats,which were transplanted with normal fecal microbe and fecal microbe of GHRS combine with pneumonia,respectively.GHRS combine with Pneumonia model was established as group FMT3,which were transplanted stool microbe from SPT group.The general condition of the rat,the pathology tissue of lung and colon were observed;the organ index was calculated;the level of IL-6,TGF-? in serum and 5-HT level in serum were detected by ELISA;gut microbes was detected by 16S rDNA amplicon sequencing,the levels of short-chain fatty acids in serum and intestinal contents were detected by targeting the metabolome.Results1.Antibiotic rats(1)General conditions:The rats are in poor mental state,slow in activity,curled up and piled up,their fur is lack of smooth and gloss,and their stools are yellow and shapeless.(2)The gut microbesa.The results of OTU clustering and species annotation showed that the number of gut microbes OTU of group FMT1 and FMT2 decreased after modeling.b.The results of species composition at each taxonomic level showed that Bacteroides and Akkermansia increased,and Prevotella and Lactobacillus decreased after modeling in group FMT1 and FMT2 at the genus level,which means that the structure of the gut microbes changed.c.The analysis of bacterial diversity showed that the Shannon coefficient,Ace coefficient and Chao1 coefficient of group FMT1 and FMT2 were significantly reduced after modeling compared with before(P<0.01).The Simpson coefficient of FMT1 group has a downward trend after modeling,and the Simpson coefficient of FMT2 group was significantly lower after modeling(P<0.01),and the meanwhile the diversity of flora decreased.2.The situation after fecal microbe transplantation(1)Organ index:Compared with group FMT1,the liver,spleen,and thymus indexes of the group FMT2 all increased;compared with group SP,the liver index of group FMT3 increased significantly(P<0.01),and the spleen index was on a downward trend,the thymus index in group FMT3 decreased significantly(P<0.01).(2)Histopathology of lung and colona.Lung histopathology:The lung pathological changes of group FMT2 and group SP were similar.Both the alveolar structure was unclear,the alveolar wall was obviously thickened,and there was a large amount of inflammatory cell infiltration.The degree of inflammatory cell infiltration in group FMT2 was slightly less than that in group SP.There are alveolar fractures and fusions.The lung pathological changes of group FMT3and group SPT were similar.It can be seen that the alveolar structure became clear,the alveolar wall became thinner,the rupture and fusion phenomenon was reduced,and the degree of inflammatory cell infiltration was significantly reduced compared with the group FMT2 and SP.b.Colonic histopathology:The structure of the epithelial cells in the colon tissue of rats in groupFMT2 was clear,and the number of irregularly arranged goblet cells was more than that of group SP and less than that of group FMT1,and more inflammatory cell infiltration was seen The colonic pathological changes of group FMT3 and group SPT were similar.It can be seen that the epithelial cell structure of the colon tissue was clear,the goblet cells were arranged less regular and the number was small,and the inflammatory cell infiltration was reduced.(3)Inflammatory factors in serum:Compared with group FMT1,serum IL-6 and TGF-? of rats in group FMT2 had a tendency to increase.Compared with the SP group,there was no significant difference in the serum IL-6 level of group FMT3,but the TGF-? level had a decreasing trend.(4)5-HT:Compared with the group FMT1,the serum 5-HT level of group FMT2 was reduced(P<0.05).Compared with group SP,the serum 5-HT level of group FMT3 were significantly reduced(P<0.01).(5)Short-chain fatty acids:Compared with group FMT1,the fecal and serum levels of acetic acid,propionic acid,butyric acid and total short-chain fatty acids of rats in group FMT2 all had an increasing trend.Compared with group SP,the fecal acetic acid and propionic acid levels of rats in group FMT3 showed an upward trend,butyric acid levels had a downward trend,and the total short-chain fatty acid levels had an upward trend;serum acetic acid levels increased(P<0.05),propionic acid level had a rising trend,the butyric acid level is significantly reduced(P<0.01),and the total short-chain fatty acid level had a rising trend.Conclusions1.Gut microbes of GHRS combine with pneumonia can trigger a pathological reaction in the body,and Yinlai Decoction can exert a therapeutic effect through the gut microbes.2.GHRS combine with pneumonia can lead to disorders of structure and function of gut microbes,which cause metabolic disorders of its metabolite SCFAs,thus affects the secretion of 5-HT.Furthermore,it can affect the body's inflammatory response through the brain-gut axis.Yinlai Decoction can intervene in the same way and exert a therapeutic effect.