Protective Effect Of Fasudil On Human Umbilical Vein Endothelial Cells (HUVEC) Injury Induced By Iodixanol And Its Mechanism

As the promotion and popularity of Coronary arteriography, which serve as the gold standard of the diagnosis of coronary atherosclerotic heart disease (CHD), the requirement of contrast media (CM) is increasing. An increasing reports[l] showed CM could injury the kidney, whose mechanism is indefinite. Previous researches demonstrated[2] that anisosmotic CM could lead to the morphological alterations of the renal vascular endothelial cells. Then the damage of kidney by the osmotic of anisosmotic CM was avoided for the appearance of isotonic CM. Nevetheless, the destruction was not totally avoided. For more avoidance to the renal damage, scholars detected[3] that isotonic CM might interfere the secretion of nitric oxide(NO) after handling endothelial cells. In addition, researchers found[4] isotonic CM might induce apoptosis of endothelial cells. As mentioned above, CM can lead to the injury of kidney. Furthermore, multiplicative use of CM owing to the differential diagnosis of CHD is a serious threaten to the endothelial cells, which is located in total body. However, the detailed mechanism is not clear, which leaves a serious hidden danger for the prophylaxis of damage by CM.RhoA/ROCK pathway which serve as a frequent inflammatory mechanism is related to the development of many kinds of diseases, including hypertension, pulmonary hypertension, atherosclerosis, coronarospasm, hypertrophic cardiomyopathy, diabetes and so on[5]. Besides, it also induced the apoptosis[6]. Bax, which is interrelated to apoptosis, could be restrained by Bcl-2. It predict the raise of apoptosis that the increasing expression of Bax, decreasing expression of Bcl-2 and the multiplicative Bax/Bcl-2 ratio [7].After interfering HUVEC by isotonic CM(Iodixanol), we aim to check out the level of ROCK-1、Bax、Bcl-2 by Western Blotting、rt-PCR. The results showed that HUVEC of experimental groups did a significant, concentration dependent, increasing expression of ROCK-1, decreasing expression of Bcl-2, which implicated Iodixanol could induce apoptosis by RhoA/ROCK-1 pathway. To obtain further evidences, we handled the HUVEC after handling by Iodxianol by Fasudil, and we finally found that the HUVEC after treatment did a significant, concentration dependent, decreasing expression of ROCK-1, increasing expression of Bcl-2, which could explain that Iodixanol induce apoptosis by RhoA/ROCK-1 pathway, which provided rationale for the protection of endothelial cells.Part 1 To Research The Function of RhoA/ROCK-1 Pathway in The Damage of HUVEC by IodixanolPurpose To research the function of RhoA/ROCK-1 pathway in the danmage of HUVEC by IodixanolMethod Separate the secondary HUVEC to five groups:A(control group), B(treated with 16mg/ml concentration Iodixanol), C(treated with 32mg/ml concentration Iodixanol), D(treated with 48mg/ml concentration Iodixanol), E(treated with 64mg/ml concentration Iodixanol), which was distinguishly cultivated with DMEM containing 10% Fetal Bovine Serum, DMEM containing 10% Fetal Bovine Serum and 16mg/ml concentration Iodixanol, DMEM containing 10% Fetal Bovine Serum and 32mg/ml concentration Iodixanol, DMEM containing 10% Fetal Bovine Serum and 48mg/ml concentration Iodixanol and DMEM containing 10% Fetal Bovine Serum and 64mg/ml concentration Iodixanol for 2 hours,4 hours,10 hours and 24 hours.As following will be checked out:the OD value of them for drawing the curve of growth and calculating the death rate; the appearance of them and the expression of ROCK-1, Bax and Bcl-2 by Western-Blotting and rt-PCR.Result1. The morphological alterations of HUVEC induced by Iodixanol:Compared with the control group, the appearance of experimental groups showed a significant and concentration dependent alteration.2. The influence of growth rate and death rate of HUVEC by Iodixanol:Compared with control group, the growth rate of experiment group displayed an time dependent and concentration dependent decrease, especially after being cultivated for 10 hours(P<0.05); After calculating the death rate, we found that experiment group showed a time dependent and concentration dependent increasing death rate compared with control group(P>0.05).3. The expression of ROCK-1, Bax, Bcl-2 of HUVEC:The expression of ROCK-1 in experiment group was higher than control group, showing a concentration dependent change(P<0.05); In contrast, the Bcl-2 of experiment group presented a lower, concentration dependent expression(P<0.05).As displayed above, the Iodixanol could change the shape and growth rate of HUVEC concentration dependently, which could be explained by the alteration of RhoA/ ROCK-1 pathway and the expression of Bax/Bcl-2, which could induce the apoptosis of HUVEC.Part 2 To Research The Protection of HUVEC Injured by Iodixanol via The Use of FasudilPurpose To research The protection of HUVEC injured by Iodixanol via the use of Fasudil.Method Separate the secondary HUVEC to four groups:A(control group), B(treated with 32mg/ml concentration Iodixanol), C(treated with 32mg/ml concentration Iodixanol and 50umol/L concentration Fasudil), D(treated with 32mg/ml concentration Iodixanol and 100umol/L concentration Fasudil), which was distinguishly cultivated with DMEM containing 10% Fetal Bovine Serum, DMEM containing 10% Fetal Bovine Serum and 32mg/ml concentration Iodixanol, DMEM containing 10% Fetal Bovine Serum and 32mg/ml concentration Iodixanol and 50umol/L concentration Fasudil, DMEM containing 10% Fetal Bovine Serum and 32mg/ml concentration Iodixanol and 100umol/L concentration Fasudil for 2 hours,4 hours,8 hours and 24 hours.As following will be checked out:the OD value of them for drawing the curve of growth and calculating the death rate; the appearance of them; the expression of ROCK-1, Bax and Bcl-2 by Western-Blotting and rt-PCR.Result1. The morphological alterations of HUVEC induced by Fasudil:Compared with the Iodixanol group, the appearance of Fasudil group showed a significant and concentration dependent improvement.2. The influence of growth rate and death rate of HUVEC by Fasudil:Compared with Iodixanol group, the growth rate of Fasudil group displayed an time dependence and concentration dependent increase (P>0.05). Compared with Iodixanol group, the death rate of Fasudil group displayed an time dependence and concentration dependent decrease (P>0.05).3. The expression of ROCK-1, Bax, Bcl-2 of HUVEC:The expression of ROCK-1 in Fasudil group was lower than control group, showing a concentration dependent change(P<0.05); In contrast, the Bcl-2 of Fasudil group presented a higher, concentration dependent expression(P<0.05).As displayed above, the Fasudil could improve the shape and growth rate, death rate of HUVEC concentration dependently, which could be explained by the alteration of RhoA/ROCK-1 pathway, and by interfering the RhoA/ROCK-1 pathway the expression of Bax/Bcl-2 would change, which could induce the apoptosis of HUVEC.Conclusion1. Iodixanol could do a time dependent and concentration dependent damage on HUVEC, which could be inhibited by Fasudil by inhibiting the RhoA/ROCK-1 pathway.2. The expression of Bcl-2 in HUVEC could be influenced by varying expression of ROCK-1, which could be weaken by Fasudil to improve the HUVEC apoptosis.On the whole, Iodixanol could influence the expression of Bax/Bcl-2 by interfering RhoA/ROCK-1 pathway, which could induce the apoptosis of HUVEC and be inhibited by Fasudil.