基于免疫炎症-肠道微生态探讨清热解毒方对痛风小鼠治疗作用的研究  

Therapeutic effects of Qingre jiedu formula on mice with gout based on immune inflammation and gut microbiota

作　　者：何宪顺 林锟 陆舜 韦雨柔 田佳庆 江禹来 魏腾飞 林天烨 何敏聪 魏秋实 HE Xianshun;LIN Kun;LU Shun;WEI Yurou;TIAN Jiaqing;JIANG Yulai;WEI Tengfei;LIN Tianye;HE Mincong;WEI Qiushi(the Third Clinical Medical School of Guangzhou University of Chinese Medicine,Guangzhou 510378,China;Guangdong Research Institute for Orthopedics and Traumatology of Chinese Medicine,Guangzhou 510378;the Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510378)

机构地区：[1]广州中医药大学第三临床医学院,广州510378 [2]广东省中医骨伤研究院,广州510378 [3]广州中医药大学第三附属医院,广州510378

出　　处：《中国比较医学杂志》2024年第5期1-12,共12页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金面上项目(82274544);国家自然科学基金青年基金(82004392);广东省自然科学基金面上项目(2023A1515010551);广州中医药大学“双一流”与高水平大学学科协同创新团队培育项目(2021XK41);广东省新黄埔中医药联合创新研究院2022年度第一批联合创新研究项目(2022IR012);毕节市科学技术局2022年度“揭榜挂帅”项目(毕科合重大专项〔2022〕1号)。

摘　　要：目的探究清热解毒方对痛风小鼠抗炎作用及对肠道菌群的影响。方法将40只7~8周龄、体重20~22 g的SPF级C57BL/6雄性小鼠随机分为空白组(CON组)、模型组(MOD组)、别嘌醇组(ALLO组)、清热解毒方组(QRJD组),第1~35天每天早上CON组灌胃10 g/0.1 mL羧甲基纤维素,其余各组均灌胃氧嗪酸钾(500 mg/kg)+酵母膏(10 g/kg)混悬液制备高尿酸血症小鼠模型;第29天在异氟烷麻醉下,往CON组小鼠右踝关节注射50μL无菌羧甲基纤维素,其余各组小鼠右踝关节注射等量尿酸钠溶液(50 mg/mL)制备痛风性关节炎模型,同时各组每天灌服相应药物进行治疗;第35天取材,取材小鼠禁食不禁水6 h,检测血尿酸、血肌酐、尿素氮等血液指标,踝关节苏木精-伊红、番红-固绿染色;踝关节免疫组化检测白细胞介素-10(interleukin 10,IL-10)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)抗炎指标;收集小鼠盲肠内容物,采用16S rDNA高通量测序法检测肠道菌群变化。结果(1)用药治疗7 d后,与MOD组相比,QRJD组能有效降低疾病模型小鼠血尿酸(P<0.001)、肌酐(P<0.01)、尿素氮(P<0.05)浓度,有效保护肾功能。(2)病理结果提示,与MOD组相比,苏木精-伊红染色发现QRJD组治疗后,关节滑膜增生有所减少,炎症细胞浸润得到减轻;番红-固绿发现中药复方软骨排列较前有序,软骨破坏较MOD组减轻,基质未见失染。(3)踝关节免疫组化结果提示CON组、MOD组IL-10、TGF-β1未见明显升高;与MOD组相比,QRJD组的IL-10、TGF-β1表达升高(P<0.05)。(4)肠道菌群交互关系、多样性上,与CON组相比,MOD组特有的OTUs数目增加75个,[JP]而QRJD能减少MOD特有的OTUs数量,更接近CON组;4组间的α多样性未见明显差异(P>0.05),而β多样性发现QRJD组的菌群组成更趋向于CON组水平(P=0.001)。(5)肠道菌群差异分析结果显示,与CON组相比,MOD组Ruminococcaceae spp.、Dubosiella sp.、Tyzzerella sp.、Ileibacterium sp.和BacteroidalesObjective To explore the anti-inflammatory effect of Qingre Jiedu(QRJD)formula on mice with gout and its effect on gut microbiota.Methods Forty C57BL/6 mice weighing 20~22 g were divided into control(CON),model(MOD),allopurinol(ALLO),and QRJD formula(QRJD)groups,and i.g.10 g/0.1 mL carboxymethyl cellulose was administered to the CON every morning from 1 to 35 days.A hyperuricemia mouse model was prepared by intragastric injection of a potassium oxalic acid(500 mg/kg)and yeast extract(10 g/kg)suspension.On day 29,50μL sterile carboxymethyl cellulose was injected into the right ankle of mice in the CON group under isoflurane-induced anesthesia,and a gouty arthritis model was prepared by injecting the same volume of sodium urate(50 mg/mL)into the right ankle of mice in the other groups.Each group was treated with corresponding drugs every day.On day 35,samples were collected from mice that had been fasted for 6 hours without water.Blood indexes,such as uric acid,creatinine,and urea nitrogen,were assessed.Hematoxylin-eosin and saffranine O-fast green staining was performed on ankle joints.Anti-inflammatory indexes of interleukin-10(IL-10)and transforming growth factor-β1(TGF-β1)were detected by ankle joints immunohistochemical assay.The cecum contents of mice were collected,and changes in gut microbiota were analyzed by high-throughput sequencing of 16S rDNA.Results(1)After 7 days of treatment,compared with the MOD group,QRJD formula effectively reduced the blood concentrations of uric acid(P<0.001),creatinine(P<0.01),and urea nitrogen(P<0.05),and effectively protected renal functions.(2)Compared with the MOD group,HE staining showed that synovial hyperplasia and inflammatory cell infiltration were reduced in the QRJD formula group after treatment.The cartilage arrangement of the compound was more orderly than before,cartilage destruction was less than that in the MOD group,and no matrix loss was observed.(3)Immunohistochemical analysis of the ankle joint indicated that IL-10 and TGF-β1 were not significantly inc

关 键 词：清热解毒方 免疫炎症 肠道菌群 痛风 高尿酸血症 

分 类 号：R-33[医药卫生]