Protective Effect Of Huang-lian-jie-du-tang Against CLP-induced Polymicrobial Sepsis And Its Influence On Metabolome Profile In Rats

Objective:Sepsis is the systemic inflammatory response syndrome induced by infection ofmicroorganisms and its toxins. It is the systemic and harmful response of the host toinfection which will cause severe sepsis and septic shock. Sepsis mainly caused by severetrauma, burn, operation and shock. Because of its acute onset, rapid progression and highmortality rate, sepsis remains a leading cause of mortality in Intensive Care Units (ICU).The early hallmark sign of sepsis is a whole-body inflammatory state called systemicinflammatory response syndrome (SIRS). This early phase of excessive systemicinflammation can leading to multiple organ dysfunction syndrome (MODS) if not treatedin time. MODS is the late stage of sepsis with the mortality of40%-60%, once it occurs, itis very difficult to treatment. The pathogenesis of sepsis is very complicated, and it is stillnot fully understood. It is generally considered that the overexpressions ofproinflammatory factors, immune dysfunction, abnormal blood coagulation, intestinalbacterial shift and gene polymorphism are the main causes of sepsis. There is still noeffective drug for the treatment of sepsis. Previous studies have confirmed that simplyblocking the single cause of sepsis did not significantly improve the survival rate of sepsis,but the comprehensive therapy in patients with sepsis may be helpful. Traditional ChineseMedicine (TCM) is a holistic treatment of disease based on dialectics method. The use ofTCM in the treatment of sepsis maybe an effective therapy for sepsis.Huang-Lian-Jie-Du-Tang (HLJDT) is a traditional formula consisting of four herbs:Rhizomacoptidis, Radix scutellariae, Cortex phellodendri and Fructusgardeniae. Itsfunctions in Chinese medicine include clearing heat, purging fire and toxic material. Sepsisbelongs to the category of febrile diseases in Chinese medicine, so we propose that HLJDTmaybe an effective medicine for the treatment of sepsis.The source and the quality of Chinese herbs are often influenced by geographical andclimate, so the quality of the TCM are difficult to control, and the components in the TCMare very complex and the pharmacological mechanisms are unclear, so the widely use ofthe TCM in the international society is limited. It is a trend to use the effective componentsgroup of TCM to instead of the TCM. HLJDT contain flavonoids, iridoids glycosides andalkaloids. We have previously studied the pharmacodynamic material bases of HLJDT and identified13components in HLJDT decoction using high performance liquidchromatography coupled with mass spectrometric detection. The13components areBerberine, Palmatine, Jatrorrhizine, Magnoflorine, Phellodendrine, Coptisine, Baicalin,Baicalein, Wogonoside, Wogonin, Gardenoside B, Crocin and Chlorogenic acid. Based onthis study, we constitute a HLJDT component group (HLJDT-CG) of these13compounds.Here we studied the protective effect of HLJDT and HLJDT-CG on CLP-induced sepsis inrats.Metabonomics is a new technology developed after genome, transcriptome andproteome. It is an important part of systems biology. It has changed the traditional way ofexamination the disease by a single marker, but by a group of metabolites to diagnose thedisease systematically. We analyzed the metabolisms in septic rats, normal rats and thedrug-treated rats to find out the metabolic biomarkers which can be used to diagnose thesepsis early and rapidly, and the metabolic biomarkers which can be used to evaluate thetherapeutic efficacy of the HLJDT.Methods:Sepsis was induced in rats by cecal ligation and puncture (CLP). HLJDT andHLJDT-CG were administered orally2h before CLP. The protective effect of HLJDT andHLJDT-CG were determined by observing the survival rate, the pathology changes oftissues of liver and lung, and the serum levels of ALT and AST. The serum and tissue levelof cytokines were detected by ELISA, and the expression of cytokine mRNA in tissues ofliver, lung and kidney were detected by Real-Time-PCR. Th1-type cytokine IFN-γ,Th2-type cytokine IL-4and Th17-type cytokine IL-17in spleen were also detected byReal-Time-PCR. Bacterial loads were determined in peritoneal lavage fluid, liver, and lungsamples24h after CLP. Coagulation indexes of TT, PT, APTT and FIB were determinedby automatic blood coagulation instrument. Phagocytosis of rat peritoneal macrophageswas determined by flow cytometry. In our metabolomics study, we usedRHPLC-Q-TOF-MS to analyze the metabolic changes in serum of rats at2h,10h and24h after CLP in different groups to screen the biomarkers which can be used for diagnosis ofsepsis early and rapidly, and find biomarkers which can be used to evaluate the efficacy ofHLJDT in sepsis.Results:1、 Administration of HLJDT and HLJDT-CG can significantly improve the survival rate of septic rats, and there was no significant difference between HLJDT and HLJDT-CGtreatment groups. The optimum dosage of HLJDT was270mg/kg, and the optimumdosage of HLJDT-CG was12.5mg/kg.2、 Administration of HLJDT and HLJDT-CG can significantly amelioratepathology changes of liver and lung in septic rats, and significantly reduce serum ALT andAST levels in septic rats, indicating that HLJDT and HLJDT-CG have a significantprotective effect on septic rats.3、 Administration of HLJDT and HLJDT-CG could significantly decrease the content ofproinflammatory factors, including TNF-α, IL-1β, IL-6and IL-17, in septic rats serum,and significantly decrease the mRNA levels of these cytokines in liver, lung and renal ofseptic rats, but show no significant influence on anti-inflammatory cytokine IL-10;HLJDT and HLJDT-CG could significantly reverse the decrease of mRNA expression ofTh1-type cytokine IFN-γ, and significantly reverse the increase of mRNA expression ofTh2-type cytokine IL-4, indicating that HLJDT and HLJDT-CG could regulate theTh1/Th2immune imbalance induced by sepsis; HLJDT and HLJDT-CG could significantlyreduce the mRNA expression of Th17-type cytokine IL-17A, and significantly reduce themRNA expression of ROR-γt which is the transcription factor of IL-17expression; HLJDTand HLJDT-CG could significantly reduce the number of bacterial colonies in peritonealfluid, liver and lung homogenate, indicating that HLJDT and HLJDT-CG couldsignificantly decrease the quantity of bacterial in peritoneal, and significantly inhibit thetranslation of bacterial from peritoneal fluid to liver and lung; HLJDT andHLJDT-CG could significantly increase the coagulation indexes, such as PT andTT levels, in serum of septic rat, indicating that HLJDT and HLJDT-CG haveanticoagulant effect through inhibiting the activity of exogenous coagulation factors;HLJDT and HLJDT-CG could significantly increase phagocytosis of macrophages inthe rat peritoneal.4、 Our metabonomics study showed that septic rats undergo a significantmetabolic change at2h,10h and24h after CLP. Through analysis ofmetabolites significantly changed at2h after operation between sham operation group andmodel group, we identified17biomarkers which could be used for diagnosis of sepsis inthe early stage. They are L-Proline, L-Valine, Hippuric acid, Indole,4-Formyl Indole,Arachidic acid, Arachidonic Acid, Leukotriene F4, DocosahexaenoicAcid, Eicosapntemacnioc Acid, PGE3, Oleic acid, Carnitine, Palmitoylcarnitine, LysoPC, PC and Purine. Our metabonomics study also shows that HLJDTcould significantly reverse the metabolic changes induced by sepsis at2h and10h.Through analysis of metabolites significantly changed at2h after CLP between HLJDTgroup and model group, we identified10biomarkers, which could assess the efficacy ofHLJDT in the early stage. They are L-Proline, L-Valine, Arachidic acid, Arachidonic Acid,Docosahexaenoic Acid, Eicosapntemacnioc Acid, PGE3, Oleic acid, Carnitine andPalmitoylcarnitine. In addition, in order to observe the overall metabolic changes of sepsisand assess the overall effect of HLJDT, we also identified the metabolites at10h and24hafter CLP in each group, and obtained21biomarkers at10h,23biomarkers at24hsignificant different between Sham group and CLP group, and12biomarkers that HLJDTtreatment can significantly regulated. After analyzed the biological fuctions of thesebiomarkers and searched the relevant database and literature, we constructed the metabolicnetwork of sepsis, which suggested that sepsis is mainly associated with the dysregulationof amino acid metabolism, ω-3unsaturated fatty acid metabolism, ω-6unsaturated fattyacid metabolism, purine metabolism and lipid metabolism, et al. These metabolisms areassociated with energy metabolism, inflammation, oxidative stress. Among these metabolicpathways, ω-3polyunsaturated fatty acids and ω-6polyunsaturated fatty acid metabolismare related to inflammatory response. So we considered that HLJDT might exert itsprotective effects on sepsis through modulating these metabolic pathways.Conclusion:Both HLJDT and HLJDT-CG have significant pretective effects on CLP-induced sepsisin rats. The protective effects of HLJDT and HLJDT-CG on septic rats are associated withtheir intervention on several different aspects of sepsis, includinganti-inflammation, anticoagulation, antibacterial, inhibition on bacterialtranslocation, regulating immunity and enhance the phagocytosis of macrophage.Our metabonomics study has indentified17early diagnosis biomarkers for sepsis and10efficacy-evaluation biomarkers for HLJDT. Metabolic disorder in sepsis mainly involvedin amino acid metabolism, ω-3unsaturated fatty acid metabolism, ω-6unsaturated fattyacid metabolism, purine metabolism, and lipid metabolism, et al. HLJDT can regulatemultiple metabolic pathways in sepsis. HLJDT might exert its protective effects on septicrats through modulating ω-3polyunsaturated fatty acids and ω-6polyunsaturatedfatty acids metabolic pathways.