从脾虚大鼠AA-I的排泄与脏腑oatp2a1、oatp2b1的表达探讨脾肾关系

Exploring the Correlation between Pi and Shen from the Excretion of AA-I and Expressions of Organic Anion Transporting Polypeptide 2a1 and 2 b1 in Pi Deficiency Model Rats

目的通过观察脾虚大鼠马兜铃酸(aristolochic acid,AA)代谢与肾、大肠、小肠组织中有机阴离子转运肽(organic anion transporting polypeptide,oatp)2a1、oatp2b1mRNA及蛋白表达的关系,探讨中医学脾肾之间的相互关系。方法 46只SD大鼠随机分为空白组(12只)、脾虚组(22只)、AA-I组(6只)、脾虚AA-I组(6只)。脾虚组及脾虚AA-I组采用利血平5 mg/(kg·d)皮下注射连续16天造模。空白组及脾虚组选取6只大鼠行颈动脉插管,并予AA-I灌胃后5、15、30、45、60 min颈动脉取血行AA-I药代动力学检测;脾虚组选10只大鼠行肾、大肠、小肠AA-I组织浓度测定;脾虚组及空白组各取6只分别予生理盐水灌胃,AA-I组、脾虚AA-I组予AA-I灌胃后60 min时间点取肾、大肠、小肠3种组织,采用RT-PCR及Western blot方法检测各组织中oatp2a1、oatp2b1m RNA及蛋白表达。结果与空白组比较,脾虚组大鼠灌胃后15、30、45、60 min时体内AA-I血浆浓度值明显升高,差异有统计学意义(P<0.05)。脾虚组60 min时AA-I血浆浓度下降,肾及大肠组织浓度升高,小肠中组织浓度明显下降。空白组与脾虚组中上述3种组织oatp2a1、oatp2b1mRNA表达无差异;与空白组比较,AAI组小肠oatp2a1、oatp2b1mRNA表达降低,大肠oatp2a1mRNA表达降低(P<0.05,P<0.01);与脾虚组比较,脾虚AA-I组肾组织中oatp2a1、oatp2b1mRNA表达升高(P<0.05),大肠组织oatp2b1m RNA表达升高(P<0.05)。结论脾虚脾失运化状态下,AA-I代谢差异可能与肾、大肠、小肠组织oatp2a1、oatp2b1表达改变相关,并提示肾、大肠在脾虚状态下物质代谢中发挥重要协调作用,验证了中医脾肾相关理论。

Objective To explore the correlation between Pi and Shen by observing the relationship between the metabolism of aristolochic acid（AA） and mRNA and protein expression levels of organic anion transporting polypeptide（oatp） superfamily member2a1 and 2 b1（oatp2a1 and oatp2b1） in renal,small intestinal,and large intestinal tissues of Pi deficiency syndrome（PDS） model rats.Methods Totally 46 Sprague-Dawley（SD） rats were randomly divided into four groups,i.e.,the blank group（n =12）,the PDS group（n =22）,the AA-I group（n =6）,and the PDS AA-I group（n =6）.PDS model was established by subcutaneously injecting Reserpine at the daily dose of 5 mg/kg for 16 successive days.Carotid intubation was performed in 6 rats selected from the blank group and the PDS group.Pharmacokinetics of AA-I were detected at 5,15,30,45,and 60 min after gastrogavage of AA-I.AA-I concentrations in renal,small intestinal,and large intestinal tissues of 10 rats selected from the PDS group were determined.Normal saline was administered to 6 rats selected from the PDS group and the blank group by gastrogavage.Renal,small intestinal,and large intestinal tissues were collected in the AA-I group and the PDS AA-I group at 60 min after gastrogavage of AA-I.mRNA and protein expression levels of oatp2a1 and oatp2b1 in each tissue were detected using real-time polymerase chain reaction（RTPCR） and Western blot.Results Compared with the blank group,plasma concentrations of in vivo AA-I were obviously higher in the PDS group at 15,30,45,and 60 min after gastrogavage of AA-I with statistical difference（P〈0.05）.Plasma concentrations of AA-I were obviously decreased at 60 min after gastrogavage of AA-I;AA-I concentrations in renal and large intestinal tissues were elevated;AA-I concentrations in small intestinal tissues were obviously reduced in the PDS group.There was no statistical difference in mRNA expression levels of oatp2a1 and oatp2b1 in the aforesaid three tissues of rats between the blank group and the PDS group.Compared with the blank group,mRNA expression levels of oatp2a1 and oatp2b1 decreased in small intestinal tissues of the AA-I group,and the mRNA expression level of oatp2a1 in large intestinal tissues significantly decreased（P〈0.05,P〈0.01）.Compared with the PDS group,mRNA expression levels of oatp2a1 and oatp2b1 increased in renal tissues of the PDS AA-I group（P〈0.05）;mRNA expression levels of oatp2b1 increased in large intestinal tissues of the PDS AA-I group（P〈0.05）.Conclusions The difference in AA-I metabolism might be associated with changed expression levels of oatp2a1 and oatp2b1 in renal,small intestinal,and large intestinal tissues under Pi deficiency induced loss of transportation.Shen and Dachang played important roles in substance metabolism under Pi deficiency state,which proved Pi-Shen correlated in Chinese medical theories.