基于16S rDNA测序分析独活寄生汤对膝骨关节炎大鼠肠道菌群的影响  

Effects of Duhuo Jisheng Decoction on intestinal flora of rats with knee osteoarthritis based on 16S rDNA sequencing

作　　者：卢一帆 徐无忌[2] 熊辉[3] 齐新宇[3] 伍搏宇 简功辉 杨卓 段建辉 LU Yifan;XU Wuji;XIONG Hui;QI Xinyu;WU Boyu;JIAN Gonghui;YANG Zhuo;DUAN Jianhui(Changde First Hospital of Traditional Chinese Medicine,Changde,Hunan 415000,China;The Second Hospital of HunanUniversity of Chinese Medicine,Changsha,Hunan 410005,China;Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;Dongguan Hospital of Guangzhou University of Chinese Medicine,Dongguan,Guangdong 523000,China)

机构地区：[1]常德市第一中医医院,湖南常德415000 [2]湖南中医药大学第二附属医院,湖南长沙410005 [3]湖南中医药大学,湖南长沙410208 [4]广州中医药大学东莞医院,广东东莞523000

出　　处：《湖南中医药大学学报》2024年第12期2277-2285,共9页Journal of Hunan University of Chinese Medicine

基　　金：湖南省自然科学基金项目(2022JJ30087,2024JJ6342);湖南省教育厅资助科研项目(21B0377,23B0343);湖南省中医药科研计划项目(D2022033,C2023031);常德市技术研发和技术创新引导项目(CDKJJ20220357);湖南中医药大学校级科研课题(2022XYLH045,2022XYLH051)。

摘　　要：目的通过16S r DNA测序探讨独活寄生汤(Duhuo Jisheng Decoction,DHJSD)在调控肠道菌群以治疗膝骨关节炎(knee osteoarthritis,KOA)中的潜在机制。方法选取18只SD大鼠,随机分为模型组(关节腔内注射50μL浓度为40 mg/mL的碘乙酸钠)、空白组(灌胃等量生理盐水)以及DHJSD组(造模方法与模型组相同,灌胃独活寄生汤16.5 g/kg),每组6只。造模2周后开始干预,每天灌胃1次,持续4周,并观察其一般情况。给药结束后进行取材,通过HE染色观察软骨变化。ELISA法检测血清白细胞介素(interleukin,IL)-1β、IL-6、IL-17和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)炎症因子水平。RT-qPCR检测滑膜中核因子κB p65(nuclear factor-κB p65,NF-κB p65)、雷帕霉素靶蛋白(mechanistic target of rapamycin,mTOR)、AMP激活蛋白激酶(AMP-activated protein kinase,AMPK)和磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)的相对表达量。取肠道粪便进行16S rDNA测序分析肠道微生物群的差异。结果相比空白组,模型组大鼠的活动减少,毛发失去光泽,摄食量和饮水量减少。相比模型组,DHJSD组大鼠的活动性、毛发光泽和饮食均明显改善。HE染色结果表明,DHJSD能有效改善软骨损伤,减轻纤维化和炎症反应。ELISA结果表明,相比空白组,模型组的IL-1β、IL-6、IL-17和TNF-α水平升高(P<0.01)。相比模型组,DHJSD组IL-1β、TNF-α水平降低(P<0.01)。RT-qPCR结果表明,相比空白组,模型组NF-κB p65、mTOR和AMPK的相对表达量升高(P<0.05)。相比模型组,DHJSD组mTOR的相对表达量降低(P<0.05)。16S r DNA测序结果表明,DHJSD可以调控KOA大鼠的肠道菌群结构,并影响菌群的α和β多样性。乳球菌属(Lactococcus)和肠杆菌属(Enterorhabdus)分别是DHJSD组和模型组中富集最显著的菌群。结论DHJSD能有效修复软骨损伤,减缓KOA的进展,其机制可能与调控肠道菌群、维护肠道屏障完整性以及减少免疫炎症反应有关。Objective To explore the potential mechanism of Duhuo Jisheng Decoction(DHJSD)in regulating intestinal flora for the treatment of knee osteoarthritis(KOA)through 16S rDNA sequencing.Methods Eighteen SD rats were randomized into model group(intra-articularly injected with 50μL of sodium iodoacetate at a concentration of 40 mg/mL),blank group(gavaged with an equal volume of normal saline),and DHJSD group(modeled using the same method as the model group and gavaged with DHJSD at a dose of 16.5 g/kg),with six rats in each group.Intervention began two weeks after modeling,with once-daily gavage for four weeks,during which the rat general condition was observed.After the administration period,samples were collected.Cartilage changes were observed using HE staining.ELISA was used to measure serum levels of inflammatory factors including interleukin(IL)-1β,IL-6,IL-17,and tumor necrosis factor-α(TNF-α).RT-qPCR was employed to determine the relative expression levels of nuclear factor-κB p65(NF-κB p65),mechanistic target of rapamycin(mTOR),AMP-activated protein kinase(AMPK),and phospha-tidylinositol 3-kinase(PI3K)in the synovium.Fecal samples were collected for 16S rDNA sequencing to analyze differences in the intestinal flora.Results Compared with the blank group,rats in the model group exhibited decreased activity,lusterless fur,and reduced food and water intake.In contrast,the DHJSD group showed significant improvements in activity,fur luster,and food intake compared to the model group.The HE staining indicated that DHJSD effectively reduced cartilage damage,fibrosis,and inflammatory responses.The ELISA showed that,compared with the blank group,the levels of IL-1β,IL-6,IL-17,and TNF-αin the model group were significantly higher(P<0.01).However,compared with the model group,the levels of IL-1βand TNF-αin the DHJSD group were significantly lower(P<0.01).The RT-qPCR indicated that,compared with the blank group,the relative expression levels of NF-κB p65,mTOR,and AMPK significantly increased in the model group(P<

关 键 词：膝骨关节炎 16S r DNA测序 肠道菌群 独活寄生汤 菌群多样性 菌群相对丰度 

分 类 号：R285.5[医药卫生—中药学]