摘要
目的:探讨独活-秦艽对膝骨关节炎(knee osteoarthritis,KOA)模型大鼠的影响及其作用机制。方法:将56只雄性SD大鼠随机分为7组,每组8只。空白组大鼠膝关节腔注射生理盐水,其余各组均通过向膝关节腔注射碘乙酸钠溶液进行KOA造模。造模后第7天开始,塞来昔布组按0.018 g·kg^(-1)以塞来昔布药液灌胃,独活-秦艽低、高剂量组分别按2.1 g·kg^(-1)(生药量)、4.2 g·kg^(-1)(生药量)以独活-秦艽药液灌胃,独活-秦艽多糖组、独活-秦艽上清液组分别以提取的独活-秦艽多糖和独活-秦艽上清液灌胃,空白组和模型组以等量生理盐水灌胃。药物干预每天1次,持续4周。造模前后测量各组大鼠膝关节直径差(造模后直径-造模前直径);药物干预结束后取大鼠膝关节,HE染色后进行组织病理学观察;取大鼠腹主动脉血,采用ELISA技术检测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-6、IL-10、基质金属蛋白酶(matrix metalloproteinase,MMP)-1、MMP-3、MMP-13含量;收集大鼠粪便,采用16S核糖体DNA测序观察肠道菌群变化。结果:①膝关节直径差测量结果。造模后第7天,空白组的膝关节直径差小于其余6组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000)。造模后第14天,塞来昔布组的膝关节直径差小于模型组(P=0.044)。②膝关节病理组织学观察结果。模型组的改良Mankin’s评分高于空白组、塞来昔布组及独活-秦艽高剂量组、多糖组、上清液组(P=0.000,P=0.003,P=0.003,P=0.000,P=0.000);塞来昔布组的改良Mankin’s评分高于独活-秦艽高剂量组(P=0.044);独活-秦艽低剂量组的改良Mankin’s评分高于独活-秦艽高剂量组、多糖组、上清液组(P=0.000,P=0.003,P=0.012)。③血清TNF-α含量检测结果。空白组、塞来昔布组、独活-秦艽高剂量组、独活-秦艽上清液组的血清TNF-α含量均低于模型组(P=0.038,P=0.016,P=0.016,P=0.016);塞来昔布组、独活-秦艽高剂量组、独活-秦艽上清液组的血清TNF-α含量均低于独活-秦艽低剂量组、多糖组(P=0.029,P=0.034;P=0.000,P=0.000;P=0.008,P=0.009)。④血清IL-6含量检测结果。空白组、塞来昔布组、独活-秦艽高剂量组、独活-秦艽上清液组的血清IL-6含量均低于模型组(P=0.012,P=0.000,P=0.000,P=0.000);塞来昔布组、独活-秦艽上清液组的血清IL-6含量均低于独活-秦艽低剂量组、多糖组(P=0.000,P=0.000;P=0.000,P=0.000);独活-秦艽高剂量组的血清IL-6含量低于低剂量组、多糖组、上清液组(P=0.000,P=0.000,P=0.028)。⑤血清IL-10含量检测结果。空白组、塞来昔布组、独活-秦艽高剂量组、独活-秦艽上清液组的血清IL-10含量均高于模型组(P=0.001,P=0.001,P=0.000,P=0.000);塞来昔布组的血清IL-10含量高于独活-秦艽多糖组(P=0.000);独活-秦艽高剂量组、上清液组的血清IL-10含量均高于低剂量组、多糖组(P=0.010,P=0.000;P=0.000,P=0.000);独活-秦艽低剂量组的血清IL-10含量高于多糖组(P=0.004)。⑥血清MMP-1含量检测结果。空白组、塞来昔布组、独活-秦艽高剂量组、独活-秦艽上清液组的血清MMP-1含量均低于模型组(P=0.000,P=0.000,P=0.001,P=0.000);塞来昔布组的血清MMP-1含量低于独活-秦艽低剂量组、多糖组(P=0.005,P=0.017);独活-秦艽上清液组的血清MMP-1含量低于低剂量组、高剂量组、多糖组(P=0.001,P=0.022,P=0.004)。⑦血清MMP-3含量检测结果。空白组、塞来昔布组、独活-秦艽上清液组的血清MMP-3含量均低于模型组(P=0.041,P=0.003,P=0.000);塞来昔布组的血清MMP-3含量低于独活-秦艽低剂量组、多糖组(P=0.011,P=0.047);独活-秦艽上清液组的血清MMP-3含量低于低剂量组、高剂量组、多糖组(P=0.000,P=0.005,P=0.000)。⑧血清MMP-13含量检测结果。空白组、塞来昔布组、独活-秦艽高剂量组、独活-秦艽上清液组的血清MMP-13含量均低于模型组(P=0.000,P=0.007,P=0.000,P=0.001);独活-秦艽高剂量组、上清液组的血清MMP-13含量均低于低剂量组、多糖组(P=0.000,P=0.000;P=0.021,P=0.002)。⑨肠道菌群分析结果。Alpha多样性分析结果显示,7组大鼠肠道菌群的Chao1指数和Shannon指数总体比较,差异均无统计学意义。Beta多样性分析结果显示,各组肠道菌群组成的组间差异大于组内差异(R 2=0.369,P=0.014)。物种组成及LEfSe分析结果显示,在门水平上,与模型组相比,独活-秦艽高剂量组和独活-秦艽上清液组Firmicutes相对丰度均增加、Bacteroidota相对丰度均降低。在属水平上,与模型组相比,独活-秦艽低剂量组、高剂量组、多糖组及上清液组norank_f_Muribaculaceae、UCG-005、Blautia相对丰度均增加,norank_o__Clostridia_UCG-014(除独活-秦艽上清液组外)、Romboutsia相对丰度均降低;与模型组相比,塞来昔布组和独活-秦艽高剂量组Lactobacillus相对丰度均增加,塞来昔布组Blautia相对丰度增加。结论:独活-秦艽可减轻KOA模型大鼠炎症反应、保护关节软骨,其有效成分可能存在于上清液中;其作用机制可能与增加norank_f_Muribaculaceae、Lactobacillus、UCG-005、Blautia相对丰度,降低norank_o__Clostridia_UCG-014、Romboutsia相对丰度有关。
Objective:To observe the therapeutic effects of Duhuo(Angelicae Pubescentis Radix(TCD))-Qinjiao(Gentianae Macrophyllae Radix)(DH-QJ)on knee osteoarthritis(KOA)in model rats,and to explore its underlying mechanism.Methods:Fifty-six male SD rats were selected and randomized into blank group,model group,celecoxib group,DH-QJ low-dose group,DH-QJ high-dose group,DH-QJ polysaccharide group,and DH-QJ supernatant group,with 8 cases in each group.The rats in blank group were intervened by knee intra-articular injection of normal saline,while the rest were modeled by knee intra-articular injection of sodium monoiodoacetate to induce KOA.From day 7 after successful modeling,the rats in celecoxib group,DH-QJ low-dose group,DH-QJ high-dose group,DH-QJ polysaccharide group,and DH-QJ supernatant group were intervened by intragastric administration with celecoxib solution(0.018 g/kg),DH-QJ solution(crude drug:2.1 g/kg),DH-QJ solution(crude drug:4.2 g/kg),extracted DH-QJ polysaccharide,and extracted DH-QJ supernatant,respectively,while the ones in blank group and model group with the same dose of normal saline,once a day for consecutive 4 weeks.The knee diameter difference(difference between post-modeling diameter and pre-modeling diameter)of the rats in each group were measured before and after the modeling.After the end of the intervention,the rat knee joints and faeces were harvested,and the blood was drawn from the abdominal aorta of rats in each group.The knee joints were stained with HE to observe the histopathological changes,and the degree of cartilage lesions was assessed using the modified Mankin’s score.Furthermore,the serum levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-10,matrix metalloproteinase(MMP)-1,MMP-3 and MMP-13 in the abdominal aortic blood were detected by ELISA,and the microbial communities in the rat fecal samples were identified by 16S ribosomal DNA(rDNA)sequencing.Results:①The knee diameter difference.On day 7 after the modeling,the knee diameter difference was smaller in blank group compared to the other 6 groups(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).On day 14 after the modeling,the knee diameter difference was smaller in celecoxib group compared to model group(P=0.044).②The histopathological findings in knee joint.The modified Mankin’s score was higher in model group compared to blank group,celecoxib group,DH-QJ high-dose group,DH-QJ polysaccharide group,and DH-QJ supernatant group(P=0.000,P=0.003,P=0.003,P=0.000,P=0.000),was higher in celecoxib group compared to DH-QJ high-dose group(P=0.044),and was higher in DH-QJ low-dose group compared to DH-QJ high-dose group,DH-QJ polysaccharide group,and DH-QJ supernatant group(P=0.000,P=0.003,P=0.012).③The serum level of TNF-α.The serum level of TNF-α was lower in blank group,celecoxib group,DH-QJ high-dose group,and DH-QJ supernatant group compared to model group(P=0.038,P=0.016,P=0.016,P=0.016),and was lower in celecoxib group,DH-QJ high-dose group,and DH-QJ supernatant group compared to DH-QJ low-dose group and DH-QJ polysaccharide group(P=0.029,P=0.034;P=0.000,P=0.000;P=0.008,P=0.009).④The serum level of IL-6.The serum level of IL-6 was lower in blank group,celecoxib group,DH-QJ high-dose group,and DH-QJ supernatant group compared to model group(P=0.012,P=0.000,P=0.000,P=0.000),was lower in celecoxib group and DH-QJ supernatant group compared to DH-QJ low-dose group and DH-QJ polysaccharide group(P=0.000,P=0.000;P=0.000,P=0.000),and was lower in DH-QJ high-dose group compared to DH-QJ low-dose group,DH-QJ polysaccharide group,and DH-QJ supernatant group(P=0.000,P=0.000,P=0.028).⑤The serum level of IL-10.The serum level of IL-10 was higher in blank group,celecoxib group,DH-QJ high-dose group,and DH-QJ supernatant group compared to model group(P=0.001,P=0.001,P=0.000,P=0.000),was higher in celecoxib group compared to DH-QJ polysaccharide group(P=0.000),was higher in DH-QJ high-dose group and DH-QJ supernatant group compared to DH-QJ low-dose group and DH-QJ polysaccharide group(P=0.010,P=0.000;P=0.000,P=0.000),and was higher in DH-QJ low-dose group compared to DH-QJ polysaccharide group(P=0.004).⑥The serum level of MMP-1.The serum level of MMP-1 was lower in blank group,celecoxib group,DH-QJ high-dose group,and DH-QJ supernatant group compared to model group(P=0.000,P=0.000,P=0.001,P=0.000),was lower in celecoxib group compared to DH-QJ low-dose group and DH-QJ polysaccharide group(P=0.005,P=0.017),and was lower in DH-QJ supernatant group compared to DH-QJ low-dose group,DH-QJ high-dose group,and DH-QJ polysaccharide group(P=0.001,P=0.022,P=0.004).⑦The serum level of MMP-3.The serum level of MMP-3 was lower in blank group,celecoxib group,and DH-QJ supernatant group compared to model group(P=0.041,P=0.003,P=0.000),was lower in celecoxib group compared to DH-QJ low-dose group and DH-QJ polysaccharide group(P=0.011,P=0.047),and was lower in DH-QJ supernatant group compared to DH-QJ low-dose group,DH-QJ high-dose group,and DH-QJ polysaccharide group(P=0.000,P=0.005,P=0.000).⑧The serum level of MMP-13.The serum level of MMP-13 was lower in blank group,celecoxib group,DH-QJ high-dose group,and DH-QJ supernatant group compared to model group(P=0.000,P=0.007,P=0.000,P=0.001),and was lower in DH-QJ high-dose group and DH-QJ supernatant group compared to DH-QJ low-dose group and DH-QJ polysaccharide group(P=0.000,P=0.000;P=0.021,P=0.002).⑨The gut microbiota.The Alpha diversity analysis revealed that there was no statistical difference in Chao1 and Shannon indices of gut microbiota among the 7 groups in general.The Beta diversity analysis showed that the inter-group differences in gut microbial composition exceeded the intra-group variations(R 2=0.369,P=0.014).The species composition and LEfSe analysis indicated that,at the phylum level,the relative abundance of Firmicutes in the gut microbiota increased,while that of Bacteroidota decreased in DH-QJ high-dose group and DH-QJ supernatant group compared to model group;at the genus level,the relative abundance of norank_f_Muribaculaceae,UCG-005,and Blautia in the gut microbiota increased,while that of norank_o__Clostridia_UCG-014(except for DH-QJ supernatant group)and Romboutsia decreased in DH-QJ low-dose group,DH-QJ high-dose group,DH-QJ polysaccharide group,and DH-QJ supernatant group compared to model group;apart from that,the relative abundance of Lactobacillus in the gut microbiota increased in celecoxib group and DH-QJ high-dose group,and that of Blautia increased in celecoxib group compared to model group.Conclusion:DH-QJ can alleviate the inflammatory response and protect the articular cartilage in KOA model rats,potentially via active components in its supernatant.It may exert the effects by modulating the gut microbiota via enriching the beneficial taxa,e.g.,norank_f_Muribaculaceae,Lactobacillus,UCG-005,and Blautia,and suppressing the pathogenic genera,e.g.,norank_o__Clostridia_UCG-014 and Romboutsia.
作者
赵晨洋
张园媛
周娇
任育鹏
石崇荣
匡海学
王秋红
ZHAO Chenyang;ZHANG Yuanyuan;ZHOU Jiao;REN Yupeng;SHI Chongrong;KUANG Haixue;WANG Qiu-hong(College of Pharmacy,Heilongjiang University of Chinese Medicine,Harbin 150040,Heilongjiang,China;Macao Association of Chinese Medicine,Macao 999078,China)
出处
《中医正骨》
2025年第5期30-41,共12页
The Journal of Traditional Chinese Orthopedics and Traumatology
基金
全国名老中医药专家传承工作室建设项目(国中医药人教函〔2022〕75号)
第七批全国老中医药专家学术经验继承工作项目(国中医药人教函〔2022〕76号)
黑龙江省“头雁”团队支持项目(黑龙江省头雁行动领导小组文件〔2019〕5号)
中医药传承与创新“百千万”人才工程——岐黄工程首席科学家项目(国中医药人教函〔2021〕7号)
全国老药工传承工作室建设项目(国中医药人教函〔2024〕255号)。
关键词
骨关节炎
膝
大鼠
独活(中药)
秦艽
胃肠道微生物群
DNA
核糖体
osteoarthritis,knee
rats
angelicae pubescentis radix(TCD)
gentianae macrophyllae radix
gastrointestinal microbiome
DNA,ribosomal
