| Objective1.1.To understand the changes of intestinal barrier function after severe scald,and to observe the protective effect of different concentrations of modified Sijunzi Decoction on intestinal barrier function after severe scald.2.To understand the change of 16 SrDNA flora diversity of intestine after scald,to elaborate the advantage of high-throughput detection for flora analysis,and to observe the effect of modified Sijunzi Decoction on flora diversity of intestine after scald.3.Proteomics was used to screen the different proteins in the mTOR pathway after scald,and the effect of modified Sijunzi Decoction on these different proteins was observed.Method90 Japanese big eared rabbits were randomly divided into 5 groups,18 in each group.The specific grouping is as follows:Normal control group: free diet and drinking water;scald + conventional feeding group + normal saline infusion group: after the establishment of scald model,normal saline infusion was given to the stomach,10 ml / time,3 times / D;scald + conventional feeding + Sijunzi Decoction infusion group(0.2g / ml): after scald,modified Sijunzi decoction was given to the stomach,0.2g/ml-1,10 ml / time,3 times / D;scald + conventional feeding + Sijunzi Decoction infusion group(1.0g / ml)After scalding,the rats were given Sijunzi Decoction(1.0 g /ml,10 ml / time,3 times / D);after scalding + routine feeding + Sijunzi Decoction(5.0 g / ml): after scalding,the rats were given Sijunzi Decoction(5.0 g /ml,10 ml / time,3 times / D);the animals were killed on the first day,the third day,and the seventh day respectively;part of the ileum was taken for HE staining to detect the pathological changes of intestinal mucosa in each group;the Ileal mucosa was taken for ELSIA to detect TNF-α,IL-10 and IL-1 β in each group.after scalding,the rats were given modified sijunzi Decoction 1.0 g /ml,10 ml / time,3 times / day for 7 days.On the 7th day after gavage,16 S rdna of intestinal contents was detected by high-throughput technology,and the diversity of flora in intestinal contents was analyzed by correlation analysis.Proteomics technology was used to screen the differential proteins in the mTOR pathway in each group,and 6 proteins were selected for validation by fluorescence quantitative PCR and Western blot.ResultsBefore the burn,the skin of the Japanese rabbit was normal,but no large or small blisters were found immediately after the burn,the skin was injured and relaxed after 1 day burn,scald 3 days and 7 days after the black scab attachment,the epidermis was pale,hard and hollow,with no large or small blisters.HE staining of skin pathology after burn is visible: The epidermis and dermis were seriously damaged,and the hair follicle structure was disordered.A large number of inflammatory cells were found in the normal control group,and the ileum mucosa of the scald model group was not abnormal.With the extension of time,ileum mucosa inflammation became more and more serious,accompanied by a large number of inflammatory cells infiltration.Sijunzi Decoction treatment effect of the drug group was more obvious with the extension of drug concentration and time.TNF-αand IL-1βwere significantly increased and IL-10 was significantly decreased in the scald model group,Sijunzi Decoction(1.0 g/ml);Sijunzi Decoction(5.0 g/ml)concentrations of TNF-αand IL-1βdecreased with time and were lowest at 7 days;Concentration of IL-10 increased with time and reached the highest level on 7 days.299309 sequences were obtained from the three groups of samples after the removal of doubt sequences,96023 high-quality sequences were obtained from the control group,107365 high-quality sequences were obtained from the scald + conventional feeding group + normal saline gavage group,and 95921 from the scald + conventional feeding + Modified Sijunzi Decoction gavage group.With the refinement of the classification level,the differences of the composition of the three groups are gradually obvious.The composition of the three groups of samples was uniform,and the species with different contents were similar.There was no significant difference in Simpson,Chao1 and Shannon index among the three groups(P > 0.005).PCoA analysis showed that there was no difference in beta diversity among the three groups based on the relatively concentrated distribution distance of the three groups of samples.A total of 894 proteins were identified in this experiment;There were 12 differentially expressed proteins in the mTOR pathway in each group.Compared with the normal control group,4 genes were up-regulated and 8 genes were down-regulated in the model group.Compared with the model group,there were 8 up-regulated genes and 4 down-regulated genes in modified Sijunzi Decoction group.The differentially expressed proteins in each group could significantly distinguish the model group and modified Sijunzi Decoction group in the normal control group,indicating that the differentially expressed proteins in the mTOR pathway we screened could represent the differences among the three groups of samples.Compared with the normal control group,the mRNA and protein expression levels of PEBP1 and EIF4G1 in the model group were significantly increased,while the mRNA and protein expression levels of CSNK2A1 EIF3 I PPP2CA and MAP2K1 were significantly decreased(P<0.05).Compared with the model group,the mRNA and protein expression levels of PEBP1 and EIF4G1 in modified Sijunzi Decoction group were significantly decreased,while the mRNA and protein expression levels of CSNK2A1 EIF3 I PPP2CA and MAP2K1 were significantly increased(P<0.05).These results are consistent with the proteomics results.Conclusion1.The ileum mucosa of severely burned rabbits was damaged obviously and the inflammatory reaction was serious,and the longer the time was,the more obvious the injury was.Modified Sijunzi Decoction can promote the repair of ileum mucosa and reduce the inflammatory reaction.The best concentration was 1.0g/ml and the action time was the 7th day..2.Compared with the traditional method of flora analysis,high-throughput sequencing method can more accurately reflect the composition of intestinal flora.In this study,we found that there was no significant difference in the composition of flora in each group,but there were differences in the percentage of class,order,family and genus,which indicated that modified Sijunzi decoction had a certain regulatory effect on intestinal flora.3.TMT and LC-MS / MS were used to screen 894 proteins related to mTOR pathway and 12 differential proteins in each group,so as to find the target of modified Sijunzi Decoction... |
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