Neferine inhibits the progression of diabetic nephropathy by modulating the miR-17-5p/nuclear factor E2-related factor 2 axis

doi:10.19852/j.cnki.jtcm.20231204.004

Abstract

Abstract:

OBJECTIVE: To investigate the effect of Neferine (Nef) on diabetic nephropathy (DN) and to explore the mechanism of Nef in DN based on miRNA regulation theory.

METHODS: A DN mouse model was constructed and treated with Nef. Serum creatinine (Crea), blood urea (UREA) and urinary albumin were measured in mice by kits, and renal histopathological changes and fibrosis were observed by hematoxylin-eosin staining and Masson staining. Renal tissue superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) activities were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to detect the expression of nuclear factor E2-related factor 2 (Nrf2)/ heme oxygenase 1 (HO-1) signaling pathway-related proteins in kidney tissues. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-17-5p in kidney tissues. Subsequently, a DN in vitro model was constructed by high glucose culture of human mesangial cells (HMCs), cells were transfected with miR-17-5p mimic and/or treated with Nef, and we used qRT-PCR to detect cellular miR-17 expression, flow cytometry to detect apoptosis, ELISAs to detect cellular SOD, MDA, and GSH-Px activities, Western blots to detect Nrf2/HO-1 signaling pathway-related protein expression, and dual luciferase reporter gene assays to verify the targeting relationship between Nrf2 and miR-17-5p.

RESULTS: Administration of Nef significantly reduced the levels of blood glucose, Crea, and UREA and the expression of miR-17-5p, improved renal histopathology and fibrosis, significantly reduced MDA levels, elevated SOD and GSH-Px activities, and activated Nrf2 expression in kidney tissues from mice with DN. Nrf2 is a post-transcriptional target of miR-17-5p. In HMCs transfected with miR-17-5p mimics, the mRNA and protein levels of Nrf2 were significantly suppressed. Furthermore, miR-17-5p overexpression and Nef intervention resulted in a significant increase in high glucose-induced apoptosis and MDA levels in HMCs and a significant decrease in the protein expression of HO-1 and Nrf2.

CONCLUSION: Collectively, these results indicate that Nef has an ameliorative effect on DN, and the mechanism may be through the miR-17-5p/Nrf2 pathway.

Key words: diabetic nephropathies, neferine, miR-17-5p, NF-E2-related factor 2, oxidative stress

HUANG Hongmei, YANG Maojun, LI Ting, WANG Dandan, LI Ying, TANG Xiaochi, YUAN Lu, GU Shi, XU Yong. Neferine inhibits the progression of diabetic nephropathy by modulating the miR-17-5p/nuclear factor E2-related factor 2 axis[J]. Journal of Traditional Chinese Medicine, 2024, 44(1): 44-53.

Figures/Tables 5

Table 1 Blood biochemistry and blood glucose of each group of mice ($\bar{x}±s$)

Group	n	BG (mmol/L)	Crea (μmol/L)	UREA (mmol/L)	ALB (g/L)
Control	7	7.5±0.6	56.1±24.0	5.4±1.4	30.0±3.0
DN	7	22.2±0.4^a	159.5±24.0^a	18.6±3.2^a	51.3±7.3^a
DN+RSG	7	13.0±0.7^b	99.7±13.1^b	10.8±1.2^b	35.6±3.9^b
DN+Nef-L	7	17.2±0.6^b	135.3±19.8	16.3±1.2	49.8±5.5
DN+Nef-M	7	13.9±1.1^b	111.1±14.1^b	11.2±2.4^b	42.6±4.4
DN+Nef-H	7	11.5±1.1^b	89.2±10.3^bc	9.1±1.4^bc	33.5±3.6^b

Figure 1 Effect of Nef on the kidney histopathology in mice with DN A: HE staining of kidney tissue (scale bar, 200 μm and 50 μm); A1: control group (×100); A2: DN group (×100); A3: DN + RSG group (×100); A4: control group (×400); A5: DN group (×400); A6: DN + RSG group (×400); A7: DN-Nef-L group (×100); A8: DN-Nef-M group (×100); A9: DN-Nef-H group (×100); A10: DN-Nef-L group (×400); A11: DN-Nef-M group (×400); A12: DN-Nef-H group (×400). B: Masson staining of kidney tissue (×400, scale bar, 50 μm); B1: control group; B2: DN group; B3: DN + RSG group; B4: DN-Nef-L group; B5: DN-Nef-M group; B6: DN-Nef-H group. C: percentage of positive collagen fiber area (%); D: kidney weight index of the mice. DN: diabetic nephropathy; HE: hematoxylin and eosin; RSG: rosiglitazone (4 mg/kg); Nef-L: neferine low dose (60 mg/kg); Nef-M: neferine medium dose (120 mg/kg); Nef-H: neferine high dose (240 mg/kg). Data were statistically analyzed by least significant difference. Data were expressed as mean ± standard deviation (n =3). aP < 0.05, compared with the control group; bP < 0.05, compared with the DN group; cP < 0.05, compared with the DN + RSG group.

Figure 2 Effect of Nef on renal oxidative stress indicators in mice with DN A: levels of SOD in the kidney tissues of mice. B: levels of MDA in the kidney tissues of mice. C: levels of GSH-Px in the kidney tissues of mice. D: representative blot images of HO-1, Nrf2, Col I, and Col III measured by western blot. E: protein expression of HO-1, Nrf2, Col I, and Col III in the kidney tissues of mice was detected by Western blotting. DN: diabetic nephropathy; RSG: rosiglitazone (4 mg/kg); Nef-L: neferine low dose (60 mg/kg); Nef-M: neferine medium dose (120 mg/kg); Nef-H: neferine high dose (240 mg/kg); SOD: superoxide dismutase; MDA: malondialdehyde; GSH-Px: glutathione peroxidase; HO-1: heme oxygenase 1; Nrf2: nuclear factor E2-related factor 2; Col I: collagen I; Col III: collagen III. Data were statistically analyzed by least significant difference-t. Data were expressed as mean ± standard deviation (n = 3). aP < 0.05, compared with the control group, bP < 0.05, compared with the DN group, cP < 0.05, compared with the DN + RSG group.

Figure 3 Targeting relationship between Nrf2 and miR-17-5p and the effect of Nef on renal miR-17-5p expression in the mice with DN A: TargetScan (http://www.targetscan.org) prediction showing potential miR-17-5p binding sites for the Nrf2 3'-UTR, complementary pairing of target genes is indicated in red. B: miR-17-5p target sequences were fused with a luciferase reporter and transfected with miR-17-5p mimic or NC mimic into HEK-293T cells, and miR-17-5p significantly restrained the luciferase activity of the Nrf2 3'-UTR. C: mRNA level of miR-17-5p in the kidney tissues of mice. D: mRNA levels of miR-17-5p in HMCs. E: mRNA levels of Nrf2 in HMCs. F: protein expression of Nrf2 in HMCs. G: representative blot images of Nrf2. H: detection of the proliferation of HMCs treated with different concentrations of Nef. The data in qRT-PCR analysis were expressed after normalization to U6 or β-actin, while those in Western blot assays were expressed after normalization to β-actin. DN: diabetic nephropathy; Nef-L: neferine low dose (60 mg/kg); Nef-H: neferine high dose (240 mg/kg). Nrf2: nuclear factor E2-related factor 2. NC: negative control; HMCs: human mesangial cells; qRT-PCR: quantitative reverse transcription-polymerase chain reaction. Data were statistically analyzed by least significant difference-t. Data were expressed as mean ± standard deviation (n = 3). aP < 0.05, compared with the control or NC mimic group; bP < 0.05, compared with the DN group.

Figure 4 Effect of Nef-mediated miR-17-5p on oxidative stress injury in high glucose-induced HMCs A: mRNA level of miR-17-5p in HMCs; B: representative blot images of HO-1, Nrf2, Col Ⅰ, and Col Ⅲ; C: protein expression of HO-1, Nrf2, Col Ⅰ, and Col Ⅲ in HMCs; D: detection of cell apoptosis using flow cytometry; D1: control group; D2: HG group; D3: HG + Nef group; D4: HG + Nef + NC mimic group; D5: HG + Nef + miR-17-5p mimic group; E: cell apoptosis; F: levels of SOD in HMCs; G: levels of MDA in HMCs; H: levels of GSH-Px in HMCs. HG: high glucose (25 mmol/L). HG + Nef: high glucose (25 mmol/L) + Nef (1 mg/mL). HG+Nef+NC mimic: high glucose (25 mmol/L) + Nef (1 mg/mL) + NC mimic. HG + Nef + miR-17-5p mimic: high glucose (25 mmol/L) + Nef (1 mg/mL) + miR-17-5p mimic. The data from the qRT-PCR analysis were normalized with U6, while those from Western blot assays were normalized with β-actin. HG: high glucose; Nef: neferine; HO-1: heme oxygenase 1; Nrf2: nuclear factor E2-related factor 2; ColⅠ: collagenⅠ; Col Ⅲ: collagen Ⅲ; SOD: superoxide dismutase; MDA: malondialdehyde; GSH-Px: glutathione peroxidase; HMCs: human mesangial cells; NC: negative control; qRT-PCR: quantitative reverse transcription-polymerase chain reaction. Data were statistically analyzed by least significant difference-t. Data were expressed as mean ± standard deviation (n = 3). aP < 0.05, compared with the control group; bP < 0.05, compared with the HG group; cP < 0.05, compared with the HG + Nef group.

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