Study On The Dose-effect Relationship Of Dushen Decoction In Anti-shock Effects And Its Mechanism On Inhibiting HSPs Induced Endoplasmic Reticulum Stress To Alleviate Lung Injury

Objective: The application of Dushen decoction and related formulas has a long history in the treatment of “Tuo” syndrome,which means desertion disease.Recently,there have been multiple clinical trials were reported that Dushen decoction was used for critical diseases,such as cardiogenic shock and septic shock.However,the dose-response relationship and mechanism in critical diseases of Dushen decoction have not been elucidated.Herein,we aim to comprehensively evaluate the therapeutic effect of Dushen decoction on septic shock induced by cecal ligation puncture(CLP)and hemorrhagic shock induced by femoral artery hemorrhage in rats from multiple perspectives such as cardiac strengthening,blood pressure boosting,improvement of blood indicators,improvement of microcirculation and blood oxygen saturation,protection of important organs,and anti-inflammatory effects.Through the transcriptomics analysis,mechanisms were explored to reveal the scientific connotation of Dushen decoction in reviving yang for resuscitation,nourishing qi and preventing exhaustion,providing reliable experimental basis for the clinical application of Dushen decoction for shock treatment.Methods:1.Qualitative and quantitative analysis of the chemical components in Dushen decoction was conducted using the protein quantification method of 2,2-bisquinoline-4,4-dicarboxylate disodium(BCA),phenol sulfuric acid method,and vanillin sulfuric acid method;A highperformance liquid chromatography-triple quadrupole mass spectrometry(HPLC-TQMS)method was established to detect the prototype and blood components of Dushen decoction;The quality of 6 different decoction batches of Dushen decoction were detected using HPLC method;The total polysaccharides in Dushen decoction were derivatized with 1-phenyl-3-methyl-5-pyrazolone(PMP)and analyzed for their composition using HPLC.2.A CLP induced septic shock rat model was established and modeling times were optimized.Subsequently,the dose-response relationship of Dushen decoction was analyzed on CLP rats using methods such as blood pressure monitoring,lactate level detection,blood gas analysis,blood routine analysis,speckle blood flow imaging analysis,and blood oxygen saturation monitoring at 8 different doses.Next,further blood biochemical analysis,pathological analysis of liver,kidney,and lung tissues,detection of serum inflammatory factors,as well as detection of lung lactate and inflammatory factors were conducted within the effective dose range of Dushen decoction.3.A hemorrhagic shock/resuscitation(HS/R)rat model was established,and the antihemorrhagic shock effect of Dushen decoction was evaluated using methods such as blood pressure and heart rate monitoring,blood gas analysis,blood routine analysis,speckle flow imaging analysis,blood biochemistry analysis,and hematoxylin eosin(HE)staining for liver,kidney and lung tissue.4.Transcriptomics of lung tissue was performed on CLP rats.Partial least squares discriminant analysis(PLS-DA),differential expression gene screening,cluster analysis,screening and functional enrichment analysis of common differential expression genes,and protein interaction network analysis of common differential expression genes were performed based on RNA extraction and library construction.Subsequently,qPCR and Western blotting were performed on all genes in the pathway with the most enriched differential genes in the Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment analysis.Next,immunofluorescence and fluorescence colocalization analysis were used to evaluate the roles of heat shock factor(HSF)1 and HSF2 in the expression of heat shock proteins(Hsps)after administration of Dushen decoction,and the effect of Dushen decoction on the binding of HSF and HSE was detected using luciferase reporter gene technology.Subsequently,immunofluorescence staining was used to evaluate the effects of Dushen decoction on the protein expression levels of protein kinase RNA like endoplasmic reticulum kinase(PERK),transmembrane protein inositol demand enzyme(IRE)1α and transcription activating factor(ATF)6,which were respectively initiate three endoplasmic reticulum stress pathways in lung tissue of CLP rats.Finally,in a lipopolysaccharide(LPS)induced inflammation and endoplasmic reticulum stress model of A549 cells,the effect of Dushen decoction on the release of inflammatory factors in A549 cells was validated using qPCR method.Immunoblotting and immunofluorescence staining were used to evaluate the effects on the expression of PERK and IRE1α and ATF6,and preliminary screening of active ingredients was conducted.Results:1.Successfully prepared Dushen decoction,which with a saponin content of 6.98%,polysaccharide content of 47.21%,and protein content of 3.66%.In addition,we detected 11 types of saponin components,of which 3 saponins were detected in the blood of rats 12 hours after administration.There were no apparent differences in ingredients and stable quality in the six batches of Dushen decoction.Glucose is the highest content in total polysaccharide from Dushen decoction,and also mannose,rhamnose,glucuronic acid,galacturonic acid and galactose were detected.2.After 12 hours of CLP modeling,the blood pressure of rats reached its lowest level,acidosis was the most severe,red blood cell levels increased,immature reticulocytes were significantly decreased,microcirculation failure and lung,liver,and kidney injury were the most significant.Therefore,it is determined that 12 hours of modeling is the optimal modeling time for CLP induced septic shock.Dushen decoction exhibit significant anti shock effects on CLP induced septic shock rats in the dose range of 0.75～3 g/kg,can maintain blood pressure,reduce lactate production,alleviate acidosis,restore ion balance,improve microcirculation blood flow perfusion and blood oxygen saturation in CLP induced septic shock rats.Therefore,it is speculated that 0.75～3 g/kg is the effective dose range of Dushen decoction.Subsequently,it was demonstrated that Dushen decoction significantly reduced the mRNA expression of serum inflammatory factor in CLP rats within the effective range of 0.75～3 g/kg,alleviating organ injuries in the liver,kidney,and lungs of CLP rats.Among them,lung injury improved the most significantly,liver and kidney function could also be improved.Dushen decoction significantly reduced the levels of lung lactate and inflammatory factors in CLP rats.3.Dushen decoction significantly maintained normal blood pressure and heart rate in HS/R induced hemorrhagic shock rats,reduced acidosis in HS/R rats,restored ion balance to a certain extent,restored normal levels of red blood cells,white blood cells,and platelets in HS/R rats,increased microcirculation blood flow perfusion in HS/R rats,reduced organ injury including liver,kidney,and lungs,and improved liver and kidney function indicators.4.Functional enrichment analysis of differentially expressed genes was conducted through lung transcriptomics results,indicating that Dushen decoction may exert an antishock effect by regulating the endoplasmic reticulum protein processing pathway.Ten types of HSPs were enriched in the endoplasmic reticulum protein processing pathway,and after mRNA and protein level validation,it was found that the trend was consistent with the transcriptome results.The immunofluorescence staining results showed that administration of Dushen decoction inhibit the expression and nuclear translocation of HSF1 and HSF2 in the lung tissue of CLP rats.In addition,Dushen decoction inhibit the binding of HSF and heat shock elements(HSE).Moreover,Dushen decoction inhibited the levels of endoplasmic reticulum stress-related proteins induced by CLP in rat lung tissue.5.In the LPS induced inflammation model of A549 cells,Dushen decoction significantly reduce the expression of IL-1β,IL-6,and TNF-α in A549 cells,inhibit the three endoplasmic reticulum stress pathways of PERK,IRE1α,and ATF6.The polysaccharides and saponin components from Dushen decoction,and the major blood entering saponin components,restore the cell viability in LPS induced A549 cells.Both the saponin components and the major blood entering saponin components of Dushen decoction inhibit the luciferase activity of HEK293 T cells which transfected in plasmids with HSE.The effect of Rb2 on cell viability and luciferase activity is most significant.Conclusions:1.Dushen decoction alleviate sepsis and hemorrhagic shock,increase blood pressure,strengthen the heart,alleviate acidosis and ion disorders,improve microcirculation,inhibit inflammation,and reduce organ damage.2.0.75-3 g/kg is the effective dose range of Dushen decoction for treating septic shock,with 3 g/kg being the optimal dose.3.Dushen decoction reduces the transcription level of Hsps in lung tissue of CLP rats by inhibiting the expression and nuclear translocation of HSF1 and HSF2,as well as the binding of HSF and HSE,thereby inhibiting endoplasmic reticulum stress and alleviating lung injury in CLP rats;4.Ginsenoside Rb2 significantly restores the cell viability of LPS induced A549 cells and significantly inhibits the binding of HSF and HSE,which may be the main active ingredient for the anti shock effect of Dushen decoction.