| ObjectiveTo investigate the effects on release of blood-serum cytokines and on lung tissues by inhibition of complement activation with cobra venom factor (CVF) in septic rats which were induced by cecal ligation and puncture(CLP).Methods1.Experimental groupsOne hundred and forty-four SD rats(clean,male,250-300g) were randomly divided into three groups(each n=48):sham group (group A): abdomen was opened and closed later but without cecal ligation and puncture(CLP);sepsis group(group B):sepsis was induced by CLP;CVF pretreatment group (group C):sepsis was induced by CLP after pretreatment with CVF.Twenty-four hours before the operation,rats were given a dose of either 50μg/kg CVF in group C,an equal volume of saline solution in group A and group B.2. Model establishmentThe septic model was established by cecal ligation and puncture.The setups were as below,1 ml/kg of pentobarbital(3%) administered in abdominal cavity to anesthetize the rat.The rat was fixed,depilated and sterilized subsequently.Then we made a incision(1.5cm) on the abdominal skin,moved the feces of the ascending colon to the cecum, ligated the distal end of cecum, stabbed the proximal end of cecum twice and detained a piece of rubber(0.5cm×3cm) in the proximal puncture sub,sutured the incision and prevented shock by fluid replacement(60ml/kg) subcutaneously sequently. In the end,the rat was taken to the cage, free to act, drink and eat.3. The effects of inhibition of complement activation on the general stateActivity, food taking, piloerection, respiration, reaction, distentionof intestine, hyperemia, ascites,omental adhesion to the cecum,lung tissues and pleural effussion were observed after the operation.4. The effects of inhibition of complement activation on the cytokines Eight rats were killed at 2,4,8,12,16, 24 hours points after operation, and blood samples were harvested before the animal killed.The serum levels of TNF-αand IL-6 were detected by radio immunoassay,and the serum levels of IL-10 were detected by enzyme linked immunosorbent assay(ELISA).5. The effects of inhibition of complement activation on the myeloperoxidaseThe lung tissues were harvested and detacted the changes of MPO contents which could reflect the aggregation and infiltration of neutrophil in the lung tissues.6. The effects of inhibition of complement activation on the pathologyThe lung tissues were harvested and detacted the changes of pathology which could reflect the lung tissues injury.Results1. The degree of manifestations of sepsis were different in three groupsThe manifestations of sepsis of group B were severe than that of other groups.2. The effects of inhibition of complement activation on the cytokines(1)TNF-αThe levels of TNF-αof three groups rose to the peak quickly after operation, and descended after 2h point, but rose again respectively, and the levels in group B were higher than that of other groups significantly except the 8h point(P< 0.05 or P< 0.01).(2)IL-6The levels of IL-6 of three groups rose to the peak quickly after operation, and descended quickly after 4h point, and the levels in group B were higher than that of other groups significantly(P< 0.05 or P< 0.01).(3)IL-10The levels of IL-10 of group B which remained a low level were lower than that of group C at all points except 2h point and 24h point(P< 0.05 or P< 0.01).3.The effects of inhibition of complement activation on the myeloperoxidaseThe MPO levels of lung tissues of group A remained low in all points. However, the levels of group B and group C rose quickly after operation and were higher than that of group A significantly except the 2h point(P< 0.05 or P< 0.01).The levels of group C were lower than that of group B significantly(P< 0.05 or P< 0.01). 4. The effects of inhibition of complement activation on the pathologyThe pathology of lung tissue indicated that the lung injury were severe in group B than that of group A and were ameliorated in CVF group compared to that of group B.Conclusions1. CVF could lessen the over release of blood serum proinflammatory cytokines (TNF-αand IL-6) which mediated by complement activation,and increase the release of blood serum anti-inflammatory cytokines (IL-10) relatively, which could maintain the balance of cytokines.2. CVF could lessen the aggregation and infiltration of neutrophil in the lung tissues, and lighten the lung injury, which showed the protective effects on lung tissues.3. The two-peak phenomenon of TNF-αindicated that the immune state changed endlessly in the early phase of sepsis,which provided some evidences to the immune balance and opportunity of immune method intervention treatment.
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