The Prevention Effect Of Puerarin On SIRS And MODS Induced By Peritonitis In Rats

The SIRS was introduced by ACCP and SCCM since 1991.The definition of SIRS did not necessarily require the presence of bacterial infection. Clinical SIRS and MODS were diagnosed by meeting two or more of following criteria. â‘  Temperature >38℃ or<36℃; â‘¡ Heart rate >90 beats per minute;â‘¢ Respiratory rate >20 breaths per minute or PaCO₂<32 mmHg; â‘£ White blood cell count >12×10⁹/L or <4×10⁹/L or presence of >10% immature neutrophils.Altered organ function such that homeostasis needs to be maintained with intervention.SIRS is accompanied with a complex, unbalanced, fatal activation of the immune and inflammatory systems. Although the mechanisms are not yet completely explored, a variety of infectious and noninfectious conditions can lead to hyperactive inflammatory responses at the onset of SIRS, followed by immunoparalysis, compromised immune function and defective innate immune responses at a later stage.It was previously reported that both pro- and anti-inflammatory cytokines are elevated in SIRS. Cytokine mediated systemic neutrophil activation is a direct consequence of SIRS, and can lead to multiple organ dysfunction syndrome (MODS). This prospective study assessed the risk of SIRS and MODS by measuring the circulating levels of inflammatory cytokines such as IL-6, TNF-a and IL-10 as well as the neutrophil functions as a marker of organ failure. Object:To study the prevention effect of Puerarin on SIRS and MODS induced by peritonitis in rats.Materials:1. AnimalsSD mice (5-7 weeks age) weighing 160-200 g, purchased from the Animal Care and Use Center (Breeding of Laboratory Animals, Zhejiang University, Hangzhou, China), were kept in a room with controlled temperature (22 ℃) and lighting (lights on 8:00 a.m.-20:00 p.m.) and free access to food and water. All experiments were conducted between 8:00 a.m. and 4:00 p.m.2. Drugs and ReagentsPuerarin 100mg/2ml/ampZymosan A (Sigma)Mineral oilTNF-α ELISA KitIL-6 ELISA KitIL-10 ELISA KitMethods:1.The SIRS ModelZymosan A (Sigma) was suspended in mineral oil at a concentration of 100 mg/ml, and sterilized in a boiling water bath for 80 min. The zymosan in oil suspension was then sonicated in 5 ml aliquots for 20 s and frozen at-70℃ until used. Zymosan was injected intraperitoneally (ip).The SIRS models of rat were diagnosed by meeting two offollowing criteria.â‘  Compared with the normal group, the rectum temperature raisedor declined ≥1℃; â‘¡ Compared with the normal group, the total white cell amount exceeded 200% or reduced 50%.2. The Experiment GroupsBased on the successful manufacture of SIRS model in rats, SD mice were randomized (on experiment hour 0, time =0h) into five initial groups: Group A (Normal Control) received no zymosan and no puerarin. Group B1 (SIRS Control) received zymosan (1000mg/kg mouse BW t=0) and no puerarin. Group B2 (SIRS Treatment) receive zymosan (1000mg/kg mouse BW, t=0) and puerarin (62.5mg/kg mouse BW, t=0). Group C1 (SIRS Control) receive zymosan (750mg/kg mouse BW t=0) and no puerarin. Group C2 (SIRS Treatment) receive zymosan (750mg/kg mouse BW t=0) and puerarin (62.5mg/kg mouse BW, t=0). Mice were sacrificed when they were preterminal (clinical signs of shaking, shivering, or paralysis) or on Experiment Hour 48 (survivor).At the termination of the experiment, mice were anesthetized with Phenobarbital and diethyl ether, and blood was obtained for blood routine assays by severing the carotid artery and collecting the blood in eppendorf syringes (EDTA-K2 500ul). The blood was immediately centrifliged, and plasma was rapidly separated for the ELISA test of IL-10, IL-6, and TNF-α.3. The Observing Aimâ‘  The success rate of SIRS model induced by peritonitis in rats; â‘¡ The mortality in the model of SIRS were compared betweenGroup B1 and Group B2;â‘¢ The inflammatory cytokines such as IL-10, IL-6 and TNF-αwere tested and compared between Group C1 and Group C2. Both of them were compared to the control group injected i.p with normal saline. RESULT1. The SIRS models induced by peritonitis in rats were made successfully.There are two groups meeting two criteria (Group 1000mg/kg and Group 750mg/kg). (TAB1 and TAB2)TAB1: The temperature and the total white cell amount of model groupsMost rats in group 1000mg/kg were dead in 48 hours TAB2: The Mortality of model groups2. Compared with untreated mice (Group B1), puerarin-treated mice (Group B2) had significantly lower level of mortality (48h) .When control mice (Group A) were compared to Group Bl, the zymosan-exposed mice had higher morbidity scores, shorter length of survival. Functionally, the zymosan-treated mice demonstrated evidence of multiple organ system dysfunction.Mortality: Treatment of mice (Group B2) showing clinical evidence of MODS with puerarin produced a significant reduction in mortality when compared to similar mice (Group B1), which did not receive puerarin. (TAB3) TAB3: The Mortality of SIRS group mice3. Compared with untreated mice (Group C1), puerarin-treated mice(Group C2) had significantly higher level of IL-10 and lower level of IL-6 and TNF-α(P<0.01).(TAB4)TAB4: Effect of puerarin on inflammatory factors system in peritonitis-induced SIRS in rat (48h)CONCLUSION1. Intraperitoneal zymosan in a mineral oil carrier induces SIRS model in rats successfully; 2. Puerarin administration reduces mortality in the model of SIRS significantly;3. Puerarin administration inhibits the release of pro-inflammatory cytokines such as TNF-a(P<0.01), IL-6(P<0.01), promotes the release of anti-inflammatory cytokines such as IL-10(P<0.01),which may be the mechanism of puerarin's protection.Understanding the mechanism of puerarin's effects in SIRS and MODS may lead to therapeutic benefits.