基于16S rDNA测序研究当归拈痛汤对膝骨关节炎小鼠肠道菌群的影响  

Effect of Danggui Niantongtang on Intestinal Flora of Knee Osteoarthritis Mice Based on 16S rDNA Sequencing

作　　者：吴霜 袁立霞[1] 廖晴 蔡义思 钟威[2] 陈宏 WU Shuang;YUAN Lixia;LIAO Qing;CAI Yisi;ZHONG Wei;CHEN Hong(School of Traditional Chinese Medicine(TCM),State Administration of TCM Research Laboratory Level IlI Chinese Medicine Pharmacology Laboratory,Southern Medical University,Guangzhou 510515,China;Qiqihar First Hospital,Qiqihar 161000,China;Heilongjiang Academy of TCM,Harbin 150036,China)

机构地区：[1]南方医科大学中医药学院,国家中医药管理局科研三级实验室中药药理实验室,广州510515 [2]齐齐哈尔市第一医院,黑龙江齐齐哈尔161000 [3]黑龙江省中医药科学院,哈尔滨150036

出　　处：《中国实验方剂学杂志》2024年第11期9-17,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82274400);广东省自然科学基金面上项目(2022A1515011681)。

摘　　要：目的:本研究旨在通过16S rDNA探讨当归拈痛汤调控肠道菌群进而治疗膝骨关节炎(KOA)的潜在作用机制。方法:将36只C57BL/6J小鼠采用前交叉韧带切断(ACLT)的方法构建膝骨关节炎疾病模型,并随机分为假手术组、模型组、当归拈痛汤低剂量组(0.819 g·kg^(-1))、当归拈痛汤中剂量组(1.638 g·kg^(-1))、当归拈痛汤高剂量组(3.276 g·kg^(-1))、美洛昔康组(0.975 mg·kg^(-1)),每组6只。除给药组外,假手术组与模型组均予生理盐水灌胃,连续干预4周后,分别收取小鼠粪便和完整膝关节,苏木素-伊红(HE)和番红O-固绿染色观察膝关节组织形态的病理改变;采用16S rDNA测序分析给药前后肠道微生物丰度、多样性变化及相对应的功能预测。结果:高剂量当归拈痛汤和美洛昔康可以明显缓解KOA小鼠的疼痛症状,改善关节结构的紊乱,维持膝关节软骨的完整性,提高关节软骨Ⅱ型胶原a1(Col2a1)表达,降低关节软骨基质金属蛋白酶-13(MMP-13)表达。16S rDNA结果显示,高剂量当归拈痛汤可调整肠道菌群物种丰富度与菌群结构,与假手术组比较,模型组中变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、瘤胃球菌属(Ruminococcus)、拟杆菌属(Bacteroides)丰度显著提高(P<0.05),与模型组比较,当归拈痛汤组中这4种菌群丰富度显著降低;与假手术组比较,模型组疣微菌门(Verrucomicrobia)、颤螺菌属(Oscillospira)和阿克曼氏菌属(Akkermansia)丰度明显降低(P<0.05),与模型组比较,当归拈痛汤组中这三类菌群丰度明显升高(P<0.05)。对差异菌属进行功能学通路预测发现,各组间差异物种涉及到丰度较高的代谢通路主要涉及到生物合成及前体代谢物和能量的产生,包括氨基酸生物合成、核苷和核苷酸生物合成、辅因子、假体基团、电子载体和维生素生物合成。结论:当归拈痛汤能够有效保护关节软骨,延缓KOA疾病进展,可能是通过调节肠道菌群结构�Objective:This study aims to explore the potential mechanism of Danggui Niantongtang in treating knee osteoarthritis(KOA)by regulating the intestinal flora through 16S rDNA analysis.Method:Thirty-six C57BL/6J mice were subjected to anterior cruciate ligament transection(ACLT)to establish a KOA model and were randomly divided into the sham surgery group,model group,low-dose Danggui Niantongtang group(0.819 g·kg^(-1)),medium-dose Danggui Niantongtang group(1.638 g·kg^(-1)),high-dose Danggui Niantongtang group(3.276 g·kg^(-1)),and Meloxicam group(0.975 mg·kg^(-1)),with 6 mice in each group.Except for the treatment groups,the sham surgery group and model group were given normal saline by gavage.After 4 weeks of continuous intervention,feces and intact knee joints of the mice were collected.Hematoxylin-eosin(HE)staining and Safranin O-Fast Green staining were performed to observe the pathological changes in knee joint tissue morphology.The 16S rDNA sequencing was used to analyze changes in the abundance and diversity of intestinal microorganisms before and after treatment,along with corresponding functional predictions.Result:High-dose Danggui Niantongtang and Meloxicam significantly relieved pain symptoms in KOA mice,improved the disorder of joint structure,maintained the integrity of knee articular cartilage,increased the expression of typeⅡcollagen alpha 1(Col2a1)in articular cartilage,and decreased the expression of matrix metalloproteinase-13(MMP-13).The results of 16S rDNA sequencing showed that high-dose Danggui Niantongtang could adjust the abundance and structure of intestinal microbial species.Compared with the sham surgery group,the abundance of Proteobacteria,Actinobacteria,Ruminococcus,and Bacteroides was significantly increased in the model group(P<0.05),while in the Danggui Niantongtang group,the abundance of these four flora was significantly reduced compared with the model group.Compared with the sham surgery group,the abundance of Verrucomicrobia,Oscillospira,and Akkermansia was significantly d

关 键 词：膝骨关节炎 当归拈痛汤 肠道菌群 16S rDNA高通量测序 关节软骨 

分 类 号：R2-0[医药卫生—中医学] R33R289R684.3