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基于16S rRNA技术分析黄连酒蒸前后对正常及2型糖尿病大鼠肠道微生物的影响
Effect of Coptidis Rhizoma on Gut Microbiota of Normal and Type 2 Diabetic Rats Before and After Being Steamed with Rice Wine Based on 16S rRNA Technique
- 中国实验方剂学杂志 2019年25卷第22期 页码：92-102
- 作者机构：
1.成都中医药大学 药学院，国家中医药管理局 中药炮制技术重点研究室，成都 611137；
2.雅安迅康药业有限公司，四川 雅安 625600
- 作者简介：
王瑾，在读博士，助理研究员，从事中药炮制研究，E-mail：wangjin0816@126.com
黄勤挽，博士，教授，从事中药炮制研究，Tel：028-61800231，E-mail：huangqinwan@cdutcm.edu.cn
- 基金信息：四川省中医药管理局中医药基础研究项目(2018JC011);成都市科技局科技惠民技术研发项目(2015-HM01-00401-SF)
DOI：10.13422/j.cnki.syfjx.20192447
中图分类号： R22; R28; C37; R943.1; TS193收稿日期：2019-08-07，
网络出版日期：2019-09-04，
纸质出版日期：2019-11-20
- 稿件说明：
移动端阅览
王瑾, 冉倩, 王琳, 等. 基于16S rRNA技术分析黄连酒蒸前后对正常及2型糖尿病大鼠肠道微生物的影响[J]. 中国实验方剂学杂志, 2019,25(22):92-102.
Jin WANG, Qian RAN, Lin WANG, et al. Effect of Coptidis Rhizoma on Gut Microbiota of Normal and Type 2 Diabetic Rats Before and After Being Steamed with Rice Wine Based on 16S rRNA Technique[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(22): 92-102.
王瑾, 冉倩, 王琳, 等. 基于16S rRNA技术分析黄连酒蒸前后对正常及2型糖尿病大鼠肠道微生物的影响[J]. 中国实验方剂学杂志, 2019,25(22):92-102. DOI： 10.13422/j.cnki.syfjx.20192447.
Jin WANG, Qian RAN, Lin WANG, et al. Effect of Coptidis Rhizoma on Gut Microbiota of Normal and Type 2 Diabetic Rats Before and After Being Steamed with Rice Wine Based on 16S rRNA Technique[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(22): 92-102. DOI： 10.13422/j.cnki.syfjx.20192447.
摘要
目的：
2
基于16S rRNA技术研究黄连酒蒸前后对正常和2型糖尿病大鼠肠道菌群的影响，从肠道菌群的角度探讨黄连酒蒸炮制“减毒增效”的原理。
方法：
2
采用高脂饮食结合链脲佐菌素复制2型糖尿病大鼠模型。正常和模型大鼠分别按0.8 g·kg
－1
(按生药量计)每天灌胃生黄连生品或酒蒸黄连水煎液，阳性药组按0.25 g·kg
－1
每天灌胃二甲双胍溶液，连续给药30 d。每周测1次血糖，于给药第27天进行口服糖耐量的测定。于第30天取粪便后麻醉大鼠，取血进行酶联免疫吸附试验(ELISA)检测，解剖取结肠进行苏木精-伊红(HE)染色。基于16S rRNA基因测序技术对各个组大鼠肠道菌群的alpha多样性，beta多样性，菌群丰度及构成，及其与抗炎抗氧化的相关性等进行综合评价。
结果：
2
正常大鼠给予黄连生品或酒蒸黄连后，出现了一定的炎性反应，氧化-抗氧化系统失衡，结肠病理性损伤以及肠道菌群改变，表现出了一定的毒副作用，但酒蒸黄连的毒副作用低于生黄连。黄连酒蒸前后均能降低2型糖尿病大鼠的空腹血糖以及提高糖耐量，虽然酒蒸后作用有增强的趋势，但酒蒸前后无显著差异。2型糖尿病大鼠出现轻度的炎症反应和氧化-抗氧化系统失衡，表现为白细胞介素(IL)-6，核转录因子(NF)-
κ
B和丙二醛(MDA)含量显著升高(
P
<
0.05，
P
<
0.01)，谷胱甘肽过氧化物酶(GSH-Px)，还原型谷胱甘肽(GSH)，超氧化物歧化酶(SOD)，过氧化氢酶(CAT)活力明显降低(
P
<
0.01)，黄连酒蒸前后均能抑制炎症反应以及氧化-抗氧化系统失衡，且酒蒸黄连作用优于黄连生品；2型糖尿病大鼠出现明显的结肠病理性损伤，而黄连酒蒸前后均能减轻这种改变，且酒蒸黄连作用优于生黄连。2型糖尿病大鼠出现明显的肠道菌群紊乱，包括下调拟杆菌门(Bacteroidetes)，厚壁菌门(Firmicutes)和Kiritimatiellaeota，上调变形菌门(Proteobacteria)，放线菌门(Actinobacteria)，酸杆菌门(Acidobacteria)，绿弯菌门(Chloroflexi)，蓝细菌门(Cyanobacteria)和疣微菌门(Verrucomicrobia)，黄连酒蒸前后均能减轻肠道菌群紊乱，且黄连对Kiritimatiellaeota，Cyanobacteria和Verrucomicrobia的调节作用强于酒蒸黄连，酒蒸黄连可以显著降低Chloroflexi水平，而黄连生品无此作用。肠道菌群可能与黄连酒蒸前后的抗氧化能力具有一定的相关性。
结论：
2
黄连酒蒸前后可通过调节肠道菌群而对2型糖尿病大鼠起到治疗作用，且酒蒸黄连疗效更佳；黄连酒蒸后对肠道菌群的“毒副作用”降低。黄连酒蒸“减毒增效”的炮制机制与肠道菌群密切相关。
Abstract
Objective:
2
To investigate the effect of Coptidis Rhizoma on intestinal microflora of normal and type 2 diabetic rats before and after being steamed with rice wine based on 16S rRNA sequencing technology.
Method:
2
The type 2 diabetic rat model was established by high-fat diet and streptozotocin. Normal and model rats were given the decoction of Coptidis Rhizoma or Coptidis Rhizoma steamed with rice wine every day (0.8 g·kg
-1
according to raw drug quantity)
the positive drug group was given metformin solution every day (0.25 g·kg
-1
)
continuous administration for 30 d. Blood glucose was measured once a week and oral glucose tolerance was measured on the 27
th
day of administration. On the 30
th
day
feces were taken and the rats were anesthetized. Blood was taken for enzyme linked immunosorbent assay (ELISA). Colon was dissected for hematoxylin-eosin (HE) staining. Based on 16S rRNA gene sequencing technology
the alpha diversity
beta diversity
abundance and composition
and the correlation with anti-inflammatory and antioxidant activities of intestinal microflora were comprehensively analyzed and evaluated.
Result:
2
After giving normal rats Coptidis Rhizoma or Coptidis Rhizoma steamed with rice wine
there were some inflammatory reactions
imbalance of oxidation-antioxidant system
pathological damage of colon and changes of intestinal microflora
which showed some toxic and side effects
but the toxic and side effects of Coptidis Rhizoma steamed with rice wine were lower than those of Coptidis Rhizoma. Coptidis Rhizoma could reduce fasting blood glucose and improve glucose tolerance in type 2 diabetic rats before and after steaming. Although the effect of Coptidis Rhizoma steamed with rice wine had an increasing trend
there was no significant difference between before and after processing. Type 2 diabetic rats appeared mild inflammation and imbalance of oxidation-antioxidant system
characterized by significant increase of contents of interleukin (IL)-6
nuclear transcription factor (NF)-
κ
B and malondialdehyde (MDA) (
P
<
0.05
P
<
0.01)
significant decrease of glutathione peroxidase (GSH-Px)
superoxide dismutase (SOD)
catalase (CAT) and reduced glutathione (GSH) activities (
P
<
0.01). Coptidis Rhizoma could inhibit inflammation and imbalance of oxidation-antioxidant system
and alleviate pathological damage of colon before and after steaming in type 2 diabetic tats
the effect of Coptidis Rhizoma steamed with rice wine was better than that of Coptidis Rhizoma. Significant intestinal flora disorders were observed in type 2 diabetic rats
including down-regulation of Bacteroidetes
Firmicutes and Kiritimatiellaeota
up-regulation of Proteobacteria
Actinobacteria
Acidobacteria
Chloroflexi
Cyanobacteria and Verrucomicrobia. Coptidis Rhizoma could alleviate intestinal microflora disturbance before and after steaming
and the regulation effect of Coptidis Rhizoma on Kiritimatiellaeota
Cyanobacteria and Verrucomicrobia was stronger than that of Coptidis Rhizoma steamed with rice wine. Coptidis Rhizoma steamed with rice wine could significantly reduce the level of Chloroflexi
but Coptidis Rhizoma had no such effect. The intestinal microflora may be correlated with the antioxidant capacity of Coptidis Rhizoma before and after steaming.
Conclusion:
2
Coptidis Rhizoma can treat type 2 diabetic rats by regulating intestinal microflora before and after steaming with rice wine
and the effect of Coptidis Rhizoma steamed with rice wine is better. The toxic and side effects of Coptidis Rhizoma on intestinal flora are reduced by steamed with rice wine. The processing mechanism of increasing efficacy and decreasing toxicity of Coptidis Rhizoma steamed with rice wine is closely related to intestinal microflora.
关键词
Keywords
references
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