The Basic Study On Intestinal Endotoxemia In Acute Hepatic Injury Rats And The Effect Of Dachengqi Decoction

At present, the relationship between intestinal dysbacteriosis of hapatopathy and intestinal endotoxemia (IETM) has become more and more important. In the process of hapatopathy, there is intestinal dysbacterisis and obvious proliferation of some G- bacteria, which will increase the incidence of IETM. Endotoxemia plays an important role in the process of hepatopathy, and will aggravate it. Therefore, hepatopathy-intestinal dysbacteria-IETM forms an infernal circle.At first, the study based on IETM models of acute hepatic injury by thioactamide (TAA) injection in rats, will explore the mechanism and effect of IETM. In the second, it will explore the effect and mechanism for NF-ΚB in the process of acute hepatic injury by TAA. At last, Dangchengqi Decoction is used to prevent and treat it.1. The study on mechinasim for IETM in the process of acute hepatic injury by TAA injection.Twenty-two Wistar rats were randomly divided into two groups: normal group and TAA model group. Rats in TAA group were given TAA at a dose of 300mg/kg subcutaneously, and repeated after 24 hours. The normal group of rats were given Sodium Chloride at the same dose. After 48 hours, the acute hepatic injury models were completed.After the rats were killed, the plasma endotoxin,ALT,AST,TBIL,tumor necrosis factor(TNF) and D-lactate were measured. The ileum of rat was put into the Sodium Chloride and cultured for bacteria. The mesenteric lymph nodes(MLN), liver, spleen and intestinal contents were collected for bacteriological study . The plasma D-lactic was measured for intestinal permeability. After being fixed with 10% formalin and embeded with paraffin wax, some liver tissues and ileums were examined for the pathological changes.We found that the levels of ALT,AST and TBIL were obviously increased (P<0.01) in TAA model group compared with normal rats. The plasma D-lactate was increased significantly(P<0.01),which indicated high intestinal permeability. There was intestinal dysbacteriosis in TAA model group. In normal group, the bacteria were cultivated only in MLN and the displacement rate is 27.27%. However, in TAA group ,the bacteria were cultivated not only in MLN, but also in the liver and spleen. The displacement rate of MLN was 90.00%, which was the highest. Under light microscopy, we found that there were punctiform necrosis of liver tissues,central vein infiltrated by inflammatory cells and sinus hepaticus destroyed in TAA model group rats. We also found that the lamina propria became thin , villus intestinalis became shorten significantly ,destroyed and infiltrated by inflammatory cells.The results indicate that intestinal dysbacteriosis,changes in the intestinal membrane structure,high permeability and bacteria displacement are important factors in the intestinal endotoxemia. When intestinal endotoxemia activate the network system of cytokin, the endotoxin-cytokin network system plays an important role in the acute hepatic injury(the second attack theory), which worsens hepatic injury and leads to liver necrosis.2. The study on mechanism and effect of NF-kB in the process of acute hepatic injury78 Wistar rats were randomly divided into 3 groups: (1) TAA model group: 30 rats were injected TAA at a dose of 300mg/kg subcutaneously, repeated after 24 hours and completed after 48 hours. (2) PDTC pretreated group: 30 rats were injected PDTC at a dose of 100mg/kg before making the acute hepatic injury models. (3) Normal group: 18 rats were injected Sodium Chloride at the same dose. In the first and the second groups, every 10 rats was killed at 6,24 and 48 hours after the models completed, And 6 rats were killed each time in the normal group.The plasma endotoxin, TNF-αand IL-6 were measured. The RT-PCR was used to assess the expression of the ICAM-1 in the hepatic tissue,hepatic pathology and immunohistochemistry were measured .Here is our outcome:(1)the level of endotoxin was elevated in TAA group at 6,24,48 hour compared with the normal group(P<0.01). It increased as time going on and were obviously increased each time compared with PDTC group. (2)Compared with those normal and PDTC groups, the levels of plasma TNF-αand IL-6 at any time were increased in TAA group(P<0.01), with beginning to increase after 6 hours and reaching the peak level at 24 hours .(3)Compared with the normal group, the NF-kB was activated obviously at any time in TAA group(P<0.01), with beginning to increase after 6 hours and reaching the peak level at 24 hours. Compared with the TAA group, activation of NF-kB was obviously reduced in the PDTC group (P<0.01). (4)Compared with the normal group, the expression of ICAM-1mRNA was obviously increased at any time(P<0.01). And it increased as time going on. Compared with PDTC group, the expression of ICAM-1mRNA was obviously increased at 12 hours and 24 hours in TAA group(P<0.01) with no differences at 6 hours(P>0.05). It indicated that the expression of ICAM-1mRNA was decreased because NF-kB was inhibited by PDTC. (5)The liver pathological section of the TAA group at 6 hours indicated that the liver tissues necrosed partly,liver cells swelled and the cytoplasm became puffed ,the liver tissues were infiltrated by inflammatory cells, lobule of liver were still clear. The pathological section at 24 hours indicated that liver cells necrosis areas became larger than those at 6 hours, liver cells swelled obviously and some cells appearing vacuolus apomorphosis, the liver tissues were infiltrated by inflammatory cells and hepatic cords arranged irregularly. The pathological section at 48 hours indicated that necrosis areas became larger than those at 24 hours and the structure of hepatic lobules wasn't clear. Liver cells nuclei crushed and the liver tissues were infiltrated by a lot of inflammatory cells.The findings indicated that NF-kB aggravated the hepatic injury due to increasing the expression of TNF-α, IL-6 and ICAM-1 in intestinal endotoxemia in the process of acute hepatic injury.3. The study on intervention of Dachengqi Decoction in endotoxin biological effectiveness in the process of acute hepatic injury.50 Wastar rats were randomly divided into 5 groups: normal group (A group),TAA model group (B group), treatment group of Dachengqi Decoction at low dose (C group),treatment group at moderate dose(D group),treatment group at high dose (E group).There were 10 rats in every group. The rats in Group C ,D and E were given Decoction throug lavage two days before making model ,those in Group A and B were given the same dose Sodium Chloride. Then the rats in Group B,C ,Dand E were injected TAA at a dose of 300mg/kg subcutaneously, repeated after 24 hours and the acute hepatic injury models were completed after 48 hours. Those in C ,D and E groups were continually given Decoction for three days.After completing the models , the plasma endotoxins,TNF and ALT,TBIL were measured and part of left liver tissue was observed for pathological changes and measured by immunohistiochemical methodsHere is our outcome: (1)Compared with the normal group, the levels of ALT,TBIL,TNF-αand endotoxin were obviously elevated in Group TAA. Compared with Group TAA, there was obvious difference in all indexes in Group D and E(P<0.01).(2) There was intestinal dysbacteriosis in TAA model group. Compared with TAA model group, C,D ,E group's dysbacteria situations were improved.(3) Compared with the normal group, the level of plasma D-lactate was obviously elevated in Group TAA(P<0.01),and the level of plasma D-lactate in D,E groups was decreased(P<0.01,P<0.05).(4)Compared with the normal group, NF-kB and CD14 were obviously activated in Group TAA(P<0.01).Compared with Group TAA, the activations of NF-kB and CD14 were reduced in each Decoction treatment group(P<0.01).(5)Liver pathological section indicated that liver tissues necrosed and were infiltrated by many inflammatory cells, hepatic sinusoid tissue was destroyed in TAA Group. The pathological findings were better in Group C,D and E ,and it was almost normal in Group E. According to the study, we find that Dachengqi Decoction can improve intestinal dysbacteriosis and intestinal permeability, reduce the activations of NF- kB , CD14 and the level of plasma endotoxin, improve the liver pathological changes, and has effective intervention in biological effectiveness of intestinal endotoxemia in the process of acute hepatic injury. It can protect rats from acute liver injury. High-dosed drug can improve IETM situation significantly, which indicate the effect is more obvious with drug dose inceasing.