| Objective To study the mechanism of CVF and Liangge San on rat liver injury with traumatic hemorrhagic shockMethod By withdrawal from carotid artery afer fracture to make the model of traumatic hemorrhagic shock.20 Wistar male rats received the attack to be observed the changes of some indicators and the liver injury.130 healthy male rats were randomly divided into 13 groups:sham group, model group 1.3,6,24 h, CVF (cobra venom factor) +model group 1h,3h,6h.24h.Liangge San+model group 1h,3h,6h. 24h,10 rats in each group. Rats were sacrificed in 1h,3h,6h,24h separately:ollect part of liver and the blood. Immunohistochemistry was used to detect membrane attack complex (C5b-9)deposition, the expression of bax, bc12, fas, fasL protein. caspase3 protein and C5a receptor C5a-R in the liver; Apoptosis of liver were then examined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Western blot was used to detect C5a-R and caspase3; mRNA expresion of bax, bcl-2, fas, fasL were measured by reverse transcriptase-polymerase chain reaction (RT-PCR); C5b-9.CH50,C5a in plasma were measured by by enzyme-linked immunosorbent assay, and rate method was used for determination of plasma aspartate aminotransferase(AST). Paraffin sections were used to observe the pathological damage of liver.Result The first part:establish rats'traumatic hemorrhagic shock model and control the mortality under 20%. AST increased and liver cells became edema, necrosis, inflammatory cell infiltration into liver.The second part:1.changes of model groups?changes of complement in model groups:In the modellh,3h.6h, plasma CH50 increased significantly compared with sham group and reached the highest value in model 1h, and 24h reached the lowest point; we can detect small amounts of plasma C5b-9 in the sham group. In the model 1h plasma C5b-9 increased significantly and peaked(compared with 3hours,6hours,24hours, respectively P <0.05), but in the model 3hours, began to decline. In 24h, it reduced to the minimum; plasma C5a increased significantly in the model 3h,6h,24h (compared with sham group, P<0.05) and peaked in 24h; aspartate aminotransferase(AST) in the model 1h, 3h,6h and 24h increased significantly( compared with other groups, respectively P <0.05), peaked in 24 h. In liver, in the sham group, the model 1h and 6h no membrane attack complex was found in liver, but in the model 3h a large number of liver parenchyma cells around central veins were found with membrane attack complex, A small amount were found in the model of 24 hours; in the model 1h,3h,6h and 24h mRNA of C5a-R in liver increased significantly compared with sham group and peaked in model 6h and 24h (there were no significant difference between the two groups). Protein of C5a-R increased in model 3h,6h, and 24h, peaked in model 24h;?apoptosis:no apoptotic cell was found in sham group, and in model 1h,6 h and 24h, there were scattered apoptotic cells. In model 3h around central veins apoptotic cell count increased significantly and peaked; the expression of bax, fas, bax/bcl-2 ratio, caspase3 peaked in model 3h and fasL peaked in model 6h.2. Changes of CVF+model groups:?changes of complement:plasma CH50 and C5b-9 decreased significantly in lh,3h,6h, compared with model groups, but increased in CVF24h compared with model 24h; plasma C5a increased significantly in CVFlh compared with model 1h, but there were no statistic difference between 3h,6h,24h and model groups at same time points. No membrane attack complex was found in liver in CVF groups. The C5a-R mRNA in 1h,3h and protein in 1h,3h,6h, increased significantly (compared with model groups), AST decreased in all the CVF groups compared with model groups at same time point. Pathological scores were decreased in CVF 3h, 6h,24h, compared with model groups at same time point.?Changes of apoptosis: liver parenchymal cells apoptosis was found in CVF 1h, and cell count was higer than model 1h; apoptotic lymphocytes were found in the sinusoid in CVF 3h, and the count was lower than model group; the levels of bax mRNA, protein, ratio of bax to bcl-2 protein and caspase3 decreased significantly in 3h and 6h compared with model groups at same time point. The level of bcl-2 mRNA and protein decreased in 3h and 24h. Fas and fasL did not found in liver. The third part:Changes of rats given Liangge San+model groups?Changes of complement:plasma CH50, C5b-9 decreased significantly in 1h,3h,6h compared with model groups at same time points, but increased in 24h compared with model 24h. C5a increased in 1h and 3h compared with model groups, but decreased in 24h compared with model 24h. The values of AST decreased in all groups given Liangge San compared with model groups, pathological scores decreased in 3h,6h and 24h.?Apoptotic lymphocytes were found in the sinusoid in Liangge San 3h and 6h,cell count in 3h is lower than model group; the value of bax, ratio of bax to bcl-2, and caspase3 decreased significantly in Liangge San 3h and 6h vs model groups, Liangge San had no effect on expression of bcl-2. Fas and FasL expression were not found in liver.Conclusions Acute liver injury occurred in rats when received traumatic hemorrhagic shock attack, and complements were activated, membrane attack complex deposited in liver cells. Apoptotic parenchymal cells in liver were found CVF(cobra venom factor) and Liangge San inhibited parenchymal cells apoptosis, promoted lymphocytes apoptosis, and protect liver from injury. Liangge San can inhibit the complement excessive activation in early stage, but maintain proper activity of complement so that protect liver from injury. The degree of reduction of pathological injury in Liangge San was greater than CVF group. |
PDF Full Text Request