Effects Of Active Compounds Of Pulsatill Decoction On The Abnormality Of The Secretion Function And The Expression Of CAMs Induced By LPS In RIMEC

In order to illustrate the anti-inflammation mechanisms of Pulsatill Decoction, we investigated the effect of LPS on secretion function and the expression of CAMs in rat intestinal microvascular endothelial cell (RIMEC) in vitro, and the effect of active compounds (berberine, esculetin, anemonin) of Pulsatill Decoction on the abnormality of secretion function and the expression of CAMs induced by LPS.1. RIMECs were isolated by mechanical compression from the jejunum of rat and cultivated. RIMEC cultures were recognized by microscopic morphological features, expression of factor Ⅷ -associated antigen by fluorescence microscopy. As underlay the physiological and pathological studies of RIMECs.2. We focused on the effect of LPS (1μg/ml, 5μg/ml and 10μg/ml) on secretion of nitric oxide (NO), Endothelin-1 (ET-I) and expression of ICAM-1 and E-selectin from RIMECs. The results showed that the LPS enhanced the secretion of NO and ET-1 and the expression of ICAM-1 and E-selectin. These results suggest that LPS directly induced RIMECs activation may play an important role in the pathogenesis of colibacillosis. Supernatants NO level was determined by chemiluminescence, detection of ET-1 and sICAM-1 by the ELISA, ICAM-1 and E-selectin expression by immunohistochemical evaluations.3. The effects of anemonin, berberine and esculetin on RIMECs production of NO, ET-1, ICAM-1 and E-selectin in response to LPS were investigated. After LPS (0,1μg/ml) stimulation for 12h with or without berberine, esculetin, anemonin (1, 5, 10μg/ml) pretreatment for 2h, secretion of the ET-1 and NO, expression of ICAM-1 and E-selectin from unstimulated and stimulated RIMECs were assessed. The results showed:① Berberine did not alter the production of NO, sICAM-1 and expression of E-selectin, in non-LPS treated control cultures, but ET-I expression was significantly increased (p<0.01). After LPS (1μg/ml) stimulation with berberine pretreatment, the secretion of ET-I was significantly increased (p<0.01), the levels of NO, sICAM-1 and E-selectin expression were significantly reduced than LPS group (p<0.01).② Anemonin did not alter the production of NO, ET-I, sICAM-1 and expression of E-selectin, in non-LPS treated control cultures. After LPS (1μg/ml) stimulation with anemonin (10μg/mL, 5μg/mL, 1μg/mL ) pretreatment, the levels of NO were significantly reduced than LPS group (p<0.01). The secretion of ET-I was inhibited by anemonin pretreatment. Anemonin at the concentration of 1 μg/ml could not inhibited sICAM-1 expression elevation after LPS stimulation. However anemonin at the concentration of 5μg/ml and 10μg/ml inhibited sICAM-1 expression. Anemonin at the concentration of 10μg/ml significantly inhibited E-selectin expression (p<0.01).③ Esculetin did not alter the secretion of NO and ET-I in non-LPS treated control cultures, sICAM-1 expression was significantly increased (p<0.05). After LPS (1μg/ml) stimulation with esculetin pretreatment, the levels of NO and sICAM-1 were significantly reduced than LPS group (p<0.01). Esculetin could not inhibited sICAM-1 expression elevation after LPS stimulation.These findings suggest that anemonin significantly inhibited of the secretion of NO and ET-1 and the expression of ICAM-1 and E-selectin induced by LPS, Berberine could lower the expression of ICAM-1 and E-selectin induced by LPS and esculetin mainly lowered the secretion of NO and the expression of ICAM-1.Our results indicate that Pulsatill Decoction may exert their anti-inflammation action, at least in part, by inhibiting the secretion of NO, ET-I and expression of ICAM-1 and E-selectin in RIMECs, preventing intestinal microvascular dysfunction.