基于药桑叶抗急性肾损伤潜在活性成分及作用机制的UPLC-Q-TOF/MS和网络药理学分析及其实验验证

doi:10.13481/j.1671-587X.20260107

Abstract

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

CLC Number:

R692.5

Huaimin LIANG,Jiacheng JIN,Wenhua CHEN,Yuyao LI,Hangyu WANG,Ke ZHANG,Jinhui WANG. UPLC-Q-TOF/MS and network pharmacology analysis and experimental verification based on potential active ingredients and mechanisms of medicinal Mulberry Leaves in anti-acute kidney injury[J].Journal of Jilin University(Medicine Edition), 2026, 52(1): 56-69.

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References 21

[1]	WILSON F P， YAMAMOTO Y， MARTIN M， et al. A randomized clinical trial assessing the effect of automated medication-targeted alerts on acute kidney injury outcomes［J］. Nat Commun， 2023， 14（1）： 2826.
[2]	STARKEL S， GOODBERLET M， SCHULER B， et al. Evaluating the incidence of acute kidney injury and gentamicin synergy dosing for endocarditis［J］. JAC- Antimicrob Resist， 2024， 6（1）： dlad144-dlad144.
[3]	ZHU J， YUAN A N， LE Y F， et al. Yi-Qi-Jian-Pi-Xiao-Yu formula inhibits cisplatin-induced acute kidney injury through suppressing ferroptosis via STING-NCOA4-mediated ferritinophagy［J］. Phytomedicine， 2024， 135： 156189-156189.
[4]	贺水花，杨玲. 新疆特色植物药桑多酚的研究进展［J］. 食品研究与开发， 2021， 42（20）： 167-172.
[5]	LI R L， WANG J， LIU J Y， et al. Mulberry leaf and its effects against obesity： A systematic review of phytochemistry， molecular mechanisms and applications［J］. Phytomedicine， 2024， 128： 155528.
[6]	PAN Y， SONG Y S， ZHAO M J， et al. Mulberry leaf extract ameliorates high-fat diet-induced obesity in mice by regulating the gut microbiota and metabolites［J］. Food Biosci， 2024， 62： 105359.
[7]	HU Y X， ZHANG Y， CUI X A， et al. Structure-function relationship and biological activity of polysaccharides from mulberry leaves： a review［J］. Int J Biol Macromol， 2024， 268（P1）： 131701.
[8]	YAO Q F， ZHANG X Y， WANG L F， et al. Therapeutic potential of p-coumaric acid in alleviating renal fibrosis through inhibition of M2 macrophage infiltration and cellular communication［J］. Phytomedicine， 2025， 139：156507.
[9]	李炳钦. 新疆药桑叶化学成分和生物活性研究［D］. 阿拉尔：塔里木大学， 2019.
[10]	马志，孟庆海，蒯美玉，等. 桑酮碱对2型糖尿病肾病大鼠的保护作用及机制［J］. 中成药， 2016， 38（6）： 1215-1220.
[11]	寇婕，石梦茜，吕晨慧，等. 茶多酚通过抑制线粒体凋亡改善糖尿病肾病大鼠肾功能的研究［J］. 营养学报， 2023， 45（1）： 70-75.
[12]	CHEN Y L， HUANG J Y， WANG H X， et al. Polysaccharides from Sacha Inchi shell reduces renal fibrosis in mice by modulating the TGF-β1/Smad pathway and intestinal microbiota［J］. Int J Biol Macromol， 2024， 280： 136039.
[13]	WALKOWSKI B， KLEIBERT M， MAJKA M， et al. Insight into the role of the PI3K/Akt pathway in ischemic injury and post-infarct left ventricular remodeling in normal and diabetic heart［J］. Cells， 2022， 11（9）： 1553.
[14]	黄丹，周斌，彭盛亮，等. PI3K-Akt信号通路在大鼠肾缺血再灌注损伤的作用及白黎芦醇的干预研究［J］. 吉林医学， 2024， 45（12）： 2897-2900.
[15]	万金艳，龙宇，张羽璐，等. PI3K/Akt信号通路在糖尿病肾病中的作用及中药干预的研究进展［J］. 中草药， 2021， 52（12）： 3705-3716.
[16]	郭晨雪，刘笑芬，曲星伊，等. 细胞凋亡在药物诱导的急性肾小管坏死中的作用［J］. 中国感染与化疗杂志， 2023， 23（2）： 254-260.
[17]	雷鸣琤. 电针通过抑制肾脏细胞凋亡和炎症反应减轻急性肾损伤的作用机制研究［D］. 石家庄：河北医科大学， 2024.
[18]	陈雪，王莺，康晶，等. 毛蕊花糖苷对热损伤睾丸间质细胞凋亡的保护作用［J］. 中国兽医杂志， 2021， 57（6）： 70-75.
[19]	QI H， YIN Y S， YIN Z Y， et al. Mitochondrial outer membrane permeabilization and inner membrane permeabilization in regulating apoptosis and inflammation［J］. J Theor Biol， 2023， 571： 111558.
[20]	李瑞杰，项瑞君，郑树眺，等. 基于NF-κB/NLRP3/Caspase-1信号通路探究大黄素对草酸钙肾结石模型大鼠的影响［J］. 解放军医学院学报， 2024， 45（11）： 1159-1166.
[21]	芮翊馨，谢红潇，李丹，等. 中乌宁通过BAX/BCL-2/Caspase3信号通路抑制细胞凋亡改善慢性肾衰竭大鼠肾纤维化［J］. 中药药理与临床， 2024， 40（5）： 42-48.

Number	Retention time (t/min)	Compound name	Molecular formula	Mass-to-charge ratio(m/z)	Error (ppm)
1	0.56	Kuwanol D	C₂₅H₂₈O₅	453.192 5	1.43
2	9.04	Wittifuran U	C24H₂₆O₆	455.172 1	2.15
3	0.58	Melezitose	C₁₈H₃₂O₁₆	527.159 9	3.16
4	7.23	Kaempferol-3-O-rutinoside	C₂₇H₃₀O₁₅	595.167 7	3.23
5	4.29	Aurantiamide	C₂₅H₂₆N₂O₃	425.182 7	-2.09
6	3.18	Bakuchalcone	C₂₀H₂₀O₅	363.120 6	0.84
7	3.36	Wittifuran B	C24H₂₄O₅	415.152 8	2.96
8	2.62	Mulberroside F	C₂₆H₃₀O₁₄	567.171 1	0.40

Number	Retentiontime (t/min)	Compound name	Molecular formula	Mass-to-charge ratio(m/z)	Error (ppm)
1	0.56	Mulberrofuran Y	C₂₅H₂₈O₅	453.1910	-1.91
2	3.17	Albanin F	C₄₀H₃₆O₁₁	737.2242	0.39
3	10.47	Eicosenoic acid	C₂₀H₃₈O₂	309.2787	-3.94
4	3.73	Kynurenine	C₁₀H₁₂N₂O₃	253.0832	0.90
5	3.73	Morrole G	C₁₁H₁₂N₂O₄	281.0788	3.26
6	3.14	Kuwanon W	C₄₅H₄₂O₁₁	757.2656	0.16
7	0.56	2',4'-dihydroxy-7'-methoxy-8-prenylflavan	C₂₁H₂₄O₄	363.1574	2.04
8	2.75	2-O-α-D-galactopyranosyl-1-deoxynojirimycin	C₁₂H₂₃NO₉	326.1432	-4.26
9	8.73	Behenic acid	C₂₂H₄₄O₂	341.3417	0.76
10	7.50	Kuwanol C	C₂₅H₂₆O₆	445.1623	0.39
11	7.72	Sanggenol N.	C₂₅H₂₆O₆	445.1618	-0.82
12	0.58	D-pantothenic acid	C₉H₁₇NO₅	220.1184	2.17
13	5.29	Isobaisseoside.	C₂₁H₂₆O₁₃	509.1277	2.33
14	4.77	Kuwanol B	C₃₄H₂₆O₈	563.1676	-4.28
15	0.59	N-(3-Amino-3-oxopropyl 3,4-DHP	C₈H₁₆N₂O₄	205.1173	-4.98
16	6.80	Palmitoleic acid	C₁₆H₃₀O₂	277.2128	-3.68
17	2.81	Euchrenone a7	C₂₀H₂₀O₅	363.1198	-1.40
18	9.02	Sanggenon G	C₄₀H₃₈O₁₁	695.2457	-4.31
19	7.80	Sanggenon N	C₂₅H₂₆O₆	445.1635	2.94

Group	n	CRE [c_B/(μmol·L^-1)]	BUN [c_B/(mmol·L^-1)]
Blank	10	65.76±5.50	3.78±1.08
Modeling	60	211.27±42.71^*	14.56±4.02^*

Group	CRE [c_B/(μmol·L^-1)]	BUN [c_B/(mmol·L^-1)]	UA [c_B/(μmol·L^-1)]	UP [ρ_B/(g·L^-1)]
Blank	53.76±6.08	4.57±0.88	35.40±15.32	370.75±103.10
Model	68.43±6.54^*	10.32±0.88^*	88.33±23.56^*	626.00±149.44^*
Medicinal Mulberry Leaves
Low dose	67.28±5.57	4.81±0.37^△	93.18±15.09	396.60±122.26^△
Medium dose	64.76±8.09	3.65±0.46^△	78.07±15.48	384.80±68.93^△
High dose	59.21±6.94	3.29±0.94^△	65.31±8.38	390.48±92.64^△
Medicinal Mulberry Leaves enrichment substance	52.20±2.45^△	5.44±0.57^△	47.84±7.57^△	545.77±52.62
Positive drug	63.36±5.02	3.72±0.67^△	66.75±14.50	379.37±54.87^△

Group	β2-MG[ρ_B/(μg·L^-1)]	ALB[ρ_B/(mg·L^-1)]	IP-10[ρ_B/(ng·L^-1)]	KIM-1[ρ_B/(μg·L^-1)]	NGAL[ρ_B/(ng·L^-1)]
Blank	0.85±0.04	36.84±10.93	93.90±19.66	0.77±0.11	395.78±81.77
Model	1.12±0.11^*	52.43±9.95^*	414.18±128.44^**	6.07±0.88^**	1 815.37±123.92^**
Medicinal Mulberry Leaves
Low dose	1.04±0.06	50.35±1.67	300.64±107.85	5.25±1.00	691.43±48.83^△
Medium dose	1.06±0.17	47.74±6.61	191.90±43.80^△	3.06±0.62^△	408.77±48.67^△
High dose	0.98±0.23	42.96±5.90	99.06±29.89^△	1.31±0.45^△	173.26±60.34^△
Medicinal Mulberry Leaves enrichment substance	1.08±0.16	42.73±5.33	214.07±62.52^△	1.68±0.53^△	390.15±93.91^△
Positive drug	0.91±0.08	46.33±5.84	100.55±28.92^△	5.13±1.42	470.71±118.28^△

UPLC-Q-TOF/MS and network pharmacology analysis and experimental verification based on potential active ingredients and mechanisms of medicinal Mulberry Leaves in anti-acute kidney injury

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Group	TNF-α	IL-6
Blank	13.03±5.62	33.66±22.85
Model	58.72±13.11^*	94.98±30.90^*
Medicinal Mulberry Leaves
Low dose	40.68±13.66^△	73.49±8.36
Medium dose	14.75±5.64^△△	70.17±11.44
High dose	16.56±6.89^△△	68.87±17.72
Medicinal Mulberry Leaves enrichment substance	22.42±7.42^△△	75.31±13.11
Positive drug	29.67±10.78^△△	55.73±8.45^△△

Group	GSH [m_B/（μmol·g^-1）]	MDA [m_B/(mmol·g^-1)]	GSH-Px [λ_B/(U·mg^-1)]	SOD [λ_B/(U·mg^-1)]	NOS [λ_B/(U·mg^-1)]
Blank	0.31±0.02	2.32±0.17	757.43±38.93	211.17±30.56	0.17±0.03
Model	0.16±0.01^*	5.17±0.22^*	434.49±54.53^*	97.43±8.73^*	0.87±0.04^*
Medicinal Mulberry Leaves
Low dose	0.19±0.01	4.76±0.09	562.66±47.25^△	130.27±6.93	0.76±0.03^△
Medium dose	0.20±0.01^△	4.19±0.37^△	598.96±49.79^△	129.07±12.68	0.68±0.04^△
High dose	0.28±0.01^△	3.77±0.24^△	726.85±36.76^△	147.10±20.53^△	0.65±0.03^△
Medicinal Mulberry Leaves enriched substance	0.21±0.02^△	4.13±0.10^△	637.29±27.84^△	161.12±13.17^△	0.60±0.04^△
Positive drug group	0.23±0.02^△	3.27±0.27^△	656.88±41.47^△	178.57±29.65^△	0.37±0.04^△

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