Effect Of Angiotensin Ⅱ On NFκB Activation In Vascular Smooth Muscle Cells Cultured In High Glucose Media

Diabetic vascular complication is a major death cause for patients suffering from diabetes, proliferation and migration of vascular smooth muscle cells play a very important role during the development of diabetic large vessels complication such as Atherosclerosis and high - glucose and Angiotensin II ( Ang II ) are two independent stimulus of VSMCs proliferation. NFkB is a pleiotropic oxidant -sensitive transcriptional factor and the effect of many stimulus on VSMCs proliferation was mediated by NFkB. Some studies indicated that both high - glucose and Ang II can induce NFkB activation. In order to definitude the role of Ang II in diabetic vascular complication, we studied the effect of Ang II on NFkB translocation in VSMCs cultured in media containing higher or lower glucose. During the course of NFkB activation, the degradation of IkBa: is an initiate step of NFkB nuclear translocation, so we also examined the effect of Ang II on the degradation of IkBa in VSMCs cultured in media containing higher or lower glucose. .Methods8w male wistar rats (weighting 200 -250g) were anesthetized with 10% aquachloral, then thoracic aortae were prepared for VSMCs . Classic tissue expanding method was used to acquire VSMCs, and the third passage cells were used for experiment. After fully grew on slide or culture bottle, VSMCs were made quiescent with serum depletion medium containing higher or lower glucose for 24h then the cells were exposed to 10-7mol/L Ang II for 0, 1, 2, 6, 12h in media containing higher or lower glucose. Immunocytochemistry was employed to observe NFkB nuclear translocation and Western Blot was used to exanimateIkBa protein quantity change in different time point before and after the treatment, Metamorph image analyses system was employed to take on image analy-ses.ResultsTo be observed with inverted phase contrast microscope, the primary cultured cells show a long fusiform shape, peak - gorge growth pattern. Immunocy-tochernistry stain show that in VSMCs cultured in low glucose type DMEM, before the cells were exposed to Ang II , the NFkB p65 show a median positive stain in cytoplasm, after the cells were exposed to Ang II for 1 h, the NFkB p65 in cytoplasm turn to a weak positive stain; in VSMCs cultured in high glucose type DMEM, the NFkB p65 show a weak positive stain in cytoplasm, before the cells were exposed to Ang II , the NFkB p65 in cytoplasm turn to a negative stain, after the cells were exposed to Ang II for 1 h; both the cells cultured in high glucose type and low glucose type media show a negative stain of NFkB p65 in cytoplasm after be exposed to Ang II for 2, 6, 12h. all the sample show negative stain of NFkB p65 in nuclear. Western Blot indicate that before VSMCs were exposed to Ang II, the IkBo: protein quantity in VSMCs cultured in high glucose type DMEM significantly is lower than that in VSMCs cultured in high glucose type DMEM. After be exposed to Ang II, the IkBa protein quantity in VSMCs cultured in high glucose type DMEM all is lower than that VSMCs cultured in low glucose type DMEM to every time point. The IkBa protein quantity change both in VSMCs cultured in high glucose type DMEM and low glucose type DMEM show time dependent tendency, but this tendency is more significant in VSMCs cultured in low glucose type DMEM.Conclusions1) Ang II and higher glucose induce the cytoplasmic NFkB of VSMC decreased.2) Higher glucose induce IkBa: protein quantity decreased in VSMCs.3 ) Ang II induce IkBo: protein quantity decreased in VSMCs and the IkBa protein quantity change show a time dependent tendency. This tendency is more significant in VSMCs cultured in low glucose type DMEM.