Diabetes Metab J.  2025 Jan;49(1):34-48. 10.4093/dmj.2024.0024.

Shionone Inhibits Glomerular Fibirosis by Suppressing NLRP3 Related Inflammasome though SESN2-NRF2/ HO-1 Pathway

• Xiao T
• Zhao H
• Wang Y
• Chen M
• Wang C
• Qiao C

• KMID: 2563268
• DOI: http://doi.org/10.4093/dmj.2024.0024

Abstract

Background
Diabetic nephropathy (DN) is the most common and serious complication of diabetes mellitus. Shionone (SH), an important triterpenoid compound in the root extract of Aster, might exert a protective effect in DN mice and high glucose cultivated glomerular podocytes. The current study aimed to unravel the underlying mechanism by which SH mitigates DN. We postulate that SH stimulates the expression of sestrin-2 (SESN2), a pivotal stress-inducible protein in the anti-inflammasome machinery.
Methods
We utilized high-fat diet combined with streptozotocin (55 mg/kg intraperitoneal) for DN mice model, and high glucose (30 mM, 48 hours) cultured glomerular podocytes for DN cell model to evaluate the effect of SH. We also preformed experimentation on SESN2 deficiency models (SESN2 knockout mice and SESN2 siRNA in cells) to further prove our hypothesis.
Results
The results demonstrated that SH effectively suppressed glomerular fibrosis, induced adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, and inhibited NLR family pyrin domain containing 3 (NLRP3) activation. Furthermore, our findings revealed that SH exerted its anti-inflammatory effect through Sesn2-dependent nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and subsequent activation of its downstream target heme oxygenase-1 (HO-1).
Conclusion
In summary, our findings suggest that SH serves as a promising therapeutic agent for the treatment of DN-related glomerular fibrosis. SH enhances the expression of SESN2, attenuates α-smooth muscle actin accumulation, and suppresses NLRP3-related inflammation through the Nrf2/HO-1 signaling pathway.

Keyword

NLR family, pyrin domain-containing 3 protein; Sesn 2 protein; Shionone

Figure

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  Fig. 1. Shionone (SH) improved weight change and the function of kidney in diabetic mice. Four weeks after streptozotocin injection, diabetic models were examined based on general indicators. (A) Body weight of each group of mice. (B) Kidney index of each group of mice, (C) blood urea nitrogen (BUN), (D) random blood glucose (RBG), (E) urine protein, (F) urine albumin, and (G) serum interleukin 1β (IL-1β) were tested following the instructions of the kits. Data are expressed as the mean±standard error of the mean. aP<0.05, bP<0.01, cP<0.001, compared with the control group; dP<0.01, eP<0.001, compared with the type 2 diabetes mellitus (T2DM) group; fP<0.05, gP<0.01, compared with the irbesartan group.

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  Fig. 2. Shionone (SH) improved glomerulus fibrosis and inflammatory in diabetic mice. (A) The histopathology analysis of glomeruli using Masson staining, ×200. (B) Quantitative analysis of Masson staining. (C) The histopathology analysis of glomeruli using Periodic acid–Schiff (PAS) staining, ×200. (D) Quantitative analysis of PAS staining. (E) Subcellular structure analysis of podocytes detected by transmission electron microscopy, ×10,000. (F) Expression of α-smooth muscle actin (α-SMA), NLR family pyrin domain containing 3 (NLRP3), interleukin 1β (IL-1β), phosphorylated adenosine monophosphate-activated protein kinase (P-AMPK) in glomeruli through immunohistochemistry, ×200. (G) α-SMA, NLRP3, IL-1β, P-AMPK proteins from wild-type mice detected by Western blot. (H) Quantitative analysis of Western blot. Data are expressed as the mean±standard error of the mean. aP<0.05, bP<0.01, cP<0.001, compared with the control group; dP<0.05, eP<0.01, fP<0.001 compared with the type 2 diabetes mellitus (T2DM) group; gP<0.05, compared with the irbesartan group.

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  Fig. 3. Shionone (SH) activated nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) via sestrin-2 (SESN2) in diabetic mice. (A) Expression of SESN2 in glomeruli through immunohistochemistry, ×200. (B) SESN2, NRF2, HO-1 proteins from wild-type mice detected by Western blot. (C) Quantitative analysis of Western blot. (D) Effect of SH on the nucleus expression of NRF2 in type 2 diabetes mellitus (T2DM) mice kidney was detected by Western blot. (E) Effect of SH on the mRNA level of SESN2, NRF2 in T2DM mice. (F) SESN2, NRF2 in detected by confocal scanning microscopy, ×200, ×500. The position indicated by the white arrows is the site of nuclear import of NRF2. Data are expressed as the mean±standard error of the mean. DAPI, 4′,6-diamidino-2-phenylindole. aP<0.05, bP<0.01, compared with the control group; cP<0.01, dP<0.001 compared with the T2DM group; eP<0.001, compared with the irbesartan group.

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  Fig. 4. Shionone (SH)’s anti-fibrosis effect was sestrin-2 (SESN2) dependent in diabetic mice. (A) The histopathology analysis of SESN2-/- glomeruli using Masson staining, ×200. (B) Quantitative analysis of Masson staining. (C) The histopathology analysis of SESN2-/- glomeruli using Periodic acid–Schiff (PAS) staining, ×200. (D) Quantitative analysis of PAS staining. (E) Subcellular structure analysis of podocytes in SESN2-/- mice detected by transmission electron microscopy, ×10,000. (F) Expression of SESN2, phosphorylated adenosine monophosphate-activated protein kinase (PAMPK), NLR family pyrin domain containing 3 (NLRP3), interleukin 1β (IL-1β), α-smooth muscle actin (α-SMA) in SESN2-/- glomeruli through immunohistochemistry, ×200. (G) P-AMPK, NLRP3, IL-1β, α-SMA proteins from SESN2-/- mice detected by Western blot. (H) Quantitative analysis of Western blot. (I) SESN2, nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase- 1 (HO-1) proteins from SESN2-/- mice detected by Western blot. (J) Effect of SH on the nucleus expression of NRF2 in type 2 diabetes mellitus (T2DM) SESN2-/- mice kidney was detected by Western blot. (K) Quantitative analysis of Western blot. (L) SESN2, NRF2 in detected by confocal scanning microscopy in SESN2-/- mice, ×200, ×500. The position indicated by the white arrows is the site of nuclear import of NRF2. Data are expressed as the mean±standard error of the mean. DAPI, 4´,6-diamidino-2-phenylindole. aP<0.05, bP<0.01, cP<0.001, compared with the control group; dP<0.05, eP<0.01, fP<0.001 compared with the SESN2-/- group.

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  Fig. 5. Shionone (SH) could inhibit NLR family pyrin domain containing 3 (NLRP3) via sestrin-2 (SESN2)-nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) in high glucose (HG) treated mouse podocyte clone 5 (MPC-5) cells. (A) Expression of SESN2, phosphorylated adenosine monophosphate-activated protein kinase (P-AMPK), NRF2, HO-1, NLRP3, interleukin 1β (IL-1β), α-smooth muscle actin (α-SMA) in MPC-5 cells detected by Western blot. (B) Quantitative analysis of Western blot. (C) Effect of SH on the mRNA level of SESN2, NRF2, NLRP3, α-SMA in MPC-5 cells. Quantitative analysis of Western blot. Data are expressed as the mean±standard error of the mean. aP<0.05, bP<0.01, cP<0.001, compared with the control group; dP<0.05, eP<0.01, fP<0.001 compared with the SESN2-/- group; gP<0.05, hP<0.01, iP<0.001 compared with the SESN2+/+ group.

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