Renoprotective Effect of Formononetin by Suppressing Smad3 Expression in Db/Db Mice

Abstract

Purpose: Glomerular sclerosis and renal interstitial fibrosis are the most important pathologies in the development of kidney damage under diabetic conditions. Smad3 plays antagonistic roles in high glucose-induced renal tubular fibrosis, which is an important treatment target for diabetic nephropathy (DN). Formononetin (FMN) has multiple effects on diabetic vascular complications including DN. However, whether it plays an anti-fibrosis role by regulating smad3 is unclear. The purpose of this study was to evaluate the renoprotective effect of FMN by suppressing smad3 expression in db/db mice.

Methods: FMN was orally administered to db/db mice with a dose of 25 or 50 mg/kg/day for 8 weeks. At the end of the study, serum, urine, and kidney samples were collected for biochemical and pathological examinations. The expressions of proteins and mRNA associated with renal fibrosis were determined by biochemical, histological, immunofluorescence, and real-time PCR analysis.

Results: The results showed that FMN substantially improved the glucolipid metabolism, reduced the oxidative stress, and protected renal function in db/db mice. Meanwhile, protein and mRNA expression of smad3 and related regulatory factor of extracellular matrix deposition were significantly suppressed.

Conclusion: The present study suggested that FMN has a good renoprotective effect in DN, which plays an anti-fibrosis role in db/db mice by suppressing the expression of smad3.

Keywords: antioxidants; db/db mice; diabetic nephropathy; expression of smad3; formononetin; renal fibrosis.

Figure 1

Chemical structure of FMN.

Figure 2

Effect of FMN on FBG (A, B), FINS (C), ISI (D), IRI (E), TG (F), and TC (G) in db/db mice. Data were expressed as Mean±SEM (n=6). ***P<0.001, **P<0.01, *P<0.05 vs db/+ mice; ^###P<0.001, ^##P<0.01, ^#P<0.05 vs non-treated db/db mice.

Figure 3

Effect of FMN on 24hUP (A), UCr (B), BUN (C), SCr (D), ACR (E), and BUN/SCr (F) in db/db mice. Data were expressed as Mean±SEM (n=6). ***P<0.001, **P<0.01, *P<0.05 vs db/+ mice; ^###P<0.001, ^##P<0.01, ^#P<0.05 vs non-treated db/db mice.

Figure 4

Effect of FMN on histopathologically (200X) (A), CVF (B) in db/db mice. Data were expressed as Mean±SEM (n=6). ***P<0.001 vs db/+ mice; ^###P<0.001, ^#P<0.05 vs non-treated db/db mice.

Figure 5

Effect of FMN on SOD (A), MDA (B), GSH-Px (C), CAT (D) in db/db mice. Data were expressed as Mean±SEM (n=6). **P<0.01, *P<0.05 vs db/+ mice; ^###P<0.001, ^##P<0.01, ^#P<0.05 vs non-treated db/db mice.

Figure 6

Continued.

Figure 6

The fluorescent immunogram (A, B), the expressions of COLI (C), COLIII (D), and α-SMA (E) of pancreas tissues on db/db mice between different study groups. Data were expressed as Mean±SEM (n=3). ***P<0.001 vs db/+ mice; ^###P<0.001, ^##P<0.01 vs non-treated db/db mice.

Figure 7

Effect of FMN on COL1a1 (A), COL3a1 (B), α-SMA (C), and smad3 (D) in db/db mice. Data were expressed as M(P₂₅, P₇₅) (n=3). **P<0.01 vs db/+ mice; ^##P<0.01 vs non-treated db/db mice.