颐年降压饮含药血清对自发性高血压大鼠内皮细胞增殖及PPAR-γ mRNA表达的影响

Effects of Yinian Jiangya Decoction Contained Serum on Endothelial Cell Proliferation and PPARγ mRNA Expression in Spontaneously Hypertensive Rat

目的观察颐年降压饮含药血清对体外原代培养的自发性高血压大鼠(spontaneously hypertensiverats,SHR)内皮细胞增殖及PPAR-γmRNA表达的影响。方法取SHR主动脉内皮细胞进行原代培养,取3代后内皮细胞用于实验,SD大鼠40只随机分为正常血清对照组和颐年降压饮高、中、低剂量组,给与高脂饮食喂养,分别灌服生理盐水和高、中、低剂量颐年降压饮(分别含生药5.2 g/mL、2.6 g/mL、1.3 g/mL),给药20天麻醉后开始收集血清,经灭活后作用各组内皮细胞。MTT法检测不同浓度血清作用各组2、4、8、16、24、48 h细胞活性;RT-PCR法检测作用4、8、16、24 h内皮细胞PPAR-γmRNA表达情况。结果MTT检测OD值发现,4、8 h颐年降压饮高、中、低剂量组OD值均高于正常血清对照组,差异有统计学意义(P<0.05),而颐年降压饮3剂量组间差异无统计学意义(P>0.05);16 h颐年降压饮中剂量组OD值上升低于其他组,差异有统计学意义(P<0.05);24 h各组OD值均下降,但颐年降压饮高、中剂量组下降比低剂量、正常血清对照组明显,差异有统计学意义(P<0.05);48 h各组OD值继续下降,颐年降压饮高剂量组比正常血清对照组下降更明显(P<0.05)。RT-PCR检测不同血清作用内皮细胞4 h时,各组PPAR-γmRNA表达差异无统计学意义(P>0.05);8 h时颐年降压饮高剂量组PPARγmRNA表达明显低于其他各组(P<0.05);16 h颐年降压饮各剂量组PPAR-γmR NA表达比正常血清对照组高(P<0.05),其中颐年降压饮高剂量组PPARγmR NA表达最高,与其他组比较,差异有统计学意义(P<0.01);24 h各组PPAR-γmR NA表达有所下降,但颐年降压饮低剂量组PPAR-γmRNA表达仍高于正常血清对照组(P<0.05)。结论颐年降压饮对内皮细胞增殖呈双向调节作用,或许与调节PPAR-γmR NA表达有关。颐年降压饮所具有的上调及维持PPAR-γmRNA表达作用,可能是该方药具有保护血管内皮功能,降低血压的机制之一。

Objective To observe the effects of Yinian Jiangya （YNJY） Decoction contained serum on cell proliferation of primary cultured endothelial cells of spontaneously hypertensive rat （SHR） and the peroxisome proliferator-activated receptor-γ （PPAR-y） mRNA expression in the cells. Methods SD rats were fed with high-lipid diet and different doses （ high, medium and low, containing crude drug of 5.2 g/mL, 2.6 g/mL, 1.3 g/mL respectively） of YNJY respectively and the serum contained different doses of YNJY （S-YNJY） was prepared by collecting the rats＇ serum after 20 days of feeding. The serum obtained from SD rats fed with normal diet, i.e. drug free serum （S-free） was taken for control. Primary cultured endothelial cells of SHR were treated respectively with S-YNJY in different doses （treated groups） and S-free （control group）, the cell activity and the mRNA expression of PPARγ in cells of all groups were detected by MTT and RT-PCR respectively at different time points ： 2, 4, 8, 16 and 24 h after treatment. Results MTT test showed that at time points of 4 and 8 h, the OD value raised in the treated groups with insignificant difference among them （ P 〉0.05）, but was higher than that in the control group （ P 〈 0.05） ; at 16 h, it increased but showed a smaller increment in the medium-dose treated group （P〈0.05） ; at 24 h, it decreased in all groups, but the decrement in the high- and medium-dose treated groups was more remarkable （ P 〈 0.05） ; at 48 h, it decreased continuously, with the decrement more significant in the high-dose group than in the control group （ P 〈0.05）. RT-PCR detection showed that at 4 h, the expression of PPARy mRNA was not changed significantly in all groups （P 〉0.05） ; at 8 h, it was remarkably lower in high-dose group than in others （P〈0.05） ; at 16 h, it was higher in the three treated groups than in the control group （ P 〈0.05）, with a highest level shown in the high-dose treated group （ P 〈0.01 ） ; at 24 h, the expression decreased in all groups, but the level in the low-dose treated group was still higher than that in the control group （ P 〈 0.05）. Conclusions YNJY Decoction shows a two-way regulation on endothelial cells proliferation, and which is negatively dose-dependent in the late stage. The regulation is perhaps relevant to the regulating of the PPARy mRNA expressions. The PPARy mRNA expression up-regulating and maintaining effects of YNJY Decoction may be one of the mechanisms for its vascular endothelial cell protection and blood pressure suppressing.