Panax notoginseng saponins alleviate diabetic retinopathy by inhibiting retinal inflammation: Association with the NF-B signaling pathway

Ethnopharmacological relevance: Diabetic retinopathy (DR) is a neurovascular disease that causes blindness in adults and is the most serious and common complication of diabetes mellitus. Retinal inflammation is an early stage of DR, and it is believed to play a crucial role in the development of DR. Panax notoginseng saponins (PNS) are the major active constituent in the main root of P. notoginseng , and they exhibit various biological activities, including anti-inflammatory, antioxidant, neuroprotective, and immunomodulatory functions. However, the protective effects and underlying mechanisms of PNS against DR remain unclear.Aim of the study: This study aimed to investigate the alleviation effects of PNS on DR and the mechanisms involved. Furthermore, it intended to explore the major components that exert efficacy in vivo. Materials and methods: Streptozotocin (STZ) was administered intraperitoneally to Sprague Dawley rats, and PNS was administered orally for 1 month after 2 months of STZ injection. The morphological structure of the retina and retinal acellular capillaries were assessed via hematoxylin and eosin (H&E) staining assay. The disruption of the blood-retinal barrier (BRB) was detected through Evans blue dye leakage assay, and retinal leukocyte adhesion was achieved via fluorescein isothiocyanate-coupled concanavalin A lectin labeling assay. Immuno-fluorescence staining and Western blot assays were conducted to detect the expression of tight junction proteins, adhesion molecules, and the ionized calcium-binding adapter molecule-1 (Iba-1) in the retina. Enzyme-linked immunosorbent assay was performed to detect the levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1 beta in serum. In addition, the protein expression levels of nuclear factor (NF)-kappa B p65, phosphorylated I kappa B kinase (p-IKK), phosphorylated NF-kappa B inhibitor (p-I kappa B), and phosphorylated NF-kappa B p65 (p-p65) were measured using Western blot assay. The ocular tissue distribution of PNS in normal and diabetic rats was determined through ultra-performance liquid chromatography-tandem mass spectrometry. The in vitro anti-inflammatory effects of PNS, notoginsenoside (NGR1), ginsenoside Rg1, Re, Rb1, and Rd (GRg1, GRe, GRb1, and GRd) were evaluated on human Muller (MIO-M1) cells.Results: PNS increased the reduction in retinal inner nuclear layer thickness, reduced the increase in retinal acellular capillaries, and attenuated elevated BRB disruption by upregulating the decrease in protein expression of claudin-1 and occludin. Furthermore, PNS significantly abrogated microglial cell activation and reversed the increase in leukocyte adhesion by downregulating the increase in the protein expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Moreover, PNS reduced the elevated levels of TNF-alpha, IL-6, and IL-1 beta in serum and inhibited the increased protein expression of p-IKK, p-I kappa B, and p-p65, and the nuclear translocation of p65. The tissue distribution results revealed that NGR1, GRg1, GRe, GRb1, and GRd were detected in the ocular tissue, while GRg1 and GRb1 were found at the highest levels compared with the other components. The cellular results showed that PNS, NGR1, GRg1, GRe, GRb1, and GRd suppressed the devel-opment of cellular inflammatory responses by inhibiting the activation of the NF -KB signaling pathway in MIO-M1 cells and that their anti-inflammatory effects were comparable. Conclusion: PNS suppressed retinal inflammation by inhibiting the activation of the NF -KB signaling pathway, alleviating DR. GRg1 and GRb1 may be the primary components that exert anti-inflammatory effects in vivo.