益生菌补充改善吡嗪酰胺致大鼠肝损伤及肠道菌群紊乱的效果

Probiotics Improve Pyrazinamide-Induced Liver Injury and Intestinal Flora Disturbance in Rats

目的:吡嗪酰胺作为抗结核药物治疗结核病可引起患者肝损伤及肠道菌群紊乱,本研究拟探讨益生菌(干酪乳杆菌(Lactobacillus casei,Lc S))补充对吡嗪酰胺致大鼠肝损伤及肠道菌群紊乱的影响。方法:将40只成年雄性SD大鼠随机分为4组,即正常对照组(NC组)、吡嗪酰胺组(L0组)、低剂量Lc S组(L1组)、高剂量Lc S组(L2组)。L0组、L1组和L2组均给予吡嗪酰胺处理,以建立肝损伤模型;L0为阳性(药物肝损伤)组;L1组和L2组大鼠每天分别给予10、20 m L/(kg·d)Lc S(10~8 CFU/m L)灌胃,持续10周。苏木精-伊红染色观察各组大鼠肝脏组织病理学变化;速率法检测各组大鼠血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天冬氨酸氨基转移酶(aspartate aminotransferase,AST)水平;实时荧光定量聚合酶链式反应技术对大鼠粪便中的双歧杆菌、乳酸杆菌及大肠杆菌16S r DNA V3可变区进行定量分析。结果:经吡嗪酰胺处理10周后,L0组大鼠苏木精-伊红染色切片显示肝细胞中度水肿,出现明显的气球样变,肝索结构消失并伴有炎性细胞浸润,病理学评分达到3.20分,血清ALT及AST水平分别升高到95.90 U/L和188.60 U/L,显著高于正常对照组的73.90 U/L和139.20 U/L,说明肝损伤造模成功。经过不同剂量的Lc S干预10周后,大鼠肝小叶结构均较L0组得到明显改善,病理学评分、血清ALT和AST水平均降低到正常对照组水平。实验菌株定量分析结果显示,大鼠经过不同分组及干预时间的延长,3种代表菌株在组间及组内均具有统计学差异。从分组来看,高剂量Lc S组大鼠在干预第6周末及干预第10周末,双歧杆菌和乳酸杆菌的量均较L0组明显升高,大肠杆菌的量明显降低(P<0.05);从干预时间来看,高剂量Lc S组大鼠在干预第10周末,双歧杆菌和乳酸杆菌的量均较干预前显著增加,分别达到干预前的1.18倍和1.03倍。结论:Lc S对吡嗪酰胺诱导的肝损伤及肠道菌群紊乱具有一定的改善作用,且随着时间的延长及剂量的增加,效果更明显。其作用机制可能与Lc S维持肠道内环境稳态、调节肠道菌群有关。

Objective： Pyrazinamide（PZA）, an important first-line drug for tuberculosis, can cause liver injury and intestinal flora disorder in patients. In this study, we intended to explore the effect of dietary probiotic（Lactobacillus casei, Lc S） supplementation on PZA-induced liver injury and intestinal flora disorder in rats. Methods： A total of 40 adult male SD rats were randomly divided into 4 groups： normal control group（NC）, PZA group（L0）, low-dose Lc S group（L1） and highdose Lc S group（L2）. Liver injury was induced in rats in the L0, L1 and L2 groups, and those in the L1 and L2 groups were subjected to Lc S（10~8 CFU/m L） intervention at a dose of 10 and 20 m L/（kg·d） by gavage daily for 10 consecutive weeks. Hematoxylin-eosin（HE） staining was used to observe the pathological changes of liver tissues in rats. The serum levels of alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were detected. Quantitative analysis of Bifidobacteria, Lactobacillus and Escherichia coli in rat feces was conducted by real-time fluorescence quantitative polymerase chain reaction（q PCR） based on 16 S r DNA V3 variable region. Results： After 10 weeks of treatment with pyrazinamide, HE staining of liver tissues revealed that liver cells showed moderate edema and marked ballooning degeneration with the disappearance of hepatic cord accompanied by the occurrence of inflammatory cell infiltration. The pathological score was 3.20 points, and serum ALT and AST levels were significantly increased to 95.90 and 188.60 U/L compared with those in the control group（73.90 and 139.20 U/L）, suggesting successful establishment of the liver injury model. After Lc S intervention at different doses for 10 weeks, examination of liver tissue sections showed the structure of hepatic lobules was obviously improved compared with the PZA group and pathological score as well as serum ALT and AST levels were reduced to normal. The quantities of 3 representative intestinal strains were statistically different between different groups and changed with prolonged intervention time. At the end of the sixth and the tenth week of intervention, the amounts of Bifidobacteriaand Lactobacillus in the high-dose Lc S group were significantly increased compared with the PZA group, while the number of Escherichia coli was significantly decreased（P〈0.05）. At the end of the tenth week, the amounts of Bifidobacteria and Lactobacillus in the high-dose Lc S group were significantly increased by 1.18 and 1.03 times compared with those before intervention, respectively. Conclusions： Lc S time-and dose-dependently has a protective effect on liver injury and intestinal flora disturbance induced by pyrazinamide. The underlying mechanism may be related to the maintenance of intestinal homeostasis and the modulation of the intestinal microbiota in the presence of Lc S.