The Laboratory And Clinical Research On The Cardiopulmonary Protective Effect Of Penehyclidine Hydrochloride

Completion of great majority of cardiac procedures requires cardiopulmonary bypass (CPB). However, CPB-induced systemic inflammatory response syndrome (SIRS) can cause multi-organ dysfunction, including heart or lung failure. Myocardial injury, which inevitablely happens during CPB, can cause postoperative cardiac dysfunction, manifested as complications such as arrhythmia and heart failure. Acute lung injury (ALI) is a mainstay of SIRS at lungs, characterized by spread of pulmonary infiltration of inflammatory cells, increased pulmonary capillary vascular permeability succeeded by hypoxemia. The incidence of acute respiratory insufficiency after open-heart surgery is up to 15%-30%. The number of deaths caused by acute respiratory insufficiency accounted for 30% of the total number of deaths during postoperative period. At the molecular level, ALI is manifested as the excessive release of a variety of inflammatory mediators and inflammatory factors and the imbalance of pro-inflammatory and anti-inflammatory response. On the other hand, at the cell level, ALI shows the accumulation and infiltration of neutrophils, macrophages and other inflammatory cells. The clinical manifestations of ALI stand for the diffuse injury of alveolar vascular endothelial cell, pulmonary edema, pulmonary atelectasis and other pathological features. In severe cases, ALI can lead to acute respiratory distress syndrome (ARDS). Therefore, recent clinical researches are focused on exploring effective methods to protect hearts and lungs from CPB-induced systemic inflammatory response syndrome.Drug intervention plays an important role in the prevention and treatment of CPB-related SIRS. At present, the mainstay of drugs includes steroid drugs, ulinastatin, heparin, glucosamine glycan, phosphodiesterase inhibitors such as Milrinone and Amrinone, anti-oxidants, and sodium nitroprusside. However, the clinical application of these drugs is not satisfactory because of their poor efficacy and/or adverse reaction. Anticholinergic medicines have been used in the treatment of ALI for many years as well. In recent years, Anisodamine, has also made remarkable achievements in the prevention and treatment of SIRS. The clinical application of traditional anticholinergic drugs such as Anisodamine, nevertheless, is limited because of non-selection on cholinergic receptor subtypes as well as side effects. Penehyclidine hydrochoride (PHC) selectively acts on the M1 and M3 receptor which most exist in the visceral smooth muscle and glands while it has no or less action on the M2 receptors located on presynaptic membrane of myocardium. Recent studies have shown that PHC has demonstrated effects on improving microcirculation, reducing capillary wall permeability, and decreasing the release of lysosomal discharge. These effects exert cell protection. PHC may have a therapeutic effect on ALI and CPB-related SIRS.Currently, there are more and more reports on the organ protective effects of PHC, but little on their mechanism. Recent years, LPS signal transduction pathway becomes a hot research. Animal experiments showed that PHC could inhibit mRNA expression of nuclear factor-KB (NF-κB) in ALI rats, but effects of PHC on endotoxin receptor CD 14 and TLR4 have not been investigated. As the endotoxin receptor CD14 and TLR4 signal transduction pathway in the most upstream, their activation will lead to the activation of a series of downstream molecules, ultimately lead to activation of NF-κB, transcription and expression of inflammatory cytokines, causing the occurrence of SIRS or ALI or ARDS and MODS. As CD 14 and TLR4 in the signal transduction pathway upstream, they are in a "valva"key positions. CD 14 and TLR4 are ideal therapeutic targets and have an important role in treating ALI. Therefore, this study was designed to investigate the expression of myocardial NF-κB and the plasma concentration of tumor necrosis factor-α(TNF-α), soluble intercellular adhesion molecule-1 (sICAM-1) and cardiac troponin I (cTnI) in patients undergoing valve replacement under CPB to explore the myocardial protective effect of PHC; meanwhile, monitor respiratory index (RI), oxygenation index (OI) and pulmonary dynamic compliance (Cd) during the CPB were measured to investigate the pulmonary protective effect of penehyclidine hydrochloride. On the basis of the clinical research, this study was to observe the effect of PHC on oxidative stress and the mRNA expression of CD 14, toll-like receptor-4 and NF-κB on ALI through the establishment of rat model of endotoxin-induced ALI, and further to explore the protective mechanism of PHC and dose-effect relationship used in ALI. This study was composed of three parts.Materials and MethodsSixty patients undergoing valve replacement under CPB were randomly divided into three groups(20 patients for each group):PHC group 1 (P1), PHC group 2 (P2) and control group (C) to receive 0.05 mg/kg,0.1 mg/kg of PHC (diluted to 10mL with saline) and equal volume of saline at beginning of operation via central vein, respectively. Slices of right atricle tissue were collected at prior to CPB (T1),30min after aortic clamping (T2) and 30 min after aortic unclamping (T3) to check the expression of nuclear transcription factor-k B (NF-κB) with immunohistochemistry. Artery blood was drawn at before intravenous injection of PHC(T1),30min after aortic clamping (T2),30 min (T3),4h (T4) and 24h (T5) after aortic unclamping. Concentrations of tumor necrosis factor-α(TNF-α), soluble intercellular adhesion molecule-1 (sICAM-1) and cardiac troponin I (cTnl) were measured. Hemodynamic parameters such as heart rate (HR), mean arterial pressure (MAP) and central venous pressure(CVP)were recorded at each measuring point. The patients, groups and drug administration in the second part were the same as the first part. Radial artery blood 1ml collected from the first part of T1-T5 time points was used to do artery blood gas analysis. Recordings of PaCO2, PaO2, and FiO2 were measured at each measuring point to calculate respiratory index (RI) and oxygenation index (OI). VT, Pmax and PEEP were recorded at after intubation lOmin,30min, the beginning of CPB, after CPB 30min and 60min to calculate pulmonary dynamic compliance (Cd).Thirty-two male SD rats weighing 230-280g were randomly divided into 4 groups(n=8 each):control group; group M(received LPS only); group L(LPS+low does PHC); group H(LPS+high does PHC); group L and H received intraperitoneal PHC 0.3mg/kg or lmg/kg 30 min before LPS administration. LPS 5mg/kg was administered intravenously in Group M, L and H. The rats were killed at 6 h after LPS administration. The lungs were removed immediately for determination of W/D lung weight ratio and water content. Reverse-transcription polymerase chain reaction (RT-PCR) was used to evaluate expression of CD14 mRNA, TLR4 mRNA, and NF-κB mRNA. Level of TNF-a was analyzed by enzyme- labeled immunosorbent assay (ELISA). Content of myeloperoxidase (MPO), malondialdehyde (MDA), activity of superoxide dismutase (SOD), lactate dehydrogenase (LDH) in lung tissue and blood was detected by spectrophotometer. Morphologic change of lung tissue was observed under light microscope and electron microscope. Results1. Compared with group C, myocardial injury was slight in group PI and P2; cellular swell was inconspicuous; areas of necrosis were relatively smaller.2. Compared with T1, NF-κB in three groups significantly increased at T2 and T3(P<0.01). NF-κB in group P1 and group P2 was much lower than those in group C (P<0.05). NF-κB in group P1 and group P2 had no significant difference(P>0.05).3. Compared with T1, TNF-αin three groups significantly increased at T3-T5 (P <0.01). TNF-αin group P1 and group P2 were much lower than those in group C (P<0.05). TNF-αin group P1 and group P2 had no significant difference(P>0.05).4. Compared with T1, sICAM-1 in three groups significantly increased at T3-T5 (P <0.01). sICAM-1 in group P1 and group P2 was much lower than those in group C (P<0.05). sICAM-1 of group PI and group P2 had no significant difference(P>0.05).5. Compared with T1, cTnI in three groups significantly increased at T2-T5 (P< 0.01). cTn-I in group P1 and group P2 was much lower than those in group C (P<0.05). cTn-I in group P1 and group P2 had no significant difference(P> 0.05).1. There were no significant differences regard to RI among three groups at T1(P >0.05). RI in group P1 and group P2 was much lower than those in group C at T2-T5(P<0.01), while there were no significant differences between group PI and group P2(P> 0.05).2. There were no significant differences in OI among three groups at T1(P>0.05). 01 in group P1 and group P2 was much higher than those in group C at T2-T5(P <0.01), while there were no significant differences between group P1 and group P2(P> 0.05).3. There were no significant differences in Cd among three groups at after intubation 10min (P>0.05). Cd in group P1 and group P2 was much higher than those in group C at after intubation 30min, before and after CPB (P<0.01), while there were no significant differences between group P1 and group P2 (P >0.05).1. Expression of CD 14 and TLR4 mRNA in the lung tissue of rats:Compared with group C, expression of CD 14 and TLR4 mRNA in group M, L and H increased significantly (P<0.05); compared with group M, expression of CD14 and TLR4 mRNA in group L and H decreased significantly (P<0.05), while there were no significant differences between group L and group H(P>0.05).2. Expression of NF-κB mRNA in the lung tissue of rats:Compared with group C, expression of NF-κB mRNA in group M, L and H increased significantly (P< 0.05); compared with group M, expression of NF-κB mRNA in group L and H decreased significantly (P<0.05), while there also were significant differences between group L and group H(P<0.05).3. TNF-a levels in serum and lung tissue of rats:Compared with group C, TNF-αlevels in group M, L and H increased significantly (P<0.05); compared with group M, TNF-αlevels in group L and H decreased significantly (P<0.05), while there also were significant differences between group L and group H(P<0.05).4. MPO activities in the lung tissue of rats:Compared with group C, MPO activities in group M, L and H increased significantly (P<0.05); compared with group M, MPO activities in group L and H decreased significantly (P<0.05), while there also were significant differences between group L and group H(P< 0.05).5. MDA levels in serum and lung tissue of rats:Compared with group C, MDA levels in group M, L and H increased significantly (P<0.05); compared with group M, MDA levels in group L and H decreased significantly (P<0.05), while there also were significant differences between group L and group H(P<0.05).6. SOD activities in serum and lung tissue of rats:Compared with group C, SOD activities in group M, L and H increased significantly (P<0.05); compared with group M, SOD activities in group L and H decreased significantly (P<0.05), while SOD activities in group H were much higher than those in group L(P< 0.05).7. LDH activities in serum of rats:Compared with group C, LDH activities in group M, L and H increased significantly (P<0.05); compared with group M, LDH activities in group L and H decreased significantly (P<0.05), while LDH activities in group H were much lower than those in group L(P<0.05).8. W/D lung weight ratio and water content in rats:Compared with group C, W/D lung weight ratio and water conten in group M, L and H increased significantly (P <0.05); compared with group M, W/D lung weight ratio and water conten in group L and H decreased significantly (P<0.05), while W/D lung weight ratio and water conten in group H were much lower than those in group L(P<0.05).9. Pathological changes of lung tissue:Observation by light microscope and electron microscope was that the structure of lung tissue was normal in group C, seriously injured in group M, slightly injured in group L and H; meanwhile, the injury in group H was much lighter than that in group L.Conclusion1. PHC protects myocardium against injury induced by CPB-related SIRS in patients undergoing heart valve replacement under CPB through inhibiting increase of NF-κB transcript activity in myocardium and plasma concentration of TNF-α, sICAM-1, cTn-I.2. Penehyclidine hydrochloride, which significantly reduces RI and increases OI and Cd, can improve pulmonary function and reduce acute lung injury.3. Pretreatment with PHC can attenuate LPS-induced ALI by inhibiting expression of CD 14m RNA, TLR4 mRNA, NF-κB mRNA, reducing concentration of TNF-α, MPO and MDA, and impairing activity of LDH and enhancing activity of SOD.