广藿香醇对幽门螺杆菌酸抵抗能力的影响及其机制探讨

摘要

目的 观察广藿香醇对于幽门螺杆菌(Hp)酸抵抗能力的影响,并探讨其机制.方法 体外培养并鉴定Hp后随机分为5组,受试药高、中、低剂量组分别加入20、10、5 mg/L广藿香醇,阳性对照组加入尿素酶(Ure)抑制剂AHA,空白对照组加入等体积DMSO;干预0.5 h后应用琼脂稀释法测定Hp菌落数,观察其抗酸能力.通过大肠杆菌系统表达和纯化得到Hp尿素酶A(UreA)、UreB、UreG,取UreA/UreB蛋白混合溶液体外模拟Ure成熟过程,将UreG蛋白单体溶液和镍离子(Ni2+)溶液混合,均分别以20、10、5 mg/L广藿香醇(受试药高、中、低剂量组)和等体积DMSO处理(对照组);采用Berthelot显色法检测Ure活性,判断Ure成熟度;以原子吸收光谱法检测Ni2+浓度,判断UreG携带Ni2+的能力.结果 在中性环境中,受试药各剂量组和阳性对照组与空白对照组Hp菌落数比较无统计学差异;在酸性环境中,受试药各剂量组和阳性对照组均较空白对照组Hp菌落数减少(P均<0.01),且受试药高剂量组、阳性对照组<受试药中剂量组<受试药低剂量组(P均<0.01).受试药高、中、低剂量组和对照组Ure活性分别为54.89％±11.47％、63.99％±9.46％、83.88％±11.81％、101.53％±2.55％,Ni2+浓度分别为(0.77±0.24)、(0.80±0.22)、(1.12±0.35)、(1.56±0.42)μmol/L,受试药高、中剂量组<受试药低剂量组<对照组(P均<0.01).结论 广藿香醇可以降低Hp的酸抵抗能力,可能与阻断Ni2+的传递途径和Ure的成熟过程进而抑制Ure活性有关.%Objective To investigate the effect of patchouli alcohol on acid resistance of Helicobater pylori (Hp) and its mechanism.Methods After Hp were cultured in vitro and identified, Hp were divided into 5 groups.The high-, medium-, and low-dose drug groups were added with 20, 10, and 5 mg/L patchouli alcohol;the positive control group was added with urea enzyme inhibitor AHA;the blank control group was added with the same amount of DMSO.Agar dilution method was used to detect the bacterial survival rates in the acidic (pH=1.0) and neutral (pH=7.0) conditions after 0.5 hour.We obtained the Hp urease protein A (UreA) , urease protein B (UreB), and urease protein G (UreG) by E.coli expression system, and mixed up UreA and UreB protein.Then, we took the UreA/UreB protein mixed solution to simulate the Ure maturation process and mixed the UreG protein monomer solution with the nickel ion (Ni2+) solution.The Ure activity was detected by Berthelot method to determine the Ure maturity.The concentration of Ni2+ was measured by atomic absorption spectrometry to determine the ability of UreG carrying Ni2+.Results In the neutral condition, there was no significant difference in the Hp colony forming units among the high-, medium-, and low-dose drug groups, positive control group, and blank control group.Compared with the blank control group, the Hp colony forming units of the high-, medium-, and low-dose drug groups and positive control group decreased in neutral conditions (all P<0.05), and the order was as follows: the high-dose drug group, positive control group < medium-dose drug group < low-dose drug group (all P<0.01).The urease activities of the high-, medium-, low-dose drug groups and the control group were 54.89％±11.47％, 63.99％±9.46％, 83.88％±11.81％, and 101.53％±2.55％;the concentrations of Ni2+ were (0.77±0.24), (0.80±0.22), (1.12±0.35), (1.56±0.42) μmol/L, and the order was as follows: the high-dose drug group, medium-dose drug group < low-dose drug group < control group (all P<0.01).Conclusions Patchouli alcohol can decrease the acid resistance of Hp, which may be associated with blocking the transmission pathway of Ni2+ and Ure''s maturation process and thus inhibiting Ure activity.