Xanthohumol ameliorates diabetic kidney disease through suppression of renal fibrosis by regulating SNHG10/miR-378b

Abstract

Content: Diabetic kidney disease (DKD), commonly termed diabetic nephropathy (DN), is characterized by oxidative stress and renal tubular epithelial cells apoptosis driven by high glucose (HG).

Objective: To explore the protective effects and underlying mechanism of xanthohumol in DN mice and HG-induced HK-2 cells.

Materials and methods: The STZ-treated mice and HG stimulated HK-2 cells were applied to establish in vivo and in vitro DN models. The concentrations of blood glucose, serum creatinine, BUN and urine creatinine, and β-n-acetylglucosaminidase (NAG) activity was determined. The pathological changes of renal tissues were evaluated by Masson and periodic acid schiff (PAS) staining. TNF-α, IL-1β and IL-6 levels were detected using ELISA. Furthermore, CCK-8 assay and flow cytometer analysis were applied for determining HK-2 cells viability and apoptosis, respectively. Gene and protein levels was evaluated by qRT-PCR analysis and western blot/IHC. The relationship between lncRNA SNHG10 and miR-378b was confirmed by luciferase reporter assay.

Results: Xanthohumol effectively improves DN-stimulated kidney structural and functional abnormalities. LncRNA SNHG10 was downregulated in the renal tissues of DN mice and HG induced HK-2 cells, while this inhibition was reversed by xanthohumol treatment. We also noted that xanthohumol remarkably reversed HG induced HK-2 cells injury. Upregulation of lncRNA SNHG10 also improved DN in mice. Meanwhile, downregulation of SNHG10 reversed the effects of xanthohumol on HG-induced HK-2 cells. Additionally, miR-378b directly targeted lncRNA SNHG10.

Conclusion and discussion: Xanthohumol inhibited the progression of DN by regulating SNHG10/miR-378b, indicating a novel understanding of xanthohumol in DN progression and providing a latent therapeutic target for DN therapy.

Keywords: SNHG10/miR-378b; diabetic nephropathy; inflammatory response; interstitial fibrosis; renal fibrosis; xanthohumol.

FIGURE 1

Effects of xanthohumol on renal function in STZ-induced DN mice. STZ-induced DN mice were intraperitoneal injection of 25 mg/kg xanthohumol, followed by the harvest of blood samples and renal tissues. (A,B) The body weights and the K/B weight ratio were calculated. (C) Blood glucose level was analyzed by Glucose meter. Urinary β-n-acetylglucosaminidase (NAG) activity (D), urinary albumin (ALB) (E), serum creatinine levels (F), urine creatinine levels (G), and blood urine nitragen (H) were measured using an automatic biochemistry analyser. (I–K) ELISA analysis of serum levels of TNF-α, IL-1β and IL-6. N = 3. **P < 0.01.

FIGURE 2

Effects of xanthohumol on pathological changes and renal interstitial fibrosis in the kidneys of DN mice. (A) PAS staining of pathological changes of renal tissues. (B) Renal tissue sections are stained with Masson and the pathological changes of renal tissues were assessed. Scale: 100 μm. (C) IHC analysis of FN, a-SMA, Col-I and E-cadherin in mouse renal tissues. N = 3. **P < 0.01.

FIGURE 3

Effects of xanthohumol on the expression of lncRNA SNHG10 in the kidney tissues of DN mice. qRT-PCR analysis was used to measure lncRNA SNHG10 level in renal tissues from DN mice. N = 3. **P < 0.01.

FIGURE 4

Effects of xanthohumol on cells viability, apoptosis, renal fibrosis and inflammatory response in high glucose-induced HK-2 cells. HK-2 cells were exposed to high glucose and treated with 50 uM xanthohumol. (A) CCK-8 assay of cell viability. (B) Cell apoptosis was detected by Flow cytometry assay. (C) Quantification of apoptotic cells. (D,E) The expression of fibrotic proteins and epithelial cell markers, including FN, a-SMA, Col-I and E-cadherin, were determined by Western blot. (E) Quantification of FN, a-SMA, Col-I and E-cadherin expression relative to β-action. (F) The secretion of TNF-α, IL-1β and IL-6 were determined using ELISA. (G) The levels of lncRNA SNHG10 in HK-2 cells were measured by qRT-PCR analysis. N = 3. *P < 0.05, and **P < 0.01.

FIGURE 5

Effects of SNHG10-plasmid on renal function and inflammatory response in STZ-induced DN mice. STZ-induced DN mice were intraperitoneal injection of SNHG10-plasmid, followed by the harvest of blood samples and renal tissues. (A) The levels of lncRNA SNHG10 were measured by qRT-PCR analysis. (B) Body weights, (C) kidney weight/body weight ratio, (D) blood glucose concentrations, (E) NAG activity, (F) 24-h urinary albumin, (G) serum creatinine levels, (H) urine creatinine levels, and (I) blood urine nitragen in STZ-induced DN mice were determined by automatic biochemistry analyser. ELISA analysis of serum levels of TNF-α (J), IL-1β (K) and IL-6 (L). N = 3. *P < 0.05, and **P < 0.01.

FIGURE 6

Effects of SNHG10-plasmid on renal interstitial fibrosis in the kidneys of in STZ-induced DN mice. (A) Pathological changes of renal tissues were detected by PAS staining. (B) Pathological changes of renal tissues were detected by PAS staining. Scale: 100 μm. (C) IHC analysis of FN, a-SMA, Col-I and E-cadherin in mouse renal tissues. scale: 100 μm. N = 3. *P < 0.05, and **P < 0.01.

FIGURE 7

Downregulation of SNHG10 reversed the effects of xanthohumol on renal fibrosis and inflammatory response HK-2 cells. The high glucose-induced HK-2 cells were treated with xanthohumol or SNHG10-plasmid, followed by shRNA-SNHG10 stimulation. (A,B) The levels of SNHG10 in different groups were determined by qRT-PCR analysis. (C) CCK-8 assay of cell viability. (D) Cell apoptosis was detected by Flow cytometry assay. (E) Quantification of apoptotic cells. (F,G) Detection of fibrotic proteins and epithelial cell markers using Western blot assay. (H) ELISA analysis of serum levels of TNF-α, IL-1β and IL-6. N = 3. **P < 0.01.

FIGURE 8

MiR-378b directly targets lncRNA SNHG10 (A) The conserved target sites of miR-378b binding to SNHG10 3′-UTR were shown. (B) Luciferase activities in 293T cells transfected with WT- SNHG10 or MUT-SNHG10 and miR-378b mimic. N = 3. *P < 0.05.