牛珀至宝微丸对内毒素休克相关基因表达调控的影响
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摘要
1、研究目的
牛珀至宝微丸临床治疗感染性休克疗效显著，一氧化氮(NO)及其相关基因在内毒素休克中对血压调节、脏器损伤与修复起重要作用。为探讨牛珀至宝微丸治疗感染性休克的机理是否与一氧化氮及其相关基因相关，本研究从整体水平、细胞、信号转导与基因调控各个层次对牛珀至宝微丸调控一氧化氮及其相关基因进行研究。
2、研究内容、方法
本实验分在体实验与离体实验二部分。
整体实验主要观察牛珀至宝微丸调控NO对内毒素休克血压、脏器损伤及修复的影响。用静脉注射脂多糖内毒素(LPS)1.5mg／kg、腹腔注射D-氨基半乳糖(D-GaLN)100mg／kg造成内毒素休克模型，用牛珀至宝微丸和一氧化氮合酶抑制剂氨基胍(AG)作干预处理，用BL-410生理仪监测血压，用OlympusAU2700型全自动生化分析仪进行肝功能检测，用HE染色观察各脏器的病理形态，用硝酸还原酶法检测血浆NO浓度，同位素标记法测组织匀浆一氧化氮合酶(NOS)活性，免疫组化、Western blot方法检测诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)在各脏器组织内的表达，用Van Gieson法染色检测胶原纤维在肺组织内的表达，免疫组化方法检测肺组织高迁移蛋白(HMGB_1)、转化生长因子(TGF-β_1)及其受体的表达，并用激光共聚焦扫描显微镜进一步观察高迁移率族蛋白(HMGB_1)的表达。
离体实验从细胞信号转导与基因调控水平观察牛珀至宝微丸动物血清对LPS刺激RAW264.7iNOS基因转录调控的影响。用硝酸还原酶法检测测定蛋白激酶C(PKC)抑制剂对LPS诱导的NO生成作用，用PKC活性测定法研究LPS对PKC的激活作用；用基因重组技术构建iNOS荧光素酶报告基因载体；用基因转染及报告基因检测等方法研究牛珀至宝微丸调控LPS刺激RAW264.7细胞对iNOS启动子活性的诱导作用及其与PKC的关系，以明确牛珀至宝微丸对LPS刺激RAW264.7细胞iNOS启动子活性的诱导作用及PKC的影响。
3、研究结果
牛珀至宝微丸对内毒素休克与NO影响结果显示：LPS注射即时血压迅速下降成稳定的低血压，AG有显著的升压作用，用药30min后血压上升接近正常水平，至240min甚至高于原基础血压；牛珀至宝微丸血压回升稳定而缓慢，AG+牛珀至宝微丸有升压作用，但并未叠加，而是使升压作用缓和。LPS组NO浓度持续上升，AG可明显使NO浓度下降迅速而且稳定，注射后30min即回复至正常水平，240min后仍有继续下降趋势。牛珀至宝微丸组NO缓慢上升，其上升幅度高于LPS组而低于正常组。提示牛珀至宝微丸可使内毒素休克鼠血浆NO浓度缓慢下降，抑制NO的过量生成。AG+牛珀至宝微丸的作用叠加无显著意义。iNOS蛋白活性检测结果显示：LPS可致iNOS活性显著增强，AG可抑制iNOS，牛珀至宝微丸可抑制iNOS活性，AG+牛珀至宝微丸其作用有增强趋势。iNOS Western Blotting结果显示：正常组iNOS条带缺如，LPS组iNOS强阳性表达，AG组iNOS表达较弱，牛珀至宝微丸iNOS表达也较弱，但不如AG组明显，AG+牛珀至宝微丸其作用有增强趋势。
牛珀至宝微丸对内毒素休克各脏器损伤的形态、功能影响与iNOS关系结果显示：AG对内毒素性肝损伤血清ALT、AST的浓度变化无影响，牛珀至宝微丸则可以明显改善以上肝功能指标及肝脏病理损坏，并能减少肝细胞、枯否氏细胞、内皮细胞的iNOS表达。内毒素休克心脏iNOS阳性细胞分布于心脏外膜与心肌，牛珀至宝微丸能明显降低内毒素所致iNOS表达，并能改善内毒素休克的心脏损害。内毒素休克脑iNOS阳性细胞分布于大脑皮质、纹状体、脑干、小脑等，牛珀至宝微丸能明显降低内毒素脑iNOS表达，并能改善内毒素休克脑损害。内毒素休克肾组织iNOS表达增强，牛珀至宝微丸能明显降低内毒素肾iNOS表达，并能改善内毒素休克肾损害。
牛珀至宝微丸对内毒素休克脏器损伤修复与NOS表达关系结果显示：牛珀至宝微丸降低肺组织iNOS活性，使肺内iNOS表达减弱、而且同时能使eNOS表达增强，肺损伤减轻。对内毒素急性肺损伤的修复表明，内毒素肺损伤肺组织胶原纤维染色显著增强，牛珀至宝微丸能减轻肺损伤，并能减弱肺组织胶原纤维染色。内毒素肺损伤与修复的相关因子结果显示：牛珀至宝微丸可增强内毒素休克晚期关键核因子高迁移蛋白(HMGB_1)的表达，增强肺组织转化生长因子-β_1及其受体表达而不产生纤维化。
离体实验从细胞信号转导与基因调控水平研究结果显示：牛珀至宝微丸可负调节LPS刺激RAW264.7细胞引起的PKC磷酸化激活和膜移位，并可延长PKC抑制剂H7下调NO作用时间。荧光素酶报告基因实验结果显示，牛珀至宝微丸可抑制LPS刺激诱导的iNOS启动子的转录活性，并可增强H7和钙离子通道阻滞剂维拉帕米的作用。牛珀至宝微丸抑制LPS刺激RAW264.7细胞所致的NO生成增加，其机制之一是量效与时间两方面抑制PKC激活和细胞内钙增加，从而影响iNOS启动子的转录活性。
4、研究结论
AG过快、无限制的升压可能对机体不利。牛珀至宝微丸治疗内毒素休克与NO途径相关，但不同于单纯的一氧化氮合酶抑制剂，升压稳定而缓慢，可能更有利于机体恢复，这也说明牛珀至宝微丸有平稳的升压作用不只是单纯的NO作用途径，而且存在某种稳定NO浓度的机制。牛珀至宝微丸治疗内毒素休克各脏器损伤与减少iNOS表达相关，与一氧化氮合酶抑制剂不同的是能使eNOS表达增强；对于内毒素损伤与修复表明，牛珀至宝微丸其机理可能与增强纤维化因子TGF-β_1及其受体表达相关，能增强内毒素休克晚期关键核因子HMGB_1的表达，其损伤与修复之间可能是通过核因子HMGB_1而发生作用。鉴于iNOS的表达主要取决于基因转录水平，研究表明牛珀至宝微丸抑制iNOS启动子转录活性，细胞信号研究表明牛珀至宝微丸可增强PKC抑制剂H7下调NO的作用。本研究的最终结果表明：牛珀至宝微丸对内毒素休克NO影响是一个独特的整体调节机制，而且其靶点主要在细胞核。
1.Objective
Niu-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 method
Research 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.Result
The 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.Conclusion
Compared 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.
牛珀至宝微丸临床治疗感染性休克疗效显著，一氧化氮(NO)及其相关基因在内毒素休克中对血压调节、脏器损伤与修复起重要作用。为探讨牛珀至宝微丸治疗感染性休克的机理是否与一氧化氮及其相关基因相关，本研究从整体水平、细胞、信号转导与基因调控各个层次对牛珀至宝微丸调控一氧化氮及其相关基因进行研究。
2、研究内容、方法
本实验分在体实验与离体实验二部分。
整体实验主要观察牛珀至宝微丸调控NO对内毒素休克血压、脏器损伤及修复的影响。用静脉注射脂多糖内毒素(LPS)1.5mg／kg、腹腔注射D-氨基半乳糖(D-GaLN)100mg／kg造成内毒素休克模型，用牛珀至宝微丸和一氧化氮合酶抑制剂氨基胍(AG)作干预处理，用BL-410生理仪监测血压，用OlympusAU2700型全自动生化分析仪进行肝功能检测，用HE染色观察各脏器的病理形态，用硝酸还原酶法检测血浆NO浓度，同位素标记法测组织匀浆一氧化氮合酶(NOS)活性，免疫组化、Western blot方法检测诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)在各脏器组织内的表达，用Van Gieson法染色检测胶原纤维在肺组织内的表达，免疫组化方法检测肺组织高迁移蛋白(HMGB_1)、转化生长因子(TGF-β_1)及其受体的表达，并用激光共聚焦扫描显微镜进一步观察高迁移率族蛋白(HMGB_1)的表达。
离体实验从细胞信号转导与基因调控水平观察牛珀至宝微丸动物血清对LPS刺激RAW264.7iNOS基因转录调控的影响。用硝酸还原酶法检测测定蛋白激酶C(PKC)抑制剂对LPS诱导的NO生成作用，用PKC活性测定法研究LPS对PKC的激活作用；用基因重组技术构建iNOS荧光素酶报告基因载体；用基因转染及报告基因检测等方法研究牛珀至宝微丸调控LPS刺激RAW264.7细胞对iNOS启动子活性的诱导作用及其与PKC的关系，以明确牛珀至宝微丸对LPS刺激RAW264.7细胞iNOS启动子活性的诱导作用及PKC的影响。
3、研究结果
牛珀至宝微丸对内毒素休克与NO影响结果显示：LPS注射即时血压迅速下降成稳定的低血压，AG有显著的升压作用，用药30min后血压上升接近正常水平，至240min甚至高于原基础血压；牛珀至宝微丸血压回升稳定而缓慢，AG+牛珀至宝微丸有升压作用，但并未叠加，而是使升压作用缓和。LPS组NO浓度持续上升，AG可明显使NO浓度下降迅速而且稳定，注射后30min即回复至正常水平，240min后仍有继续下降趋势。牛珀至宝微丸组NO缓慢上升，其上升幅度高于LPS组而低于正常组。提示牛珀至宝微丸可使内毒素休克鼠血浆NO浓度缓慢下降，抑制NO的过量生成。AG+牛珀至宝微丸的作用叠加无显著意义。iNOS蛋白活性检测结果显示：LPS可致iNOS活性显著增强，AG可抑制iNOS，牛珀至宝微丸可抑制iNOS活性，AG+牛珀至宝微丸其作用有增强趋势。iNOS Western Blotting结果显示：正常组iNOS条带缺如，LPS组iNOS强阳性表达，AG组iNOS表达较弱，牛珀至宝微丸iNOS表达也较弱，但不如AG组明显，AG+牛珀至宝微丸其作用有增强趋势。
牛珀至宝微丸对内毒素休克各脏器损伤的形态、功能影响与iNOS关系结果显示：AG对内毒素性肝损伤血清ALT、AST的浓度变化无影响，牛珀至宝微丸则可以明显改善以上肝功能指标及肝脏病理损坏，并能减少肝细胞、枯否氏细胞、内皮细胞的iNOS表达。内毒素休克心脏iNOS阳性细胞分布于心脏外膜与心肌，牛珀至宝微丸能明显降低内毒素所致iNOS表达，并能改善内毒素休克的心脏损害。内毒素休克脑iNOS阳性细胞分布于大脑皮质、纹状体、脑干、小脑等，牛珀至宝微丸能明显降低内毒素脑iNOS表达，并能改善内毒素休克脑损害。内毒素休克肾组织iNOS表达增强，牛珀至宝微丸能明显降低内毒素肾iNOS表达，并能改善内毒素休克肾损害。
牛珀至宝微丸对内毒素休克脏器损伤修复与NOS表达关系结果显示：牛珀至宝微丸降低肺组织iNOS活性，使肺内iNOS表达减弱、而且同时能使eNOS表达增强，肺损伤减轻。对内毒素急性肺损伤的修复表明，内毒素肺损伤肺组织胶原纤维染色显著增强，牛珀至宝微丸能减轻肺损伤，并能减弱肺组织胶原纤维染色。内毒素肺损伤与修复的相关因子结果显示：牛珀至宝微丸可增强内毒素休克晚期关键核因子高迁移蛋白(HMGB_1)的表达，增强肺组织转化生长因子-β_1及其受体表达而不产生纤维化。
离体实验从细胞信号转导与基因调控水平研究结果显示：牛珀至宝微丸可负调节LPS刺激RAW264.7细胞引起的PKC磷酸化激活和膜移位，并可延长PKC抑制剂H7下调NO作用时间。荧光素酶报告基因实验结果显示，牛珀至宝微丸可抑制LPS刺激诱导的iNOS启动子的转录活性，并可增强H7和钙离子通道阻滞剂维拉帕米的作用。牛珀至宝微丸抑制LPS刺激RAW264.7细胞所致的NO生成增加，其机制之一是量效与时间两方面抑制PKC激活和细胞内钙增加，从而影响iNOS启动子的转录活性。
4、研究结论
AG过快、无限制的升压可能对机体不利。牛珀至宝微丸治疗内毒素休克与NO途径相关，但不同于单纯的一氧化氮合酶抑制剂，升压稳定而缓慢，可能更有利于机体恢复，这也说明牛珀至宝微丸有平稳的升压作用不只是单纯的NO作用途径，而且存在某种稳定NO浓度的机制。牛珀至宝微丸治疗内毒素休克各脏器损伤与减少iNOS表达相关，与一氧化氮合酶抑制剂不同的是能使eNOS表达增强；对于内毒素损伤与修复表明，牛珀至宝微丸其机理可能与增强纤维化因子TGF-β_1及其受体表达相关，能增强内毒素休克晚期关键核因子HMGB_1的表达，其损伤与修复之间可能是通过核因子HMGB_1而发生作用。鉴于iNOS的表达主要取决于基因转录水平，研究表明牛珀至宝微丸抑制iNOS启动子转录活性，细胞信号研究表明牛珀至宝微丸可增强PKC抑制剂H7下调NO的作用。本研究的最终结果表明：牛珀至宝微丸对内毒素休克NO影响是一个独特的整体调节机制，而且其靶点主要在细胞核。
1.Objective
Niu-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 method
Research 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.Result
The 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.Conclusion
Compared 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.
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