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. 2025 Aug 5;10(32):35488–35496. doi: 10.1021/acsomega.4c11098

The Effect of Cordyceps sinensis Polysaccharide Combined with Cisplatin on LLC1 Lung Cancer

Xiaodong Wang a,b, Wei Chen b, Dongfang Zhuang b, Wenbin Deng c,*
PMCID: PMC12368679  PMID: 40852277

Abstract

Although cisplatin (DDP) is commonly used in the clinical treatment of lung cancer, the associated side effects remained serious. Chinese medicine can be used to increase the sensitivity of the body to antitumor drugs while reducing their adverse effects. Cordyceps sinensis polysaccharide (CSP), which is derived from C. sinensis, has already been demonstrated to regulate immune function, but the effects of CSP in combination with DDP have not yet been reported. In this study, we explore the synergistic and attenuated toxicity effects of CSP combined with DDP. Our results revealed that DDP and DDP + CSP combination treatment both induced cytotoxicity in LLC1 lung cancer cells and SK-MEL-28 melanoma cancer cells, and that CSP significantly increased tumor cell death. Moreover, cell toxicity in DDP-treated RAW 264.7 cells was suppressed by CSP. In a mouse model generated on LLC1 tumor-bearing mice, CSP and DDP combination treatment significantly increased DDP-induced tumor cell death. Western analysis revealed that this was achieved via CSP regulation of the expression levels of proteins involved in pyroptosiscaspase-1 and gasdermin D (GSDMD). In addition, CSP reduced oxidative stress levels to help alleviate the acute renal toxicity caused by DDP. In summary, our study provides further evidence of the efficacy of CSP and it lays a foundation for the clinical use of CSP as an adjuvant chemotherapy drug.

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Introduction

Although cisplatin is commonly used for the clinical treatment of lung cancer, it often causes serious side effects in treated patients, including immunosuppression, nephrotoxicity, hepatotoxicity and sarcopenia. − To address this issue, researchers have increasingly focused on improving the efficacy of cisplatin while reducing its side effects. Traditional Chinese medicine has been shown to enhance the sensitivity of the body to antitumor drugs, to improve the immune function of cancer patients, and to reduce the adverse reactions of radiotherapy and chemotherapy, reducing the overall risk of death. A number of Chinese herbal extracts that significantly improve the antitumor efficacy of cisplatin have already been identified. − Additionally, several extracts have been shown to attenuate the toxicity of cisplatin in the body. ,

An important component of these traditional Chinese medicines is polysaccharide, and polysaccharides generally account for a large proportion of the content in water extracts of traditional Chinese medicines. During cotreatment protocols, pharmacologically active polysaccharides in water extracts of traditional Chinese medicines have been reported to enhance the inhibitory effects of cisplatin on various cancers, and also to reduce its toxic side effects. , Examples include the water-soluble polysaccharide derived from Ganoderma lucidum, which was demonstrated to both increase the therapeutic effectiveness of cisplatin and to reduce its cytotoxicity. Similarly, Cudrania tricuspidata fruit-derived polysaccharides were demonstrated to alleviate the side effects of chemotherapeutic drugs.

The traditional Chinese medicine C. sinensis has many active components and a range of pharmacological effects. , Again, polysaccharides are the major pharmacologically active compounds of C. sinensis. , Some studies have shown that CS has inhibitory effect on both lung cancer and melanoma, such as the growth of B16-BL6 cells, LLC cells and hematogenic metastasis of B16–F1 cells were inhibited by cordycepin. , WECS, a water extract of CS, reduced HGF-accelerated B16–F0 cell invasion in a concentration-dependent manner. CS extract can inhibit the proliferation of lung cancer cell A549 by inducing apoptosis and activating immunosuppression. CME-1, a polysaccharide, purified from the mycelia of CS, could inhibit MMP-1 expressions in B16F10 melanoma cells thereby inhibiting cell migration. Moreover, extracts containing these CSPs can help regulate the body’s immune system by balancing helper T cells (Th1) and enhancing Th2 activity for immune regulation, thereby reducing cytotoxicity. − To date, most of the polysaccharides used in research were derived from C. militaris or the mycelium of C. sinensis. − And various medicinal products derived from fermented C. sinensis commonly employed in clinical practice, they are utilized in the treatment of conditions such as chronic bronchitis, chronic renal insuffciency, hyperlipidemia, cirrhosis and other diseases. In particular, only a few studies have been performed on the natural and ecological breeding products of C. sinensis. Furthermore, although we previously demonstrated that extracts of C. sinensis could enhance the antitumor activity of cisplatin and reduce its toxicity, additional evidence of the effects of CSP when combined with cisplatin is essential.

In the present study, the synergic effects of CSP and DDP were investigated to provide additional evidence for CSP enhancement of the tumor-killing effects of DDP and CSP attenuation of DDP cytotoxicity, and to understand CSP regulatory mechanisms. We report that CSP significantly enhanced the inhibitory effects of DDP in vitro, and that CSP helped alleviate DDP-induced cytotoxicity. In a mouse model, CSP increased tumor cell death by modulating pyroptosis. In addition, CSP significantly reduced DDP-induced renal toxicity in our mouse model. In summary, our study provides further evidence of the efficacy of C. sinensis and its polysaccharide active component, and it also lays a foundation for the clinical use of C. sinensis extracts as an adjuvant chemotherapy drug.

Results and Discussion

CSP Coapplication Increased DDP-Induced Cytotoxicity in Cancer Cells and Increased Survival in RAW 264.7 Cells

To investigate the effects of CSP combined with DDP in vitro, cell viability after CSP, DDP, or DDP+CSP treatment of different tumor cells was analyzed using a CellTiter-Glo (CTG) assay kit. As shown in Figure A, CSP (111.11 μg/mL) had a synergistic effect on LLC1 tumor cells when coapplied with DDP (10 μM), eliciting a statistically significant increase in cell inhibition (from 56.5% to 91.2%). Moreover, cell inhibition of SK-MEL-28 tumor cells by DDP was increased 25% following coapplication of CSP (Figure B). Hence, CSP demonstrated marked dose-dependent, synergistic activity on the proliferation of LLC1 and SK-MEL-28 tumor cells in vitro. However, cell inhibition of RAJI and NCI-H460 tumor cells had no significant increase when DDP coapplied with CSP (Figure C and D). To investigate the effects of CSP in vivo, LLC1 tumor cells were used to generate a mouse model of cancer.

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Effects of DDP and CSP combination treatment in vitro. Co-application of CSP and DDP (10.0 μM) to (A) LLC1 tumor cells, (B) SK-MEL-28 tumor cells, (C) RAJI tumor cells, (D) NCI-H460 tumor cells. (E) CSP inhibits DDP-induced RAW 264.7 cytotoxicity. Treatment groups compared with CSP (0 μg/mL) group, **p < 0.01, ***p < 0.001, ****p < 0.0001.

In addition, the effect of CSP on DDP-induced cytotoxicity in RAW 264.7 cells was determined using a CTG assay kit. As shown in Figure E, CSP application significantly promoted the proliferation of RAW 264.7 cells, causing an increase in the proliferation rate to 122.3%. Conversely, DDP application significantly reduced the survival rate of RAW 264.7 cells in a dose-dependent manner (establishing our experimental model). Importantly, the survival rate of RAW 264.7 cells was significantly increased with CSP coapplication (in the DDP+CSP group), yielding a 30% increase compared with DDP treatment only. Together, these results provide evidence that CSP can alleviate DDP-induced cytotoxicity in RAW 264.7 cells.

CSP and DDP Combination Treatment Inhibited Tumor Growth in LLC1 Tumor-Bearing Mice

Next, we verified the antitumor effect of CSP and DDP combination treatment in an in vivo model established using LLC1 tumor-bearing mice. The measured changes in in situ tumor volume over time in each treatment group are shown in Figure A. While DDP was observed to significantly inhibit tumor growth (as expected), the inhibitory effect of CSP and DDP combination treatment was significantly higher (compared with DDP only). At completion of the experiment (after harvesting of the tumor tissue), mean tumor volume in the DDP+CSP group was significantly smaller than mean tumor volume in the DDP group (Figure B), providing further confirmation of the results shown in Figure A. While mean tumor weight in the DDP+CSP group was decreased compared with mean tumor weight in the DDP group (Figure C), this decrease did not reach significance. This discrepancy can be explained by the observation that some tumor tissues were liquefied or hollow, confounding the correlation between volume and weight.

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Effects of CSP combined with DDP on mice. (A) The changes in tumor volume with different treatments. (B) Representative photographs of tumor after excision on day 36 (n = 5). (C) Mean weights of excised tumors (D) Body weights of mice during evaluation period after various treatments. Treatment groups compared with control group, ##p < 0.01, ###p < 0.001. DDP group compared with DDP+CSP group, *p < 0.05.

The body weights of Control group and Model group mice did not decrease significantly during the experiment (a slight upward trend was observed). While body weights in DDP group and DDP+CSP group mice did decrease gradually after DDP treatment (Figure D), this decrease was not significant (and was likely a side effect of DDP treatment). As expected, survival rate in the Model group was low (only 20%) compared with survival rate in the Control group (100%) in the absence of further treatment (Figure S1A). With DDP treatment, survival rate was increased to 80% in the DDP group, and to 100% in the DDP+CSP group. Additionally, DDP-induced toxicity was attenuated in mice receiving DDP and CSP combination treatment (see Figure S1B–1D). These figures graph the results of major organ index, liver function, and kidney function analyses, and provide evidence of CSP attenuation of DDP-induced toxicity. In summary, CSP with DDP combination treatment significantly enhanced the antitumor effects of DDP.

CSP Treatment Promoted the Expression of Proteins Involved in Programmed Cell Death

To explore the synergistic mechanism underlying CSP enhancement of DDP-induced inhibition of cell proliferation, we analyzed the expression levels of proteins related to pyroptosis pathway using Western blot analysis. As shown in Figure A and Figure B, the expression levels of pyroptosis-related Caspase-1 protein and GSDMD protein were significantly increased in the DDP+CSP group (when compared with the DDP group and Control group), providing evidence that CSP may promote pyroptosis in tumor cells. In summary, CSP significantly increased tumor cell death in DDP-treated mice through pyroptosis. By modulating the mechanisms of programmed cell death, CSP can synergistically enhance the tumor cell proliferation inhibition effects of DDP.

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Western blot analysis of protein expression in harvested tumor tissue samples (n = 5). (A) Representative staining bands, and (B) Relative protein levels of Caspase-1 and GSDMD. Treatment groups compared with model group, #p < 0.05, ##p < 0.01. DDP group compared with DDP+CSP group, **p < 0.01, ***p < 0.001.

Effects of CSP on DDP-Induced Acute Toxicity

To confirm the generalizability of our previous observation that CSP attenuates DDP-induced acute toxicity in RAW 264.7 cells, we established a mouse model of acute DDP-induced toxicity for in vivo studies. According to body weight measurements, mice in the Control group exhibited normal growth (Figure A), while mice in the DDP and DDP+CSP groups exhibited a significant decrease in body weight. Hence, while DDP treatment induced a decrease in body weight, CSP treatment could not alleviate this change. However, CSP cotreatment did alleviate other indices of DDP induced-toxicity. For example, we observed a decrease in relative thymus weight for the DDP group, and significant increases in the relative liver weight and relative kidney weight. CSP cotreatment significantly reduced the DDP-induced increases in relative liver weight and relative kidney weight (Figure B). These improvements are readily observable by simply comparing the corresponding relative organ weight in the DDP+CSP and DDP groups. Furthermore, although the AST index of liver function and the UREAL and CREA indices of renal function were significantly increased in the DDP group (Figure C and Figure D), these indices were significantly lower in the DDP+CSP group. Together, these results provide evidence that CSP can alleviate renal toxicity caused by DDP, and to a lesser extent hepatic toxicity.

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Effects of CSP on DDP-induced acute toxicity in mice (n = 3). (A) body weight, (B) relative organ weight, (C) ALT and AST levels, (D) UREAL and CREA levels. Treatment groups compared with model group, #p < 0.05, ###p < 0.001, ####p < 0.0001. DDP group compared with DDP+CSP group, **p < 0.01, ****p < 0.0001.

The Effects of CSP on Oxidative Stress and Nephrotoxicity

To understand how CSP alleviated DDP toxicity, we analyzed several indicators of oxidative stress. As shown in Figure A– C, MDA levels in the DDP group were significantly increased (compared with the control group), and the enzymatic activities of GSH-Px and SOD were significantly decreased. However, MDA levels in the DDP+CSP group were significantly decreased compared with the DDP group, and the enzymatic activities of GSH-Px and SOD were significantly increased. These results provide evidence that CSP significantly reduced DDP-induced oxidative stress.

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Oxidative stress indicators and TIM-1 protein expression levels following DDP treatment and CSP+DDP combination treatment (n = 3). (A) Enzymatic activity of SOD, (B) Levels of MDA, (C) Enzymatic activity of GSH-Px, (D) Representative immunohistochemical images of kidneys stained with anti-TIM-1 antibody, (E) TIM-1 protein levels (% positive staining). Treatment groups compared with model group, ##p < 0.01, ####p < 0.0001. DDP group compared with DDP+CSP group, *p < 0.05, **p < 0.01, ****p < 0.0001.

To further investigate DDP nephrotoxicity, we analyzed expression levels of TIM-1 protein, a known biomarker of acute kidney injury. TIM-1 protein levels were significantly increased in the DDP group (positive rate, 13.41%) compared with the Control group (positive rate, 0%). However, TIM-1 levels were significantly decreased in the DDP+CSP group (positive rate, 6.97%) compared with the DDP group (Figure D and Figure E). This result provides further evidence that kidney injury associated with DDP treatment was improved by CSP. As shown in Figure , the H&E staining results were generally consistent with the above results, and with the organ index results shown in Figure B. DDP caused obvious kidney injury, which manifested itself as diffuse tubular degeneration/necrosis (transparency type), in addition to effects on the liver and thymus tissue. Importantly, CSP helped to alleviate these changes. In summary, CSP reduced oxidative stress levels and TIM-1 protein expression levels (a biomarker of kidney injury), providing evidence that CSP can help reduce DDP-induced renal damage.

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Representative H&E stained images of major organsincluding the heart, liver, spleen, thymus, and kidneysharvested from mice with DDP-induced acute toxicity.

Discussion

Long-term use of DDP causes a series of adverse reactions in body, and drug resistance eventually limits its efficacy. Therefore, novel mechanisms that enhance the effects of DDP while mitigating its side effects are a top research priority. , A large number of studies have reported that polysaccharides can help alleviate the adverse reactions caused by DDP. − For example, fucoidan was demonstrated to alleviate ototoxicity induced by cisplatin, and polysaccharides from Annona muricata leaves were demonstrated to alleviate macrophage toxicity induced by cisplatin. In previous studies, the water extract of C. sinensis was demonstrated to enhance the antitumor effects of cisplatin while reducing treatment-related toxicity. From a consideration of the basic theory of traditional Chinese medicine, we have explored the synergic effects of CSP and DDP in the present study, investigating CSP enhancement of the antitumor effect of cisplatin and CSP alleviation of treatment-related toxicity. CSP significantly enhanced the in vitro efficacy of DDP in inhibiting tumor cell proliferation, and it also reduced DDP-induced toxicity in RAW 264.7 cells. These results are consistent with those obtained using other polysaccharides. In addition, CSP significantly enhanced the in vivo efficacy of DDP in a mouse model, while reducing its overall cell toxicity.

Previous studies have shown that inducing tumor cell death is a meaningful therapeutic strategy. , Cell death can be classified as active cell death, such as apoptosis, pyroptosis, etc., or passive cell death, specifically cell necrosis. To further understand the effects of CSP combined with DDP, we used Western blot analysis on LLC1 tumor-bearing mice to show that CSP enhanced the tumor inhibition effect of DDP by regulating the expression of proteins related to pyroptosis. Pyroptosis is a type of programmed cell death, although it differs from apoptosis in several ways. Pyroptosis is mediated through either the classical pathway (which relies on Caspase-1) or the nonclassical pathway. In the classical pathway, the cell recognizes an external stimulus, and Caspase-1 is activated to cleave GSDMD protein. This releases peptide fragments at the amino terminal end of GSDMD that encode a pore-forming domain, which eventually induces membrane perforation, cell rupture, the release of cell contents, and inflammation. , Our present results reveal that CSP combined with DDP increased cell death in tumor cells by up-regulating the expression levels of proteins involved in pyroptosis (Caspase-1 and GSDMD).

In addition, CSP reduced oxidative stress by regulating the activities of oxidative stress-related enzymes, and it also reduced nephrotoxicity, as assessed by a reduction in TIM-1 protein expression. TIM-1 (also known as KIM-1, kidney injury molecule 1) is a transmembrane glycoprotein involved in regulating immune cell activity, and it is commonly used as a biomarker of acute kidney injury. This is because TIM-1 expression is increased in the proximal tubular cells during acute kidney injury. A decrease of TIM-1 production in the proximal tubular cells after acute kidney injury has been shown to reflect an improvement of kidney function. Hence, CSP helps to alleviate DDP-induced acute renal toxicity in mice.

Conclusion

The findings presented in this study provide evidence that CSP inhibits the growth of tumor cells by regulating the expression levels of proteins involved in pyroptosis, processes that ultimately lead to tumor cell death. In addition, CSP helps alleviate DDP-induced nephrotoxicity by decreasing oxidative stress levels and relieving kidney injury (as assessed by a reduction in TIM-1 expression). Although we did not conduct an in-depth exploration of other DDP-induced toxicity processes in this study (with the exception of nephrotoxicity), we can conclude that CSP demonstrates great potential.

Although C. sinensis has been collected by the Chinese Pharmacopoeia, the quality control of CSP still remains at the level of total sugar content determination, resulting in a serious shortage of clinical efficacy. In this paper, the combined treatment of CSP and DDP on lung cancer was studied, and found that CSP could increase the efficacy and reduce the side effects of DDP. There is still more work to be done in the clinical application of CSP, but this finding still provides certain guidance and directions for future clinical research of CSP. In conclusion, clinical CSP and DDP combined use may enhance the efficacy of DDP in patients and reduce its side effects.

Materials and Methods

Materials

C. sinensis specimens were provided by Dongguan HEC Cordyceps R & D Co., Ltd. DDP was procured from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and Trypsin-EDTA (0.25%) were procured from Gibco Life Technologies. The BCA kits were from Thermo Fisher Scientific Co., Ltd. CellTiter-Glo2.0 Reagent was purchased from Promega Biotech Co., Ltd. Anti-GSDMD Rabbit pAb, and Anti-Caspase-1 Rabbit pAb were purchased from Servicebio Technology Co., Ltd. RIPA lysate and supersensitive ECL chemiluminescence kits were from Beyotime Biotechnology Co., Ltd. The 10% ExpressCast PAGE Gel Preparation kit was purchased from NCM Biotech Co., Ltd. The Superoxide Dismutase (SOD) assay kit, Glutathione Peroxidase (GSH-PX) assay kit, and Malondialdehyde (MDA) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. Anti-TIM-1 antibody was provided by Abcam (Shanghai) Trading Co., Ltd.

Preparation of CSP

CSP preparation was performed using the method of Wang et al. Briefly, the fat-soluble components were removed from crushed C. sinensis by reflux heating in 80% ethanol. The residue was then mixed with water and extracted three times (for 2 h each time) by reflux at 100 °C. The three water-soluble extractions were combined and then concentrated under reduced pressure. After adding ethyl alcohol, the extract was precipitated for 24 h and then centrifuged. The resulting precipitate was redissolved in water (with heating), and subsequently deproteinized using the Sevage method. After dialysis for 48 h, the solution was filtered through a 0.45 μm membrane. Finally, the resulting filtrate was mixed with ethanol, centrifuged, precipitated, redissolved, and freeze-dried to obtain CSP.

Cell Culture

LLC1 cells (a lung cancer cell line), SK-MEL-28 cells (a melanoma cell line), and RAW264.7 cells (a monocyte/macrophage-like cell line) were purchased from the American Type Culture Collection (ATCC). All three cell types were cultured at 37 °C under 5% carbon dioxide in dulbecco’s modified eagle medium (DMEM) which contains 10% Foetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin Solution (changed every 2 days). To ensure optimal subculturing, the cells were observed under an inverted microscope every day, and then subcultured as the growth conditions required. Once the cells reached their recommended confluency, the spent medium was discarded, the cell layer was washed twice with PBS, and the cells were digested with pancreatic enzyme digestion solution. Fresh culture medium was added to stop the digestion process after the cells became rounded and floated upward. The dissociated cells were then transferred to a centrifuge tube, gently centrifuged, and subcultured at their appropriate cell passage densities.

Effects of CSP on Different Cells

Cells in logarithmic growth phase were resuspended with complete culture medium to an appropriate concentration. The cells were then inoculated into 96-well plates and incubated at 37 °C for 24 h. After administration of CSP for 1 h, DDP was added to the appropriate wells. All drug treatments were performed in triplicate, and control and background wells were included. After incubation at 37 °C for 24 h, CTG was used to detect cell activity. The results were then used to calculate the effect of drugs on cell proliferation.

Animals

Male C57BL/6J mice (SPF) were purchased from Hunan Silaikejingda Laboratory Animal Co., Ltd. (Hunan, China). All animals were housed under control conditions (temperature range, 20 ∼ 26 °C; humidity range, 40 ∼ 70%) and were provided clean drinking water and sufficient feed. The animal experiments were performed in strict compliance with all relevant animal welfare and ethics regulations. This study was approved by SUNSHINE LAKE PHARMA CO., Ltd. (IAEC-K-231016–01, IAEC-K-231204–01).

Enhancement Effect of CSP Combined with DDP In Vivo

Twenty mice were randomly divided into four groups: a Control group; a Model group; a DDP group; and a DDP+CSP group. The Control group, Model group, and DDP group were administered ultrapure water once a day (by intragastric loading), while the DDP+CSP group was administrated CSP (100 mg/kg) once a day (by intragastric loading). On the eighth day, mice in the Model group, DDP group, and DDP+CSP group were subcutaneously inoculated with LLC1 cells. From the ninth day onward, saline was administered intraperitoneally every 2 days to mice in the Control group and Model group (until the end of the experiment). In parallel (every 2 days from the ninth day), DDP (2 mg/kg) was administered intraperitoneally to mice in the DDP group and the DDP+CSP group (until the end of the experiment).

Starting from the 19th day, subcutaneous tumor size was monitored (twice a week) in all mice within each group using Vernier calipers. The longest diameter (a) and shortest diameter (b) of the tumors were measured, and tumor volume (V) was determined from these measurements (using V = ab2/2). If the calculated tumor volume was greater than 2000 mm3, the mouse was immediately euthanized. The quality of life (QoL) of every mouse in each group was also monitored twice a week, and the weights of individual mice were recorded. At completion of the experiment, all mice were ethically euthanized, and the subcutaneous tumor tissue was harvested for further experiments.

Acute Toxicity by DDP on Mice

Fifteen mice were randomly divided into three groups: a Control group; a Model group; and a CSP group. Mice in the CSP group were administered CSP (100 mg/kg) once a day (by intragastric loading) throughout the course of the experiment, while mice in the Control and Model groups were administered ultrapure water (by intragastric loading) once a day. A mouse model of acute toxicity was immediately established in the Model and CSP groups on the first day of the experiment (1 h after administering the test material) through intraperitoneal injection of DDP (20 mg/kg); for comparison, mice in the Control group were administered saline by intraperitoneal injection. Throughout the course of the experiment, mouse QoL was observed and recorded in each group (once per day). Additionally, the weight of each mouse was monitored once a day. On the final day of the experiment, the kidney tissues of individual mice were harvested, processed, and stained with TIM-1 to observe kidney damage. To evaluate histopathological changes in the major organs, hematoxylin and eosin (H&E) staining was also performed.

The Indices of Serum Biochemistry and Organs

On the final day of the experiment, blood was collected from the orbital veins of each mouse using capillary tubes. The collected blood samples were allowed to clot and then centrifuged. The serum samples obtained were collected and analyzed on a Cobas C311 clinical chemistry analyzer (Roche) for blood routine tests and to detect ALT, AST, UREA, and CREA. The main organs were then extracted and rinsed repeatedly in saline. After drying on filter paper, the organs were weighed and relative organ weight (mg/g) was calculated as relative heart/liver/spleen/kidney/thymus weight (mg/g)= heart/liver/spleen/kidney/thymus mass (mg)/body weight (g).

Western Blot Analysis

Total protein was extracted from tumor tissue samples using complete RIPA lysis buffer. The concentration of total protein in individual samples was then determined using a BCA kit. The total protein samples were denatured by boiling, and subsequently separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% ExpressCast PAGE gel. Next, protein bands were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane using standard techniques. The membrane was subsequently blocked using a rapid sealing solution and incubated with primary and then secondary antibodies. Finally, a chemiluminescence gel imaging system was used to detect the target proteins.

Evaluation of Oxidative Stress in Mice

Mouse kidney tissues were harvested, and a 10% tissue homogenate was prepared (in a saline ice bath). Individual supernatants were obtained for further analysis by centrifugation (4 °C, 15 min) of the homogenate at 3000 rpm. The levels of malonaldehyde (MDA) and antioxidant enzymesglutathione peroxidase (GSH-Px) and superoxide dismutase (SOD)were detected using a chemical assay kit (according to the manufacturer’s protocol).

Statistical Analysis

Data are expressed as Mean ± SD of the measured results, and analyzed using GraphPad Prism8.0 software. The normal distribution and homogeneity of variance were tested. Consistent with normal distribution (p > 0.10) and homogeneity of variance (p > 0.10), one-way ANOVA was used and multiple comparisons were made by LSD method, p < 0.05 were considered statistically significant. Non-normal distribution or inconsistent variance: the Kruskal–Wallis H method of nonparametric test was adopted. If the result of Kruskal-wallis H test was significant (p < 0.05), Dunn’s was used for pound-to-group comparison, and p < 0.05 was considered statistically significant.

Supplementary Material

ao4c11098_si_001.pdf (358.2KB, pdf)

Acknowledgments

We are grateful for the financial support from the Shenzhen Science and Technology Program (KQTD20190929173853397), Shenzhen Key Laboratory of Neural Cell Reprogramming and Drug Research (ZDSYS20230626091202006), the National Natural Science Foundation of China (81971081), and Guangdong Major Project of Basic and Applied Basic Research (2023B0303000026).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c11098.

  • Effects of CSP combined with DDP on mice (Figure S1), survival rate, major organ indices, ALT and AST levels, UREAL and CREA levels; original Western blot of GSDMD and β-actin (Figure S2); original Western blot of caspase-1 and β-actin (Figure S3) (PDF)

X.W. and W.C. contributed equally to this study. Xiaodong Wang: conceptualization, methodology, project administration and writing (review and editing); Wei Chen: methodology, formal analysis, visualization, data curation, writing (original draft), and writing (review and editing); Dongfang Zhuang: validation, formal analysis and investigation; Wenbin Deng: conceptualization, resources, supervision and funding acquisition.

The authors declare no competing financial interest.

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