蛋白质精氨酸甲基转移酶1-非对称性二甲基精氨酸-二甲基精氨酸二甲胺水解酶1路径在肝纤维化、肾小管间质纤维化大鼠组织中的作用及意义

Role and Significance of PRMT1-ADMA-DDAH1 Pathway in Rats with Hepatic and Renal Tubulointerstitial Fibrosis

目的观察蛋白质精氨酸甲基转移酶1(PRMT1)-非对称性二甲基精氨酸(ADMA)-二甲基精氨酸二甲胺水解酶1(DDAH1)代谢轴在肝纤维化、肾小管间质纤维化大鼠模型体内的变化情况,并探讨血清ADMA水平与肝纤维化、肾小管间质纤维化程度间的相关性。方法 2015年9—12月,从40只SPF级Sprague-Dawley雄性大鼠中随机抽取20只,将其平均分为溶剂对照组(A组)和肝纤维化模型组(B组);再将剩余的20只SpragueDawley大鼠平均分为假手术组(C组)和模型组(D组),各10只。采用四氯化碳(CCl4)慢性染毒和单侧输尿管结扎的方法分别建立大鼠肝纤维化模型(B组)和肾小管间质纤维化模型(D组)。采用皮下注射等量花生油和单侧输尿管只分离不结扎的方法分别建立溶剂对照模型(A组)和假手术模型(C组)。A、B组大鼠分别于干预8周后,C、D组大鼠分别于干预2周后,采用Masson三色染色后计算肝、肾胶原纤维面积百分比;采用化学比色法检测肝、肾组织羟脯氨酸(Hyp)水平;采用改良的高效液相色谱法检测血清ADMA水平;采用蛋白质免疫印迹法检测肝、肾组织PRMT1、DDAH1表达水平。结果 A组、B组、C组、D组大鼠血清ADMA水平分别为(0.43±0.07)、(1.10±0.21)、(0.45±0.03)、(1.26±0.21)μmol/L。B组大鼠血清ADMA水平高于A组(t=6.32,P<0.05);D组血清ADMA水平高于C组(t=6.96,P<0.05)。血清ADMA水平与肝、肾胶原纤维面积百分比均呈正相关(r值分别为0.913、0.934,P<0.05)。血清ADMA水平与肝、肾组织Hyp水平均呈正相关(r值分别为0.856、0.878,P<0.05)。B组大鼠肝组织PRMT1表达水平高于A组,DDAH1表达水平低于A组(P<0.05)。D组大鼠肾组织PRMT1表达水平高于C组,DDAH1表达水平低于C组(P<0.05)。结论在肝纤维化、肾小管间质纤维化状态下,PRMT1-ADMA-DDAH1代谢轴中PRMT1表达增高,而DDAH1表达减低,可导致血清ADMA水平升高,引起肝血窦和肾小管周围毛细血管内皮细胞的损伤,从而参与纤维化进程的启动和病理改变的发生。

Objective To study the changes of protein - arginine N - methyltransferases 1 （PRMT1） - asymmetrical dimethylarginine （ADMA） - dimethylarginine dimethylaminohydrolase 1 （DDAH1）axis in rat models of hepatic and renal tubulointerstitial fibrosis,and discuss the correlation between serum ADMA levels and degree of fibrosis. Methods From September to December 2015,20 Sprague - Dawley rats were randomly selected from 40 male SPF Sprague - Dawley rats,and equally divided into control group of solvent （group A）and model group of hepatic fibrosis （group B）;the remaining 20 Sprague - Dawley rats were randomly divided into sham operation group （group C）and model group （group D）,each had 10 rats. The rat models of hepatic fibrosis （group B）and tubulointerstitial fibrosis （group D）were established by chronic exposure to carbon tetrachloride （CCl 4 ）and unilateral ureteral obstruction. The solvent control model （group A）and sham operation model （group C）were established by subcutaneous injection of the same amount of peanut oil and unilateral ureteral separation without ligation respectively. After 8 - week and 2 - week intervention for rats in group A and B,group C and D respectively, Masson trichrome staining method was calculated percentages of areas of hepatic and renal collagen fibers;the levels of hydroxyproline （Hyp）in hepatic and renal were detected by chemical colorimetry. Serum ADMA levels were measured by the modified HPLC method. The expression levels of PRMT1 and DDAH1 in hepatic and renal tissues were detected by Western blotting. Results The serum ADMA levels of group A,group B,group C and group D were （0. 43 ± 0. 07） （1. 10 ± 0. 21） （0. 45 ± 0. 03） （1. 26 ± 0. 21）μmol/ L respectively. The serum ADMA level of group B was higher than that of group A （t= 6. 32,P 〈 0. 05）. The serum ADMA level of group D was higher than that of group C （t = 6. 96,P 〈 0. 05）. There was a positive correlation between the ADMA level in serum and the percentage of areas of hepatic and renal collagen fibers （r = 0. 913, 0. 934 respectively,P 〈 0. 05）. There was a positive correlation between the ADMA level in serum and the level of Hyp in hepatic and renal tissues （r = 0. 856,0. 878 respectively,P 〈 0. 05）. The expression level of PRMT1 in hepatic tissues of group B was higher than that of group A,while its expression level of DDAH1 was lower than that of group A （P 〈 0. 05）. The expression level of PRMT1 in renal tissues of group D was higher than that of group C,while its expression level of DDAH1 was lower than that of group C （P 〈 0. 05）. Conclusion Under the condition of hepatic and renal fibrosis,increased expression of PRMT1 and decreased expression of DDAH1 in PRMT1 - ADMA - DDAH1 can lead to elevated levels of serum ADMA,cause damages of capillary endothelial cells around hepatic sinusoids and renal tubules,and thus involve in the initiation of and pathological changes of the fibrosis process.