Effects Of Andrographitis On Intestinal Permeability, Intestinal Bacterial Overgrowth And Bacterial Translocation In Cirrhotic Rats

PART 1. Establishment of HPLC/RID method for detection of intestinal permeability in cirrhotic ratsObjective Intestinal permeability plays an important role in bacterial translocation in cirrhosis. To establish a high performance liquid chromatography/refractive index detector (HPLC/RID) method for detection of intestinal permeability in cirrhotic rats by using two-sugar absorption test.Method Liver-cirrhosis model in rats was induced by carbon tetrachloride. Thirty urine samples of the cirrhotic rats and the healthy control were collected respectively after test solution (50mg mannitol and 100mg lactulose) administrated 8 hours. Those samples were removed proteins, positive and negative ions by using boiling and supplementing ion-exchange resin until HPLC/RID examination. The chromatographic condition was described as follow: chromatographic column: NH2 reversed-phase column (4.6mm×250mm×5um, Warters), Temperature of column: ambient temperature. Mobile phase: acetonitrite/water (70/30, v/v). Flow rate: 1.0 ml/min. Injection volume: 20μl.Result Under these conditions, the retention time of mannitol and lactulose were 6.271min and 9.242min respectively. The recovery rate ranged from 92.7 % to 98 % for mannitol and 91.3%～97.4% for lactulose. A significant rise in 8-hour urinary L/M excretion ratios was found in the cirrhotic rats (0.03578±0.01245) compared to that of control (0.02563±0.00871) (P <0. 05).Conclusion A high performance liquid chromatography/refractive index detector (HPLC/RID) method for detection of intestinal permeability in cirrhotic rats has been established, and the intestinal permeability is increased in cirrhotic rats. PART 2. Effects of andrographitis on intestinal permeability, intestinal bacterial overgrowth and bacterial translocation in cirrhotic rats.Objective To evaluate the effects of andrographitis on intestinal permeability, intestinal bacterial overgrowth and bacterial translocation in experimental cirrhotic rats.Method Liver-cirrhosis model in rats was induced by carbon tetrachloride. Thirty cirrhotic rats were randomized to divide into four groups: group AN (n=8,andrographitis 0.5g/kg*d),group NF(n=8 Norfloxin0.3g/kg*d),group AN+NF (n=8,androgtaphitis 0.5g/kg*d + Norfloxacin 0.3g/kg*d),group CN(n=6,9% Sodium Chloride 4ml/kg*d). At the end of two weeks treatment, the mortality of every group was analyzed. Blood specimens were collected from peripheral vein and detections of serum level of ALT, AST, TNF-a, NO were performed. The MLNs, serum, liver, spleen and contents of ileum were obtained and cultured. Intestinal permeability was measured by HPLC/RID. Parts of liver and ileum were kept at -20℃until histological examination.Result Compared with group CN,the serum levels of TNF-a and NO of group AN,group NF and group AN+NF were decreased significantly(P<0.05), the intestinal permeability of group AN and group NF was also decreased remarkably(P<0.05). Total ileum bacterial contents and bacterial translocation in group CN were significantly higher than them in other groups (P< 0. 05). The ileum histological appearance of group CN was obviously abnormal; villi were shortened and damaged with submucosal inflammatory infiltration.Conclusion Administration of andrographitis resulted in a reduction of intestinal bacterial overgrowth, bacterial translocation and a protection of intestinal permeability, which suggests that oral andrographitis may be useful in preventing infection caused by intestinal bacterium in cirrhotic patients.