Explore The Material Basis And Mechanisms Of Simiao Yong’an Decoction Combined With Antibiotic In The Treatment Of Sepsis

Purpose:Sepsis was a life-threatening organ dysfunction caused by infection and a threat to patients’life with a high mortality rate.Studies explored that the three obvious pathological features of sepsis patients were:inflammatory imbalance,abnormal coagulation function,and microcirculation dysfunction.Simiao Yong’an Decoction(SMYAD)was one of the classic prescriptions in ancient China.Modern research showed that it had effects of anti-inflammatory,clotting improvement,and microcirculation.However,the material basis and mechanism of action of SMYAD in treating sepsis are still unclear.Therefore,this article aims to explore the quality markers(Q-markers),biomarkers(B-markers),and potential mechanisms effect of SMYAD in combination with antibiotic(imipenem/cilastatin sodium)in the treatment of sepsis.Methods:1.Study on the profile of compounds in extract and medicated serum of SMYADSMYAD was composed by Lonicerae Japonicae Flos(90 g),Scrophulariae Radix(90 g),Angelicae Sinensis Radix(60 g),Glycyrrhizae Radix et(30 g).The extraction solution for three times was combined to obtain the freeze-dried powder of SMYAD and dissolved in methanol to prepare 0.2 g/m L solution;Linear Trap LC/MS~nwas used to analyze the chemical composition of the extract from SMYAD;Subsequently,male SD rats(250±20 g)were selected and given SMYAD solution(12.5g/kg)by gavage for three consecutive doses.After the last administration,blood was taken from the orbital venous plexus and centrifuged to obtain a serum sample containing the drug.Finally,the serum absorbed components were analyzed.2.Q-markers of SMYAD were screened based upon network pharmacology and molecular docking The ingredients and corresponding targets in SMYAD were collected from the TCMSP database and so on,and sepsis corresponding targets about inflammation,coagulation,and microcirculation were collected from Genecards etc.,database.The common targets of SMYAD and sepsis of different mechanisms were obtained through Venn diagrams.Then,protein-protein interaction(PPI)network,GO and KEGG analysis were performed based on the common targets.Molecular docking was performed on the active ingredients and key proteins on the signaling pathway of SMYAD obtained from the database screening.The Q-markers was determined by the affinity,serum medicinal chemistry and Q-markers screening principles.The structure were visualized through Pymol and Autodock Vina,and the connections between main signaling pathways and Q-markers were constructed using Cytoscape based on the"active ingredients-common targets-signaling pathways"network.3.The quality control of SMYAD was carried out The standard substances were harpagide,chlorogenic acid,sweroside,ferulic acid,angoroside C,and glycyrrhizic acid.The mixed standard solution were prepared.The methodology validation were determined simultaneously using the HPLC-DAD method;Subsequently,an appropriate amount of SMYAD freeze-dried powder was weighted and prepared 0.2 g/m L solution of SMYAD for HPLC detection;Finally,the external standard method was used to quantify the content of Q-markers in SMYAD and established its quality control method.4.Screening of B-markers by metabolomicsSixty male SD rats were purchased and divided into six group:control group,sham group,model group,antibiotic group,antibiotic and SMYAD high-dose group,antibiotic and SMYAD high-dose group.Rats were modeled by cecal ligation and puncture(CLP)except control and sham group.After the last administration,blood was collected from the orbital venous plexus and centrifuged to obtain a serum sample containing the drug;After protein precipitation,nitrogen blowing concentration,and derivatization,serum samples were detected using GC-MS;Qualitative and quantitative analysis of metabolites based on internal standard method and database comparison;Multivariate statistical analysis was used to screen the endogenous differential metabolites in Metabo Analyst.Besides,metabolic pathway analysis and joint-pathway analysis were obtained based on the B-markers.5.Explore the mechanism of SMYAD united imipenem/cilastatin sodium in sepsis.The animal grouping and administration method were the same as above.Except for taking blood from the orbital venous plexus,the rats were euthanized and their organs were weighed and preserved.Firstly,the Kaplan-Meier survival curve was drawn to reveal the effect of SMYAD combined with antibiotic on the mortality rate of SD rats for sepsis.Each organ was weighed and the organ coefficient was calculated when the rats were sacrificed.In order to further explore the organ function damage induced by sepsis,HE staining was employed to observe the histopathological changes.Then,the serum biochemical indicators related to inflammation(IL-2,TNF-α,IL-4,IL-10),coagulation(TAT and LA),and microcirculation function(s P-selection)were detected.Besides,Western blot(WB)was used to explore the relationships between key proteins and main signaling pathways including IL-17A/IL-17 signaling pathway,C3/C5/complement-coagulation cascades signaling pathway and STAT3/VEGFR2/VEGF signaling pathway.Furthermore,the m RNA expression levels of IL-17A,C3,C5a,VEGFR2 and STAT3were measured using real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR).Results:1.Through the firstly and secondary mass spectrometry of precursor ions and their fragment ions in the positive/negative ion mode of linear trap LC/MS~n,combined with standard substance comparison and reference literatures,a total of 113chemical components were analyzed in the extract of SMYAD,including 36flavonoids,19 iridoids,17 organic acids,12 triterpenes,10 phenylpropanoids,5phthalides,4 nucleotides,4 coumarins,3 alkaloids,and 3 sterols;In addition,25serum migrant ingredients were detected in the bleeding serum,mainly flavonoids and iridoid compounds,to preliminarily elucidate the chemical profiles in the water extract of SMYAD and serum.2.In network pharmacology research,93 chemical components in SMYAD were selected from the TCMSP database,corresponding to 664 targets;2098,1550 and 686disease targets for sepsis-related to inflammation,coagulation,and microcirculation were screened.After Venn analysis,SMYAD shared 200,166 and 125 gene targets with sepsis-inflammation,sepsis-coagulation and sepsis-microcirculation,respectively.In addition,KEGG results showed that the signaling pathways closely related to the three mechanisms were the IL-17 signaling pathway,complete-coagulation cascades signaling pathway and VEGF signaling pathway.Therefore,93 active components were docking with the key protein targets(IL-17A,C3,C5a,VEGFR2 and STAT3)on these three signal pathways to determine the Q-markers.Finally,the chlorogenic acid and sweroside in JYH,angoroside C and harpagide in XS,ferulic acid in DG,and glycyrrhizic acid in GC were selected as Q-markers of SMYAD.What’s more,the Q-markers network diagram showed that both sweroside and ferulic acid were the main active components in the three signaling pathways;Glycyrrhizic acid may have significant effects on inflammatory response and alleviated microcirculation dysfunction;Chlorogenic acid and angoroside C were closely related to VEGF signaling pathways while harpagide mainly regulated inflammatory reactions.3.The methodological validation results of the content determination of Q-markers in HPLC were as follows:standard curve’s R2of Q-markers both reached0.999,which showed a good feasibility and linear relationship.In the precision experiment,the RSD values were between 0.447%and 1.746%,indicating that the instrument had good precision results and reliable data;The RSD values detected in the stability experiment were<3%,suggesting that the solution of SMYAD had good stability within 0-24 hours.The RSD values detected in the repeatability test were<2%,showing good repeatability of the experiment.It was verified that the HPLC detection method was accurate and reliable according to the standard addition recovery rate(between 96%and 110%)and the RSD values(less than 3%).Finally,the content of Q-markers was determined,and the detection results showed that the contents of harpagide,chlorogenic acid,sweroside,ferulic acid,angoroside C,and glycyrrhizic acid were 3.62,3.45,1.20,0.28,0.78,and 0.61 mg/g,respectively.4.In metabolomics research,OPLS-DA screened eight potential B-markers,namely L-serine,phosphoric acid,cyclohexane,2-Oleoylglycerol,Myo-Inositol,ascorbic acid,citric acid and deoxyuridine(VIP>1.0 and P<0.05);Metabolic pathway analysis showed that when SMYAD combined with antibiotic for sepsis,the citric acid cycle/tricarboxylic acid cycle(TCA cycle)metabolic pathway played an important role(Impact>0.1,P<0.05).Besides,a total of 256 joint pathways were enriched in the analysis of joint pathways,among which the IL-17 signaling pathway,C-type lectin receptor signaling pathway,and VEGF signaling pathway(Impact>0.1,P<0.05)are closely related to sepsis inflammation,coagulation,and microcirculation,and also correspond to KEGG enrichment analysis results.5.In SD rats,the mortality rate of sepsis induced by CLP within 72 hours was as high as about 70%,and the organ coefficients of the lungs,kidneys,and spleen in the model group were significantly increased,suggesting that edema might occur in these organs after modeling.HE staining results manifested that the lung,kidney,and spleen were damaged to varying degrees:alveolar swelling,structural irregularities,and other phenomena appeared in the lung tissue;A large number of glomerular atrophies,interstitial edema,and inflammatory cell infiltration could be seen in renal tissue;In spleen tissue,pathological areas such as spleen corpuscle deformation and fragmentation occurred.Among them,the score of renal injury was the highest.Luckily,the histopathological damage of these organs was significantly improved after the combined treatment of SMYAD and antibiotic.The measurement of serum biochemical indicators revealed that CLP modeling dramatically increased the level of inflammatory cytokines(P<0.001),activated the levels of TAT,LA,and s P-selection(P<0.01),and each treatment group alleviated sepsis-induced inflammatory reactions,abnormal coagulation function,and microcirculation dysfunction.The high-dose combination treatment group showed the best therapeutic effect(P<0.05).Additionally,WB and q RT-PCR results demonstrated that the levels of IL-17A,C3,C5a,VEGFR2 and STAT3 proteins and m RNA in the model group were highly raised(P<0.01),indicating that the IL-17 signaling pathway,complete-coagulation cascades signaling pathway,and VEGF signaling pathway were activated,and SMYAD could inhibit the activation of the signaling pathway by inhibiting related proteins(P<0.01).Conclusions:The classic formula of SMYAD combined with imipenem/cilastatin sodium effectively improved the survival rate of septic mice and improve multiple organ damage caused by sepsis.Chlorogenic acid,Angeloside,Angeloside C,Habanoside,Ferulic acid,and Glycyrrhetinic acid were important Q-markers for the anti-sepsis effects.By regulating the TCA cycle metabolic pathway,inhibiting the IL-17 signaling pathway,complement-coagulation cascade signaling pathway,and VEGF signaling pathway,SMYAD exerted an important role in inhibiting the activation of the inflammatory response,regulating coagulation dysfunction,and improving microcirculation abnormalities to combat sepsis.