Paeonol Regulate PPARgamma Corepression Transcription Via HMGB1 Treatment Septic Shock

Objective:Sepsis(sepsis)is a severe acute systemic inflammatory response caused by the pathogen of acute infection(SIRS).And the shock is the final pathological process after acute exacerbation of sepsis.The mortality rate is about 43%,and the disease itself is a big threat to the global health.Due to the infection caused by shock,it is also known as sepsis or septic shock,mostly the infections are caused by gram negative bacteria.The lipopolysaccharide(LPS)of the bacteria is not only the basic structure of gram-negative bacterial cell membrane structure,but also become one of the most common endotoxin and the basic unit of stimulation of inflammation.Therefore,many experimental studies directly use LPS stimulation to study the inflammatory response mechanism,and the resulting shock is named as endotoxic shock.Although the antibiotics can effectively remove or inhibit the bacteria,it also induces the release of endotoxin and aggravates endotoxemia.The rate of drug resistance and antibiotic failure is even faster than the development of the new drugs.Researches on repression of anti-inflammatory cytokines treatment do not progress very well.On the contrary,the monoclonal antibody of TNF-alpha(infiximab and adalimumab)and soluble TNF-receptor,such as the use of etanercept in animal experiments are always accompanied by an increase of organ injury and mortality.In recent years,it has been found that the nuclear receptor transcriptional co repressor of innate or adaptive immune responses is more effective and safe.Relates to the glucose and lipid metabolic disorder,the peroxisome proliferator activated receptors(Peroxisome proliferater-activated receptor,PPAP)is a ligand activated nuclear receptor.And one of them is PPARγ,which studied the most.In the inflammatory reaction,generation of PPARγ plays an important role in inhibiting inflammation,which can inhibit the inflammatory factor signaling pathway via inflammatory mediators’ competition.Studies have found that PPARγ activator rosiglitazone can directly inhibit lipopolysaccharide(Lippolysaccharide,LPS)in RAW264.7 cells induced by endogenous toxic factor secretion.It can effectively improve the survival rate of sepsis animal.PPARγ is the transcriptional regulatory factor in the nucleus,which inhibits the transcription of inflammatory factors with a co-repression mechanism and does not affect the innate immune response.The preliminary study found that paeonol could upregulate PPARγ about 5.46 times more than normal.Paeonol upregulates PPARγ may regulate the co-repression of PPARγ/NF-κ pathway.Paeonol is Paeonia(dried root bark of Paeonia suffruticosa Andr Aaeoina suffruticosa)and Cynanchum paniculatum(dried root and rhizome of ceropegias xuchangqing Cynanchum paniculatum)of the main active ingredients.Mu’dan is a traditional Chinese herbal medicine for activating blood circulation,dry root bark of Ranunculaceae peony.Mu’dan is slightly cold,bitter,acrid,heart,liver and kidney,with heat and cooling blood,blood circulation of the effect,for the treatment of warm poison spots,hematemesis Nvxue,carbuncle sore,amenorrhea and dysmenorrhea,traumatic injury disease.Modern pharmacological research found that paeonol has anti-inflammatory,anti-cancer,anti-ischemia reperfusion injury,anti-atherosclerosis,anti-allergic asthma and neuroprotective function and other pharmacological effects.The current study found that the main effective components of Paeonia is paeonol and other compounds,with the treatment of heat-clearing,protect the liver and kidney,analgesic antispasmodic and sedative and other pharmacological activity.In the application for the treatment of septic shock,paeonol often with other drugs can play a good effect.Based on the previous research of paeonol by regulating expression of NF-κB,inhibited the expression of TNFalpha and HMGB1 in lung tissues of rats,inhibit the nuclear translocation of HMGB1 to achieve anti-inflammatory effect."Wenbingtiaobian" refers as there is a ventral fire in the middle,which causes the fire to return to the Yin,and the book emphasizes the characteristics of Paeonol could decrease internal heat and removing toxic characteristics."materia medica words" refers the general cold soup,heat and cooling blood,good blood stasis eliminating carbuncle.Treatment of burn flourishing,boils with the study of Paeonol in.In the early stage of the project,the therapeutic effect of endotoxin shock rats was studied.It has been found that paeonol can improve the livability of endotoxin shock rats,reduce the infiltration of pulmonary tissue hyperemia edema and inflammatory cells,and serum HMGB1 concentration.In the lung tissue,the expression of PPARγ was enhanced by the paeonol.On the contrast,not only the expression and nuclear translocation of NF-κB were inhibited,but also inhibited the expression of inflammatory factors such as TNF-α and HMGB1 at the same time when the inflammation was increased after the PPARγ inhibitor was applied.On the basis of the previous study,the effect of the effective component of the hematopoietic drug paeonol on HMGB1 and PPARγ,the mechanism of the co-repression of HMGB1 and PPARγ is going to be discussed.Methods:1.The use of LPS stimulated RAW264.7 cell model,detect the effects of Paeonol on PPARγ expression and activity.The LPS stimulated rat macrophage RAW264.7 cells as a model,using paeonol(0.2,0.6,1mM)to detect the expression of mRNA and protein of PPARγ and NF-κB.Construction of the promoter report gene of PPRE NF-κB was investigated on PPRE activity and NF-κB promoter.2.To detect Paeonol on effect of activity and expression of PPARγ By,it used Tet-on-HMGB1 as overexpression of the RAW264.7 cell model.LPS stimulates the overexpression of Tet-on-HMGB1 in RAW264.7 cells model,using paeonol(0.2,0.6,1mM)with PPAR gamma inhibitor GW9662 as intervention,the expression of WB,qRT-PCR,ELISA,and the method of detection of PPARγ,P65,P50 and inflammatory factors.3.To detect the effects of Paeonol on PPAR expression and activity,it used the Tet-on-HMGB1 mutation in RAW264.7 cell model.LPS stimulates the point mutation of Tet-on-HMGB1 in RAW264.7 cells model,using paeonol(0.2,0.6,1mM)with PPAR gamma inhibitor GW9662 intervention,the expression of qRT-PCR and ELISA by WB,PPAR,P65,and the gamma detection method of P50 and inflammatory factors.4.To construct the mammalian hybrid system,it detects the effect of Paeonol on the combination of PPARγ and HMGB1 and the combination of HMGB1 and P65/P50.Results:1.Paeonol promotes the expression of PPARγmRNA and protein in RAW264.7 cells,reporter gene activity of PPRE,showing that paeonol can promote PPARγ expression and biological activity.Paeonol inhibited the expression of mRNA of NF-kappa B and protein,reporter gene activity of NF-kappa B,and the secretion of inflammatory factor TNF alpha and IL-1 beta,suggesting that paeonol may repress expression of NF-K B activity and the inflammatory factor TNF alpha and IL-1 beta by PPARγ.2.Via the stimulation of LPS to the expression of Tet-on-HMGB1 in the RAW264.7 cell model,paeonol enhanced PPARγmRNA and inhibited NF-kappa Bp65mRNA expression and the secretion of inflammatory factor TNF alpha and IL-1 beta.The inhibitor of PPARγ,GW9662,and the inhibitor of HDAC3,RGFP966,could reverse the inhibitory effect of Paeonol on the expression of NF-K B and the secretion of TNF alpha and IL-1 beta.3.Via the stimulation of LPS to the expression of Tet-on-HMGB1 in the RAW264.7 cell model,paeonol enhanced the combination of PPARγ and HMGB1 and the combination of PPARγ and NF-KBp65,while the GW9662 could inhibit the combination of PPARγ,HMGB1,and NF-kappa Bp65.The results of mammalian hybrid system test show that the paeonol could enhanced the combination of PPARγ and HMGB1,and the combination of HMGB1 and NF-KBp65.Conclusion:By producing anti-inflammatory effect on PPARγ co-repressor mechanism,paeonol shows its biological activity which can promote the expression of PPARγ;HMGB1,PPAR y and HDAC3 may lead to the formation of co-repressor complexes,inhibiting the activity and expression of NF-κB.Paeonol can affect the combination among HMGB1,PPAR,and HDAC3through the anti-inflammatory effect co-repressor mechanism.