Comparison Of The Effects Of Penehyclidine Hydrochloride And Anisodamine On Acute Lung Injury Induced By "Two-hit" In Rats

Systemic inflammatory response syndrome (SIRS) that followed by variety of serious stimulus such as trauma, blood loss and infections often results in excessive release of cytokines and inflammatory medators.If the inflammatory response is out of control and develops into immune disorders, cells, tissue and organ will be damaged or become disfunction.Eventually it might cause multiple organ dysfuncion (MODS) inevitably and even death. The pathological process of MODS is very comlex, involving activation of generous cells, inflammatory mediator, as well as blood coagulation system. Liver, lung, small intestine are the major target organs for damage. The highest incidence in MODS is respiratory dysfunction,which appears earlier than other incidences, generally at 24-72h after onset, with clinical manifestations of acute lung injury (ALI). ALI is susceptible to acute respiratory distress syndrome (ARDS) with a high mortality and poor prognosis. Accordingly, it is very important to study pathogenesis of ALI, explorating an effective way of prevention and treatment against ALI.In recent year, the response of uncontrolled inflammatory directly or indirectly caused the damage of alveolar-capillary membrane.It is resported that, after hemorrhagic shock, neutrophils plays a critical role in inducing tissue damage even multiple organ dysfunction. On one hand, ischemia can activate the complement system; on the other hand, endothelial cells can release oxygen free radicals during reperfusion. These two process will result in chemotactic metabolites such as C3 and leukotriene, activation and multiplication of leukocytosis. Neutrophils are activated, producing more oxygen free radicals through respiratory burst, with increasing oxygen consumption. Some inflammatory cells and endothelial cells can release cytokines and inflammatory mediators, including tumor necrosis factor (TNF-a) interleukins (IL-1,IL-8,IL-6), oxygen free radicals,thromboxane, which woule not only damage the capillary endothelial cells but destroy the permeability of vessel wall. Currently, IL-1,IL-8 and TNF-a are the hottest objects in pro-inflammatory cytokine research. They can cause strong inflammatory response, leading to organ damages and secondary injury in distant organs. We can reduce ALI, if the way of inflammatory cytokines participation was blocked properly.Hemorrhagic shock often rusults in peripheral and visceral circulation disorder, leading to gastrointestinal and mucosal barrier dysfunction. Bacteria and endotoxin displacement, Gut-derived bacteremia and endotoxemia.Gram-negative bacterial infection plays an important role in clinical inflammatory. The endotoxin is the main toxic component. LPS cause obvious mutiple organ dysfunction. Once ischemia-reperfusion and endotoxemia were treated promptly and properly, it will lower the ability of monocyte-macrophage cells to present antigen. The activated cells release inflammatory mediators. Excessive inflammatory cytokine secretion leads to vicious cycle,causing single or multiple organs dysfunction or failure. Recent studies supported a "two-hit" hypothesis in the pathogenesis of lung injury in trauma or hemorrhagic shock patients, namely an initial stimulus may prime for subsequent organ damage in response to a second, often minor, insult. The base of two-hit therory is that SIRS caused by sever trauma and infection is imbalance between inflammarion and imflammatory reactions, inducing MODS.Experiments on animals showed that anticholinergic drugs such as anisodamine inhibit the production of inflammatory cytokines,scavenging oxygen free radicals, reducing lipid peroxidation and reduce acute lung inury.However, the short action of anisodamine as no selective anticholinergic agents limits its clinical use and development.Penehyclidine hydrochloride is a new type selective anticholinergic agents, which selectively resists M1,M3 and N1,N2 cholinergic receptors.Because of no significant effect on the M2 receptor, penehyclidine hydrochloride has been found to increase heart rate only a little and show the stronger anticholinergic effect and long duration compared with hyoscyamine. Few scholars researched the relationships between anticholinergic drugs and organ injury induced by hemorrhagic shock and endotoxin in rats at home and abroad, Liu jian researched that anisodamine can reduce the incidence of MODS caused by hemorrhagic shock with endotoxemia. Inhibiting the over-expression of lipopolysaccharide binding protein (LBP), Anisodamine can protect rats against acute lung injury induced by "acid-LPS" two-hit with blocking the sensitization LPS by LBP.Our previous study has showed that Penehyclidine hydrochloride has protective effects on acute lung injury induced by hemorrhagic-endotoxin in rats.Comparison of the effects of penehyclidine hydrochloride and traditional anticholinergic compound on acute lung injury induced by "two-hit" has not been reported. We developed a "two-hit" lung injury model characterized by hemorrhagic shock with resuscitation, followed by intratracheal LPS administration.The aim of this study was to compare the therapeutic effects of penehyclidine hydrochloride and anisodamine on acute lung injury induced by "two-hit" in rats.Materials and methods1. Animals and groupsFifty-four healthy adult Wistar rats weighing 180～220g, male or female, were provided by department of laboratory animal center of Southern Medical University.Certificate number:SCXKyue 2006-0015.The fifty-four rats were randomly divided into six groups(n=9):group C(normal control); group M(ALI model),group A1(anisodamine 5mg.kg-1),group A2(anisodamine 10mg.kg-1)group P1(penehyclidine hydrochloride lmg.kg-1) and group P2(penehyclidine hydrochloride 2mg·kg-1).2.two-hit modelHemorrhagic shock rats were injected with lipoplysaccharide(LPS) through femoral vein as the second hit to bulid up ALI model. The animals were fasted 12 hours prior to the experiments with water given ad libitum prior to anesthesia. Adult Wistar rats were anesthetized with urethan(lg/kg body wt) intraperitoneally. Additional urethan was injected to maitain the depth of anesthesia according to the situation. A left inguinal incision was performed.After heparinization, a polyethylene catheter was inserted into the left femoral artery to record the mean arterial pressure(MAP).When blood pressure is stable in 10min after operation,hemorrhagic shock was initiated by blood withdraw and reduction of the MAP to 35±5mmHg within 15min. This blood pressrue was maintained by further blood withdrawn or infusion. The left femoral vein was cannulated for reinfusion of shed blood and additional Ringer's lactate.After hypotension period of 60min, animals were resuscitated by transfusion of the shed blood and RL in a volume equal to that of shed blood,over a period of 90 min.Sham animals underwent cannulation without shock or resuscitation and served as negative controls. Animals were resuscitated by transfusion of shed blood and equivalent amount of Ringer's lactate for 90 min.30min later,Rats were injected with lipoplysaccharide(LPS) through femoral vein as the second hit to bulid up ALI model. Anisodamine or penehyclidein hydrochoride was injected respectively through femoral vein 1h after the treatment of LPS. Group C and Group M were injected the same amount of saline.Two hours later, the artery blood samples and lung tissues were collected for further determination. 3. Experimental indexesThe concentration of TNF-α, IL-1, IL-8 in serum was measured using Luminex with 100 liquid chip machine. The middle lobe of the right lung was taken for histological analysis.The inferior lobe of the right lung was used for detection of W/D.The other lung were maitaned in -80℃refrigerator preparing for the SOD activity and MPO activity dectect.4.Statistical analysisSPSS 13.0 for windows were used to analyze the data. All parameters were expressed as mean values±the standard error of the mean(mean±SE).Test of homogeneity of variances was analyzed firstly,then analysis of one-way-anova and Welch test were used to evaluate the datas. Dunnett's T3 test were used when heterogeneity of variance.A significant difference was presumed for a probability value of <0.05.Rusults1.IL-1,IL-8 and TNF-αThe concentration of serum IL-1,IL-8 and TNF-ain group A1,A2, P1 and P2 were higher than those in group C (p<0.01) and lower than those group M (p<0.05). Compared with group A1,the concentration of serum IL-1,IL-8 and TNF-ain group A2 decreased (P=0.014,P=0.048, P=0.001). The concentration of serum IL-1,IL-8 and TNF-αin group P1 were lower than those in group P2 (P=0.048, P=0.022, P=0.018).The serum IL-1,IL-8 and TNF-ain group P1 also decreased compared with those in goup A2 (P=0.001,P=0.007,P=0.000).Compared with anisodamine, penehyclidine hydrochloride can reduce the levels of IL-1, IL-8 and TNF-α..2. SOD,MPO and W/DIn order to evaluate the effect of PHC and anisodamine on the enzyme released,the method described above was used.W/D and MPO activity of lung tissues in group A1,A2, P1 and P2 were obviously higher than those in group C (P <0.01) and lower than those group M (P<0.05). Compared with group A1, W/D and MPO activity of lung tissues in group A2 decreased (P=0.006,.P=0.029),while the activity of SOD showed no statistical difference between group A1 and A2 (P =0.258). W/D and MPO activity of lung tissues in group P1 were significantly lower than those in group P2(.P=0.013,P=0.000) while the SOD activity increased in group P1 (P=0.038).W/D of lung tissues in group P1 also decreased compared with those in goup A2(P=0.038),there were no significant differences in the activities of MPO and SOD between group A2 and group P1 (P=0.256, P=0.212).3. Histopathology of lung tissueLight microscopic findings in group C demonstrated the normal appearance of lung specimens;the appearance of lungs from group M noted the marked inflammatory infiltrate,alveolar-capillary membrane thickening, and areas of alveolar edema;optical microscopy demonstrated milder inflammatory cell and interstitial edema in the lung tissues in group A1,A2, P1 and P2 than in group M. Focal destruction, part of the alveolar wall dissoluted and ruptured, some alveolar edema fluid accumulation, inflammatory cells was increased in group A1. Part of the alveolar walls widened slightly, showing a mild interstitial pneumonia to change in group A2, P1 and P2.Conclusions1. Pehyclidine hydrochloride and anisodamine can reduce the level of the serum IL-1, IL-8 and TNF-α, and decrease W/D and the activity of MPO in the lung, as well as can increase the activity of SOD.So that, they can reduce the pathologic damage of lung tissues in rats with hemorrhagic shock and endotoxemia and have protective effects.2. 10mg.kg-1 of anisodamine showed better protective effect against acute lung injury induced by hemorrhagic shock and endotoxemia two-hit in rats than 5mg.kg-1. Penehyclidine hydrochloride lmg.kg-1 was significantly better than 2mg.kg-1.3.1n contrast to anisodamine (10mg.kg-1), penehyclidine hydrochloride (1mg.kg-1) has better lung protective effect.