关键词: 药桑叶, 急性肾损伤, 化学成分, 抗炎, 细胞凋亡, 网络药理学

Abstract:

Objective To discuss the active components of Xinjiang characteristic plant Medicinal Mulberry Leaves against acute kidney injury （AKI） and to clarify the mechanism through ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF/MS） technology， network pharmacology， and animal experimental verification. Methods UPLC-Q-TOF/MS technology was used to analyze the chemical components in 70% ethanol percolation extract of Medicinal Mulberry Leaves and in serum of the rats at 0 and 60 min after intragastric administration； the SMILES numbers of the blood-entering active components of Medicinal Mulberry Leaves were obtained from PubMed， and then their target genes were obtained from the SIB database； meanwhile， using “acute kidney injury” as the keyword， the AKI-related targets were retrieved from databases such as the Human Gene database （GeneCards）， the Disease Gene Network database （DisGENET）， and the Online Mendelian Inheritance in Man （OMIM） disease database； the two were compared and intersected to discover the gene targets of Medicinal Mulberry Leaves for treating AKI. A total of 70 SD rats were randomly divided into blank group （10 rats） and model establishment group （60 rats）； the rats in model establishment group were intraperitoneally injected with gentamicin to establish the model for 7 d， the rats in blank group were injected with same amount of normal saline； after modeling， the serum creatinine （CRE） and urea nitrogen （BUN） levels of the rats were measured to determine whether the model was successfully prepared. A total of 60 successfully modeled rats were randomly divided into model group， low dose of Medicinal Mulberry Leaves group （120 mg·kg^-1 Medicinal Mulberry Leaves）， medium dose of Medicinal Mulberry Leaves group （300 mg·kg^-1 Medicinal Mulberry Leaves）， high dose of Medicinal Mulberry Leaves group （750 mg·kg^-1 Medicinal Mulberry Leaves）， Medicinal Mulberry Leaves enrichment substance group （750 mg·kg^-1 Medicinal Mulberry Leaves）， and positive drug group （15 mg·kg^-1 verapamil）； after treated for 14 d， the 24 h urine of the rats in various groups was collected， and the blood and kidney tissue of the rats in various groups were collected for later detection. Hematoxylin-eosin （HE） staining was used to detect the pathomorphology of kidney tissue of the rats in various groups； colorimetric method was used to detect the levels of urine protein （UP）， uric acid （UA）， β2-microglobulin （β2-MG）， albumin （ALB）， 10 kDa interferon γ-induced protein （IP-10）， kidney injury molecule-1 （KIM-1）， and neutrophil gelatinase-associated lipocalin （NGAL） in urine of the rats in various groups； microplate method was used to detect the serum creatinine （CRE） level of the rats in various groups； urease method was used to detect the serum urea nitrogen （BUN） level of the rats in various groups； ELISA method was used to detect the serum levels of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） of the rats in various groups and the activities of superoxide dismutase （SOD） and nitric oxide synthase （NOS） and the levels of malondialdehyde （MDA）， reduced glutathione （GSH）， and glutathione peroxidase （GSH-Px） in kidney tissue of the rats in various groups； Western blotting method was used to detect the expression levels of apoptosis and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （AKT） signaling pathway-related proteins in kidney tissue of the rats in various groups. Results The UPLC-Q-TOF/MS results showed that a total of 159 chemical components were identified， and 27 blood-entering components were screened out， including 8 prototype products and 19 metabolites， mainly flavonoids， benzofurans， and organic acids. The network pharmacology results showed that AKT， B-cell lymphoma-2 （Bcl-2）， and cysteinyl aspartate specific proteinase-3 （Caspase-3） were the key targets of Medicinal Mulberry Leaves for treating AKI， and the mechanism mainly involved multi-pathway linkage and integrated regulation including the PI3K/AKT signaling pathway. Compared with model group， the levels of UP and NGAL in urine of the rats in low， medium， and high doses of Medicinal Mulberry Leaves groups were significantly decreased （P<0.01）； compared with model group， the levels of KIM-1 and IP-10 in urine of the rats in medium and high doses of Medicinal Mulberry Leaves groups were significantly decreased （P<0.01）. Compared with model group， the levels of BUN and TNF-α in blood of the rats in low， medium， and high doses of Medicinal Mulberry Leaves groups were significantly decreased （P<0.05 or P<0.01）. Compared with model group， the levels of GSH and GSH-Px in kidney tissue of the rats in medium and high doses of Medicinal Mulberry Leaves groups were significantly increased （P<0.01）； compared with model group， the level of MDA and the activity of NOS in kidney tissue of the rats in medium and high doses of Medicinal Mulberry Leaves groups were significantly decreased （P<0.01）. The Western blotting results showed that compared with model group， the expression level of PI3K protein in kidney tissue of the rats in high dose of Medicinal Mulberry Leaves group was significantly decreased （P<0.01）； compared with model group， the expression levels of AKT protein in kidney tissue of the rats in low， medium， and high doses of Medicinal Mulberry Leaves groups were decreased （P<0.05 or P<0.01）； compared with model group， the level of Bcl-2 in kidney tissue of the rats in high dose of Medicinal Mulberry Leaves group was significantly increased （P<0.05）， and the levels of Caspase-3 and Bcl-2-associated X protein （Bax） were significantly decreased （P<0.05 or P<0.01）. Conclusion Medicinal Mulberry Leaves are identified to contain a total of 159 chemical components， including 27 blood-entering components， mainly flavonoids； Medicinal Mulberry Leaves have an ameliorative effect on gentamicin-induced AKI in rats， and the mechanism may be related to the inhibition of PI3K/AKT and apoptosis signaling pathways by Medicinal Mulberry Leaves.

Key words: Medicinal Mulberry Leaves, Acute kidney injury, Chemical components, Anti-inflammatory, Apoptosis, Network pharmacology