Protective Effects Of Penehyclidine Hydrochloride Against Acute Renal Injury Induced By Hemorrhagic Shock-endotoxin In Rats

If hemorrhagic shock is not treated properly or timely, it can be the first hit to the body in the state of systemic inflammatory response syndrome (SIRS), then the infection caused by a second hit to amplify the existing inflammation occurres over the SIRS and even multiple organ dysfunction (MODS).Acute kidney injury (AKI) is a common pathological process during shock, infection, trauma induced MODS,its occurrence plays an important role in prognosis of MODS.Hemorrhagic shock decrease the renal blood.Total renal blood flow is reduced but the blood flow to the cotex is reduced out of the proportion to that of the medulla. Since 80% of the functional glomeruli in the cotex.A shift away from these glomeruli results in a significantly decreased glomerular filtration rate.Tubular obstruction also may occur because of either swelling of tubular epithelial cells due to isehemia or direct effect of tubular toxins.-As the tubules swelling, they occlude the tubular lumen, thus causing a blockage of the flow of flitration..The increase tubule permeability results in back leakage of filtrate from the tubular lumen into the interstitium, In ischemia accumulation of hypoxanthine as a result of ATP degradation.Reperfusion of this ischemic organ causes the release of OFR.which are known to further exacerbate the primart injury induced by ischemia.Excess generation of OFR or reduction in antioxidant levels have been imphcated as the causative factors of oxidative injury.a great variety of cytokines play an important role in pathogenesis of acute renal ischemia reperfusion injury such as IL-1,IL-6,IL-8,IC AM-1,TNF-a. which can induce the cascade of inflammation and amplify inflame. Matory reaction and cause leukocytes to accumulate in the vasarecta of the outer medulla.so aggravate renal injury.Traditional anti-cholinergic drugs which be had relieved vasospasm. Improved microcirculation, inhibited calcium overload and membrane lipid peroxidation.Inhibitde the production and release of inflammatory mediators, play a protective role on cells. It is widely used in clinical since 1960s.But bceause of the large application dose and many adverse reactions,the application is limited Compared with traditional anti-cholinergic drugs, Penehyclidine hydrochloride selectively blocks M receptor subtype,the major selective effects on M1andM3receptors but few on M2 receptor-associated cardiovascular. penehyclidine hydrochloride on acute lung injury caused by various reasons, has a protective effect. For heart valve replacement patients, they can significantly reduce lipid peroxidation and excessive inflammatory response and reduce myocardial damage after myocardial ischemia reperfusion. on ischemia-reperfusion injury in heart, brain, kidney and other organs it has a protective effect.According to Liu et al, anisodamine protects rabbits from organic damages caused by hemorrhagic-endotoxin hit, Although this short-action drug does not have a selective anticholinergic effect. Previous study shows that, PHC also processes the protection against hemorrhage-endotoxin hit in rats. In this study, we evaluated the differences of TNF-a, IL-8, IL-1, Cr, BUN, ICAM-1 and NF-κB in three PHC groups with a rat model of renal injury induced by hemorrhagic shock and LPS.Materials and methods:1.Animals 45 male wistar rats,weight 180-230g,were provided by the experimental animal Center of Southern Medical University. Certificate number:SCXKyue 2006-00152. Animal groups Wistar rats were randomly divided into five groups.each group consisted of 9 rats:①sham operated group(S group)②odel group (M group):hemorrhagic shock+lipopolysaccharide (LPS,2 mg/kg)+0.9%NS(lml)③PHC 1 mg/kg group (PHC1 group):hemorrhagic shock+LPS(2 mg/kg)+PHC (1 mg/kg)④HC 2 mg/kg group (PHC2 group):hemorrhagic shock+LPS (2 mg/kg)+PHC (2 mg/kg)⑤HC 3 mg/kg group (PHC3 group):hemorrhagic shock+LPS (2 mg/kg)+PHC (3 mg/kg)3.Two-hit model In accordance with Fan's Method to build up the model, the first attack was made by (hemorrhagic shock+reperfusion) and the second attack(endotoxemia).Animals were fasted the night before, normal water drinking, weighed the next morning. anesthetized by intraperitoneal injectionwith 20% urethane (5 mL/kg),then fixed on the rat supine board, controlled body temerature at (37±1)℃by using aheat lamps. Isolated and intubated the left femoral artery and femoral vein under sterile conditions. and the use of heparin saline flush catheter, femoral artery to sensor connection to monitor blood pressure. blood pressure stable lasted 15 min. Uniform open femoral artery catheter slowly bled inl5min(Heparinized blood stored for transfusion),mean arterial pressure (MAP) fell to 35±5 mmHg(Bled about 30% of the total blood volume), the formation of hemorrhagicshock. Through Intermittent bleeding through the femoral vein catheter and blood transfusion to maintain 1h, and then fluid resuscitation, time of 90 min. autologous transfusion heparinized blood and 1.5 times Ringer's lactate from the femoral vein. blood pressure returned to the blood of 97% or more of the former as success. After 30 min. of the success Femoral vein injected 2 mg/kg (LPS). After 60 min.. M group through the femoral vein injection of saline 1ml. PHC1,2,3 mg/kg were injected through the femoral vein in PHC1 group, PHC2 group, PHC3 group. Were diluted to lml before injection.S group is not blood only femoral artery, femoral vein cannulation.2 h after injection, collected 2 ml blood samples for centrifugation, the plasma frozen at-80 degrees to be detected the level of plasma TNF-α, IL-1, IL-8, Cr, BUN and then all animals are sacrificed. Kidney put in 10% formalin fixed. And renal tissues were collected to measure the expressions of ICAM-1 and NF-κB and to observe the pathological changes respectively. Renal tissue samples were obtained for histological evaluation.4. Index determination(1) Liquidchip determination to test the TNF-α, IL-1, IL-8 concentration Beforeanimals are sacrificed. collected arterial blood 2ml, standing after 30 min,4℃, 3000 r/min, the plasma obtained after centrifugation 15 min, operating in accordance with the kit instructions, Luminex 100 liquid chip machine to measure the concentration of TNF-α, IL-1, IL-8 in plasma.(2) To determine the concentrations of urine creatinine (Cr) and blood urine nitrogen (BUN) Using automatic biochemical analyzer which come to Southern hospital laboratory.(3)Determination of renal ICAM-1 and NF-κB Immunohistochemical method be used for detection. The expression of ICAM-1 and NF-κB be observed by the microscope. Divided according to the degree of positive staining cells(Antigen content):Weakly positive (+)---1score.Medium positive (++)---2score.Strong positive (+++)---3score. Divided according to the number of positive cells:Weakly positive(+The total number of positive cells<25%).Medium positive (++The total number of positive cells 26～49%). Strong positive (+++The total number of positive cells<50%).To be used Comprehensive measurement of integral:Formula (+)%×1+(++)%×2+(+++)%×3. Total value of<0.5 for the (+).0.5-1.0 for the (++).1.0-1.5 for the (+++).> 1.5 for the (++++).were observed at least 5-10 HPF.Result(1)The change of serum TNF-α,IL-1,IL-8,Cr,BUNTNF-α, IL-1, IL-8, Cr, BUN in M group and PHC1, PHC2, PHC3 group were obviously higher than those in S group(P<0.05).Compared with M group and PHC1, PHC2, PHC3 group. PHC1, PHC2, PHC3 group is lower(P<0.05).PHC1 group was lower than PHC2 group PHC3 group(P<0.05). No significant difference between PHC2 and PHC3 (P>0.05)(2)The expression of 1CAM-1 and NF-κB in renalA large number of the expression of ICAM-1 mainly in the glomerular capillaries, mesangial area and interstitial, renal tubules also expressed. Showing uneven brown granular material in M group. The expression of ICAM-1 in glomerular capillaries and, mesangial.a small amount in interstitial.expressed as uneven light yellow granular material in PHC1, PHC2, PHC3 group. Almost no positive expression showed in S group. The expression of ICAM-lin five group were significantly different(P<0.05).The expression of NF-κB mainly in renal tubular epithelial cells. also in vascular endothelial cells.showed uneven brown granular material. In particular, It was significantly in M group.A small amount of brown granular material can be seen in renal tubular epithelial cells in PHC1, PHC2, PHC3 group. Almost no positive expression showed in S group. The expression of NF-κB in five groups were significantly different(P<0.05).(3)The morphological changes of renal tissueRenal tubular epithelial cell swelling, nuclear enrichment vacuolar degeneration, necrosis, large lumen and tubular cells, proteins and shedding of tubular cells, stromal vascular dilation, congestion, inflammatory cell infiltration in M group. The swelling of renal tubular epithelial cells was significantly reduced.A little interstitial congestion and infiltration of inflammatory in PHC1, PHC2, PHC3 group. S group were normal renal tissues.Conclusion1.Penehyclidine hydrochloride has a protective effect on on rat kidney injury caused by two-hit2.Treatment of 1mg/kg PHC has a significant protective effect than other PHC group.