Yishen Huashi Granules Ameliorated the Development of Diabetic Nephropathy by Reducing the Damage of Glomerular Filtration Barrier

FIGURE 1. Fingerprint chromatogram of Yishen Huashi Extract.

FIGURE 2. Effect of YSHS on biochemical parameters in DN rats. Changes in body weight (A), blood glucose (B), and urine volume (C) throughout the experimental period. Changes in kidney index (D) and parameters of blood HbA1c (E), glucose (F), LPS (G), insulin (H), C-peptide (I), MDA (J), and SOD (K) at the end of the experiment. Changes of serum BUN-s (L), Cr-s (M), and mALB (N), TP-u (O), Cr-u (P), α1-MG (Q), and β2-MG (R) within urine at the end of the experiment. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, vs. Normal; & p < 0.05, & p < 0.01, and &&& p < 0.001, vs. DN.

FIGURE 3. Effects of YSHS on histopathologic changes of DN rats. TEM (A,B), H&E (C), and PAS (D) staining of YSHS in different groups on DN. (E) PAS-positive area was analyzed by the IPP software. Values are presented as means ± SEM. **p < 0.01, vs. Normal; & p < 0.05, && p < 0.01, &&& p < 0.001, vs. DN.

FIGURE 4. Effects of YSHS on renal fibrosis in DN rats. Podocin and fibronectin expression was detected by immunohistochemical staining (A). IHC was quantitated for fibronectin (B) and podocin (C) using the IPP software. Podocin and fibronectin expression was determined by WB (D). Relative expression of fibronectin (E) and podocin (F) was calculated using ImageJ software. Protein expression of CD31, α-SMA, TIMP-1, and GST-PI in the DN rat was determined by WB (G). Relative expression of TIMP-1(H), α-SMA (I), GST-PI (J), and CD31 (K) was analyzed by ImageJ software. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, vs. Normal; & p < 0.05, && p < 0.01, vs. DN.

FIGURE 5. Network pharmacology analysis and differential metabolite analysis of urine. Venn diagram of diseases and YSHS drugs (A). Compound target disease (CTD) network of YSHS (B); the green circles represent compound targets, and the blue circles represent diseases. The PPI network (C) was sorted in descending order from the bottom of the figure in a circular layout, on the basis of the number of degrees; the size of the target and disease nodes reflects the number of degrees. Biological processes related to targets of YSHS (D). Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (E). Metabolomic analysis of rat urine: Separation of rat urine in a PCA score plot (F); loading S-plot generated by OPLS-DA analysis of the metabolome in rat urine (G); differential metabolite pathway analysis (H) differential metabolite heat map of the urine among groups (I).

FIGURE 6. Western blot analysis of PI3K/AKT/m-TOR signaling pathways (A). Relative expression and activation of mTOR (B), PI3K (C), and AKT (D) was analyzed by the ImageJ software. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, vs. Normal; &p < 0.05, &&p < 0.01, &&&p < 0.001, vs. DN.

FIGURE 7. Influence of YSHS on viability of the GEnC and HPC cells. Viability analysis of GEnCs on drug administration (A–E); viability analysis of HPCs on drug administration (F,G). Data are expressed as the mean ± SEM. Each experiment was repeated at least three times. (A–C,F,G) *p < 0.05, **p < 0.01, ***p < 0.001, vs. control. (D), *p < 0.05, **p < 0.01, ***p < 0.001, vs. 24 h control; ## p < 0.01, ### p < 0.001, vs. 48 h control. (E), *p < 0.05, **p < 0.01, ***p < 0.001, vs. control. (D), & p < 0.05, && p < 0.01, &&& p < 0.001, vs. H-Glu + Ins.

FIGURE 8. Effect of YSHS on oxidative stress, mitochondrial membrane potential (Δψm), and apoptosis in GEnC cells and HPC cells. Intracellular ROS levels within GEnC (A) and HPC (D,E) cells were measured by flow cytometry. JC-1 levels on GEnCs (B) and HPCs (F) cells were measured by flow cytometry. Apoptosis of GEnC (C) and HPC (G) cells were measured by flow cytometry. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, vs. control; & p < 0.05, && p < 0.01, &&& p < 0.001, vs. H-Glu + Ins.

FIGURE 9. Effect of YSHS on the glomerular filtration barrier in GEnC cells. Immonofluorescence assay for GEnCs tight junction protein VE (A), ZO-1 (C), claudin-5 (E), and occludin (G) under a laser scanning confocal microscope; and relative fluorescence intensity for them were determined (B,D,F,G). YSHS decreased VEGF (I), VEGFR1 (K), fibronectin (M), and collagen1 (O) expressions, and relative fluorescence intensity for them (J), (L,N), and (P) were determined. Values are presented as means ± SEM. ***p < 0.001, vs. control; & p < 0.05, && p < 0.01, &&& p < 0.001, vs. H-Glu + Ins.

FIGURE 10. Effect of YSHS on the glomerular filtration barrier in HPC cells. Immonofluorescence assay for HPC tight junction protein VE (A), ZO-1 (C), claudin-5 (E), and occludin (G) under a laser scanning confocal microscope; and relative fluorescence intensity for them (B,D,F,G) were determined. YSHS increased podocin (I) and CD31 (K) expressions, and relative fluorescence intensity for them (J,L) were determined. YSHS decreased fibronectin (M) and collagen1 (O) expressions, and relative fluorescence intensity for them (N,P) were determined. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, vs. control; & p < 0.05, && p < 0.01, &&& p < 0.001, vs. H-Glu + Ins.

FIGURE 11. Proposed mechanism of YSHS on protecting the glomerular filtration barrier under DN settings.