The Inhibitory Effect And The Mechanism Of Action Of Danbiqing Granules On Hepatocyte Apoptosis Following Mice Intestinal Ischemia/reperfusion Injury

BACKGROUND: Intestinal ischemia/reperfusion (I/R) injury is one of common tissue lesions in surgery practice. It is important in the pathologic progress of serious infections, trauma, shock, cardiorespiratory dysfunction, et al. It not only can elicit topical lesion in alimentary tract, but also can lead to translocation of bacteria and endotoxin from intestine into systemic circulation, which can elicit series of reactions in mononuclear phagocyte system, and then a great amount of associated intermediate and cytokine will be released. Sometimes multiple organ dysfunction syndrome (MODS) may even happen. Chinese traditional compound formula-Danbiqing granules is made from Rhubarb, Salviae Miltiorrhizae Radix, Artemisiae Capillaris Herba, Pulsatillae Radix, Baijiang Herba Cum Radice and Cyrrhizae Radix. It can invigorate blood and transform stasis and eliminate evil heat and detoxication. Clinical practice and animal experiments have demonstrated that it is used to treat acute fulminant cholangitis and acute abdomen accompanying abdominal infections. But its inhibitory effect on hepatocyte apoptosis in I/R injury has not been testified. OBJECTIVE: To study the inhibitory effect and the mechanism of action of different doses of Danbiqing granules on hepatocyte apoptosis following mice intestinal I/R injury. METHODS: (1) Model Establishment: The superior mesenteric artery was clamped for 1 hour and reperfused for 1 hour as I/R injury model. (2) Groups: Mice were randomly divided into four groups: sham group, model group, and Danbiqing therapeutic groups (3.12g/kg; 6.24g/kg). Before operation, Danbiqing granules were given to the mice of the therapeutic groups for three consecutive days, and saline was given to the mice of the sham and model groups. For the sham group, superior mesenteric artery was only isolated without clamping. For the model and Danbiqing therapeutic groups, superior mesenteric arteries were isolated and clamped. (3) Items of Observation (1)Apoptotic hepatocytes were detected by TUNEL method. (2)Douche liver, collect hepatocytes, incubate fiepatocytes with Fura-2/AM, and then detect [Ca2+]i by Shimadzu RF-510 spectrofluorometer. (3)Detect the level of malondialdehyde (MDA) and (4)Detect the level of myeloperoxidase (MPO) in hepatic tissue by 722-type spectrophotometer. RESULTS: (1) Hepatocyte apoptosis: In the model group, the number of apoptotic hepatocytes induced by I/R injury increases significantly. Compared with the sham group, the statistical difference is significant. Compared with the model group, the large and middle dosages of Danbiqing granules have great inhibitory effect on hepatocyte apoptosis. (2) [Ca2+] in hepatocytes: Compared with the sham group, [Ca2+]i increases significantly in the model group. Compared with the model group, it decreases significantly in the Danbiqing granules group. (3) The level of MDA in hepatic tissue: It increases more significantly in the model group as compared with the sham group. Danbiqing granules decrease the level of MDA. The large dosage group decreases it significantly (p<0.01). (4) The level of MPO in hepatic tissue: It increases more significantly in the model group as compared with the sham group. Danbiqing granules decrease the level of MPO. The middle dosage group decreases it significantly (p<0.01). CONCLUSIONS: (1) The model was successful. After ischemia for 1 hour and reperfusion for 1 hour, hepatocyte apoptotic index, [Ca2+]i and the levels of MDA and MPO increase. (2) Danbiqing granules inhibit hepatocyte apoptosis and decrease [Ca2+]i and the levels of MDA and MPO. (3) The inhibitory effect of Danbiqing granules on hepatocyte apoptosis was concerned with their decreasing [Ca2+]i to lessen hepatic injury and their decreasing the levels of MDA and MPO to lessen lipid peroxidation injury.