Luteolin Alleviates Diabetic Nephropathy Fibrosis Involving AMPK/NLRP3/TGF-β Pathway

Abstract

Purpose: Luteolin is a promising candidate for diabetic nephropathy due to its potential anti-inflammatory and anti-fibrotic properties. This study explored the molecular mechanisms through which luteolin combats fibrosis in DN.

Methods: Potential targets affected by luteolin and genes associated with DN were collected from databases. Overlapping targets between luteolin and diabetic nephropathy were identified through Venn analysis. A protein-protein interaction network was constructed using these common targets, and critical pathways and targets were elucidated through GO and KEGG analysis. These pathways and targets were confirmed using a streptozotocin-induced mouse model. Luteolin was administered at 45 mg/kg and 90 mg/kg. Various parameters were evaluated, including body weight, blood glucose levels, and histopathological examinations. Protein levels related to energy metabolism, inflammation, and fibrosis were quantified.

Results: Fifty-three targets associated with luteolin and 36 genes related to diabetic nephropathy were extracted. The AGE-RAGE signaling pathway was the key pathway impacted by luteolin in diabetic nephropathy. Key molecular targets include TGF-β, IL-1β, and PPARG. Luteolin reduced body weight and blood glucose levels, lowered the left kidney index, and improved insulin and glucose tolerance. Furthermore, luteolin mitigated inflammatory cell infiltration, basement membrane thickening, and collagen deposition in the kidney. Luteolin up-regulated the protein expression of p-AMPKα (Th172) while simultaneously down-regulated the protein expression of p-NF-ĸB (p65), NLRP3, TGF-β1, α-SMA, and Collagen I.

Conclusion: Luteolin mitigated renal fibrosis by alleviating energy metabolism disruptions and inflammation by modulating the AMPK/NLRP3/TGF-β signaling pathway.

Keywords: TGF-β; diabetic nephropathy; fibrosis; inflammation; network pharmacology.

Figure 1

Luteolin targets and diabetic nephropathy disease genes. (A) Structural formula of luteolin. (B) Veen analysis of luteolin targets. (C) Venn analysis of diabetic nephropathy disease genes. (D) The network of luteolin-diabetic nephropathy.

Figure 2

Luteolin-diabetic nephropathy interaction network of targets. (A) PPI network of luteolin-diabetic nephropathy common targets. (B) Upset plot of top 30 targets in luteolin-diabetic nephropathy common targets by MCC, MNC, DMNC algorithm.

Figure 3

GO and KEGG enrichment analysis of target proteins. (A) GO enrichment analysis of the top gene in luteolin-diabetic nephropathy common targets. (B) KEGG enrichment analysis of the top gene in luteolin-diabetic nephropathy common targets.

Figure 4

Luteolin attenuated diabetic nephropathy injury in diabetic nephropathy mice. (A) The process of animal experiment. (B) Body weight in diabetic nephropathy mice(n=6). (C) Fasting blood glucose in diabetic nephropathy mice(n=6). (D) Left kidney index in diabetic nephropathy mice(n=6). (E) The curve of IPITT in diabetic nephropathy mice(n=6). (F) IPITT AUC in diabetic nephropathy mice(n=6). (G) The curve of IPGTT in diabetic nephropathy mice(n=6). (H) IPGTT AUC in diabetic nephropathy mice(n=6). ^#P<0.05, ^##P<0.01, ^###P<0.001 shows compared with N group, *P<0.05, **P<0.01, ***P<0.001 shows compared with STZ group.

Figure 5

Luteolin relieved renal fibrosis in diabetic nephropathy mice. (A) Staining of HE, PAS and Masson of kidney in diabetic nephropathy mice(arrows indicate structural derangements of glomerulus). (B) Kidney damage score(n=6). (C) Fibrosis damage score(n=6). (D–F) The protein expression levels of AMPK and p-AMPK(Th172) of kidney in diabetic nephropathy mice, and were expressed as Mean ± SEM (n=6). (G) Representative protein blotting results of kidney in diabetic nephropathy mice. ^#P<0.05, ^##P<0.01, ^###P<0.001 shows compared with N group, *P<0.05, **P<0.01 shows compared with STZ group.

Figure 6

Luteolin alleviated renal fibrosis by down-regulating the protein expression of NF-κB and TGF-β1 in mice with diabetic nephropathy. (A–G)The protein expression levels of NF-κB, p-NF-κB(p65), NLRP3, ASC, Cleaved caspase1 and Cleaved IL-1β of kidney in diabetic nephropathy mice, and were expressed as Mean ± SEM(n=6). (H and I)Representative protein blotting results of kidney in diabetic nephropathy mice. (J–L)The protein expression levels of TGF-β1, α-SMA and Collagen1 of kidney in diabetic nephropathy mice, and were expressed as Mean ± SEM(n=6). (M) Representative protein blotting results of kidney in diabetic nephropathy mice. ^#P<0.05, ^##P<0.01, ^###P<0.001 shows compared with N group, *P<0.05, **P<0.01, ***P<0.001 shows compared with STZ group.

Figure 7

Luteolin alleviated renal fibrosis in STZ-induced diabetic nephropathy mice involving AMPK/NLRP3/TGF-β signaling pathway.