The Regulation And Its Mechanism Of Polydatin On The β-adrenoreceptor In Cardiac Myocytes Stimulated By LPS

Polydatin (PD) is a small molecule substance purified from Chinese traditional herb Polygonum cuspidatum, which was found to have a good effects in the treatment against hemorrhagic shock, burn shock and septic shock in animals and patients. The previous works have demonstrated that PD in vivo can dilate blood vessels, increase pulse pressure, enhance the contractile force of cardiac muscle. These suggested that the effects of PD are related to the contractibility of vascular smooth muscle cells (VSMC) and myocardiocytes, but the molecular and cellular mechanism is not clear. Some studies suggested that lipopolysaccharide (LPS) could induce the contractile dysfunction of cardiomyocytes. It is well known that p-adrenoreceptor (P-AR) is a important messenger in cells, it can regulate the function of myocardiocyte. It is likely that PD regulate the constriction of cardiomyocytes through P-AR- AC-ATP signal pathway. In order to investigate the mechanism of PD, we observed the changes of activities and quantities of P-AR in cultured rat cardiomyocytes which are stimulated by LPS to mimic the conditions and the damages of septic shock and the P-AR functions changes induced by PD.There are two parts in our experiments. First, we observed the effect of PD on the activities of P-AR in cardiomyocytes under the stimulation of LPS. Second, to observe the influence of LPS on changes of filamentous actin (F-actin) of rat cardiac myocytes and the intervening action of PD. To make clear if there were some changes induced by LPS and PD.In the first experiment , cardiac myocytes were isolated and cultured from 3d newborn SD rat in vitro, then divided to four groups: control group(D-Hank's 30min), LPS group (100ng/ml 30min) , LPS+PD group (100ng/ml LPS 30min , 0.2mmol/L PD 10min) and PD group (0.2mmol/L lOmin). Radioligand binding assay was used to detect the function of P-AR. In control group, P-AR number (Bmax) is 4.14+1.41 fmol/105 and P-AR affinity (Kd) is 4.02+0.59 nmol/L. In LPS group, P-AR number downregulated (Bmax=1.78+0.12 fmol/105) and the affinity decreased(Kd=23.88+2.34 nmol/L). In LPS+PD group, PD could reverse the effects of LPS. P-AR number upregulated to normal (Bmax=3.37+0.36 fmol/105) and the affinity increased (Kd=3.44+0.44 nmol/L). But the PD group P-AR functions have no significant discrimination with normal group (Bmax=2.76+0.32 fmol/105 and Kd=4.63+1.54 nmol/L). These results suggested that LPS induce the contractile dysfunction of cardiomyocytes. is related to the function of P-AR with its quantity downregulated and affinity decreased. PD could reverse the changes caused by LPS with increased number and affinity. It is presumed that P-AR pathway is one of the mechanisms of PD to have better effects in septic shock treatment.In order to make clear the reason why LPS induced P-AR function decline. Immunofiuorescent assay was used. As anti-B1-AR was marked with FTTC, the distribution of B1-AR in cells could be seen by fluorescent microscope. The results indicated that LPS induce pi-AR displacement from cell membrane to cytoplasm and the staining was fainter. While in normal group, Pi-AR located on the surface membrane and staining very strong. When giving PD, Pi-AR recycled and the distributed similar to normal group. These results indicated that LPS may induce receptor internalization, and PD may prevent the effect of LPS from the internalization.P-AR mediates adrenergic control of cardiac musle contraction process by the myosin and actin interaction. F-actin distributed mainly in cardiomyocytes, and it is the important protein in cells. So, in our experiment, Myocardial F-actin was analyzed by immunofluorescent staining to detect the changes made from LPS and PD. In control group, F-actin was localized in the cortex of cardiac myocytes and the cells were filled with F-actin organized into reticular structures. After LPS stimulation, the staining for F-actin was faint or even invisible in the cortex, with the formation of stress fibers observed in the cells, which disappeared upon the 10-mi...