Structural Characterization and Anti-Gouty Nephropathy Potential of Polysaccharides from Atractylodes chinensis

Abstract

Polysaccharides derived from Atractylodes chinensis (DC.) Koidz. (ACP), a traditional Chinese medicine, were extracted and analyzed for their structural characteristics and anti-gouty nephropathy (GN) activity. Sprague-Dawley (SD) rats were divided into six groups: control, model, positive control, and three treatment groups (ACP-60-L, ACP-60-M, and ACP-60-H). Treatment significantly reduced inflammatory responses and renal damage, as evidenced by decreased levels of uric acid (UA), creatinine (Cr), and blood urea nitrogen (BUN), alongside modulation of NOD-like receptor protein 3 (NLRP3) expression in renal tissues. ACP-60 was fractionated into three polysaccharides, including ACP-60-A (Mw 9.18 kDa), ACP-60-B (Mw 58.21 kDa), and ACP-60-C (Mw 109.01 kDa) using DEAE-52 cellulose column chromatography. Monosaccharide analysis revealed that ACP-60-A predominantly comprised fructose (Fru) and glucose (Glc), ACP-60-B contained rhamnose (Rha), galactose (Gal), Fru, and mannose (Man), and ACP-60-C included Man, Gal, Rha and xylose (Xyl). In vitro studies using HK-2 cells confirmed the anti-GN activity of all three fractions, with ACP-60-A demonstrating the highest efficacy. Structural elucidation of ACP-60-A identified its main glycosidic linkages as a →1)-β-Fruf-(2→ backbone with α-Glcp-(1→ and β-Fruf-(2→ branches. The underlying mechanism of ACP-60-A's anti-GN activity is associated with inhibition of the NLRP3 inflammasome signaling pathway, suppression of downstream inflammatory factor release, and downregulation of NLRP3, ASC, and Caspase-1 protein expression. Further studies demonstrated that the superior activity of ACP-60-A is attributable to its lower molecular weight, specific monosaccharide composition, and unique glycosidic bond arrangement. ACP-60-A shows potential for increased anti-GN efficacy through purification or modification, laying the groundwork for developing novel therapeutic agents for GN.

Keywords: Atractylodes chinensis (DC.) Koidz.; NLRP3; gouty nephropathy; polysaccharide; structural characterization.

Figure 1

Elution curves of ACP-60-A, ACP-60-B, and ACP-60-C on DEAE-52 cellulose column.

Figure 2

Molecular weights of ACP-60-A, ACP-60-B, and ACP-60-C.

Figure 3

Analysis of monosaccharide composition by HPLC for ACP-60-A, ACP-60-B, and ACP-60-C. A: mixed monosaccharide standards 1. Rha; 2. Fuc; 3. Xyl; 4. Ara; 5. Fru; 6. Man; 7. Glc; 8. Gal; B: ACP-60-A; C: ACP-60-B; D: ACP-60-C.

Figure 4

FT-IR spectra of the polysaccharide fractions ACP-60-A, ACP-60-B, and ACP-60-C.

Figure 5

¹H NMR spectra of ACP-60-A.

Figure 6

DEPT-135 spectra of ACP-60-A.

Figure 7

¹H-¹H COSY spectra of ACP-60-A.

Figure 8

HSQC spectra of ACP-60-A.

Figure 9

HMBC spectra of ACP-60-A.

Figure 10

NOESY spectra of ACP-60-A.

Figure 11

Effects of ACP-60-A, ACP-60-B, and ACP-60-C on the survival rate of HK-2 cells. ($\stackrel{-}{\mathrm{x}}$ ± s; n = 3) ^## p < 0.01 vs. the Con group, ** p < 0.01 vs. the Mod group.

Figure 12

Plot of the morphology of HK-2 cells (100×).

Figure 13

Expression of NLRP3, ASC, and Caspase-1 proteins in HK-2 cells. ($\stackrel{-}{\mathrm{x}}$ ± s; n = 3) ^## p < 0.01 vs. the Con group, * p < 0.05, ** p < 0.01 vs. the Mod group.

Figure 14

Effect of ACP-60-L, M, H on the urinary protein content of GN rats. ($\stackrel{-}{\mathrm{x}}$ ± s; n = 3), ^## p < 0.01 vs. the Con group, * p < 0.05, ** p < 0.01 vs. the Mod group.

Figure 15

Effect of ACP-60-L, M, H on UA, Cr, BUN content of GN rats. ($\stackrel{-}{\mathrm{x}}$ ± s; n = 3) ^## p < 0.01 vs. the Con group, * p < 0.05, ** p < 0.01 vs. the Mod group.

Figure 16

Effect of ACP-60-L, M, H on IL-18, IL-1β content of GN rats. ($\stackrel{-}{\mathrm{x}}$ ± s; n = 3) ^## p < 0.01 vs. the Con group, * p < 0.05, ** p < 0.01 vs. the Mod group.

Figure 17

Pathological observation of renal tissue (HE staining, 400×). The glomerulus and tubules are individually marked by black and red arrows.

Figure 18

Expression of NLRP3, ASC, and Caspaes-1 proteins in renal tissue. ($\stackrel{-}{\mathrm{x}}$ ± s; n = 3) ^## p < 0.01 vs. the Con group, * p < 0.05, ** p < 0.01 vs. the Mod group.

Figure 19

mRNA expression of NLRP3, ASC and Caspaes-1 in renal tissue. ($\stackrel{-}{\mathrm{x}}$ ± s; n = 3) ^## p < 0.01 vs. the Con group, * p < 0.05, ** p < 0.01 vs. the Mod group.

Figure 20

Diagram of the mechanism of ACP-60 (drawn using figdraw).