摘要
目的 分析N6-甲基腺嘌呤(m6A)甲基化转移酶3(METTL3)在肾脏缺血再灌注损伤(IRI)大鼠动物模型中的动态变化.方法 取健康成年雄性SD大鼠20只,根据随机数字法将大鼠分为IRI组及假手术组,IRI组根据缺血再灌注后不同时间分为IRI 24 h组、IRI 48 h组和IRI 72 h组,每组5只大鼠.测定大鼠血清肌酐和尿素氮水平,并进行肾组织HE染色.采用实时定量聚合酶链式反应检测METTL3 mRNA相对转录水平,采用蛋白质印迹法检测METTL3蛋白相对表达量.使用单因素方差分析进行多组间比较,组间两两比较采用LSD法;当数据不满足正态分布和方差齐性时,使用Kruskal-Wallis H检验进行多组间比较,两两比较采用Bonfferoni检验进行分析.使用Pearson法进行相关性分析.P＜0.05为差异有统计学意义.结果 假手术组以及IRI 24 h、48 h和72 h组大鼠血清肌酐分别为(15.8±2.28)、(57.8±6.50)、(37.0± 6.00)和(29.8±5.02)μmol/L,尿素氮分别为(3.86±0.45)、(16.48±2.49)、(7.09±0.91)和(5.49±0.75)mmol/L,差异均有统计学意义(F=56.44和81.83,P均＜0.05).大鼠肾脏组织病理染色提示假手术组大鼠肾脏组织形态正常,IRI 24 h时已出现急性病变,IRI 48 h时急性病变加重,IRI 72 h时急性病变仍明显.假手术组以及IRI 24 h、48 h和72 h组大鼠总m6A相对修饰水平分别为(1.00±0.36)、(8.53±3.29)、(4.41±1.12)和(2.40±0.93),METTL3 蛋白相对表达量分别为(1.00±0.29)、(6.37±2.59)、(2.06±0.76)和(1.55±0.63),METTL3 mRNA 相对转录水平分别为(1.00±0.04)、(2.52±0.67)、(1.86±0.21)和(0.84±0.22),差异均有统计学意义(F=16.43、15.54和13.56,P均＜0.05).总m6A修饰水平与血清肌酐和尿素氮均呈正相关(r=0.7963和0.7670,P均＜0.05);METTL3 mRNA水平与血清肌酐、尿素氮和总m6A修饰水平均呈正相关(r=0.7208、0.7957和0.8313,P均＜0.05).结论 在IRI早期,METTL3表达水平和总m6A修饰水平均升高,提示METTL3和总m6A修饰水平对早期实时评估IRI可能具有重要意义.
Abstract
Objective To analyze the dynamic changes of N6-methyladenine(m6A)methyltransferase-like 3(METTL3)in renal ischemia-reperfusion injury(IRI)rat models.Methods Twenty healthy adult male SD rats were selected and divided into IRI group and sham operation group(Sham group)according to the random number method.The IRI group was further divided into three subgroups as IRI 24 h group,IRI 48 h group and IRI 72 h group according to different phases after renal ischemia-reperfusion.There are 5 rats in each group.Rat serum creatinine and urea nitrogen levels were measured,and renal tissue HE staining was performed.Real-time quantitative polymerase chain reaction was used to detect the relative transcription level of METTL3 mRNA,and Western blot was used to detect the relative expression of METTL3 protein.One-way analysis of variance was used for comparison among multiple groups,and the LSD method was used for pairwise comparisons.When the data did not meet normal distribution and homogeneity of variance,the Kruskal-Wallis H test was used for comparisons between multiple groups,and the Bonfferoni method was used for pairwise comparisons.Correlation analysis was performed using the Pearson method.P＜0.05 was regarded as statistically significant.Results The serum creatinine of rats in the Sham group and the IRI 24 h,48 h and 72 h groups were(15.8±2.28),(57.8±6.50),(37.0±6.00)and(29.8±5.02)μmol/L,respectively,and the urea nitrogen of all groups were(3.86±0.45),(16.48±2.49),(7.09± 0.91)and(5.49±0.75)mmol/L,respectively.The differences were statistically significant(F=56.44 and 81.83,P all＜0.05).Pathological staining of rat kidney tissue showed that the morphology of the rat kidney tissue in the Sham group was normal,and acute lesions had appeared in the IRI 24 h group,aggravated in the IRI 48 h group,and were still obvious in the IRI 72 h group.The relative modification levels of total m6A in the Sham group and the IRI 24 h,48 h and 72 h groups were(1.00± 0.36),(8.53±3.29),(4.41±1.12)and(2.40±0.93),respectively.Relative expression levels of METTL3 protein were(1.00±0.29),(6.37±2.59),(2.06±0.76)and(1.55±0.63),respectively.The relative transcription levels of METTL3 mRNA were(1.00±0.04),(2.52± 0.67),(1.86±0.21)and(0.84±0.22),respectively.The differences were all statistically significant(F=16.43,15.54 and 13.56,all P＜0.05).The total m6A modification level was positively correlated with serum creatinine and urea nitrogen(r=0.7963 and 0.7670,all P＜0.05).The mRNA level of METTL3 was positively correlated with serum creatinine,urea nitrogen and total m6A modification level(r=0.7208,0.7957 and 0.8313,all P＜0.05).Conclusions In the early process of IRI,the expression level of METTL3 and the level of total m6A modification increased,suggesting that the level of METTL3 and total m6A modification may be of great significance for the early real-time assessment of IRI.
基金项目
江苏省自然科学基金(BK20200180)
江苏省自然科学基金(BK20211064)
常州市科技计划(CE20235059)
常州市科技计划(CJ20230051)
常州市医学重点学科(CZXK202209)
常州市十四五卫生健康高层次人才培养工程(2022260)
NETL
NSTL
万方数据