Study Of The Effect Of Ang? Receptor Blocker Candesartan On High Glucose Induced Proliferation Of Rat Vascular Smooth Muscle Cell And Its Mechanism

[Objective] To observe the effects of candesartan(cand) on high glucose induced proliferation and secretion of rat vascular smooth muscle cells A7r5 and to explore its mechanism.[Methods] According to the concentration of glucose in the A7r5 cells were divided into two groups, normal glucose(NG, 5 m M), high glucose group(Hg, 15, 25, 35, 45 m M) and each group were cultured for 48 h, using CCK-8 method to measure the absorbance(OD). To determine the optimal concentration of glucose. According to the concentration of cultured A7r5 cells 0, 24, 48, 72 h then use CCK-8 method for the determination of OD value in order to determine the optimal culture time. The next experiment was carried out with the selected glucose concentration and incubation time. Selected the three concentrations of Cand, respectively, HG(25m M) +Cand10-7mol/L, HG(25m M) +Cand10-6mol/L, HG(25m M) +Cand10-5mol/L. OD value was determined by CCK8 method, elected to choose from a significant concentration for next step experiment. Finally divided into NG(5m M), HG(25m M), HG(25m M) + Cand10-5mol/L three groups, respectively, using the cell count method, flow cytometry to study the effects of Cand on the proliferation of A7r5 induced by high glucose. ELISA assay was used to detect Ang II,cyto-immunofluorescence to detect AT1 R on the cell membrane surface. Finally, Western blot method was used to detect the expression levels of p-c-Raf, p-MEK1/2 and p-ERK1/2 to explore the mechanism of Cand inhibiting A7r5 proliferation and Ang II secretion.[Results] 1.(1) Determination of optimal glucose concentration by CCK-8 method: cells cultured for 48 h, NG(5 m M) group compared to HG(15, 25, 35, 45 m M) concentration OD value was significantly increased, and the HG(25m M) group with OD(1.044 ± 0.074) increased the most obvious, campared with NG(5 m M) OD(0.681 ± 0.055) was significant difference(P < 0.01);(2) CCK-8 method for the determination of the optimal culture time: HG(25m M) cells were cultured for 0 h(0.436 ± 0.006) compared to the cells cultured for 24h(0.717±0.018), 48h(1.044± 0.074) and 72h(1.062 ± 0.067) OD values were significantly higher, the differences were significant difference(P < 0.01). And compared to 48 h and 72 h, the OD value was no significant difference(P > 0.05);(3) Determination of optimum Cand concentration by CCK-8 method: A7r5 cells after cultured for 48 h, HG(25m M)+Cand10-5mol/L group OD for(0.920±0.010), compared with HG(25m M) group with OD(1.126 ± 0.080) have significant difference(P < 0.01), and HG(25m M)+Cand10-7mol/L, HG(25m M)+Cand10-6mol/L group campared HG(25m M) have no significant difference(P > 0.05).(4) Cell counting results: A7r5 cells cultured for 48 h, compared with HG(25m M) group cell count(77.33 ± 2.52). HG(25m M)+Cand10-5mol/L cell number(64.67 ± 116) decreased significantly, the difference between the two groups has statistical significance difference(P < 0.05).(5) Flow cytometry results show: NG(5 m M) group compared Hg(25m M) group A7r5 cell proliferation activity was significantly enhanced, cell cycle in S phase ratio [(30 ± 503 1. 521)] increased significantly(P < 0. 01). Compared with HG(25m M) group, HG(25m M) +Cand10-5mol/L group A7r5 cell proliferation activity decreased, the proportion of cell cycle S(21.677 ± 1.690)% decrease, the difference between the two groups was statistically difference(p<0.01). 2(1) ELISA test Ang II results show that NG(5 m M) cell secretion of Ang II for the OD values were measured with HG(25m M)(55.268 ± 0.310) cells secreted Ang II compared to the measured OD values(59.934±0.121) statistically significant difference(P < 0.01). Group HG(25m M)+Cand10-5mol/L campared with HG(25m M) group, hace no significant difference(p>0.05).(2) Immunofluorescencestaining results: Compared with NG(5m M) group, the expression of AT1 R on the cell membrane of HG(25m M) group was increased, and the expression of AT1 R was decreased in HG(25m M) +Cand10-5mol/L group compared with HG(25m M) group. 3.Western blot results showed that campared with NG(5 m M) group p-c-Raf(0.836 ± 0.039), p-MEK1 / 2(1.032 ± 0.034), p-ERK1 / 2(1.472 ± 0.089), HG(25m M) group p-c-Raf(1.060 ± 0.087), p-MEK1 / 2(115.5 ± 0.064), p-ERK1 / 2(1.924 ± 0.065) protein expression levels were significantly increased(P < 0.01). Compared with HG(25m M) group, HG(25m M) +Cand10-5mol/L group p-c-Raf(0.658 ± 0.039), p-MEK1/2(0.821 ± 0.049), p-ERK1/2(0.795 ± 0.028) protein expression was regulated(p<0.01).[Conclusion] 1.High glucose promoted A7r5 proliferation and increased the secretion of Ang?. 2.Candesartan inhibited the proliferation of A7r5 cells induced by high glucose, but had no effect on the secretion of Ang?. 3.Candesartan inhibit high glucose induced proliferation of A7r5 cells and the mechanism may be related to the inhibition of p-c-Raf, p-MEK1 / 2 and p-ERK1 / 2 protein expression level.