Dahuang Mudan Decoction And Its Active Ingredients Regulate HMGB1 To Prevent And Treat Sepsis

ObjectiveSepsis(sepsis)is a systemic inflammatory response syndrome(SIRS)caused by infection.At present,supportive treatment and antibiotic treatment are still the main treatment methods for sepsis.However,due to the complex and diverse causes of the disease,the pathology The process is very repetitive,and the mortality rate remains high.Sepsis has become the main cause of death in intensive care units except for cardiogenic diseases.It has a serious impact on human life and health and may cause immeasurable effects on aging society in the future.Causing the heavy social and economic burdens.Once the course of sepsis is not intervened in time,it may lead to poor prognosis such as septic shock or even multiple organ failure,and even death.Therefore,in the early stage of sepsis,when the source of infection has not been clarified,the use of broad-spectrum antibiotics has become an irreplaceable help.The foreseeable direct consequences are the abuse and misuse of antibiotics and the promotion of drug-resistant bacteria,which will eventually lead to repeated infections,prolonged illness,and even life-threatening.Because of its unique compatibility law,small side effects and no drug resistance,traditional Chinese medicine prescriptions have great potential in the treatment of acute infectious diseases,waiting to be tapped.In recent years,Chinese medicine monomers have attracted much attention in various disease fields.However,the research on the monomers of traditional Chinese medicine often overlooks the characteristics of traditional Chinese medicine in the formulation and compatibility of multiple drugs.This kind of rational compatibility of multiple drugs and ultimately exerting multi-target regulation may be the unique advantage of traditional Chinese medicine.Therefore,discovering the compatibility of the active ingredients of Chinese medicine may be a key issue in the study of Chinese medicine,Only by grasping the key issues can we maximize the advantages of traditional Chinese medicine in the treatment of acute infectious diseases.High mobility group protein B1(HMGB1)is currently considered to be an endogenous damage-related molecular model(DAMP),as a late-stage inflammatory factor that is released from cells in the late stages of the disease.When HMGB1 is released outside the cell,it will stimulate the cell to release more pro-inflammatory factors TNF-α(tumor necrosis factor α)and IL-6(interleukin 6),which constantly triggers and maintains the downstream inflammatory response,causing an inflammatory cascade..HMGB1 can be used as the main biological marker for sepsis,and has important value to the diagnosis of sepsis and the evaluation to the prognosis of the disease.Therefore,the regulation of HMGB1 may become an important means for the treatment of sepsis.The preliminary experimental study of the research group found that Niupozhibao pellets,whose main active ingredients are also emodin and paeonol,can reduce the expression of HMGB1 and inhibit the release of related inflammatory factors and play a role in the treatment of endotoxic shock.Emodin can alleviate the pathological symptoms of lipopolysaccharide(LPS)-stimulated endotoxin shock model rats,and has a good therapeutic effect.At the same time,paeonol also showed a good protective effect on LPS-stimulated acute kidney injury mouse model kidney.In addition,previous studies have also shown that whether Dahuang Mudan Decoction and its active ingredients emodin and paeonol can regulate the mechanism of HMGB1 to prevent and treat sepsis remains to be further studied.In this study,hematoxylin-eosin staining and immunohistochemical staining were used to observe the pathological changes of the liver and lungs and the expression level of HMGB1 in the cecal ligation and perforation(CLP)model group mice prepared after the intervention of Dahuang Mudan Decoction.Then the concentration ratio of emodin and paeonol in the decoction of Dahuang Mudan Decoction was determined by high performance liquid chromatography,and the ratio was adjusted and controlled by gradient.Through in vitro cell experiment and LPS model in vivo experiment,research Dahuang Mudan Decoction and its effectiveness The components regulate the molecular mechanism of HMBG1 to prevent and treat sepsis.Methods1.Prepare cecal ligation and perforation(CLP)model mice,and use Dahuang Mudan Decoction for drug intervention.The experiment is divided into control group(0.2ml/20g/d,0.9%NaCl)and model group(0.2ml/20g/d,0.9%NaCl),Dahuang Mudan Decoction high-dose group(1g/mL),Dahuang Mudan Decoction medium-dose group(0.5g/mL),Dahuang Mudan Decoction low-dose group(0.25g/mL).Hematoxylin-eosin staining was used to observe the pathological changes of liver and lungs of CLP model mice.At the same time,the expression level of HMGB1 was detected by immunohistochemical staining.2.Use high performance liquid chromatography(HPLC)to detect the content of the effective ingredients emodin and paeonol in the Dahuang Mudan Decoction,To determine the best concentration ratio of emodin and paeonol in the Dahuang Mudan Decoction for follow-up Prepare for the experiment.3.Use emodin and paeonol,the active ingredients of Dahuang Mudan Decoction,to conduct drug intervention on Raw264.7 cells stimulated by LPS.The experiment is divided into control group,LPS model group,LPS+paeonol group(PAE1μM),LPS+paeonol group(PAE10μM),LPS+Paeonol group(PAE50μM),LPS+Paeonol group(PAE100μM),LPS+Emodin group(EMO500nM),LPS+Emodin group(EMO1μM),LPS+Emodin group(EMO2μM),LPS+Paeonol+Emodingroup(PAE10μm:EMO2μM5:1),LPS+paeonol+emodin group(PAE10μM:EMO1μM 10:1),LPS+paeonol+emodin group(PAE10μM:EMO500nM 20:1).Western-blot was used to detect the effect of emodin and paeonol,the effective components of Dahuang Mudan Decoction,on the expression of HMGB1.4.Prepare LPS model mice,use Dahuang Mudan Decoction and its active ingredients for drug intervention treatment,the experimental groups are divided into control group,LPS model group,emodin(1mg/kg)group,paeonol(20mg/kg)group,Paeonol(5mg/kg)+emodin(1mg/kg)group,paeonol(1Omg/kg)+emodin(1mg/kg)group,paeonol(20mg/kg)+emodin(1mg/kg),Dahuang Mudan Decoction group.Hematoxylin-eosin staining was used to observe the pathological changes in the liver and lungs of LPS model mice.At the same time,the expression level of HMGB1 was detected by immunohistochemical staining.Results1.In the results of the survival rate of in vivo experimental mice,the survival rate of CLP modelled sepsis mice was 20%.Compared with the model group,the survival rate of the middle-dose group of Dahuang Mudan Decoction was 80%(P<0.005),Dahuang Mudan Decoction The survival rate of the middle-dose group was 70%(P<0.005),and the survival rate in the middle-dose group of Dahuang Mudan Decoction was 40%(P>0.005).In the results of in vivo hematoxylin-eosin staining,Dahuang Mudan Decoction improved the pathological conditions of the lungs and livers of CLP model sepsis mice.In the results of in vivo immunohistochemical staining,Dahuang Mudan Decoction can significantly reduce the expression of HMBG1 in the lung and liver.2.In the results of high performance liquid chromatography(HPLC),the concentration ratio of the effective ingredients paeonol and emodin in the Dahuang Mudan Decoction is 9.8:1.3.In the results of in vitro western blotting,compared with the LPS model group,LPS+paeonol group(PAE1μM),LPS+paeonol group(PAE10μM),LPS+paeonol group(PAE50μM),LPS+paeonol group(PAE100μM),can significantly down-regulate the expression of HMGB1(P<0.001),and it is concentration-dependent,that is,the higher the concentration of paeonol,the lower the expression of HMGB 1 protein.LPS+Emodin group(EMO500nM),LPS+Emodin group(EMO1μM),LPS+Emodin group(EMO2μm)can significantly down-regulate the expression of HMGB1(P<0.001),but the expression of LPS+Emodin group(EMO1μM)is the lowest(P<0.001).The LPS+paeonol+emodin group(PAE10μM:EMO2μM 5:1),the LPS+paeonol+emodin group(PAE10μM:EMO1μM 10:1)and the LPS+emodin group(EMO2μM)down-regulated the expression of HMGB1 to the same extent(P<0.001).However,the LPS+paeonol+emodin group(PAE10μM:EMO500nM 20:1)had the lowest HMGB1 protein expression in all drug groups(P<0.001).Compare with the control group,the LPS group can reduce the expression of the anti-inflammatory factor TGF-β1(P<0.001).Compared with LPS group,TGF-β1 in LPS+paeonol group(PAE1μM),LPS+paeonol group(PAE10μM),LPS+paeonol group(PAE50μM),LPS+emodin group(EMO500nM),LPS+emodin group(EMO1μM)There was no significant difference in the expression of TGF-β1(P>0.005).The expression of TGF-β1 in the LPS+paeonol group(PAE100μM)increased to a certain extent(P>0.005),while in the LPS+emodin group(EMO1μM)and LPS+emodin group(EMO2μM),the expression of TGF-β1 was significantly increased(P>0.001).In the LPS+paeonol+emodin group(PAE10μM:EMO2μM 5:1),the LPS+paeonol+emodin group(PAE10μM:EMO1μM 10:1),the LPS+paeonol+emodin group(PAE10μM:EMO500nM 20:1)In the three groups,the expression of TGF-β1 was significantly increased(P>0.001).Among them,the LPS+paeonol+emodin group(PAE10μM:EMO500nM 20:1)had the best effect,second only to the LPS+emodin group(EMO1μM)and LPS+emodin group(EMO2μM)two groups.4.In the results of the survival rate of in vivo experimental mice,Dahuang Mudan Decoction and its active ingredients improved the survival rate of LPS model sepsis mice.Among them,Dahuang Mudan Decoction and paeonol(20mg/kg)+emodin(1mg)/kg)The survival rate of mice in the group was 50%.In the results of the in vivo hematoxylin-eosin staining method,Dahuang Mudan Decoction improved the pathological conditions of the lung and liver of the LPS model sepsis mice,and the effect of the paeonol(20mg/kg)+emodin(1mg/kg)group Second.At the same time,in the results of in vivo immunohistochemical staining,Dahuang Mudan Decoction and its active ingredients can significantly reduce the expression of HMBG1 in the liver of mice.The Dahuang Mudan Decoction group had the best effect than the paeonol(20mg/kg)+emodin(1mg/kg)group.In the Western blotting results,compared with the LPS model group,the EMO(1mg/kg)group can reduce the expression of HMGB1 to a certain extent(P<0.005),while in the LPS+PAE(20mg/kg)group,LPS+PAE(5mg/kg)+EMO(1mg/kg)group,LPS+PAE(10mg/kg)+EMO(1mg/kg)group,LPS+PAE(20mg/kg)+EMO(lmg/kg)group can significantly down-regulate HMGB1 Expression(P<0.0001),and down-regulated the expression of HMGB1 to a similar degree.Among all drug groups,the HMGB1 protein expression of the Dahuang Mudan Decoction group was the lowest(P<0.0001).Conclusion1.The concentration ratio of effective ingredients paeonol and emodin in Dahuang Mudan Decoction is 9.8:1.2.Dahuang Mudan Decoction can highly improve the survival rate of sepsis mice CLP model.3.Dahuang Mudan Decoction can effectively alleviate the pathological damage to lung tissue and liver tissue of CLP model sepsis mice.4.Dahuang Mudan Decoction can significantly reduce the expression of HMGB1 in lung tissue and liver tissue of CLP model sepsis mice.5.The combination of paeonol and emodin,the active ingredients of Dahuang Mudan Decoction,can significantly down-regulate the expression of HMGB1 in Raw246.7 cells than paeonol or emodin alone.6.The combination of paeonol and emodin,the effective ingredients of Dahuang Mudan Decoction,can significantly up-regulate the expression of TGF-β1 in Raw246.7 cells compared with paeonol alone,and its effect is second only to the high-dose emodin group.7.Dahuang Mudan Decoction and its active ingredients can improve the survival rate of LPS model sepsis mice.8.Dahuang Mudan Decoction and its active ingredients can effectively alleviate the pathological damage to lung tissue and liver tissue in LPS model sepsis mice,but the effect of Dahuang Mudan Decoction is better than its effective ingredient ratio.9.Dahuang Mudan Decoction and its active ingredients can reduce the expression of HMGB1 in the liver tissue of LPS model sepsis mice,but the effect of Dahuang Mudan Decoction is better than its effective ingredient ratio.