基于初级胆汁酸合成探讨乳香醋炙对溃疡性结肠炎的增效机制

Study on efficiency enhancing mechanism of vinegar processed Olibanum on ulcerative colitis via primary bile acids synthesis

目的从对初级胆汁酸合成的影响,探讨醋炙增强乳香改善溃疡性结肠炎(ulcerative colitis,UC)的作用机制。方法基于2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid,TNBS)法建立UC大鼠模型,采用超高效液相色谱-质谱联用(UPLC-TQ-MS)法测定大鼠血浆中6种初级胆汁酸含量;进一步借助L-02肝细胞模型,围绕初级胆汁酸合成场所肝脏,探究乳香醋炙前后及醋炙前后差异成分3-乙酰-9,11-去氢-β-乳香酸(3-acetyl-9,11-dehydro-β-boswellic acid,ADHBA)的干预作用,以ELISA法考察胆汁酸前体胆固醇变化,采用q RT-PCR法测定游离型初级胆汁酸合成酶胆固醇7α-羟化酶(cholesterol 7α-hydroxylase,CYP7A1)、甾醇12α-羟化酶(sterol 12α-hydroxylase,CYP8B1)、甾醇-27-羟化酶(27-hydroxycholesterol,CYP27A1)、氧甾醇-7α-羟化酶(oxysterol-7α-hydroxylase,CYP7B1)、结合型初级胆汁酸合成酶胆汁酰基辅酶A合成酶(bile acyl-CoA synthetase,BACS)和胆汁酸-辅酶A:氨基酸正酰基转移酶(bile acid-CoA:amino acid N-acyltransferase,BAAT)m RNA表达水平。结果乳香醋炙后,对UC模型大鼠血浆中的初级胆汁酸总量和游离型、结合型初级胆汁酸总量的回调水平更接近正常组。乳香、醋乳香、醋炙前后差异成分ADHBA均可降低胆汁酸合成前体胆固醇水平,但以醋乳香及ADHBA的调控作用更加明显。醋乳香对游离型初级胆汁酸合成经典途径中CYP7A1的抑制和CYP8B1的上调作用以及对结合型初级胆汁酸合成酶BACS、BAAT的上调作用强于乳香组,乳香联合ADHBA组同醋乳香组上调作用一致。结论乳香醋炙后对初级胆汁酸合成调控作用主要是基于其对游离型初级胆汁酸经典合成途径中限速酶CYP7A1的抑制,回调机体在UC病理状态下的初级胆汁酸水平,进而增强乳香对UC的改善作用;ADHBA可能为乳香醋炙增效的物质基础之一。

Objective To investigate the efficiency enhancing mechanism for ulcerative colitis(UC) of vinegar processed Ruxiang(Olibanum) from the aspect of primary bile acids synthesis. Methods UPLC-TQ-MS was used to determine the contents of six primary bile acids in plasma of UC rats induced by 2,4,6-trinitrobenzenesulfonic acid(TNBS). At the cellular level, L-02 cell line was applied to compare the effects of Olibanum, vinegar processed Olibanum and the different components of 3-acetyl-9,11-dehydro-β-boswellic acid(ADHBA) on synthesis of primary bile acids. The content of bile acid synthesis precursor cholesterol was measured by ELISA. m RNA expressions of cholesterol 7α-hydroxylase(CYP7A1), sterol 12α-hydroxylase(CYP8B1), 27-hydroxycholesterol(CYP27A1), oxysterol-7α-hydroxylase(CYP7B1), bile acyl-CoA synthetase(BACS) and bile acid-CoA: amino acid N-acyltransferase(BAAT) were determined by qRT-PCR. Results Level of total amount of primary bile acids and free and conjugated primary bile acids in the plasma of UC rats fed with vinegar processed Olibanum was closer to that of normal rats. Olibanum, vinegar processed Olibanum and ADHBA could reduce cholesterol level, but the effect of vinegar processed Olibanum and ADHBA were more obvious.The inhibition of vinegar processed Olibanum on the classical pathway of free primary bile acid synthesis CYP7A1, the up-regulation of CYP8B1, BACS and BAAT were higher than those of Olibanum. Olibanum combined with ADHBA exhibited the same effect with vinegar processed Olibanum. Conclusion The inhibition of rate-limiting enzyme CYP7A1 in the classical synthesis pathway plays a significant role on regulation of primary bile acid synthesis by vinegar processed Olibanum, which resulted in the reduction of primary bile acid level in pathological state of UC. ADHBA is proved to be one of the material foundations of processing efficiency.