Regulation of Laminaria Polysaccharides with Different Degrees of Sulfation during the Growth of Calcium Oxalate Crystals and their Protective Effects on Renal Epithelial Cells

Abstract

The original Laminaria polysaccharide (LP0) was sulfated using the sulfur trioxide-pyridine method, and four sulfated Laminaria polysaccharides (SLPs) were obtained, namely, SLP1, SLP2, SLP3, and SLP4. The sulfated (-OSO₃ ^-) contents were 8.58%, 15.1%, 22.8%, and 31.3%, respectively. The structures of the polysaccharides were characterized using a Fourier transform infrared (FT-IR) spectrometer and nuclear magnetic resonance (NMR) techniques. SLPs showed better antioxidant activity than LP0, increased the concentration of soluble Ca²⁺ in the solution, reduced the amount of CaOx precipitation and degree of CaOx crystal aggregation, induced COD crystal formation, and protected HK-2 cells from damage caused by nanometer calcium oxalate crystals. These effects can inhibit the formation of CaOx kidney stones. The biological activity of the polysaccharides increased with the content of -OSO₃ ^-, that is, the biological activities of the polysaccharides had the following order: LP0 < SLP1 < SLP2 < SLP3 < SLP4. These results reveal that SLPs with high -OSO₃ ^- contents are potential drugs for effectively inhibiting the formation of CaOx stones.

Figure 1

Characterization of SLPs with different -OSO₃⁻ content. (a, b) GC-MS analysis. (c) FT-IR spectrum. (d) The absorption peak intensity of S=O asymmetric stretching vibration and C-O-S stretching vibration varies with the -OSO₃⁻ content of polysaccharides. (e) ¹H NMR spectrum of LP0. (f) ¹H NMR spectrum of SLP2. (g) ¹³C NMR spectrum of LP0. (h) ¹³C NMR spectrum of SLP2. (i) LP0 sulfation reaction equation.

Figure 2

XRD spectra of CaOx crystals induced by SLPs at different concentrations. (a) LP0. (b) SLP1. (c) SLP2. (d) SLP3. (e) SLP4. (f) The percentage of COD in CaOx crystals formed in the presence of SLPs at various concentrations. Polysaccharide concentration in Figs.: (a) 0; (b) 0.4; (c) 0.8; (d) 1.2; (e) 1.6; (f) 2.4 g/L. Those marked with ^∗ are COD diffraction peaks, and those without ^∗ are COM diffraction peaks.

Figure 3

FT-IR spectra of CaOx crystals induced by SLPs at different concentrations. (a) LP0. (b) SLP1. (c) SLP2. (d) SLP3. (e) SLP4. In Figs. (a) 0.4; (b) 0.8; (c) 1.2; (d) 1.6 g/L.

Figure 4

(a) SEM images of CaOx crystals, (b) soluble Ca²⁺ ions concentration in the supernatant, and (c) CaOx precipitate amount in the presence of SLPs at 1.6 g/L. (d) The influence of SLPs on the Zeta potential of CaOx crystals formed. Compared with the blank group, ^∗P < 0.05; ^∗∗P < 0.01.

Figure 5

In vitro antioxidant capacity of SLPs. (a) Scavenging •OH free radicals; (b) Scavenging DPPH free radicals. Comparison of the ability of SLPs to protect HK-2 cells from oxidative damage. (c) Cell viability was detected by the CCK-8 method. Compared with the COD group, ^∗P < 0.05; ^∗∗P < 0.01. (d) Cell cytotoxicity was detected by the CCK-8 method. Compared with the NC group, ^∗P < 0.05; ^∗∗P < 0.01. (e) The relationship between cell viability and the –OSO₃⁻ content of polysaccharide. NC: normal control; COD concentration: 200 μg/mL; damage time: 12 h; protection time: 12 h.

Figure 6

ROS level and cell morphology were detected before and after SLPs protect HK-2 cells. (a) ROS fluorescence microscope images. (b) Quantitative histogram of ROS fluorescence intensity. (c) Cell morphology. NC: normal control; polysaccharide concentration: 80 μg/mL; COD concentration: 200 μg/mL; damage time: 12 h; protection time: 12 h. Scale bars: 50 μm. Compared with the COD group, ^∗P < 0.05; ^∗∗P < 0.01.

Figure 7

The mechanism diagram of SLPs inhibiting kidney stones formation.