Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR-30a-3p/TRIM16 axis

Abstract

Objective: The molecular mechanism of the protective effect of Cordyceps cicadae polysaccharides (CCPs) on renal tubulointerstitial fibrosis in diabetic nephropathy (DN) is still unclear. This study aims to further understand the molecular mechanisms behind the therapeutic benefits of CCP on diabetic nephropathy.

Methods: Mice were randomly assigned into six groups (n = 8). Cordyceps cicadae polysaccharide dissolved in 5% dimethyl sulfoxide was administered by gavage for 12 consecutive weeks. The CCP doses were divided into low, medium, and high, 75, 150, and 300 mg/kg/day, respectively. The efficacy of CCP was determined by assessing the renal function and histological alterations in diabetic db/db mice. The degree of glomerular mesangial dilatation and sclerosis was evaluated using semiquantitative markers. Cell viability, apoptosis, epithelial-mesenchymal transition (EMT), inflammation, oxidative stress, and mitochondrial reactive oxygen species (ROS) in high glucose (HG)-cultured MPC5 podocytes were determined. The interaction of miR-30a-3p and tripartite motif-containing protein 16 (TRIM16) was examined by luciferase reporter assay. Western blotting, reverse transcription-polymerase chain reaction, and immunofluorescence were used to analyze gene and protein expressions.

Results: The in vivo findings illustrated that CCP may protect mice with type 2 diabetes from inflammation and oxidative damage (P < 0.05). Furthermore, CCP has a therapeutic value in protecting renal function and morphology in diabetic nephropathy by reversing podocyte EMT. The in vitro results indicated that CCP dose-dependently inhibited HG-induced apoptosis, EMT, inflammation, oxidative stress, and mitochondrial ROS levels in MPC5 podocytes (P < 0.05). Luciferase reporter assay confirmed the interaction between miR-30a-3p and TRIM16 in MPC5 podocytes cultured in high glucose (P < 0.05).

Conclusion: The protective effect of CCP on HG-induced MPC5 can be achieved by miR-30a-3p/TRIM16 axis.

Keywords: CCP; Diabetic nephropathy; miR-30a-3p.

Figure 1

Protective effect of cordyceps cicadae polysaccharides treatment on renal function and morphological changes in diabetic nephropathy mice. (a) Kidney weight. (b) The pathological changes of kidney tissues were observed by periodic acid‐Schiff staining. Scale bar = 50 μm. (c, d) Quantitative analysis of glomerular size and mesangial expansion scores. (e) Renal fibrosis areas were determined by Masson's staining. Scale bar = 50 μm. (f) Relative fibrosis area of Masson's staining was quantified and shown. Data shown are representative of five panels per animal. Data are expressed as mean ± SD, n = 8. *P < 0.05, **P < 0.01.

Figure 2

Effects of cordyceps cicadae polysaccharides on the parameters of oxidative stress and inflammatory cytokine production in kidney. (a–c) Expression levels of multiple pro‐inflammatory cytokines were analyzed in the kidney tissues of administered mice using ELISA assay. (a) IL‐6, (b) IL‐1β. (c) TNF‐α. (d–f) Oxidative stress parameters of each group, renal superoxide dismutase activity (d), renal catalase activity (e), renal malondialdehyde content (f). (g) 8‐OHdG levels in urine detected by ELISA assay. Data are presented as mean ± SD, n = 8. *P < 0.05, **P < 0.01.

Figure 3

Cordyceps cicadae polysaccharides administration attenuated cell apoptosis and epithelial–mesenchymal transition in the kidneys of diabetic nephropathy mice. (a, b) At the end of 12 weeks of treatment, the expression of miR‐30a‐3p and TRIM16 was determined in the kidney tissues of mice in each group by RT‐PCR, n = 8. (c) The protein expression level of TRIM16 in kidney tissues of mice was analyzed by western blot, n = 5. (d, e) Protein expression of TRIM16 in the kidney tissues using IHC staining, scale bar = 50 μm, n = 5. (f) TUNEL staining was used to detect cell apoptosis in the kidney tissues, scale bar, 20 μm, n = 5. (g) Protein expression of Bcl‐2 and Bax in the kidney tissues were detected by IHC staining, scale bar = 50 μm, n = 5. (h) Representative images of immunofluorescence staining for P‐cadherin (red), N‐cadherin (green), and DAPI (blue), scale bar, 50 μm, n = 5, scale bar = 20 μm. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01.

Figure 4

Cordyceps cicadae polysaccharides protects MPC5 podocytes from apoptosis and epithelial–mesenchymal transition induced by high glucose. MPC5 podocytes in all groups were cultured in normal glucose, mannitol and high glucose for 24 h. (a) Cell viability in each group was determined by MTT assay. (b, c) Apoptosis of podocytes was detected by the flow cytometry. (d) mRNA level of BAX and BCL‐2 was detected by RT‐PCR. (e) Western blotting was used to detect apoptosis related markers (Cleaved‐caspase‐3, Caspase‐3, Bax, and Bcl‐2) in MPC5 podocytes. The graph represents the densitometric analysis normalized to β‐Actin. (f) Representative western blot analysis of Nephrin, P‐cadherin, Desmin, and Vimentin expression in MPC5 podocytes. The graph represents the densitometric analysis normalized to β‐Actin. (g) Representative immunofluorescence images of E‐caherin (E‐cad, green) and N‐cadherin (N‐cad, red). Cell nuclei were labeled in blue by DAPI. Scale bar, 50 μm. Data are presented as mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Cordyceps cicadae polysaccharides (CCP) treatment inhibited HG‐induced oxidative stress in MPC5 podocytes. (a–c) The levels of inflammatory cytokines (IL‐6, IL‐1β, and TNF‐α) were examined using commercially ELISA kits, respectively. (d, e) The effect of CCP on MPC5 podocytes ROS production was detected by a fluorescent MitoSox probe, scale bar, 20 μm. (f–h) The content of malondialdehyde and the activities of superoxide dismutase and catalase were measured by the commercial assay kits, respectively. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Expression of miR‐30a‐3p and TRIM16 in MPC5 podocytes treated with high glucose. The MPC5 podocytes were treated with normal glucose, mannitol, and high glucose conditions for 6, 12, and 24 h. (a) The expression level of miR‐30a‐3p in mouse podocyte was detected by RT‐qPCR. (b, c) The mRNA and protein expression of TRIM16 was determined by RT‐PCR and western blotting. *P < 0.05, **P < 0.01.

Figure 7

miR‐30a‐3p regulates TRIM16 in MPC5 podocytes. (a) TRIM16 is a predicted target of miR‐30a‐3p by TargetScan. (b) A luciferase reporter plasmid containing wildtype or mutant TRIM16 was cotransfected into MPC5 podocytes with miR‐30a‐3p or miR‐NC. Luciferase activity was determined at 48 h after transfection using the dual‐luciferase assay and was normalized to Renilla activity. (c–e) RT‐PCR and western blot analysis of TRIM16 in podocytes transfected with miR‐30a‐3p or miR‐NC or miR‐30a‐3p inhibitor or inhibitor of negative control. Data are presented as mean ± SD, n = 3. **P < 0.01, ***P < 0.001. ^## P < 0.001.

Figure 8

Cordyceps cicadae polysaccharides protect mouse podocyte from high glucose induced apoptosis and epithelial–mesenchymal transition by controlling miR‐30a‐3p expression. (a) Relative miR‐30a‐3p and TRIM16 expression were detected by RT‐PCR and western blotting analysis. (b) Cell viability was determined by MTT assay. (c, d) The apoptosis of cells as detected by flow cytometry. (e) Representative images of TUNEL staining to illustrate apoptotic MPC5 podocytes in each group, cell nuclei were labeled in blue by DAPI, scale bar, 50 μm. (f) Representative immunofluorescence images of E‐cadherin (E‐cad, red) and N‐cadherin (N‐cad, green), cell nuclei were labeled in blue by DAPI, scale bar, 20 μm. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9

Cordyceps cicadae polysaccharides protect MPC5 podocytes from high glucose induced inflammation and oxidative stress by controlling miR‐30a‐3p expression. (a) The levels of inflammatory cytokines (IL‐6, IL‐1β, and TNF‐α) were examined using commercially ELISA kits, respectively. (b, c) ROS production was detected by a fluorescent MitoSox probe, scale bar, 20 μm. (d) The levels of oxidative stress‐related markers (superoxide dismutase, catalase, glutathione, and malondialdehyde) were measured by the commercially assay kits. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.