植物乳酸杆菌对小鼠草酸钙结石的干预效果

Effect of Lactobacillus plantarum on calcium oxalate stone in mice

摘要:

【背景】草酸钙结石是一种临床常见且易复发的疾病，由于结石质地坚硬，只能通过外科手术的方法治疗，给患者带了很大的痛苦。已有研究证实，肠道菌群可影响草酸钙结石的形成，降低草酸钙结石的发病率。【目的】探究植物乳杆菌对小鼠草酸钙结石的干预效果。【方法】体外实验：在MRS培养基中加入0.02 mol/L草酸钠，制备菌株筛选培养基(MRS-OX)。接种200 μL的3.48×10¹² CFU/L植物乳杆菌悬液至MRS-OX制备含菌培养基(B+MRS-OX)。将等体积MRS-OX和B+MRS-OX于37℃恒温培养2 d，测剩余草酸浓度。体内实验：以10周龄雄性昆明小鼠为实验动物，随机分为对照组、植物乳杆菌组、结石组和植物乳杆菌干预组，每组5只小鼠。通过乙醛酸诱导小鼠建立草酸钙结石模型，并给予200 μL的3.48×10¹² CFU/L植物乳杆菌进行干预治疗以观察其预防小鼠草酸钙结石的效果。实验结束后，绘制各组小鼠平均体重变化趋势图并计算小鼠肾脏脏器指数，检测每只小鼠血液学指标和氧化应激指标总超氧化物歧化酶(superoxide dismutase，SOD)和丙二醛(malondialdehyde，MDA)，对肾脏进行病理组织学观察并计算小鼠肾脏结晶评分，通过以上实验初步探究植物乳杆菌对小鼠草酸钙结石的干预效果。【结果】体外实验表明，植物乳杆菌在菌株筛选培养基(MRS-OX)中的草酸分解效率为10%；体内实验表明，植物乳杆菌可有效改善小鼠体重下降且降低血肌酐、尿素氮和血草酸水平。通过病理组织学观察发现，植物乳杆菌干预组小鼠肾脏结晶明显减少，结晶评分与结石组相比存在极显著差异(P<0.01)，并有效缓解因草酸钙结石造成的肾脏氧化应激损伤。【结论】植物乳杆菌可缓解草酸钙结石对肾脏的损伤，减少肾脏结晶的产生，有望预防草酸钙结石的发生。

Abstract:

[Background] For calcium oxalate stone, a common clinical disease with high recurrence rate, surgery is the only solution due to the hardness of the stones, from which, however, patients suffer a lot. It has been verified that intestinal flora affects the formation of calcium oxalate stones and thus reduces the incidence. [Objective] To investigate the effect of Lactobacillus plantarum (BNCC 194165) on calcium oxalate stones in mice. [Methods] For in vitro experiment, a total of 0.02 mol/L sodium oxalate was added to the MRS broth to prepare the screening medium (MRS-OX) and then 200 μL 3.48×10¹² CFU/L L. plantarum suspension was inoculated into the MRS-OX to yield the bacteria-containing medium (B+MRS-OX). MRS-OX and B+MRS-OX at equivalent volume were cultured at 37 °C for 2 days and the concentration of residual oxalic acid was measured by the oxalic acid kit. As for the in vivo experiment, 10-week-old male Kunming mice were randomized into the control (C) group, L. plantarum group (B), glyoxylic acid (GA) group, and L. plantarum+glyoxylic acid (B+GA) group, with 5 in each group. Glyoxylic acid was used to induce calcium oxalate stone in mice. A total of 200 μL 3.48×10¹² CFU/L L. plantarum suspension was given to the model mice and then the effect on calcium oxalate stones was observed. After the experiment, the variation of body weight in each group was plotted and kidney index was calculated. Then we detected the haematological indexes (creatinine, blood urea nitrogen, blood concentration of oxalic acid) and oxidative stress indexes of total superoxide dismutase (SOD) and malondialdehyde (MDA). Moreover, the pathological sections of kidney were observed and renal crystallization score was calculated. Through the above experiments, the effect of L. plantarum on calcium oxalate stone formation in mice was preliminarily explored. [Results] In the in vitro experiment, the oxalic acid-decomposing efficiency of L. plantarum in MRS-OX was 10%. In the in vivo experiment, L. plantarum significantly inhibited the weight loss of mice and reduced the levels of plasma creatinine, urea nitrogen, and oxalic acid. Pathological observation showed that the renal crystals of B+GA group were significantly reduced and the crystallization score was different from that of the GA group (P<0.01). Moreover, the oxidative stress damage of kidney caused by calcium oxalate stones was alleviated in the B+GA group. [Conclusion] L. plantarum can relieve the damage of calcium oxalate stones to kidney and reduce the generation of kidney crystals, which can be used for preventing the formation of calcium oxalate stones.