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 aging and prevent or treat CKD.

Chronic inflammation is a hallmark of aging³ and an important driver of many age-related diseases. Pro-inflammatory cytokines released by senescent cells are collectively known as the senescence-associated secretory phenotype (SASP)⁴. SASP promotes sustained inflammation, induces senescence in neighboring cells, and contributes to progressive functional decline⁵. The D-galactose (D-gal)-induced senescence model is one of the most widely used experimental aging models. Previous studies have shown that D-gal treatment leads to intracellular accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, disruption of redox homeostasis, and induction of stress-related cellular senescence⁶. Compared with natural aging models, which are time-consuming and exhibit high inter-individual variability, the D-galactose-induced model offers advantages including simplicity, rapid induction, and relatively low intra-group variability. This model is applicable to both in vivo and in vitro studies. Although it does not fully recapitulate slow, pathology-free natural aging, it is well suited for modeling renal senescence, given the kidney's high metabolic activity and susceptibility to oxidative stress. From a mechanistic perspective, D-galactose promotes cellular senescence primarily through oxidative stress, consistent with investigations focused on ROS generation and mitochondrial dysfunction during aging.

Cordyceps sinensis is a traditional Chinese medicinal fungus widely used in the treatment of kidney-related diseases⁷. It contains multiple bioactive components, including polysaccharides, adenosine, amino acids, cordycepin, and ergosterol. Extracts and fermented preparations of Cordyceps sinensis exhibit anti-inflammatory, antioxidant, and anti-aging properties^8,9,10,11. For example, Cordyceps polysaccharides have been shown to reduce inflammatory infiltration in an asthma mouse model by inhibiting the TGF-β1/Smad signaling pathway¹². In addition, Cordyceps polysaccharides can suppress MAPK pathway phosphorylation and reduce the expression of inflammatory cytokines such as IL-6 and IL-1β in RAW264.7 cells¹³. Previous studies have demonstrated that Cordyceps polysaccharides (CCP), the principal bioactive constituent of Cordyceps sinensis, can attenuate senescence in renal tubular epithelial cells by regulating autophagy and suppressing SASP production. However, the molecular mechanisms by which CCP counteracts ROS-mediated damage remain incompletely characterized.

The present study investigates the protective effects of CCP against renal aging in mice and examines its effects on inflammation, oxidative stress, and senescence using a D-galactose-induced model of renal tubular epithelial cell aging. We further explore whether CCP modulates ROS production, mitochondrial function, and downstream signaling pathways, including MAPK phosphorylation, NF-κB nuclear translocation, and NLRP3 inflammasome activation. Together, this work aims to provide experimental evidence supporting the use of CCP in studies of renal senescence and age-related kidney injury. The overall experimental workflow and proposed mechanism are summarized in Figure 1.

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 intragastric administration of 0.2 mL of saline per mouse; (2) Model group: Received daily subcutaneous injections of D-galactose (250 mg/kg), administered as 0.2 mL of a 25 mg/mL solution per mouse; (3) Low-dose CCP group: Received D-galactose injections combined with intragastric administration of CCP at 50 mg/kg (0.2 mL of a 5 mg/mL solution per mouse); (4) High-dose CCP group: Received D-galactose injections combined with intragastric administration of CCP at 100 mg/kg (0.2 mL of a 10 mg/mL solution per mouse); (5) Vitamin E group: Received D-galactose injections combined with intragastric administration of vitamin E at 100 mg/kg (0.2 mL of a 10 mg/mL solution per mouse). After 8 weeks of treatment, mice were euthanized, and serum, urine, and renal tissue samples were collected for subsequent analyses.

Renal function analysis

Renal function was assessed using commercial assay kits to measure creatinine, uric acid, and urinary protein according to the manufacturers' instructions. Absorbance values were measured at 505 nm for creatinine, 510 nm for uric acid, and 585 nm for urinary protein using a multifunction microplate reader.

Tissue section staining

Paraffin-embedded renal tissues were sectioned at a thickness of 4 µm. The sections were deparaffinized, incubated with hematoxylin for 5 min, rinsed with running water, differentiated using 1% acid alcohol for 5 s, and rinsed once more. Following a 1 min phosphate-buffered saline (PBS) wash, sections were counterstained with eosin for 3 min, dehydrated, and mounted. For Masson's trichrome staining, sections were deparaffinized and stained with hematoxylin as described above, followed by Ponceau S for 5 min and aniline blue for 5 min. Sections were then dehydrated and mounted.

Cell viability assay

HK-2 cells were seeded into 12-well plates at 1 × 10⁵ cells per well. Following 24 h exposure to D-galactose and Cordyceps polysaccharides, 10% CCK-8 solution was added to each well¹⁴. After incubation at 37 °C for 30 min, absorbance was recorded at 450 nm using a microplate reader. Quality control documentation for Cordyceps polysaccharides and STR authentication of HK-2 cells are provided in Supplemental File 1.

Reverse transcription quantitative polymerase chain reaction

After 24 h of cell culture, the culture medium was removed, and total RNA was extracted using the extraction reagent. RNA concentration was determined using a microplate spectrophotometer, and RNA integrity was verified by 2% agarose gel electrophoresis. Genomic DNA was removed, and cDNA was synthesized by reverse transcription. Primer sequences are listed in the Table of Materials.

Measurement of intracellular ROS levels

HK-2 cells were seeded into 12-well plates at 1 × 10⁵ cells per well. Upon reaching 60-70% confluence, cells underwent pretreatment with D-galactose for 12 h. Following 24 h of drug exposure, the medium was discarded and cells were rinsed once with PBS. Intracellular ROS was detected by incubation with a ROS-sensitive fluorescent probe at 37 °C for 20 min, and fluorescence images were captured at excitation/emission wavelengths of 488/525 nm¹⁵.

Western blot

Cellular proteins were lysed using RIPA buffer and clarified by centrifugation at 13,000 × g for 15 min at 4 °C. Proteins were denatured by boiling for 10 min, and equal amounts of protein (20 µg per sample) were subjected to electrophoretic separation and transferred to PVDF membranes. After blocking for 1 h, membranes were incubated overnight with primary antibodies. Subsequent PBST washes were followed by incubation with HRP-conjugated secondary antibodies, and detection was performed using an enhanced chemiluminescence (ECL) substrate¹⁶. Antibody details and reagent identifiers are listed in the Table of Materials.

SA-β-gal senescence staining

HK-2 cells were plated in 12-well plates at a density of 1 × 10⁵ cells per well. When cells reached 60-70% confluence, they were pretreated with D-galactose for 12 h. SA-β-gal staining was carried out according to the manufacturer's protocol, and stained cells were observed using an optical microscope¹⁷.

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential was evaluated using the JC-1 fluorescent probe in accordance with the supplier's instructions. JC-1 monomers were detected at excitation/emission wavelengths of 490/530 nm, whereas JC-1 aggregates were detected at 525/590 nm. Images were acquired when clear red or green fluorescence signals were observed.

Statistical analysis

Data were presented as bar or dot plots and analyzed using the referenced software. Western blot band intensities were quantified using ImageJ. Data were analyzed by analysis of variance (ANOVA) with subsequent t-tests for between-group comparisons. Statistical significance was defined as a P value < 0.05. Symbols indicate comparisons with the control group (*) or the D-gal group (#) (*P < 0.05, **P < 0.01, ***P < 0.001).

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’