A neutral polysaccharide from Ophiocordyceps lanpingensis restrains cisplatin‐induced nephrotoxicity

Abstract Ophiocordyceps lanpingensis is an edible mushroom distributed over the south‐eastern part of the Tibet Plateau, which is also recognized as an effective ethnomedicine to alleviate diseases. This study explored the effects of a kind of Ophiocordyceps lanpingensis neutral polysaccharide (ONP) on RAW264.7 macrophages and cisplatin‐induced nephrotoxicity. The results showed that ONP relieved the inflammatory response of RAW264.7 macrophages by increasing the expression level of anti‐inflammatory factor IL‐10. Furthermore, ONP treatment significantly prolonged the survival of the mice treated by cisplatin through decelerating pathological progress and alleviating damaged functions of the kidneys. Compared with the cisplatin group, ONP reduced the oxidative stress of the renal cells and the expression levels of pro‐inflammatory factors. Apoptosis of renal cells was also weakened in the ONP treatment group. These findings indicated that ONP alleviated cisplatin nephrotoxicity mainly by inhibiting oxidative stress, inflammation, and apoptosis in the kidneys, underscoring the potential of ONP supplementation to alleviate the side effects of cisplatin chemotherapy.

and plant polysaccharides have the potential to treat diseases Ma et al., 2020). In our previous study, we also found that polysaccharides might be the main bioactive ingredients in O.
Cisplatin (cis-diamminedichloroplatinum II, DDP), which is used to treat more than 20% of cancer patients, is one of the most effective chemotherapy drugs for solid tumors (Dasari and Tchounwou, 2014). However, the usage of DDP-based chemotherapy is limited by its tissue toxicities, especially the nephrotoxicity (Khairnar et al., 2020). Serious side effects of acute kidney injury (AKI) may be incurred when DDP is administered (Holditch et al., 2019). Giving amifostine before DDP injection is currently considered as an efficient approach to prevent DDP-induced nephrotoxicity; nevertheless, amifostine also has certain toxicities such as hypotension and nausea/vomiting (Bukowski, 1996). Therefore, it is necessary to explore dietary supplements suitable for alleviating nephrotoxicity induced by DDP with no apparent side effects for cancer patients.
Based on the DDP-induced nephrotoxicity and the traditional usage of O. lanpingensis in protecting urinary system, it is intriguing to investigate whether O. lanpingensis has pharmacological activity to attenuate DDP-induced nephrotoxicity.
In this study, we isolated and characterized the O. lanpingensis neutral polysaccharide (ONP). RAW264.7 macrophages were used to preliminarily explore the anti-inflammatory activity of ONP.
Furthermore, the effects of ONP on DDP-induced nephrotoxicity in mice were investigated. The alleviation of DDP-induced nephrotoxicity by ONP was confirmed based on the changes of physiological and biochemical indices, as well as the histopathological parameters.
The mechanisms for the remission of DDP-induced nephrotoxicity by ONP treatment in mice were discussed, including antioxidative, anti-inflammation, and anti-apoptosis activities.

| Analysis of polysaccharides
The ONP was extracted from O. lanpingensis according to the description in previous report (Chen et al., 2016;Zhang et al., 2017;Zhou et al., 2020). The crude polysaccharide was separated and purified according to the previous method, and ONP was obtained after ethanol precipitation and DEAE-Sepharose TM Fast Flow column purification. The ONP was determined to be a neutral polysaccharide using a pH meter.
The phenol sulfuric acid method was used to determine the total carbohydrate content (Albalasmeh et al., 2013). Fourier transform infrared spectrometer (FT-IR, Bruker, Germany) was employed to study the main functional groups of the ONP .
FT-IR measurement was performed on freeze-dried ONP, and the FT-IR spectrum was recorded in the range of 4000-400 cm -1 .
The remaining solid was redissolved in water, followed by mixing with equal volumes of 0.5 M PMP methanol solution and 0.3 M NaOH solution, and kept at 70°C for 2 hr. The reaction was stopped by adding 0.3 M HCl and then washed three times with chloroform, and the aqueous layer was collected for UPLC-MS analysis, which was performed using an Agilent Eclipse C18 column (2.1 × 50 mm, 2.7 μm). The Waters UPLC instrument was used at 25°C. The detection was performed with ammonium acetate buffer (0.02 M, pH 7.0, mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.4 ml/min. The analytes were detected using multireaction monition (MRM) in a positive ionization mode (Waters Xevo TQ-S Micro, Massachusetts, USA). Electrospray ionization voltage was 2.01 kV, ion source temperature was 150 °C, and desolvation gas (nitrogen) temperature was 500 °C. The flow rate of the nebulizer gas (nitrogen) was 1,000 L/h, and the cone hole voltage was 30 V .
The molecule weight (Mw) and number average molecular weight (Mn) of ONP were concluded by gel permeation chromatography (GPC) with the Breeze system (Waters 1525, USA) accompany with the PL aquagel-OH MIXED column kept at 30 °C and coupled to the refractive index (RI) detector (Cao et al., 2020)[4]. ONP was made elution with 0.2 M NaNO 3 and 0.01 M NaH 2 PO 4 at a flow rate of 1.0 ml/ min. The narrow distribution dextran with molecular weights from 100 to 1,214,000 (Mol Wt, Polymer Standards Service, USA) is used to estimate the Mw of ONP ( Figure S1). The calibration curve was obtained by plotting the elution volumes against the logarithm of their respective Mw. According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), the polydispersion index (PDI) is calculated as follows: Dm=Mw/Mn Wang et al., 2020).

| Cell culture
Mouse macrophage cell line RAW264.7 was purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). RAW264.7 cells were cultured in DMEM containing 10% FBS at 37°C in a 5% CO 2 incubator. The cell viability was determined by trypan blue dye exclusion method and direct counting with a hemocytometer. RAW264.7 cells (5 × 10 6 cells) were inoculated into a 25 cm 3 tissue culture flask and incubated for 24 hr in a 37% humid environment containing 5% CO 2 . The attached cells were incubated with ONP and 1.0 μg/mL LPS in 37% humid environment containing 5% CO 2 for 24 hr.
Mice were reared under constant temperature and humidity (12 hr light/dark cycles). After acclimating for 2 weeks, the mice were di- On the 14th day, 6 mice in each group were anesthetized with and sacrificed by cervical dislocation to test their biological activity; the remaining mice were used for survival curve analysis, DDP injection was stopped, and ONP was continuously administered. All animal experiments were conducted in accordance with the protocols approved by the Animal Care and Use Committee of Kunming University of Science and Technology (Permit No. SCXK 2015-0001).

| Histological analysis
The kidneys of mice washed with PBS were collected. The kidney tissue is treated with 10% paraformaldehyde, ethanol, and xylene, and finally, a wax block containing tissue is made. The waxy block was cut into thin slices (4 μm) and stained with hematoxylin and eosin (H&E).
The image was captured by Panorama MIDI (Hungarian 3D HISTECH).

| Hematological assays
The blood of the mice was collected and placed in a collection tube containing anticoagulant. After centrifugation at 4,000 rpm for 10 min, serum was collected. The contents of BUN, CRE, Ca, and P in serum were determined with a diagnostic kit.

| Biochemical assays of kidney
The kidney tissue was ground into homogenate in ice physiological water. The homogenate was centrifuged at 800 × g for 5 min at 4°C to obtain a crude supernatant. The crude supernatant was further centrifuged at 500 × g for 20 min at 4°C to obtain a pure supernatant, which was used to determine the contents of MDA, SOD, and GSH-PX.

| Real-time quantitative polymerase chain reaction (qRT-PCR)
Total RNA of the RAW264.7 cell and kidney tissue was extracted and reverse-transcribed it into cDNA, and then, qRT-PCR was carried out on the LightCycler ® 96 Real-Time PCR system (Roche, Switzerland). The primer sequences are in supplementary materials Table S1 in the support information. Based on the 2 -ΔΔCt method, the β-actin gene was used as an internal reference.

| Western blot analysis
The total protein of renal tissues was extracted, and its concentration was determined. After separating the protein by SDS-PAGE, it was transferred to a solid-phase carrier polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skimmed milk powder for 2 hr at room temperature. Incubate with primary antibody overnight at 4°C, after that incubate with HRPconjugated goat anti-rabbit or anti-mouse at room temperature for 1 hr. Using an illumination imaging system (Tanon, China), the gray value of the protein band was detected by ECL kit and Image pro plus 6.0 software.

| TUNEL analysis
Kidney tissues were subjected to TUNEL staining using an in situ apoptosis detection kit in order to analyze the apoptosis of renal cells. The waxy block was cut into thin sections (4 μm) and dewaxed according to standard procedures. Place the slides in a plastic jar containing 200 ml 0.1 M citrate buffer, pH 6.0. Apply 750 W (high) microwave irradiation for 1 min. Cool rapidly by immediately adding 80 ml double-distilled water. Transfer the slides into PBS. Immerse the slides for 30 min in 0.1 M, pH 7.5 Tris-HCl, containing 3% BSA and 20% normal bovine serum. Rinse the slides twice with PBS. Add 50 μL of TUNEL reaction mixture. Incubate for 60 min at 37°C in a humidified atmosphere in the dark. Rinse slides three times in PBS for 5 min each. Finally, after color development with 3,3'-diaminobenzidine (DAB), hematoxylin was counter-stained, and then, the slides were washed 3 times in PBS, dehydrated, transparent, and mounted. Images were captured by a Pannoramic MIDI (3D HISTECH, Hungary). Image pro plus 6.0 software was used to calculate the average value of apoptosis in 10 fields selected randomly for each slice (Mahgoub et al., 2017;Xing et al., 2019).

| Statistical analysis
The data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by Tukey-Kramer test was used to analyze statistical comparisons to identify significant differences between groups.

| Analysis of polysaccharide components
Crude polysaccharides were dissolved in distilled water and loaded them on the DEAE-Sepharose TM Fast Flow (10 cm×60 cm) column.
Sodium chloride solutions at different concentrations (0 M -1.0 M NaCl) were applied in gradient elution, and the eluents were examined using the phenol sulfuric acid method (Albalasmeh et al., 2013).
As shown in Figure 1a, the O. lanpingensis neutral polysaccharide (ONP) was collected.
FT-IR spectra of carbohydrates provide information on the primary structural characteristics of polysaccharides . The FT-IR spectrum of ONP is shown in Figure 1b In addition, after precolumn derivatization of 1-phenyl-3-methyl-5-pyrazolone (PMP), the compositions and contents of monosaccharides in ONP were analyzed by UPLC-MS (Table   S2 and Figures S2-S3). The ion chromatograms of standard monosaccharides and ONP are shown in Figure 1c, which indicated that ONP was a heteropolysaccharide mainly composed of mannose (Man), glucose (Glc), galactose (Gal), and arabinose (Ara) with the molar ratio of 21.9:27.5:19.5:31.1. These four kinds of monosaccharides formed the backbone of ONP.
Mw is the total weight of the sample divided by the weight of the individual molecules, while Mn is the total weight of the sample divided by the number of molecules . The calculated Mw and Mn of ONP are 6.1 × 10 4 Da and 1.79 × 10 4 Da, respectively ( Figure 1d). In addition, the PDI of ONP was 3.42, indicating dispersed rather than uniformly sized particles.

| ONP relived the inflammatory status of LPSinduced RAW264.7 macrophage
According to previous studies, DDP-induced nephrotoxicity could increase the expression levels of pro-inflammatory factors (iNOS, COX-2, TNFα, and IL-1β) and decrease the expression level of IL-10 anti-inflammatory factors Holditch et al., 2019).
Therefore, we used the inflammatory experiment of RAW 264.7 cell induced by LPS to confirm whether ONP had anti-inflammatory activity in vitro (Hwang et al., 2020).

| The survival status of mice treated by DDP or ONP
The experiment was carried out according to Figure 3a. Compared with the control group, DDP treatments resulted in a significant weight loss and a large number of deaths (Figure 3b-3c). The loss of body weight also happened in DDP-ONP groups, but the reduced weight was more slowly compared with the DDP group.
In addition, ONP could remarkably increase the survival rate of DDP-treated mice. All the mice were dead on the 20th day after giving DDP lonely, but the survival rate was increased to 58% in of DDP shows that nephrotoxicity is one of its main side effects and may even lead to death (Casanova et al., 2020;Charlier et al., 2004). Based on the attenuation of body weight loss and the increasing of survival rate in mice, ONP was likely to have the potential to protect the kidneys.

| Effects of ONP on morphological and histopathological changes of kidney in DDPtreated mice
The morphological changes of the kidneys are shown in Figure 4a, which indicated that the appearance color was changed from dark red to white in the DDP treatment compared with the control group; nevertheless, ONP treatments could slow such a harmful change. Next, we performed pathological analysis of renal tissues.
The pathological detection of the DDP group clearly showed massive necrosis of the renal tubules and glomeruli (Figure 4b), but such pathological damage was decreased with the treatments of ONP in DDP-ONP-L and DDP-ONP-H groups. Furthermore, there was no significant difference between ONP group and control group. Overall, ONP treatment attenuated the DDP-induced kidney damage.

| ONP inhibited the renal function impaired by DDP
The concentrations of BUN, CRE, P, and Ca in serum are important biochemical indicators for evaluating renal function (Ma et al., 2017;Meurer and Hocherl, 2019). In this study, compared with the control group, the contents of BUN, CRE, and P in the DDP group were elevated more than 4, 1, and 1 times, respectively; meanwhile, the content of Ca in the DDP group was declined to about 50% ( Figure 5).

Moreover, ONP treatment diminished these pathological changes
with a dose-dependent relationship. All the serum biochemical indexes referred to above were almost remained normal in the DDP-ONP-H group.

| ONP reduced oxidative stress and inhibited the expression of inflammatory factors in the kidney
MDA is considered to be a sign of oxidative stress, and endogenous antioxidant enzymes SOD and GSH-PX are the key factors to maintain redox balance Tsikas, 2017). As shown in Figure 6a As shown in Figure 6d-6e, the expression levels of NOX4, TNFα, and IL-1β in the DDP group were elevated compared with the control group, which indicated that inflammatory response might be activated by DDP in the renal tissues. On the contrary, ONP could inhibit NOX4 production and the expression levels of TNFα and IL-1β. In addition, ONP also increased the expression of the antiinflammatory factor IL-10 gene.

| D ISCUSS I ON
In recent years, natural product polysaccharides, especially fungal polysaccharides, have shown unique pharmacological effects in immune regulation, anti-inflammatory, anti-oxidation, anti-aging, and disease prevention such as polysaccharides from Ganoderma lucidum, Flammulina velutipes, or Cordyceps militaris could prevent and treat diabetic nephropathy (Geng et al., 2020;Kim, Wu, et al., 2020;Li et al., 2019;Lin et al., 2016). The inflammatory status was induced by LPS in RAW 264.7 cells.
It is known that LPS stimulates macrophages to express more nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to produce NO and prostaglandin E2 (Hwang et al., 2020)[20]. Moreover, iNOS and COX-2 activate the expression levels of several pro-inflammatory mediators, such as TNFα and IL-1β. In our study, O. lanpingensis neutral polysaccharide (ONP) inhibited the upregulated expression levels of iNOS, COX-2, TNFα, and IL-1β induced by LPS, which also increased the expression level of IL-10 in a dose-dependent manner in RAW 264.7 cells. IL-10 is a multifunctional cytokine that regulates cell growth and differentiation, participates in inflammatory reactions, and is recognized as an inflammation inhibitor (Ip et al., 2017).
Based on these results and the ethnic traditional usage of O. lanpingensis in the treatment of urinary system diseases, it is valuable to explore whether the ONP could alleviate DPP-induced nephrotoxicity.
In general, the clinical side effects of DDP mainly include nephrotoxicity, gastrointestinal toxicity, neurotoxicity, and ototoxicity, of which nephrotoxicity is particularly significant and may even lead to death (Arany and Safirstein, 2003;Oun and Rowan, 2017). DDP is mainly metabolized by glomerular filtration and proximal renal tubular epithelial cells, thus to accumulate DDP in the kidney to a large amount. The concentration of DDP in the renal tissues is about five times of that in the blood and such side effect eventually leads to renal dysfunction (Volarevic et al., 2019). A recent study revealed that AKI was a common multifactorial complication in the later stages of the course of severe COVID-19 patients, and the pathological characteristics of such AKT were similar to the AKT induced by DDP (Xia, Wen, et al., 2020). The predominant pathologic finding of COVID-19 patients was acute tubular injury, which eventually led to kidney organ failure and even death. In our study, ONP could reduce the rate of weight loss and increase the survival rate when DDP-induced AKT was occurred in mice. Maybe there is a potential application on severe COVID-19 patients by ONP, but it is just a hypothesis.
Morphological and pathological sections of mouse kidney preliminarily proved that ONP could alleviate nephrotoxicity. DDP exposure can induce the damage of renal vasculature and result in the decline of glomerular filtration rate (Arany et al., 2003)[2]. It is known that the kidney is the main excretory organ of urea. After urea is filtered by the glomerulus, it can be reabsorbed by the renal tubules. When renal function is impaired, the flow rate of urine decreases, urea is reabsorbed more by the renal tubules, and the BUN content in the blood increases (Ma et al., 2017). CRE is a product of muscle metabolism. In muscles, creatine slowly forms CRE through an irreversible nonenzymatic dehydration reaction, and then, CRE is released into the blood and excreted in the urine. When the glomerulus is damaged, the ability of filtering CRE by glomerulus will be decreased, and the content of CRE in the serum will be increased (Smith, 1988). Therefore, the concentrations of BUN and CRE are important biochemical markers for assessing glomerular filtration rate and renal function. In addition, renal failure results in the enhancement and reduction of the expression levels of P and Ca transporters, respectively (Meurer and Hocherl, 2019). In this study, ONP alleviated the increasing levels of BUN, CRE, and P, as well as inhibiting the decreasing level of Ca in the serum after the mice were treated by DDP, so that ONP could protect renal function to some extent.  TNFα and IL-1β levels would be increased . TNFα stimulates the production of genotoxic molecules (NO and ROS) and causes DNA damage and mutations (Hussain et al., 2003). IL-1β is a trigger of inflammation inducing mediators, thus leading to kidney damage (Dinarello et al., 2012). Our results showed that ONP could attenuate nephrotoxicity through inhibiting the oxidative stress, which also resonated with the similar mechanisms reported previously (Shen et al., 2020). The reduced oxidative stress and secretion of pro-inflammatory factors probably served as certain mechanisms of reducing cisplatin nephrotoxicity by ONP.
Under the oxidative stress and inflammation conditions, excessive ROS and pro-inflammatory cytokines could damage cellular proteins, lipids, and DNA, leading to apoptosis of cells (Dasari and Tchounwou, 2014;Florea and Busselberg, 2011). Generally, the pathways of apoptosis are mainly divided into extrinsic pathway and intrinsic pathway (Nunez et al., 1998). TNFα mediates caspase-8 to participate in the extrinsic pathway (Dasari and Tchounwou, 2014;Herbein and O'Brien, 2000). Cell stress induced by DNA damage drives mitochondria to release cytochrome c to activate caspase-9, which is an intrinsic apoptosis pathway. Moreover, Bcl-2 family proteins participate in the intrinsic apoptosis pathway by regulating the release of mitochondrial cytochrome c (Ghosh, 2019). Both caspase-8 and caspase-9 can activate caspase-3 by proteolytic cleavage to perform cellular apoptosis (Thornberry and Lazebnik, 1998). In addition, mitogen-activated protein kinases (MAPK) are a family of structurally related serine/threonine protein kinases that coordinate various extracellular signals to regulate cell growth and survival (Chang and Karin, 2001). Previous studies have shown that TNFα and IL-1β are the main conditions involved in activating JNK, p38, and ERK1/2; moreover, MAPK activation can promote cell apoptosis (Herbein and O'Brien, 2000;Johnson and Lapadat, 2002;Ning et al., 2020;Qi et al., 2019).

DDP cytotoxic mode of action is mediated by its interaction
with DNA to form DNA adducts, primarily intrastrand crosslink adducts, which activate several signal transduction pathways, including Bcl-2 family proteins, caspase family proteins, and MAPK, and culminate in the activation of apoptosis (Siddik, 2003). Notably, our results showed that ONP effectively inhibits the activations of Bcl-2 family proteins, caspase family proteins, and MAPK in the kidneys of mice, so that to alleviate DDP-induced nephrotoxicity ( Figure 7). Furthermore, the result of TUNEL was consistent with the changes of apoptosis proteins in this study, thus indicating that ONP exactly weakened the apoptosis of renal cells induced by DDP.

| CON CLUS ION
In conclusion, this study revealed the protective effects of ONP on DDP-induced renal toxicity. Our findings indicated that ONP attenuated DDP nephrotoxicity mainly by reducing oxidative stress, inflammation, and apoptosis in the kidneys, underscoring the potential of ONP supplementation to improve DDP chemotherapy (Figure 9).

ACK N OWLED G M ENTS
This work was supported by "The National Natural Science Foundation of China (81860513)" and "Yunnan Ten Thousand Talents Plan (Young & Elite Talents Project, No. YNWR-QNBJ-2018-059)."

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest and all data can be used.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data, models, and code generated or used during the study appear in the submitted article.