Analysis of Mechanism of Yishen Huashi Granules for Improving Diabetic Nephropathy Based on Metabonomics and Transcriptomics

SHEN Zhen; CUI Tao; AI Shanshan; LI Xiangyu; GAO Ranran; LI Wei; HAN Cong

doi:10.13422/j.cnki.syfjx.20230565

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Analysis of Mechanism of Yishen Huashi Granules for Improving Diabetic Nephropathy Based on Metabonomics and Transcriptomics

更新时间：2023-09-21

- Analysis of Mechanism of Yishen Huashi Granules for Improving Diabetic Nephropathy Based on Metabonomics and Transcriptomics
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 20, Pages: 109-117(2023)
- 作者机构：
  
  1.山东中医药大学第一临床医学院，济南250014
  2.济南市章丘区中医医院，济南250200
  3.济宁医学院，山东济宁272067
  4.山东中医药大学附属医院，济南250014
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230565
  CLC： R22;R28;R969.1;O657
- Received：28 October 2022，
  
  Published Online：06 January 2023，
  
  Published：20 October 2023
- 稿件说明：
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申振,崔涛,艾珊珊等.基于代谢组学及转录组学的益肾化湿颗粒改善糖尿病肾病作用机制分析[J].中国实验方剂学杂志,2023,29(20):109-117.

SHEN Zhen,CUI Tao,AI Shanshan,et al.Analysis of Mechanism of Yishen Huashi Granules for Improving Diabetic Nephropathy Based on Metabonomics and Transcriptomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(20):109-117.
申振,崔涛,艾珊珊等.基于代谢组学及转录组学的益肾化湿颗粒改善糖尿病肾病作用机制分析[J].中国实验方剂学杂志,2023,29(20):109-117. DOI： 10.13422/j.cnki.syfjx.20230565.

SHEN Zhen,CUI Tao,AI Shanshan,et al.Analysis of Mechanism of Yishen Huashi Granules for Improving Diabetic Nephropathy Based on Metabonomics and Transcriptomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(20):109-117. DOI： 10.13422/j.cnki.syfjx.20230565.

摘要

目的

基于超高效液相色谱-串联质谱法（UHPLC-MS/MS）及高通量转录组测序技术（RNA-seq）探讨益肾化湿颗粒调节血清代谢物及肾脏信使核糖核酸（mRNA）表达来改善糖尿病肾病（DKD）的作用机制。

方法

SD大鼠随机分为正常组、模型组、益肾化湿颗粒组，每组8只。以链脲佐菌素腹腔注射建立DKD大鼠模型，益肾化湿颗粒组给予益肾化湿颗粒5.54 g·kg

-1

·d

-1

灌胃，正常组与模型组给予等量生理盐水，持续6周。实验期间监测大鼠体质量、血糖，末次给药24 h后麻醉，下腔静脉取血，分离血清，检测肾功能、血脂及炎症指标，取大鼠肾组织经苏木素-伊红（HE）、马松（Masson）与过碘酸雪夫（PAS）染色观察肾脏病理变化，通过UHPLC-MS/MS与RNA-seq分析血清代谢变化及肾脏mRNA表达差异，实时荧光定量聚合酶链式反应（Real-time PCR）、蛋白免疫印迹法（Western blot）检测肾脏组织差异mRNA及蛋白表达，探寻共性表达机制。

结果

与正常组比较，模型组大鼠体质量减少，血糖、尿微量白蛋白与尿肌酐比值（UACR）、尿素氮（BUN）、胱抑素-C（Cys-C）、

-微球蛋白（

-MG）、白细胞介素-6（IL-6）、甘油三酯（TG）及总胆固醇（TC）均显著增高，总超氧化物歧化酶（T-SOD）显著降低（

0.01）；与模型组比较益肾化湿颗粒组大鼠各指标均有不同程度改善（

0.05，

0.01）。与正常组比较，模型组大鼠肾脏系膜基质增生、纤维组织增生及小管空泡变性；与模型组比较，益肾化湿颗粒组大鼠肾脏病理组织得到一定程度改善。共鉴定出益肾化湿颗粒作用靶代谢物14个及靶mRNA 96个，靶代谢物主要富集于鞘脂代谢、甘油磷脂代谢、苯丙氨酸、酪氨酸和色氨酸的生物合成等20条代谢通路；靶mRNA经富集得到与糖脂代谢密切相关的前20条通路中共涉及21个差异mRNA；血清差异代谢物与肾脏差异mRNA共同富集到甘油磷脂代谢、花生四烯酸代谢、嘌呤代谢、初级胆汁酸生物合成、抗坏血酸和醛酸代谢、半乳糖代谢等6条通路，其中共涉及磷脂酰乙醇胺（PE）等7个差异代谢物及腺苷酸环化酶3（ADCY3）等7个差异mRNA。经受试者工作特征（ROC）曲线验证，7个差异代谢物有较高的预测准确性，Real-time PCR及Western blot验证结果与测序结果高度一致。

结论

益肾化湿颗粒能够改善DKD大鼠UACR、BUN等生化指标，纠正糖脂代谢紊乱，改善肾功能，其作用机制可能与调节PE等血清代谢物的水平及肾脏细胞质磷酸酶1（Phospho1）等mRNA的表达有关，其中甘油磷脂代谢等6条通路可能发挥重要作用。

Abstract

Objective

In this study， based on ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS/MS） and high-throughput transcriptome sequencing technology（RNA-seq）， we investigated the mechanism of Yishen Huashi granules in regulating serum metabolites and renal messenger ribonucleic acid（mRNA） expression to improve diabetic kidney disease（DKD）.

Method

SD rats were randomly divided into normal group ， model group and Yishen Huashi granules group， with 8 rats in each group. The rat model of DKD was established by intraperitoneal injection of streptozotocin. Yishen Huashi granules group was given 5.54 g·kg

-1

·d

-1

of Yishen Huashi granules by gavage， and the normal group and the model group were given the same amount of normal saline for 6 weeks. During the experiment， the body weight and blood glucose of rats were monitored， and the rats were anesthetized 24 hours after the last administration， blood was collected from the inferior vena cava， serum was separated， and renal function， blood lipid， and inflammatory indicators were detected. Kidney tissue of rats was fixed in neutral paraformaldehyde， and stained with hematoxylin-eosin（HE）， Masson and periodic acid-Schiff（PAS） to observe the renal pathological changes. UHPLC-MS/MS and RNA-seq were used to identify the changes of serum metabolism and the differences of renal mRNA expression， and real time fluorescence quantitative polymerase chain reaction（Real-time PCR） and Western blot were used to detect the differential mRNA and protein expression in renal tissue to explore the common expression mechanism.

Result

Compared with the normal group， rats in the model group showed a decrease in body weight， a significant increase in blood glucose， urinary microalbumin to urinary creatinine ratio（UACR）， blood urea nitrogen（BUN）， cystatin-C（Cys-C），

-microglobulin（

-MG）， interleukin-6（IL-6）， triglyceride（TG） and total cholesterol（TC）， and a significant decrease in total superoxide dismutase（T-SOD）（

0.01）. After the intervention of Yishen Huashi granules， all the indexes were improved to different degrees in rats（

0.05，

0.01）. Compared with the normal group， the model group showed renal mesangial stromal hyperplasia， fibrous tissue hyperplasia and tubular vacuolar degeneration. Compared with the model group， the renal pathology of rats in Yishen Huashi granules group was improved to a certain extent. A total of 14 target metabolites and 96 target mRNAs were identified， the target metabolites were mainly enriched in 20 metabolic pathways， including sphingolipid metabolism， glycerophospholipid metabolism， and the biosynthesis of phenylalanine， tyrosine and tryptophan. The target mRNAs were enriched to obtain a total of 21 differential mRNAs involved in the TOP20 pathways closely related to glycolipid metabolism. A total of 6 pathways， glycerophospholipid metabolism， arachidonic acid metabolism， purine metabolism， primary bile acid biosynthesis， ascorbic acid and uronic acid metabolism， and galactose metabolism， were enriched by serum differential metabolites and renal differential mRNAs， among them， there were 7 differential metabolites such as phosphatidylethanolamine（PE） and 7 differential mRNAs such as recombinant adenylate cyclase 3（ADCY3）. Seven differential metabolites had high predictive accuracy as verified by receiver operating characteristic（ROC） curve， and the results of Real-time PCR and Western blot were highly consistent with the sequencing results.

Conclusion

Yishen Huashi granules can reduce UACR， BUN and other biochemical indexes， correct the disorder of glucose and lipid metabolism， and improve renal function of DKD rats. And its mechanism may be related to the regulation of the level of PE and other blood metabolites， and expression of Phospho1 and other mRNAs in the kidney， of which six pathways， including glycerophospholipid metabolism， may play an important role.

关键词

Keywords

references

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