Effect of neferine on diabetic nephropathy by regulating SDF-1/CXCR4 signal pathway

doi:10.3969/j.issn.1674-8115.2024.02.004

Abstract

Abstract:

Objective ·To investigate the effect of neferine (Nef) on renal tissues of diabetic nephropathy (DN) rats and its related mechanism. Methods ·DN model rats were constructed by feeding high-fat diet combined with intraperitoneal injection of streptozotocin, and the successfully constructed rats were randomly divided into DN group, Nef (low, medium and high) dose groups and Nef high-dose+pathway antagonist (AMD3100) group, with 10 rats in each group. At the same time, 10 common rats were selected as the normal group. The levels of fasting blood glucose (FBG), 24 h urinary protein, serum glycosylated hemoglobin (HbA1c), serum creatinine (Scr), blood urea nitrogen (BUN) and renal index of rats in the six groups were measured. Hematoxylin-eosin (H-E) and Masson staining were used to observe the pathological changes of renal tissues. The content of malondialdehyde (MDA) in renal tissues was determined by thiobarbituric acid (TBA) method, and the activities of superoxide dismutase (SOD) and catalase (CAT) in renal tissues were determined by water soluble tetrazolium (WST-1) method and ammonium molybdate method, respectively. The mRNA and protein expressions of stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in renal tissues were detected by quantitative real-time PCR (qPCR) and Western blotting, respectively. Rat renal tubular epithelium cells NRK-52E were induced by high glucose (30 mmol/L glucose) to establish DN cell model. The cells were divided into control group, high glucose (HG) group, HG+Nef (low, medium and high) dose (i.e.HG+Nef-L, M and H) group, and HG+Nef-H +AMD3100 group. SOD and CAT activities were detected by WST-1 method and ammonium molybdate method, respectively. MDA content was detected by TBA method. The mRNA and protein expressions of SDF-1 and CXCR4 were detected by qPCR and Western blotting, respectively. CCK-8 method and flow cytometry were used to detect cell viability and apoptosis rate, respecti-vely. Results ·Compared with the DN group, the levels of FBG, 24 h urinary protein, HbA1c, Scr, BUN, renal index and MDA content in Nef (low, medium and high) dose groups and Nef high-dose+AMD3100 group were decreased, the mRNA and protein expressions of SDF-1 and CXCR4 were increased, and the activities of SOD and CAT were increased (all P<0.05). The degree of pathological damage and fibrosis of renal tissues was reduced; all of the above changes were dose-dependent. AMD3100 could weaken the renal protective effect of high-dose Nef on DN rats. Compared with the HG group, NRK-52E cell viability, SOD and CAT activities, and the mRNA and protein expressions of SDF-1 and CXCR4 were increased in HG+Nef-L, M and H groups, while apoptosis rate and MDA content were decreased (all P<0.05). AMD3100 could reverse the protective effect of Nef-H on NRK-52E cell damage. Conclusion ·Nef may control blood glucose levels on DN rats and improve antioxidant capacity by activating the SDF-1/CXCR4 signal pathway, playing a renal protective role.

Key words: diabetic nephropathy (DN), neferine (Nef), renal, stromal cell-derived factor-1/CXC chemokine receptor 4 signal pathway

CLC Number:

R587.2

WANG Ying, PING Lifeng, LIU Tongtong, LIU Shanshan, LIU Lei. Effect of neferine on diabetic nephropathy by regulating SDF-1/CXCR4 signal pathway[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(2): 183-195.

Figures/Tables 12

Tab 1 Primer sequence for qPCR

Primer	Forward sequence (5'→3')	Reverse sequence (5'→3')
Sdf-1	GACGGTAAGCCAGTCAGCCT	CAGGTACCTCTGGATCCACT
Cxcr4	ACGCACGACGTCTTCCAGTA	CCACCTGGTTCAACTCACTCC
β-actin	GCACCACACCTTCTACAATG	TGCTTGCTGATCCACATCTG

Fig 1 Comparison of the FBG and 24 h urinary protein levels of rats in each group

Fig 2 Comparison of the serum HbA1c, Scr, BUN levels and renal index of rats in each group

Fig 3 Observation of the pathological morphology of the renal tissues of rats in each group (H-E staining, ×200)

Fig 4 Observation of renal tissue fibrosis of rats in each group (Masson staining, ×200)

Fig 5 Comparison of MDA content and SOD, CAT activity in renal tissues of rats in each group

Fig 6 Comparison of Sdf-1 and Cxcr4 mRNA expression in renal tissues of rats in each group

Fig 7 SDF-1 and CXCR4 protein expression in renal tissues of rats in each group

Fig 8 Comparison of MDA content and SOD, CAT activity in NRK-52E cells of each group

Fig 9 Comparison of Sdf-1 and Cxcr4 mRNA expression in NRK-52E cells of each group

Fig 10 SDF-1 and CXCR4 protein expression in NRK-52E cells of each group

Fig 11 Comparison of NRK-52E cell activity and apoptosis rate in each group

TrendMD

References 34

1	SAGOO M K, GNUDI L. Diabetic nephropathy: an overview[J]. Methods Mol Biol, 2020, 2067: 3-7.
2	SAMSU N. Diabetic nephropathy: challenges in pathogenesis, diagnosis, and treatment[J]. Biomed Res Int, 2021, 2021: 1497449.
3	SHI Y, RIESE D J 2nd, SHEN J Z. The role of the CXCL12/CXCR4/CXCR7 chemokine axis in cancer[J]. Front Pharmacol, 2020, 11: 574667.
4	SONG A N, JIANG A N, XIONG W, et al. The role of CXCL12 in kidney diseases: a friend or foe?[J]. Kidney Dis (Basel), 2021, 7(3): 176-185.
5	ZHANG Q Z, HE L, DONG Y, et al. Sitagliptin ameliorates renal tubular injury in diabetic kidney disease via STAT3-dependent mitochondrial homeostasis through SDF-1α/CXCR4 pathway[J]. FASEB J, 2020, 34(6): 7500-7519.
6	ZHANG T T, JIANG J G. Active ingredients of traditional Chinese medicine in the treatment of diabetes and diabetic complications[J]. Expert Opin Investig Drugs, 2012, 21(11): 1625-1642.
7	BHARATHI PRIYA L, HUANG C Y, HU R M, et al. An updated review on pharmacological properties of neferine: a bisbenzylisoquinoline alkaloid from Nelumbo nucifera[J]. J Food Biochem, 2021, 45(12): e13986.
8	CEVIK M, GOBEKA H H, AYDEMIR O. Effects of neferine on retinal tissue in experimental diabetic rat model[J]. Int Ophthalmol, 2023, 43(1): 249-260.
9	LI J, CHOU H Y, LI L, et al. Wound healing activity of neferine in experimental diabetic rats through the inhibition of inflammatory cytokines and Nrf-2 pathway[J]. Artif Cells Nanomed Biotechnol, 2020, 48(1): 96-106.
10	李莉, 李松, 曹萌, 等. MiR-146a-5p对高脂饮食/链脲佐菌素诱导的糖尿病肾病大鼠肾组织的保护作用[J]. 解剖学杂志, 2021, 44(2): 108-113.
	LI L, LI S, CAO M, et al. Protective effect of miR-146a-5p on renal tissue in high-fat diet/streptozotocin-induced diabetic nephropathy rats[J]. Chinese Journal of Anatomy, 2021, 44(2): 108-113.
11	SINGH L, RANA S, MEHAN S. Role of adenylyl cyclase activator in controlling experimental diabetic nephropathy in rats[J]. Int J Physiol Pathophysiol Pharmacol, 2018, 10(5): 144-153.
12	张培培, 夏虹, 鲁科达, 等. 加味黄风汤对糖尿病肾病模型大鼠肾组织8-OHdG表达的影响[J]. 浙江中西医结合杂志, 2020, 30(8): 618-622, 696.
	ZHANG P P, XIA H, LU K D, et al. Effect of modified Huangfeng decoction on the expression of 8-OHdG in renal tissue of diabetic nephropathy rats[J]. Zhejiang Journal of Integrated Traditional Chinese and Western Medicine, 2020, 30(8): 618-622, 696.
13	WANG Y Y, WANG S Z, WANG R, et al. Neferine exerts antioxidant and anti-inflammatory effects on carbon tetrachloride-induced liver fibrosis by inhibiting the MAPK and NF-κB/IκBα pathways[J]. Evid Based Complement Alternat Med, 2021, 2021: 4136019.
14	叶泽华, 夏煜琦, 李柏均, 等. CXCR4在草酸钙晶体致肾损伤及纤维化中的作用和机制[J]. 中华泌尿外科杂志, 2022, 43(4): 285-290.
	YE Z H, XIA Y Q, LI B J, et al. The role and mechanism of CXCR4 in renal injury and fibrosis caused by calcium oxalate crystals[J]. Chinese Journal of Urology, 2022, 43(4): 285-290.
15	TAN H T, CHEN J X, LI Y C, et al. Glabridin, a bioactive component of licorice, ameliorates diabetic nephropathy by regulating ferroptosis and the VEGF/Akt/ERK pathways[J]. Mol Med, 2022, 28(1): 58.
16	BHARATHI PRIYA L, BASKARAN R, HUANG C Y, et al. Neferine modulates IGF-1R/Nrf2 signaling in doxorubicin treated H9c2 cardiomyoblasts[J]. J Cell Biochem, 2018, 119(2): 1441-1452.
17	李建坤, 苏红宁. CXCR4对PDGF-BB诱导的血管平滑肌细胞增殖、迁移的影响及白藜芦醇的抑制作用[J]. 中药新药与临床药理, 2019, 30(7): 766-770.
	LI J K, SU H N. The role of CXCR4 in proliferation and migration of vascular smooth muscle cells and the inhibition of resveratrol[J]. Traditional Chinese Drug Research and Clinical Pharmacology, 2019, 30(7): 766-770.
18	SELBY N M, TAAL M W. An updated overview of diabetic nephropathy: diagnosis, prognosis, treatment goals and latest guidelines[J]. Diabetes Obes Metab, 2020, 22(Suppl 1): 3-15.
19	LI S J, ZHANG Y Y, ZHANG J, et al. Neferine exerts ferroptosis-inducing effect and antitumor effect on thyroid cancer through Nrf2/HO-1/NQO1 inhibition[J]. J Oncol, 2022, 2022: 7933775.
20	LIN T Y, HUNG C Y, CHIU K M, et al. Neferine, an alkaloid from lotus seed embryos, exerts antiseizure and neuroprotective effects in a kainic acid-induced seizure model in rats[J]. Int J Mol Sci, 2022, 23(8): 4130.
21	YOUSIF HUSSIN ALIMAM H, ABDELATEIF HUSSEIN W, IBRAHIM S, et al. Blood glucose, HbA1c level, and its correlation with VEGF-A (+405G/C) polymorphism as biomarker predicts the risk of retinopathy and nephropathy in type 2 diabetic patients[J]. Rep Biochem Mol Biol, 2022, 11(3): 421-429.
22	李燕菲. 糖尿病患者HbA1c、β₂-MG、α₁-MG、尿微量白蛋白/肌酐变化及与早期肾功能损害的关系分析[J]. 医学理论与实践, 2023, 36(2): 293-295.
	LI Y F. Changes of HbA1c, β₂-MG, α₁-MG, urinary microalbumin/creatinine and their relationship with early renal function impairment in patients with diabetes mellitus[J]. The Journal of Medical Theory and Practice, 2023, 36(2): 293-295.
23	司珩, 李梦檀. HbAlc、CK及FBG联合检测在糖尿病微血管病变诊断中的应用[J]. 医药论坛杂志, 2021, 42(2): 138-140, 145.
	SI H, LI M T. Application of HbAlc, CK and FBG combined detection in the diagnosis of diabetic microangionopathy[J]. Journal of Medical Forum, 2021, 42(2): 138-140, 145.
24	LI H H, CHEN W H, CHEN Y S, et al. Neferine attenuates acute kidney injury by inhibiting NF-κB signaling and upregulating klotho expression[J]. Front Pharmacol, 2019, 10: 1197.
25	HERNANDEZ L F, EGUCHI N, WHALEY D, et al. Anti-oxidative therapy in diabetic nephropathy[J]. Front Biosci (Schol Ed), 2022, 14(2): 14.
26	MA X J, MA J R, LENG T, et al. Advances in oxidative stress in pathogenesis of diabetic kidney disease and efficacy of TCM intervention[J]. Ren Fail, 2023, 45(1): 2146512.
27	TANG Y S, ZHAO Y H, ZHONG Y, et al. Neferine inhibits LPS-ATP-induced endothelial cell pyroptosis via regulation of ROS/NLRP3/caspase-1 signaling pathway[J]. Inflamm Res, 2019, 68(9): 727-738.
28	ZHANG X, LIAO Y, YE T. Correlations of SDF-1 and CXCR4 levels with caspase-3 expression in the retina of rats after optic nerve injury[J]. Int J Clin Exp Pathol, 2020, 13(8): 2058-2064.
29	SIDDIQI F S, CHEN L H, ADVANI S L, et al. CXCR4 promotes renal tubular cell survival in male diabetic rats: implications for ligand inactivation in the human kidney[J]. Endocrinology, 2015, 156(3): 1121-1132.
30	TAKASHIMA S, FUJITA H, FUJISHIMA H, et al. Stromal cell-derived factor-1 is upregulated by dipeptidyl peptidase-4 inhibition and has protective roles in progressive diabetic nephropathy[J]. Kidney Int, 2016, 90(4): 783-796.
31	TANG W M, PANJA S, JOGDEO C M, et al. Study of renal accumulation of targeted polycations in acute kidney injury[J]. Biomacromolecules, 2022, 23(5): 2064-2074.
32	CHEN L H, ADVANI S L, THAI K, et al. SDF-1/CXCR4 signaling preserves microvascular integrity and renal function in chronic kidney disease[J]. PLoS One, 2014, 9(3): e92227.
33	YANG J, ZHU F M, WANG X H, et al. Continuous AMD3100 treatment worsens renal fibrosis through regulation of bone marrow derived pro-angiogenic cells homing and T-cell-related inflammation[J]. PLoS One, 2016, 11(2): e0149926.
34	魏萍, 薛春苗, 潘霖, 等. 甲基莲心碱对胰岛素抵抗的HepG2细胞降糖的影响[J]. 临床和实验医学杂志, 2020, 19(12): 1283-1286.
	WEI P, XUE C M, PAN L, et al. Effects of neferine on hypoglycemia of insulin-resistant HepG2 cells[J]. Journal of Clinical and Experimental Medicine, 2020, 19(12): 1283-1286.

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