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

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