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. 2022 Feb 10:12:801094.
doi: 10.3389/fphar.2021.801094. eCollection 2021.

Cordyceps cicadae Ameliorates Renal Hypertensive Injury and Fibrosis Through the Regulation of SIRT1-Mediated Autophagy

Affiliations

Cordyceps cicadae Ameliorates Renal Hypertensive Injury and Fibrosis Through the Regulation of SIRT1-Mediated Autophagy

Yuzi Cai et al. Front Pharmacol. .

Abstract

Hypertensive renal injury is a complication of hypertension. Cordyceps cicadae (C. cicadae) is a traditional Chinese medicine used to treat chronic kidney diseases especially renal fibrosis. Autophagy is described as a cell self-renewal process that requires lysosomal degradation and is utilized for the maintenance of cellular energy homeostasis. The present study explores the mechanism underlying C. cicadae's renoprotection on hypertensive nephropathy (HN). First, HN rat models were established on spontaneously hypertensive rats (SHRs). The expression of fibrosis-related protein and autophagy-associated protein was detected in vivo. NRK-52E cells exposed to AngII were chosen to observe the potential health benefits of C. cicadae on renal damage. The level of extracellular matrix accumulation was detected using capillary electrophoresis immunoquantification and immunohistochemistry. After treatment with lysosomal inhibitors (chloroquine) or an autophagy activator (rapamycin), the expression of Beclin-1, LC3II, and SQSTM1/p62 was further investigated. The study also investigated the change in sirtuin1 (SIRT1), fork head box O3a (FOXO3a), and peroxidation (superoxide dismutase (SOD) and malondialdehyde (MDA)) expression when intervened by resveratrol. The changes in SIRT1 and FOXO3a were measured in patients and the SHRs. Here, we observed that C. cicadae significantly decreased damage to renal tubular epithelial cells and TGFβ1, α-smooth muscle actin (α-SMA), collagen I (Col-1), and fibronectin expression. Meanwhile, autophagy defects were observed both in vivo and in vitro. C. cicadae intervention significantly downregulated Beclin-1 and LC3II and decreased SQSTM1/p62, showing an inhibition of autophagic vesicles and the alleviation of autophagy stress. These functions were suppressed by rapamycin, and the results were just as effective as the resveratrol treatment. HN patients and the SHRs exhibited decreased levels of SIRT1 and FOXO3a. We also observed a positive correlation between SIRT1/FOXO3a and antifibrotic effects. Similar to the resveratrol group, the expression of SIRT1/FOXO3a and oxidative stress were elevated by C. cicadae in vivo. Taken together, our findings show that C. cicadae ameliorates tubulointerstitial fibrosis and delays HN progression. Renoprotection was likely attributable to the regulation of autophagic stress mediated by the SIRT1 pathway and achieved by regulating FOXO3a and oxidative stress.

Keywords: Cordyceps cicadae; SIRT1; autophagy; fibrosis; hypertensive renal injury.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
C. cicadae alleviated SHR renal injury and fibrosis. (A) H&E staining (bar = 100 μm) and Masson’s trichrome staining (bar = 40 μm). (B) C. cicadae treatment significantly decreases ACR levels after 14 and 28 weeks. (C) C. cicadae treatment significantly downregulates β2-MG expression after 14 and 28 weeks. (D) C. cicadae treatment significantly reduced β2-MG level at the 28th week. (E) The representative images and statistical graph of IHC staining for α-SMA (×200). Bar = 100 μm. Apoptosis in rat renal tubules of various groups was evaluated by TUNEL assay. Scale bar: 40 μm. (F) Effect of C. cicadae and RES on TGFβ1, α-SMA, FN, and Col-1 expression. (G–J) The relative intensities of fibrosis-related protein in kidneys were calculated after normalization against GAPDH. Data were presented as mean ± SD, n = 10 rats per group. For the WKY group vs the SHR group, #p < 0.05, ##p < 0.01. For the SHR group vs the C. cicadae group, *p < 0.05, **p < 0.01. N.S. No significance.
FIGURE 2
FIGURE 2
CMS attenuated AngII-associated injury and inhibited ECM accumulation in rat renal tubular epithelial cells. (A) NRK-52E cells were exposed to various concentrations of AngII for 24 and 72 h. (B) Effects of CMS on viability for 72 h. (C) Effects of RES on NRK-52E cell viability for 72 h. (D,E) Effect of CMS on NRK-52E cell injury. The level of cell injury was examined by KIM-1 and NGAL ELISA kits. (F) Effect of CMS on the expression of Col-1 and α-SMA. (G,H) The relative intensities of fibrosis-related protein in cells were calculated after normalization against GAPDH. Data are expressed as the mean ± SD of three separate experiments. # p < 0.05, ## p < 0.01 compared with control cells. *p < 0.05, **p < 0.01, compared with the model group.
FIGURE 3
FIGURE 3
C. cicadae and RES regulated autophagic stress in hypertensive kidney damage. (A) IHC staining result (×400) of LC3II and p62 in paraffin sections. Bar = 50 μm. (B,C) Statistical results of IHC staining of LC3II and p62, respectively. The value of mean optical density (IOD) obtained by IPP software. (D) Expression of beclin-1, LC3II, and p62. The results of evaluation are shown in (E–G). # p < 0.05, ## p < 0.01: compared with controls. *p < 0.05, **p < 0.01: relative to the model group. N.S. No significance. (H) Immunofluorescence images show the levels of LC3II (green channel) and the levels of p62 (red channel) for 48 and 72 h. Bar = 20 μm. (I–K) Statistical results of immunofluorescence staining of LC3II and p62. The value of IOD obtained by IPP software. (L) Expression of beclin-1 and LC3II. The results of evaluation are shown in (M) and (N). # p < 0.05, ## p < 0.01: compared with controls, respectively. *p < 0.05, **p < 0.01: relative to the model group, respectively.
FIGURE 4
FIGURE 4
CMS attenuated fibrosis via regulating autophagic stress in vitro. (A) NRK-52E cells were treated with AngII for 72 h, in the presence or absence of CQ (10 μM). (B) CMS regulated autophagic stress. LC3II protein expression was assessed by western blotting. (C) Statistical results of LC3II. (D) Immunofluorescence images show the levels of α-SMA (green channel) for 72 h. NRK-52E cells were exposed to AngII, with or without CMS and in the presence or absence of RAP (50 nM) for 72 h. The fluorescence intensity was detected with a laser confocal microscope, bar = 20 μm. # p < 0.05: relative to untreated cells. *p < 0.05, **p < 0.01: relative to the model group.
FIGURE 5
FIGURE 5
Experimental validation of the SIRT1/FOXO3a/ROS signaling pathways in vivo and in vitro. (A) Downregulated SIRT1 and FOXO3a existed in HN patients (bar = 40 μm). (B) Capillary electrophoresis immunoquantification of SIRT1 and FOXO3a on NRK-52E cells in each group. (C) Capillary electrophoretic analysis of SIRT1 and FOXO3a expression in the rat renal cortex of various study groups. (D,E) CMS regulated SIRT1 (green channel) and FOXO3a (red channel) signaling pathways on NRK-52E cells (bar = 20 μm). (F,G) Statistical results of immunofluorescence staining of SIRT1 and FOXO3a, respectively. Densitometry was conducted, and the ratio of SIRT1 or FOXO3a to GAPDH was expressed as fold changes relative to the control. (H,I) The content of intracellular MDA and SOD in different treatment rats. (J,K) The activity of MDA and SOD in different treatment cells. #p < 0.05, ##p < 0.01: relative to the control group. *p < 0.05, **p < 0.01: relative to the model group. N.S. No significance.

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