Studies On The Protective Mechanism Of Paeoniflorin Inhibiting HMGB1 In Liver Injury

Background:Liver is one of the most important organs.On the one hand it is the center of the body metabolism,on the other hand an important immune organ.The liver injury will seriously affect people's health.According to reports,around 15%of liver diseases for genetic factors, while 85%of liver diseases were caused by environmental factors.Of which hepatitis virus,alcohol and chemical substances were three most common factors to lead to liver injury.Various causes of liver cell injury can lead to impaire liver function,eventually cause liver failure,result in irreversible pathological changes.They were great dangers to human life.Our country is a big one of liver disease.Liver injury is a common clinical problem.Liver failure' mortality rates are as high as 40%.Therefore,to treat and cure liver injury has become the main therapeutic measures for various types of liver diseases.And looking for new efficient safety drug to combat liver injury is urgent solution in the field of current medical research.Mechanism of liver injury is complex.Treatment is difficult.Recent studies have shown that non-specific inflammatory cells in the liver together is an important mechanism for liver injury.And the newly discovered cytokine --- high mobility group protein B1 plays an important role in chemotaxis of inflammatory cells in convergence.Another important mechanism of liver injury is in the liver microcirculation,the main cells involved in sinusoidal endothelial cells. The expression of cell surface adhesion molecules is in relation to platelet adhesion and aggregation of inflammatory cells of the liver.Although specific antibody and receptor antagonist have been used for liver injury in the past,they have not been in clinical use for various reasons.Long-term clinical exploration confirms traditional Chinese herbal medicine has a unique treatment for liver disease.Chinese herbal Chishao has been attention by scholars at home and abroad.The main active ingredient of Chishao is Paeoniflorin.Paeoniflorin may reduce blood viscosity,the expansion of blood vessels,anti-platelet aggregation,improve microcirculation and so on.What is the mechanism of paeoniflorin to alleviate inflammatory response and improve microcirculation?Does it inhibit the release of HMGB1?In this study,the two following parts of experiment will be confirmed.The first part of the experiment:inhibition of Paeoniflorin on the release and expression of HMGB1 in mouse peritoneal macrophagesObjective:By observing the effects of Paeoniflorin on the expression of high mobility group protein 1(HMGB1)in endotoxin-stimulated mouse peritoneal macrophages(RAW264.7),it was aimed to understand anti-inflammatory mechanism of Paeoniflorin depthly and provide a theoretical basis about further research and development of drugs to combat liver disease Methods:1.Mouse peritoneal macrophages cells cultured:After recoveried of murine macrophage-like RAW264.7 cells,they were cultured in RPMI1640 medium supplemented with 100u/ml penicillin,100u/ml streptomycin,10%newborn bovine serum,2mol/L L-glutamine and 10μg/ml polymyxin B,simultaneously placed at 37℃,5%CO₂ incubator to culture.At 80%confluence,RAW264.7 cells were digested and delivered with 0.25%trypsin+1 mmol/L EDTA solution.2.Trypan blue dye exclusion test to identify cell viability:After the stimulated cells were digested with EDTA,they were blew into a single cell suspension and fully mixed with 0.5m10.4%Trypan blue dye,0.3mlHBSS and 0.2ml cell suspension.To draw a small amount of mixture by capillary suction into a small board room blood cell count,it was counted one of the cell shading.3.Cells immunohistochemical experiments are divided into three groups:control group,LPS stimulation(LPS100ng/ml),Paeoniflorin Group(LPS100ng/ml+ Paeoniflorin 0.625mg/ml),were detected the expression of HMGB1 of mice peritoneal macrophages using immunohistochemical methods.4.The levels of HMGB1 in the culture medium were determined by Western blot method.Results:Trypan blue dye exclusion experiments show that the experimental group RAW264.7 cell viability is higher than 95%.Added a variety of laboratory material does not affect the vitality of cells.It was excluded from the passive release factor of HMGB 1 of cells necrosis.At 20h after RAW264.7 cells stimulated by LPS,immunohistochemistry results showed that:LPS group showed cell volume increased,pseudopodia protruded,nucleus HMGB 1-specific stained light,some cell nuclei stained blue,cytoplasm stained deepened.Paeoniflorin group cell volume'variety was not obvious.The release of HMGB1 of the majority of cells was inhibited. HMGB1-specific staining nuclei become darker.Cytoplasm staining shallow.The levels of HMGB1 in the culture medium determined by Western blot method:HMGB1 in the culture medium of control group was not detected;The content of HMGB1 in the Paeoniflorin group were significantly lower than the LPS group,but higher than the control group(F=827,P＜0.01). Conclusion:Paeoniflorin can alleviate liver inflammatory response and play a protective role in the acute liver injury through inhibiting the release and expression of HMGB1.The second part of the experiment:inhibition of paeoniflorin on the expression of HMGB1 and ICAM-1 in human umbilical vein endothelial cellsObjective:By observing the effects of Paeoniflorin on the expression of high mobility group protein 1(HMGB1)and intercellular adhesion molecule(ICAM-1)in LPS-induced human umbilical vein endothelial cells(ECV304),it was aimed to explore a protective way of paeoniflorin on endothelial cells,and provide a theoretical basis to further reveal the pharmacological mechanism of paeoniflorin.Methods:1.Umbilical vein endothelial cells cultured:After the recoveried of ECV304 cells, they were cultured RPMI1640 medium supplemented with 100u/ml penicillin,100u/ml streptomycin,10%newborn bovine serum,2mol/L L-glutamine and 10μg/ml polymyxin B,at the same time placed at 37℃,5%CO2 incubator to culture.At 80%confluence,ECV304 cells were digested and delivered with 0.25%trypsin+1 mmol/L EDTA solution.2.Trypan blue dye exclusion test to identify cell viability:After the stimulated cells were digested with EDTA, they were blew into a single cell suspension and fully mixed with 0.5ml 0.4%Trypan blue dye,0.3mlHBSS and 0.2ml cell suspension.To draw a small amount of mixture by capillary suction into a small board room blood cell count,it was counted one of the cell shading.3.Cell immunohistochemical experiments are divided into three groups:control group,LPS stimulation (LPS 100ng/ml),Paeoniflorin Group(LPS100ng/ml+Paeoniflorin 0.625mg/ml),were detected the expression of t tMGB1 and ICAM-1 of human umbilica endothelial cells using immunohistochemical methods.Results:trypan blue dye exclusion experiments show that the experimental group ECV304 cell viability is higher than 95%.added a variety of laboratory material does not affect the vitality of cells.It was excluded from the passive release factor of HMGB1 of cells necrosis.Umbilical vein endothelial cells HMGB1,ICAM-1 Immunohistochemistry results showed:HMGB1 of normal cultured human umbilical vein endothelial cells which were not stimulated is gathered in the nucleus and ICAM-1 weakly express;At 20h after LPS stimulation,HMGB1 of most cells shifted from the nucleus to the cytoplasm and expression of ICAM-1 was significantly increased; the release of HMGB 1 when was added Paeoniflorin liquid inhibited mostly and the expression of ICAM- 1 decreased significantly.Conclusion:Paeoniflorin can improve the liver microcirculation in the acute liver injury through inhibiting the release of ICAM-1,HMGB1 of the vascular endothelial cells.