Study On The Mechanism Of The Fusu Mixture Based On LncRNAs Regulation In Treating Sepsis-related Acute Lung Injury In Rats

Part One.To observe the effect of Fusu mixture on lung tissue injury and serum inflammation indexes in sepsis-related ALI ratsPurpose:By observing the effects of Fusu mixture on lung tissue pathology and serum levels of TNF-α and IL-1β in rats with sepsis-related ALI,the protective effect of Fusu mixture on sepsis-related ALI rats was studied.Method:（1）After adaptively feeding 44 male Wistar rats for one week,12 rats were randomly selected as the blank group（C）,and the remaining 32 rats were used as the model to be given lipopolysaccharide（3 mg/kg,injection time 5 min）.The left femoral vein was injected to induce an animal model of ALI.The rats in the blank group were injected with an equal volume of normal saline via the left femoral vein in the same way.After the lipopolysaccharide was used for 2 hours,two model rats were randomly selected from the blank group and the modeling model,and the lung tissue pathological section HE staining and lung injury score were used to determine whether the modeling was successful.（2）After the model is successful,30 model rats are divided into model group（A）,high-dose Fusu mixture（F）and medium-dose Fusu mixture group（D）according to random number table method.Rats in high-and middle-dose Fusu mixture group.The Fusu mixture solutions of different concentrations and equal volumes were administered respectively at intervals of 24 hours for a total of 2 administrations.The blank group and the model group were administered equal volumes of normal saline in the same way.After 48 hours of traditional Chinese medicine intervention,the general conditions of each group of rats were observed.At the same time,6 rats in each group were randomly selected for lung tissue pathological section HE staining and lung injury score.Enzyme-linked immunosorbent assay（ELISA）detected the general condition of each group.The serum levels of TNF-α and IL-1β in rats.Result:（1）Observation of the general condition of rats:The general condition of rats in the Fusu mixture treatment group was better than that in the model group.（2）HE staining of lung tissue pathological sections and lung injury score:After 48 hours of traditional Chinese medicine intervention,compared with the blank group,the average lung injury score of the model group increased from 1.33 to 7.67,and the difference was statistically significant（P<0.05）;compared with the model group,the lung injury of the high-and medium-dose Fusu mixture group The score decreased,the average score of the high-dose group was 3.83,the average score of the middle-dose group was 3.67,the difference was statistically significant（P<0.05）;the difference between the high-dose and middle-dose groups of Fusu mixture was not statistically significant（P>0.05）.（3）Changes in serum inflammation-related indicators:After 48 hours of traditional Chinese medicine intervention,compared with the blank group,the levels of serum TNF-α and IL-1β in the model group were significantly increased（P<0.05）;compared with the model group,the serum TNF-α and IL-1β levels of the rats in the high and medium dose groups of Fusu mixture were significantly increased.TNF-α and IL-1β levels were significantly decreased（P<0.05）;there was no significant difference in serum TNF-α and IL-1β levels between the high-dose and medium-dose groups of Fusu mixture（P>0.05）.Part two.Transcriptome high-throughput sequencing to detect the differential expression profile of LncRNA in lung tissue and the GO function of co-expressed mRNA and the enrichment analysis of KEGG pathway.Purpose:Through high-throughput sequencing technology,explore the effect of Fusu mixture on the lncRNA expression profile of lung tissue in sepsis-related ALI rats,and explore the possible targets of Fusu mixture in the treatment of sepsis-related ALI,which is the clinical application and application of Fusu mixture.In-depth mechanism research lays the foundation and basis.Method:According to the degree of rat lung injury and serum-related index levels,three rat lung tissue samples were screened from each group,total RNA of the sample was extracted,RNA quality detection,library construction and sequencing,and eXpress and Bowtie2 software were used to quantify the transcripts to obtain each The expression levels of lncRNA and coding genes（Fragments PerKilobase Million,FPKM）of the group samples were screened by DESeq software for differentially expressed lncRNA and mRNA,and P<0.05 and Foldchange≥2 were used as the threshold to evaluate the difference in lncRNA and mRNA expression.significance.Select lncRNAs and mRNAs that are more than 2 times differentially expressed,analyze the co-expression correlation between differential lncRNAs and mRNAs with the help of Pearson correlation test,and then use hypergeometric algorithm to predict the function of differential lncRNAs.Result:（1）LncRNA and mRNA expression profile of rat lung tissueHigh-throughput sequencing results showed that a total of 10428 lncRNAs and 22601 mRNAs were detected in rat lung tissues.Compared with the model group,the blank group had 209 differentially expressed lncRNAs（132 up-regulated and 77 down-regulated）and 593 differentially expressed mRNA（452 up-regulated and 141 down-regulated）.Compared with the model group,there were 133 differentially expressed lncRNAs（62 up-regulated and 71 down-regulated）and 59 differentially expressed mRNAs（26 up-regulated and 33 down-regulated）in the middle-dose group of the Fusu mixture.The results of the volcano map analysis showed that the expression of lncRNAs was significantly different between the blank group and the model group,and between the model group and the Fusu mixture medium-dose group.（2）The expression profile of LncRNAs in rat lung tissues reversed by Fusu mixtureBy intersecting the differentially expressed genes between the blank group and the model group,and the model group and the Fusu mixture medium-dose group,it was found that compared with the blank group,21 up-regulated lncRNAs in the model group were restored and down-regulated in the Fusu mixture medium-dose group.The 11 down-regulated lncRNAs were restored to up-regulated in the Fusu mixture middle-dose group.Cluster analysis showed the expression value of lncRNA in the blank group,the model group and the dose group of the Fusu mixture,and suggested that the expression patterns of the three groups were significantly different.（3）Analysis of the correlation between differential LncRNA and mRNA expressionPearson correlation test was performed on the expression data of 32 lncRNAs and differentially expressed mRNAs that were reversed by the Fusu mixture.The results showed that there were 59 co-expressed mRNAs in 23 differentially expressed lncRNAs,and the absolute values of the corresponding correlation coefficients were all greater than 0.8.P<0.05,suggesting that there is a strong correlation between the two.（4）Differential LncRNA function predictionThe GO function analysis of the co-expressed mRNA of lncRNA reversed by the traditional Chinese medicine Fusu mixture involves biological processes such as ubiquitin-dependent protein catabolism,protein transport,and intracellular mRNA localization.KEGG analysis suggests that the Fusu mixture based on lncRNA regulation treats acute lung sepsis The damaged pathway may involve signal transduction processes such as NF-κB signaling pathway,T cell receptor signaling pathway,arachidonic acid metabolism pathway,apoptosis pathway,and cell junction pathway.Among them,the NF-κB signaling pathway has the most enriched genes and the strongest significance,and the differential lncRNAs involved in the regulation of NF-κB signaling pathway include ENSRNOT00000077323,TCONS₀0011743,TCONS₀0033256,XR₀01835811.1,XR₀01838548.1,XR₀01840956.1.Conclusion:（1）Fusu mixture can improve the general condition of ALI rats induced by LPS and improve the quality of life.（2）Fusu mixture can reduce the degree of lung injury in rats,reduce the levels of serum TNF-α and IL-1β in model rats,and have a therapeutic effect on sepsis-related ALI rats.（3）The Fusu mixture caused significant changes in the lncRNA expression profile of the lung tissue of rats with sepsis-related ALI,and the GO and KEGG analysis of the co-expression of lncRNA suggested that the resuscitationmixture based on differential lncRNAs may have a possible mechanism of action in the treatment of sepsis-related ALI It involves biological processes such as ubiquitin-dependent protein catabolism,protein transport,and intracellular mRNA localization,as well as signal transduction pathways such as NF-κB signaling pathway and T cell receptor signaling pathway.Among them,the NF-κB signaling pathway has the most enriched genes and the strongest significance,and the differential lncRNAs involved in the regulation of NF-κB signaling pathway include ENSRNOT00000077323,TCONS₀0011743,TCONS₀0033256,XR₀01835811.1,XR₀01838548.1,XR₀01840956.1,suggesting the possibility of Fusu mixture By regulating the above-mentioned lncRNA involved in the regulation of the NF-κB signaling pathway,it can play a therapeutic role,and it may be used as an anti-inflammatory target of Fusu mixture in the treatment of sepsis acute lung injury.