Protection Of Artesunate Of CLP Models In Rodents

Objectives:Sepsis, systemic inflammatory response syndrome induced by infection, is a common complication of severe trauma and major surgery operations. Nearly 50-60% of Patients with sepsis will die if shock or multiple organ dysfunction syndrome occur, which makes sepsis become the leading cause of death in critical care units. On the other hand, the fact that no effective and reliable therapy is available in clinic brings great importance for development new drugs dealing for sepsis.The model of cecal ligation and puncture (CLP) is the classical animal model of sepsis. This study aims at investigating the protective roles and possible molecule mechanisms of artesunate on CLP model in mice and rats on the basis of successful establishment of CLP models.Methods:1. Establishment of the CLP model in mice and ratsDifferent needle gauges and perforation times were used to establish CLP model, mortality at 7 days were observed;2. Protection of artesunate on rodent CLP model animals: Artesunate and ampicillin-sulbactam were given to CLP animals synergistically or separately via vena caudalis or muscle. Mortalities at 7 days were observed.3. Mechanism of artesunate on CLP model mice: Dynamic blood bacteria numbers, LPS, TNF-αand IL-6 levels were tested in different treatment groups; pathological changes of lung at 12 h after artesunate treatment for 12 h were observed, too.Results:1. Using 16 gauge needle with twice perforation for rat and 12 gauge needle with twice perforation for mouse were the optimal methods to control animal mortality rate at 90% for establishing rodent CLP models. 2. Protection of artesunate on CLP models in rodent animals:In CLP rats, the survival rates were 48% for ampicillin-sulbactam and artesunate treating group, it was significantly higher than those in rats only treated with ampicillin-sulbactam or artesunate.In CLP mice, the survival rates were 40% for ampicillin-sulbactam and artesunate treating group, it was significantly higher than those in rats only treated with ampicillin-sulbactam or artesunate.3. Mechanisms of artesunate on CLP mice:3.1 Effects of synergistic effects of artesunate and ampicillin-sulbactam on blood bacteria in CLP mice:In control group, bacteria were detected in the blood at 0.5 h after surgery in control group. The bacterial numbers increased gradually within 2 h, peaked between 8 and 12 h, and then decreased in the control group. Bacteria in blood were detected from 1 h to 12 h after CLP surgery without peak in the combination group of the two drugs. The bacteria numbers were significantly lower than those in other groups at each time-point.3.2 Effects of synergistic effects of artesunate and ampicillin-sulbactam on serum LPS levels in CLP mice:Serum LPS was detected at 0.5 h after surgery in control group. Its concentration increased gradually from 0.5 h to 2 h and rapidly reached to peak at 12 h, and then decreased. Dynamic changes of serum LPS in the combination group of the two drugs were similar to those in control group. But the levels were significantly lower than those in other groups at each time-point and especially at 24 h.3.3 Effects of synergistic effects of artesunate and ampicillin-sulbactam on serum TNF-αlevels in CLP mice:Serum TNF-αwas detected at 0.5 h after surgery in control group. Its concentration increased gradually from 0.5 h to 8 h and rapidly reached to peak at 12 h, and then decreased. Serum TNF-αincreased at 1 h in the combination group of the two drugs. There was no peak. And the levels were significantly lower than those in other groups at each time-point. 3.4 Effects of synergistic effects of artesunate and ampicillin-sulbactam on serum IL-6 levels in CLP mice:Serum IL-6 was detected at 0.5 h after surgery in control group. Its concentration increased gradually from 0.5 h to 1 h and rapidly reached to a platform from 2 h to 12 h, and then sharply decreased. Serum IL-6 levels in the combination group of the two drugs were significantly lower than those in other groups at each time-point.3.5 Pathological observation of lung tissues:Lung tissues were severely injured after CLP surgery. Pathological changes such as alveolar ectasia, serous fluid effusion in alveolar space, inflammatory cell infiltration, interalveolar septum collapse, and compartment widen, congestion and cell defluvium were found. Combination of artesunate and ampicillin-sulbactam could attenuate injuries of lung tissues at 12 h with little congestion, inflammatory cell infiltration and interalveolar septum collapse.Conclusions:Combination of artesunate and ampicillin-sulbactam more effectively protected CLP rats and mice than single drug. The protection was tightly associated with reduction of serum bacteria numbers, LPS and pro-inflammatory cytokines.