Regulatory Effects Of Niu-Po-Zhi-Bo Pellet On Endotoxin Shock Related Gene Expression

1.ObjectiveNiu-Po-Zhi-Bao peUet(NPZB pellet,牛珀至宝微丸) is clinically used to treat shock inTCM, Nitric Oxide(NO) related gene expression play an important role on blood pressureregulation and vicera injure in endotoxin shock. To investigate the mechanism of NPZBpellet in treating shock is correlated to NO related gene expression or not, the effect of theNPZB pellet on regulating NO in biochemistry of signal transduction and generegulation, cell and in vivo is studied.2.Content and methodResearch includes in vivo and in vitro experiment.To explore the effect of NPZB pellet on NO expression regulated induced bylipopolysaccharide(LPS) in rats. The septic shock was induced by LPS and D-GalN,Pathological injure was tested by lung HE stained slices, the serum ALT and AST weretested; the serum NO was tested by Griess method, The expression of nitric oxide synthase(NOS) was measured by immunohistochemistry and Western blot, The activity was assayedby isotope labeling, The effect of AG and NPZB pellet on inducible nitric oxide synthase(iNOS) expression was observed, the expression of collagenous fiber was measured by VanGieson's stained slices.The expression of TGF-βand TβRⅡwas measured byimmunohistochemistry. The expression of high mobility group box-1 protein(HMGB1) wasmeasured by immunohistochemistry and double immunofluorescence staining using laserscanning confocal microscopy.To explore the gene expression of iNOS regulated by NPZB pellet through proteinkinase C (PKC) on the stimulation of LPS in macrophages. PKC activity assay was used tostudy the activation of PKC by LPS. Griess method was used to determine inhibitory effectof PKC on the induction of NO by LPS. Luciferace reporter gene system of iNOS wasconstructed using gene recombination technique. Gene transfection and reporter gene assaywere used to study the production of NO and induction of iNOS promoter transactivityregulated by NPZB pellet in RAW264.7 cell on the stimulation of LPS and the effect ofPKC.3.ResultThe influence and effect of NPZB pellet on shock and NO in plasma showed that blood pressure decreased rapidly by injecting LPS while the blood pressure rised andapproached to normal level in 30min and higher than the basic blood pressure in 240minafter injecting AG, which had action of rising blood pressure remarkably. The bloodpressure had been rised slowly by using NPZB pellet compared to LPS control group.Combination of AG and NPZB pellet cannot synergize the effect of elevation of bloodpressure. The plasma NO concentration rised consistently after injection of LPS, LPS+AGgroup's plasma NO concentration decreased rapidly; it approached to normal level in 30minand lower than the basic concentration in 240min after injection of AG. The plasma NOconcentration in NPZB pellet group decreased slowly compared to AG group, but it waslowered than LPS control group. Combination of AG and NPZB pellet cannot enhance theeffect of decreasing the plasma NO concentration. iNOS protein and activity were inducedby LPS and decrease by AG, NPZB pellet can decrease iNOS protein and activity but waslower than AG;The results of NPZB pellet on morphology and function of organs injured by septicshock and the relationship between effect of NPZB pellet and iNOS showed that iNOSimmuno-positive cells induced by LPS distributed in liver cell, Kuffers cells andendothelium cells. In heart, iNOS immuno-positive cells induced by LPS distributed inmyocardium and epicardium; In brain, iNOS immuno-positive cells induced by distributedin layer of cerebral cortex, striatum, brain stem reticular formation, cerebellar cortex. Also,iNOS immuno-positive cells induced by distributed in kindney. The number of positiveceils in LPS group were much more than that treated by NPZB pellet, there wassignificant difference between the two groups. Pathological injure was tested by lung HEstained slices, the serum ALT and AST were tested, NPZB pellet can alleviate liver injury,heart injury, brain injury and kidney injury.We also investigated repairing function following injury in lung, NPZB pelletcan dropiNOS protein activity and rise eNOS protein activity; iNOS protein also shows less positivereaction and eNOS shows stronger in NPZB pellet group than LPS group inimmunohistochemical stained slices. NPZB pellet can obviously protect lung and dropCollagenous Fiber expression. The results in relating factor of lung injury and repair showthat NPZB pellet can enhance the expression of HMGB1, which is key nuclear factor in latephase of endotoxin shock and the expression of TGF-βand TβRⅡwhile it does not causefibrosis. The expression of TGF-βand TβRⅡwas measured by immunohistochemistry.TGF-βland TβRⅡshows stronger in NPZB pellet group than LPS group inimmunohistochemical stained slices.In RAW264.7 cells following the stimulation of LPS, PKC was activated by phosphorylation and translocated to inner cell membrane, and both of them were inhibitedby NPZB pellet. The stimulation of LPS on RAW 264.7 cells increased NO product ion H7and Verapamil, a calcium ion channel blocker, showed an inhibitory effect on iNOSpromoter transactivity induced by LPS. All effects were strengthened by NPZB pellet.4.ConclusionCompared with AG, it is beneficial to body that NPZB pellet increase the bloodpressure and decrease NO concentration in plasma slowly in septic shock induced by LPS.NPZB pellet can improve the injured organ induced by LPS through adjusting iNOSexpression, including liver, heart, brain, kidney and lung. It is showed that the effect ofNPZB pellet to cure septic shock is related to NO, but has some difference from NOSinhibitor, it can stablized NO concentration, and rise cNOS protein activity. NPZB pellet canrepair acute lung injury of endotoxin shock by adjusting collagenous fiber by enhancingexpression of TGF-βland TβRⅡprotein expression. In RAW264.7 cells stimulated byLPS, NPZB pellet can inhibit activation of PKC for the induction of iNOS gene expressionwhich contribute to the production of NO. This is an important signaling mechanism of NOproduction in septic shock. NPZB pellet can enhance the expression of HMGB1, which isthe key nuclear factor in late phase of endotoxin shock, its mechanism of regulating repairmay be through HMGB1. Since the expression of iNOS mostly depend on transcriptionallevel of gene, NPZB pellet can inhibit the transcriptional activity of promoter in iNOS, andenhance the effect of H7 which is the inhibitor of PKC to down-regulation of NO in cellsignal. Taken together, the influence of NPZB pellet on NO in shock is an uniquemechanism of regulating in whole, and its target is within the nuclear.