Effects of Cordyceps Polysaccharides on D-Galactose-induced Renal Senescence

Summary

A senescence model was established in mice and HK-2 cells using D-galactose. The effects of Cordyceps polysaccharides (CCP) on renal aging were evaluated using western blotting and immunofluorescence, with mitochondrial changes assessed by reactive oxygen species detection and JC-1 staining.

Abstract

Chronic kidney disease (CKD) poses a significant global health concern. Aging of renal tubular epithelial cells (RTECs) impairs kidney regeneration and repair and promotes CKD progression through the release of the senescence-associated secretory phenotype (SASP). Cordyceps sinensis, whose key bioactive constituents include Cordyceps polysaccharides (CCP), is incorporated into multiple clinical preparations for CKD and has demonstrated therapeutic benefits. To examine the role of CCP in renal aging, a D-galactose-induced renal senescence model was established in mice, with CCP administered as an intervention. Treatment outcomes were evaluated using senescence-related staining and assessments of renal function. A D-galactose-induced aging model in HK-2 cells was further established to investigate underlying cellular mechanisms. JC-1 staining and reactive oxygen species (ROS) detection were used to assess mitochondrial changes following CCP treatment under senescent conditions. Immunofluorescence, western blotting, and quantitative PCR (RT-PCR) were performed to evaluate the effects of CCP on MAPK phosphorylation, NF-κB nuclear translocation, and NLRP3 inflammasome activation. The results show that CCP attenuates tubulointerstitial cell senescence, renal injury, and fibrosis, while improving mitochondrial function in both cellular and animal models of D-galactose-induced senescence. These effects are associated with modulation of the ROS/MAPK/NF-κB/NLRP3 signaling pathway.

Introduction

Chronic kidney disease (CKD) is one of the most common kidney disorders and affects a large population worldwide. Its occurrence is closely associated with aging¹. Renal tubules are the primary functional units of the kidney, and cellular senescence within renal tubular epithelial cells (RTECs) is a major contributor to renal functional decline². Drug exposure and other stressors can induce tubular stress-associated senescence, leading to progressive loss of renal function and eventual progression to end-stage kidney disease (ESKD). Therefore, targeting senescence in RTECs may be an effective strategy to delay renal agin....

Protocol

All animal procedures were conducted in accordance with institutional guidelines and approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (Ethics Approval No. 202108A006). All waste generated during cell culture and staining procedures was collected in sealed containers and biohazard bags and disposed of by a licensed medical waste management service.

Animal model and therapeutic interventions

To minimize interindividual variation, 40 mice were randomly assigned to five groups (n = 8 per group) and subjected to the following interventions: (1) Control group: Received in....

Results

D-galactose was used to establish a mouse senescence model, with vitamin E serving as a commonly used anti-senescence positive control. Senescent mice were treated with different doses of Cordyceps polysaccharides (CCP). Aged mice exhibited significant renal impairment, as evidenced by increased urinary protein, serum uric acid, and creatinine levels (Figure 2B-D). Both vitamin E and CCP supplementation attenuated these abnormalities. SA-β-gal staining showed reduced renal c.......

Discussion

This study demonstrates the protective effects of Cordyceps polysaccharides on renal tubular epithelial cells and provides evidence that they attenuate inflammatory senescence in HK-2 cells by modulating the ROS/MAPK/NF-κB/NLRP3 signaling pathway. These findings further clarify potential mechanisms underlying the effects of Cordyceps sinensis-derived compounds in the treatment of acute and chronic kidney diseases and offer a reference for future investigations of anti-a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Casepase1/Cleaved Rabbit pAb	WL03450	Wanlei, Shengyang, China
CCK-8 Kit	C0037	Biyotime,Shanghai,China
CCP	B21296	Yuanye Bio,Shengyang, China
Creatinine Test Kit	C011-2-1	Jiancheng,Nanjing,China
D-gal	HY-N0210	MCE, USA
ERK Rabbit pAb	4695	CST, USA	AB_390779
gapdh Rabbit pAb	ac033	Abclonal, Wuhan, China
H & E staining Kit	G1120	Solarbio,Beijing,China
JC-1 Kit	C2003S	Biyotime,Shanghai,China
Masson's staining Kit	G1340	Solarbio,Beijing,China
NLRP3 Rabbit pAb	WLH3383	Wanlei, Shengyang, China
P21 Rabbit pAb	64016s	CST, USA	AB_2892063
p38 Rabbit pAb	9212	CST, USA	AB_330713
p50 Rabbit pAb	3035	CST, USA	AB_330564
p65 Rabbit pAb	8242	CST, USA	AB_10859369
p-ERK Rabbit pAb	4370	CST, USA	AB_2315112
Phospho-SAPK/JNK mAntibody	4668	CST, USA	AB_823588
p-p38 Rabbit pAb	4511	CST, USA	AB_2139682
ROS Staining Kit	S0033S	Biyotime,Shanghai,China
SAPK/JNK Antibody	9256	CST, USA	AB_2250373
SA-β-gal Kit	C0602	Biyotime,Shanghai,China
Uric Acid Test Kit	C012-2-1	Jiancheng,Nanjing,China
Urinary Protein Detection Kit	C035-2-1	Jiancheng,Nanjing,China
Vitamin E	S27812	Yuanye Bio,Shengyang, China
Primer for pcr
Gene name	Forward Primer	Reverse Primer
Caspase-1	5’-TACAGACAAGGGTGCTGAACAA-3’	5’-CGGAATAACGGAGTCAATCAAA-3’
GAPDH	5’-AGAAGGTGGTGAAGCAGGCGTC-3’	5’-AAAGGTGGAGGAGTGGGTGTCG-3’
NLRP3	5’-GATCTTCGCTGCGATCAACAG-3’	5’-CGTGCATTATCTGAACCCCAC-3’