Combination of Active Components of Xiexin Decoction Ameliorates Renal Fibrosis Through the Inhibition of NF-κB and TGF-β1/Smad Pathways in db/db Diabetic Mice

<div><p>Xiexin decoction, a herbal therapeutic agent commonly used in traditional Chinese medicine, is recognized for its beneficial effects on diabetic nephropathy exerted through the combined action of multiple components, including Rhizoma Coptidis alkaloids (A), Radix et Rhizoma Rhei polysaccharides (P), and Radix Scutellaria flavones (F). Our previous studies have shown that a combination of A, P, and F (APF) exhibits renoprotective effects against diabetic nephropathy. This study was aimed at determining the effects of APF on renal fibrosis in diabetic nephropathy and elucidating the underlying molecular mechanisms. To evaluate the effects of APF, in vivo, db/db diabetic mice were orally administered a low or high dose of APF (300 or 600 mg/kg, respectively) once a day for 8 weeks. We evaluated the blood and urine indices of metabolic and renal function, renal tissue histopathology, renal inflammation, and fibrosis. APF treatment significantly ameliorated glucose and lipid metabolism dysfunction, decreased urinary albumin excretion, normalized creatinine clearance, and reduced the morphological changes in renal tissue. Additionally, APF administration in db/db diabetic mice reduced the elevated levels of renal inflammation mediators such as intercellular adhesion molecule-1, monocyte chemotactic protein-1, tumor necrosis factor-α, interleukin-1β, and active nuclear factor κB (NF-κB). APF treatment also reduced type I and IV collagen, transforming growth factor-β1 (TGF-β1), and TGF-β1 type II receptor expression levels, and decreased the phosphorylation of Smad2/3 in the kidneys of db/db diabetic mice. These results suggest that APF reduces renal fibrosis in diabetic nephropathy through the NF-κB and TGF-β1/Smad signaling pathways. In vitro, APF treatment reduced cell proliferation and protein expression of α-smooth muscle actin, collagen I, TGF-β1 and NF-κB in mesangial cells cultured with high glucose concentrations. Our findings indicate that treatment with multi-component herbal therapeutic formulations may be a useful approach for the treatment of diabetic nephropathy.</p></div

Effect of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on renal histopathology and ultrastructural pathology.

<p>(a-e) hematoxylin and eosin (HE) stain. (f-j) Periodic Acid Schiff (PAS) stain. Original magnification (a–j) × 400. (k-o) Electron microscopy (EM) analysis, Representative images of glomerular basement membrane thickening and mesangial matrix expansion, scale bars 2 μm, original magnification electron microscopy × 6000. (p) Ratio of the mesangial matrix area to total glomerular area (M/G) in PAS staining. Data are expressed as mean ± S.D., n = 10, **<i>p</i> < 0.01 as compared with db/db group.</p

Effect of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on renal histopathology and ultrastructural pathology.

<p>(a-e) hematoxylin and eosin (HE) stain. (f-j) Periodic Acid Schiff (PAS) stain. Original magnification (a–j) × 400. (k-o) Electron microscopy (EM) analysis, Representative images of glomerular basement membrane thickening and mesangial matrix expansion, scale bars 2 μm, original magnification electron microscopy × 6000. (p) Ratio of the mesangial matrix area to total glomerular area (M/G) in PAS staining. Data are expressed as mean ± S.D., n = 10, **<i>p</i> < 0.01 as compared with db/db group.</p

Effect of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on renal TGF-β1 and its receptor expression.

<p>(a-e) Immunohistochemistry of TGF-β1; (f) Quantitative analysis of immunohistochemical staining of TGF-β1; (g-h) Western blot analysis of TGF-β1 and α-SMA protein levels; (i-j) Real-time RCR analysis of TGF-β1 and TβRⅡmRNA levels. a: db/m, b: db/db, c: APF 300mg/kg, d: 600 mg/kg, e: metformin. Data are expressed as mean ±S.D., n = 3 for Western blot, and n = 5 for Immunohistochemistry and Real-time PCR, *p<0.05, **p<0.01 as compared with db/db group.</p

Effect of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on renal fibrosis.

<p>(a-e) Masson’s modified trichrome histological (Masson); (p) Ratio of area with collagen accumulation to total glomerular area; (f-j) Immunohistochemistry of collagen I; (k-o) Immunohistochemistry of collagen IV. Original magnification (a–o) × 400; (q) Quantitative analysis of immunohistochemical staining of collagen I (Col I); (r) glomerular of collagen IV (Col IV); (s) interstitial of collagen IV (Col IV); (t-u) Real-time RCR analysis of collagen I and collagen IV mRNA levels. Data are expressed as mean ±S.D., n = 5, **p<0.01 as compared with db/db group.</p

Effects of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on cell proliferation and fibrosis in mesangial cells incubated at high glucose.

<p>NG: normal glucose; HG: high glucose; APF 4, 8, and 16 μg /mL represent mesangial cells incubated in high glucose concentration treated with 4, 8, and 16 μg /mL APF, respectively. (a-d) Western blot analysis of α-SMA, CollagenⅠ, TGF-β1 and NF-κB protein levels; (e) Cell proliferation. Data are expressed as mean ±S.D., n = 5 for cell proliferation, and n = 3 for Western blot, *p<0.05, **p<0.01, compared to control cells incubated at high glucose concentration with no APF.</p

Effect of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on renal inflammation in db/db mice.

<p>(a-b) Western blot analysis of protein levels; (c-d) Real-time RCR analysis of mRNA levels; ICAM-1: intercellular adhesion molecule-1; MCP-1: monocyte chemotactic protein-1; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β. Data are expressed as mean ±S.D., n = 3 for Western blot, and n = 5 for Real-time PCR, *p<0.05, **p<0.01 as compared with db/db group.</p

Effect of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on renal nuclear factor-κB (NF-κB) signaling pathway.

<p>IKKα: inhibitor of nuclear factor-κB kinase subunit α; IκBα, inhibitor of nuclear factor-κB subunit α; p-IκBα: phospho-IκBα; NF-κBp65: nuclear factor-κBp65; Data are expressed as mean ±S.D., n = 3, *p<0.05, **p<0.01 as compared with db/db group</p

Nucleotide sequence of primers used in Real-time PCR.

<p>Nucleotide sequence of primers used in Real-time PCR.</p

Effect of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on glucose and lipid metabolism, urinary albumin excretion and kidney function and renal function in db/db mice.

<p>Data are expressed as mean ±S.D., n = 10</p><p>*p<0.05</p><p>**p<0.01 as compared with db/db group</p><p>Effect of a combination of Rhizoma Coptidis alkaloids, Radix et Rhizoma Rhei polysaccharides, and Radix Scutellaria flavones (APF) on glucose and lipid metabolism, urinary albumin excretion and kidney function and renal function in db/db mice.</p