广藿香醇对幽门螺杆菌酸抵抗能力的影响及其机制探讨

Effect of patchouli alcohol on acid resistance of Helicobacter pylori

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、5mg/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0.5h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(Ni^(2+))溶液混合,均分别以20、10、5mg/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Ni^(2+)浓度,判断UreG携带Ni^(2+)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%,Ni^(2+)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Ni^(2+)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 Urematuration process and mixed the UreG protein monomer solution with the nickel ion（Ni^（2＋）） solution. The Ureactivity was detected by Berthelot method to determine the Urematurity. The concentration of ^（2＋）was measured by atomic absorption spectrometry to determine the ability of UreG carrying Ni^（2＋）. 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 Ni^（2＋）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 Ni^（2＋）and Ure＇s maturation process and thus inhibiting Ureactivity.