Intervention And Mechanism Of Total Phenolic Acid In Salvia Miltiorrhiza Stem-Leaf And Combined With Tanshinone Compatible Components On Inflammatory Bowel Disease

This work was completed with the support of the National Natural Science Foundation of China"Effects of Salvia miltiorrhiza stem-leaf on Blood Circulation and Regulation of Inflammation"（81673533）and the Key Project of Jiangsu Collaborative Innovation Center of Chinese Medicine Resources Industrialization（No.ZDXM-2-5）.On the basis of previous studies,this study mainly focused on the intervention effect and mechanism of phenolic acid components of S.miltiorrhiza stem-leaf and the the combination of total phenolic acid of S.miltiorrhiza stem-leaf and tanshinone on inflammatory bowel disease,to provide scientific basis for the further development and utilization of S.miltiorrhiza stem-leaf.This study main contents and results are briefly described as follows:Chapter one,Literature researchThis chapter systematically summarized the understanding of the pathogenesis of inflammatory bowel disease（IBD）by consulting the literature,and summarized the relationship between these factors and the occurrence and development of inflammatory bowel disease from environmental factors,intestinal microorganisms,immune abnormalities,and host genetic susceptibility,which was benefited to find targeted treatments for inflammatory bowel disease.The traditional Chinese medicine（TCM）for IBD were mainly heat-clearing medicine,blood-activating and stasis-resolving medicine,tonic-deficiency medicine and damp-moisture medicine.Among them,most of the studies were concentrated on monomer components in TCM,which reflected the richness in the clinical and pre-clinical studies of TCM in treating IBD.It has shown great study potential to provide study views for the development of anti-IBD drugs with less side and positive effects from TCM.Chapter two,Intervention effect and mechanism of phenolic acid components in Salvia miltiorrhiza stem-leaf on inflammatory bowel disease1 Preparation of total phenolic acids in Salvia miltiorrhiza stem-leafThe total phenolic acids in stems and leaves of S.miltiorrhiza Beg.（JF）were extracted by refluxing with ethanol,purified by column chromatography combined with extraction technology and enriched.Protocatechualdehyde,rosmarinic acid,lithospermic acid,salvianolic acid B,salvianolic acid A,salvianolic acid C,rutin,isoquercetin,kaempferol-3-0-rutinoside and astragalin in the JF were determined by UPLC.The contents of the above components in JF were 1.54 mg·g-1、298.43 mg·g-1、14.38 mg·g-1、315.73 mg·g-1、7.30 mg·g-1、16.45 mg·g-1、6.10 mg·g-1、7.05 mg·g-1、3.07 mg·g-1 and 2.45 mg·g-1,respectively.The total amount of phenolic acids was 653.83 mg·g-1,and the total amount of flavonoids was 18.67 mg·g-1.2 Efficacy evaluation of phenolic acid components in Salvia miltiorrhiza stem-leaf on inflammatory bowel diseaseA colitis mouse model was established using free-drinking 2%（w/v）dextran sodium sulfate（DSS）for 7 consecutive days.After successful modeling,the phenolic acid components in stems and leaves of S.miltiorrhiza（PAC）,including total phenolic acids（JF）,salvianolic acid B（SAB）,rosmarinic acid（RA）and salvianolic acid B+rosmarinic acid（corresponding to the amount of JF,respectively）was administered by orally for 7 days.During the administration,the disease activity index（DAI）score of mice was recorded.After the administration,the colon was quickly dissected for length measurement and pathological analysis to evaluate the protective effect of PAC on colitis mice.The results showed that compared with the control group,the DAI score in the model group was significantly higher（P<0.001）than that in the control group when the 2%DSS solution was stopped,the colon length was significantly shortened（P<0.001）,and colonic tissues suffered severe pathological damage（P<0.05）.After the intervention of PAC administration,above conditions were improved to varying degrees,showing the protective effects on colitis3 Study on the protective mechanism of phenolic acid components in Salvia miltiorrhiza stem-leaf on inflammatory bowel disease（1）Immune barrier improvementELISA and qPCR were performed to detect the protein and gene expression levels of inflammation-related factors in colon tissues,and the effects of PAC on the network of inflammation-related factors in colonic inflammatory states were investigated to explore its regulatory effect on the immune barrier.The results showed that compared with the control group,the model group colon tissue the proinflammatory factors TNF-α,IL-6,IL-1β,COX2 and iNOS levels were significantly increased（P<0.05）,the anti-inflammatory factor IL-10 level was significantly reduced（P<0.05）,and the IL-6,COX2 and IL17A mRNA levels were significantly increased（P<0.01）.After the intervention,PAC had no effect on the level of TNF-α,and showed varying degrees of regulation on other indicators.It was showed that PAC restored the balance between pro-inflammatory and anti-inflammatory factors by reducing pro-inflammatory factor protein and gene expression levels and increasing anti-inflammatory factor protein levels,thus achieving a protective effect on colitis.Among them,RA and SAB+RA groups have similar or better effects than JF group（2）Intestinal microbiota regulation16s rRNA gene sequencing technology was used to study the effect of PAC on the gut microbiota of colitis mice.PCoA analysis showed that PAC can regulated the abnormal microbiota structure of colitis mice to some extent,in which RA and SAB+RA group significantly shorten the RD value from the control group in the score plots（P<0.05）,revealing that RA and SAB+RA were the active components in the overall gut microbial level.Further analysis by LEfse revealed that DSS-induced colitis mice had significant differences in gut microbiota from the phylum to the species level compared with the control group.After the intervention,PAC mainly down-regulated Proteobacteria,Escherichia-Shigella,Parabacteroides,Enterococcus,Staphylococcus,Streptomyces,Escherichia_coli and Parabacteroides_distasonis relative abundance and up-regulated Odoribacter,Ruminococcaceae UCG₀13 and Ruminiclostridium₆ relative abundance.In particular,PAC has the most obvious effect on reducing the relative abundance of potential pathogenic bacteria（Streptococcus,Enterococcus,Staphylococcus,and Escherichia_coli）,thereby regulating the disturbance of gut microbiota.（3）Metabolomics researchBased on the UPLC-Orbitrap MS technology,non-targeted metabolomics analysis of serum and feces was performed.The analysis by PLS-DA found that after the intervention of PAC,it was located between the control group and the model group and significantly shortened the RD value,showing a regulation effect on abnormal serum and fecal metabolism.Among them,the regulatory effect of SAB+RA group was relatively better than that of other administration groups,indicating that it was the active component of JF.It revealed the protective effect of PAC on colitis from the overall metabolomics level.Further analysis of the metabolic disorders in the model group showed that 35 endogenous biomarkers（9 in serum and 26 in feces）were identified,and these markers returned to control group levels in varying degrees after PAC intervention.These endogenous biomarkers are mainly involved in glycerophospholipid metabolism,amino acid metabolism,and purine metabolism.In addition,it found that abnormal fluctuations in the gut microbiota of colitis mice were highly correlated with intestinal metabolites by correlation analysis.PAC treatment may rebuild the colitis mice homeostasis of the intestinal environment by down-regulating the abundance of Parabacteroides to inhibit aromatic amino acids metabolism and increase the amino acid level,up-regulating the abundance of Ruminococcaceae_UCG₀13 and Ruminiclostridium₆ to promote bile acid metabolism and increase bile acid levels.Chapter three,Intervention effect and mechanism of Salvia miltiorrhiza stem-leaf total phenolic acids combination with tanshinone on inflammatory bowel disease1 Analysis of tanshinone extractThe content of tanshinone extract（DT）was detected by high-performance liquid phase method,which mainly contained tanshinone IIA and cryptotanshinone contents of 623.63 mg·g-1 and 47.42 mg·g-1 respectively,and the total amount of tanshinone reached 671.05 mg·g-1.2 Efficacy evaluation of total phenolic acids in Salvia miltiorrhiza stem-leaf and tanshinone extract,alone and in combination,on inflammatory bowel diseaseAs in the previous chapter,a colitis mice model was established using 2%DSS.After successful modeling,JF and DT alone or in combination was administered continuously for 7 days.Similarly,the protective effects of JF and DT alone or in combination on colitis mice were evaluated by the DAI score,colon length,colonic pathological damage,and colonic histological score.The results showed that compared with the model group,JF and DT alone or in combination significantly reduced the DAI score,relieved the shortening of the colon,reduced the inflammation injury of the colon,and down-regulated the histological score,showing a protective effect on colitis.Among them,at low dose,the effect of the combination of JF and DT was relatively better than JF alone,and showed obvious synergistic effect.3 Study on the protective mechanism of total phenolic acids in Salvia miltiorrhiza stem-leaf and tanshinone extract,alone and in combination,on inflammatory bowel disease（1）Immune barrier improvementELISA and qPCR were performed to detect the expression of pro-inflammatory factor protein and gene in colon tissue,and Western blot was used to detect the expression of TLR4/PI3K/AKT/mTOR pathway-related proteins and STAT3 protein in colon tissue.To study the alleviation effect of JF and DT alone or in combination intervention on gut inflammation to explore its improvement effect on immune barrier.The results showed that compared with the control group,TLR4,mTOR.p-mTOR and NF-κBp65 protein expression levels and PI3K p110α,AKT（ser473）,and STAT3 phosphorylation levels in colon tissues were significantly increased in the model group（P<0.01）,and accompanied by the pro-inflammatory factors TNF-α,IL-6,IL-1β,and COX2 and IL-1β and iNOS mRNA expression levels significantly increased（P<0.05）,indicating that the TLR4/PI3K/AKT/mTOR pathway and STAT3 were activated.After treatment with JF and DT alone or in combination,the levels of the above-mentioned pro-inflammatory protein and mRNA were reduced and the expressions of these proteins were reduced to different degrees.Among them,the effect of JF in combination with DT at low-dose was relatively the best,showing its combined synergistic effect.In short,we speculate that the anti-inflammatory activity of the combination of JF and DT is due,at least in part,to the effects of the TLR/PI3K/AKT/mTOR axis and STAT3 protein,thereby alleviating gut inflammation and protecting colitis（2）Neurotrophic factor regulationWestern blot was used to determine the expression of neurotrophic factors（BDNF and NGF）and their corresponding high-affinity receptors（TrKB and TrKA）and NF-κBp65 in mice hippocampus.Compared with the control group,the expression of BDNF,NGF,TrKB,TrKA,and NF-κBp65 in the model group mice hippocampus were significantly increased（P<0.05）.After the intervention,only the JF and DT in combination group showed a certain callback effect on the expression levels of NGF,TrKB and TrKA to the control group level,thereby affecting a certain abnormal reaction that may exist in the hippocampus of colitis mice（such as Increased neurogenesis）to indirectly improve the mental state of mice with colitis,showing obvious synergistic effect.（3）Metabolomics researchBased on UPLC/Q-TOF/MS non-metabolic metabolomics analysis of serum and brain tissues to monitor changes in small molecule metabolites in colitis mice after the intervention of JF and DT alone or in combination.The results of PLS-DA analysis showed that there were significant metabolic abnormalities in serum and brain tissue between model group and control group.After the intervention of JF and DT alone or in combination,except for individual groups,the RD values from the control group were shortened to varying degrees,showing a certain regulatory effect on serum and brain tissue metabolic disorders.Among them,the low dose of JF alone and JF combined with DT groups significantly shortened the RD value,and showed a relatively good regulatory effect on the overall metabolic level.Further analysis of the significantly disturbed metabolites in the model group found that a total of 17 potential biomarkers（10 in serum and 7 in brain tissue）were identified that were mainly involved in amino acid metabolism（histidine metabolism,tryptophan metabolism and cysteine and methionine metabolism）,arachidonic acid metabolism and purine metabolism.After the interventions,these biomarkers returned to the control group levels in varying degree,and effectively improving the metabolic disorders of serum and brain tissue in colitis mice.