Table 1.
Influences of SH on physiological parameters in non-diabetic and diabetic mice.
Table 2.
Comparison of biochemical characteristics of experimental animals.
Fig 1.
Effects of SH on the progression of diabetic nephropathy.
After the 24 h urine collected by metabolic cages, albuminuria determined (A). Representative examples of glomerular hematoxylin and eosin (H&E)-stained sections (B). Differences of glomerular volume among four groups (C). Representative transmission electron microscope image of renal ultra stuctures (D). The differences of slit pore numbers (E) and glomerular basement membrane (GBM) thickness (F) among four groups. Original magnification: H&E staining, 400× and transmission electron microscopy, 30K×. NC, normal mice group; NC+SH, normal mice with sarpogrelate hydrochloride treatment group; DB, diabetic mice group; DB+SH, diabetic mice with sarpogrelate hydrochloride treatment group; UAE, urine albumin excretion. Values shown are mean ± SEM. *p < 0.05 vs. NC, †p < 0.05 vs. DB. n = 7 per group.
Fig 2.
Western blot analysis of the renal cortex after treatment with sarpogrelate hydrochloride.
The renal expression of nephrin and VEGF was analyzed by Western blotting (A). The relative expression of nephrin (B) and VEGF (C) were analyzed by ImageJ software. The expression of renal Cldn1 and Sirt1 was analyzed by IHC (D) and western blotting (E).The difference renal nephrin and VEGF levels among four groups. NC, normal mice group; NC+SH, normal mice with sarpogrelate hydrochloride treatment group; DB, diabetic mice group; DB+SH, diabetic mice with sarpogrelate hydrochloride treatment group. Values shown are mean ± SEM. *p < 0.05 vs. NC, †p < 0.05 vs. DB. n = 6 per group.
Fig 3.
SH modulated inflammatory-macrophage accumulation and inflammatory response.
The levels of mouse mature macrophage marker F4/80 and inflammatory macrophage marker CD11c were staining of kidney sections (A). The changes of NOS mRNA (B) and protein expression (C and D) in kidney. And the inflammatory cytokines, TNF-α was analyzed by western blotting (C and E). NC, normal mice group; NC+SH, normal mice with sarpogrelate hydrochloride treatment group; DB, diabetic mice group; DB+SH, diabetic mice with sarpogrelate hydrochloride treatment group. Values shown are mean ± SEM. *p < 0.05 vs. NC, †p < 0.05 vs. DB. n = 6 per group.
Fig 4.
SH inhibition of NRK52E cell glucotoxicity depends on the AMPK-SIRT1-PGC1alpha axis and inflammatory signals.
NRK52E cells were stimulated by HG with or without SH and analyzed of phosphorylated AMPK, Sirt1, PGC1α and Cldn1 expressions (A and B). Andinflammatory molecules such as phosphorylated p38 and phosphorylated JNK changes were analyzed in HG with or without SH cultured NRK52E cells (C and D). LG, 5.5 mM D-glucose; HG, 30 mM D-glucose; SH, sarpogrelate hydrochloride. Values shown are mean ± SEM. *p < 0.05 vs. LG, †p < 0.05 vs. HG.
Fig 5.
SH reduces macrophage migration and activation.
The differences of cell migration rate (A and C) and morphological change (B) by LPS with or without SH in Raw264.7 cells. (B) Change of phosphorylated p38 and MCP1 expressions by SH treatment in macrophage cultured media stimulated NRK52E cells (D). CON, control; LPS, lipopolysaccharides; SH, sarpogrelate hydrochloride; CM, conditioned medium. Values shown are mean ± SEM. *p < 0.05 vs. CON, †p < 0.05 vs. LPS.
Fig 6.
SH inhibits the 5HT-2A receptor and MCP1 in RAW264.7 cells.
Representative immunofluorescence staining of 5HT-2A receptor (A) and western blotting of 5HT-2A receptor and MCP1 levels (B and C)in Raw264.7 cells. CON, control; LPS, lipopolysaccharides; SH, sarpogrelate hydrochloride. Values shown are mean ± SEM. *p < 0.05 vs. CON, †p < 0.05 vs. LPS.
Fig 7.
SH ameliorates renal fibrosis in DN.
Collagen accumulation in glomeruli was analyzed by picrosirius red staining in the kidney sections (A) and fibrosis-related molecules such as β-catenin, snail, and TGF-β (B, C, D and E) were analyzed by western blotting. NC, normal mice group; NC+SH, normal mice with sarpogrelate hydrochloride treatment group; DB, diabetic mice group; DB+SH, diabetic mice with sarpogrelate hydrochloride treatment group. Values shown are mean ± SEM. *p < 0.05 vs. NC, †p < 0.05 vs. DB. n = 6 per group.