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Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3

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Abstract

Objective

The main characteristics of diabetic nephropathy (DN) at the early stage are abnormal angiogenesis of glomerular endothelial cells (GECs) and macrophage infiltration. Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand, advanced glycation end products (AGEs). Catalpol, an iridoid glucoside extracted from Rehmannia glutinosa, has been found to ameliorate vascular inflammation, reduce endothelial permeability, and protect against endothelial damage in diabetic milieu. However, little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs.

Methods

Mouse GECs (mGECs) and RAW 264.7 macrophages were treated with different concentrations of AGEs (0, 50, 100, 200 and 400 µg/mL) for different time (0, 6, 12, 24 and 48 h) to determine the optimal concentration of AGEs and treatment time. Cells were treated with catalpol (10 µmol/L), GB1107 (1 µmol/L, galectin-3 inhibitor), PX-478 (50 µmol/L, HIF-1α inhibitor), adenovirus-green fluorescent protein (Ad-GFP) [3×107 plaque-forming unit (PFU)/mL] or Ad-galectin-3-GFP (2×108 PFU/mL), which was followed by incubation with 50 µg/mL AGEs. The levels of galectin-3, vascular endothelial growth factor A (VEGFA) and pro-angiogenic factors angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tunica interna endothelial cell kinase-2 (Tie-2) were detected by enzymelinked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of these cells. The expression levels of galectin-3, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and hypoxia-inducible factor-1α (HIF-1α) in mGECs and those of galectin-3 and HIF-1α in RAW 264.7 macrophages were detected by Western blotting and immunofluorescence (IF) staining. The rat DN model was established. Catalpol (100 mg/kg) or GB1107 (10 mg/kg) was administered intragastrically once a day for 12 weeks. Ad-galectin-3-GFP (6×107 PFU/mL, 0.5 mL) or Ad-GFP (6×106 PFU/mL, 0.5 mL) was injected into the tail vein of rats 48 h before the sacrifice of the animals. The expression of galectin-3, VEGFR1, VEGFR2, and HIF-1α in renal cortices was analyzed by Western blotting. The expression of galectin-3, F4/80 (a macrophage biomarker), and CD34 (an endothelium biomarker) in renal cortices was detected by IF staining, and collagen accumulation by Masson staining.

Results

The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50 µg/mL AGEs for 48 h than those in untreated cells. Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages. Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells. Catalpol could significantly decrease the levels of Ang-1, Ang-2 and Tie-2 released by AGEs-treated mGECs, which could be reversed by over-expression of galectin-3. Catalpol could significantly inhibit AGEs-induced expression of galectin-3, HIF-1α, VEGFR1, and VEGFR2 in mGECs. The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478. Moreover, catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages, which was weakened by PX-478. Additionally, catalpol significantly inhibited the expression of galectin-3, macrophage infiltration, collagen accumulation, and angiogenesis in the kidney of diabetic rats. Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol.

Conclusion

Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3. It could prevent the progression of diabetes-induced renal damage.

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Authors and Affiliations

  1. Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-animal Husbandry Vocational College, Taizhou, 225300, China

    Wei-xiang Sun

  2. Department of Chinese Pharmaceutical Technology, School of Animal Pharmacy, Jiangsu Agri-animal Husbandry Vocational College, Taizhou, 225300, China

    Wei-xiang Sun

  3. Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China

    Wei-xiang Sun, Yu-yan Gao, Jin-fu Lu, Gao -hong Lv & Hui-qin Xu

  4. Jiangsu Key laboratory of Pharmacology and Safety Evaluation for Chinese Material Medica, Nanjing University of Chinese Medicine, Nanjing, 210023, China

    Wei-xiang Sun, Yu-yan Gao, Jin-fu Lu, Gao -hong Lv & Hui-qin Xu

  5. Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine Hanlin College, Taizhou, 225300, China

    Ying Cao & Hui-qin Xu

Authors
  1. Wei-xiang Sun
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  2. Yu-yan Gao
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Corresponding author

Correspondence to Hui-qin Xu.

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All authors contributed significantly to work and agree with the manuscript’s content. There are no conflicts of interest.

Additional information

The study was supported by grants from the National Natural Science Foundation of China (No. 81374029, No. 81073111, No. 81874359), Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (No. 18KJD360002), a Project Funded by Jiangsu Agri-animal Husbandry Vocational College (No. NSF2021CB04), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) (No. JKLPSE201604).

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Sun, Wx., Gao, Yy., Cao, Y. et al. Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3. CURR MED SCI 43, 668–678 (2023). https://doi.org/10.1007/s11596-023-2750-5

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  • DOI: https://doi.org/10.1007/s11596-023-2750-5

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