Nucleosides rich extract from Cordyceps cicadae alleviated cisplatin-induced neurotoxicity in rats: A behavioral, biochemical and histopathological study

2.1 Chemicals and reagents

Cisplatin and Elisa kits for the estimation of IL-6, IL-1β and TNF-α were purchased from Abcam (Cambridge, UK). Biochemical kits for the estimation of CAT, SOD, MDA and GPx were procured from Abbkine (Wuhan, China). All other chemicals used were of analytical grade.

2.2 Biological specimen

C. cicadae was bought from Bozhou, Anhui Province, China and authenticated by Prof Dr. Jian Tang, School of Chinese Medicine, Bozhou University (voucher number: CC2019/06). The procedures used for the extraction of the nucleosides rich extract was based on our previous report (Wang et al., 2020). Briefly, C. cicadae powder was extracted with 80 % ethanol at 100 °C for 3 h under reflux. The resulting hydroalcoholic extract solution was subjected to filtration and dried with a rotary evaporator to obtain the crude extract. The extract was redissolved in distilled water and kept at 4 °C overnight. The solution was carefully decanted, centrifuged for 30 mins at 6000 rpm. The supernatant obtained was lyophilized and the light brown hygroscopic powder obtained was designated as CCNE and stored at 4°Cuntil further use.

2.3 Experimental animals and treatment protocol

Healthy male Wistar rats (7 weeks old) were maintained in stainless cages with corn hob as beddings in an experimental house facility with standard conditions of temperature, relative humidity and 12-hour dark/light cycle. Standard pelleted chow and water were freely accessible to the animals throughout the acclimatization period of seven days as well as the experimental duration. Animal experimental procedures agreed with the requirements of the National Institute of Health and ratified by the Ethics Committee of the First People's Hospital of Lianyungang (approval number: 202000845678). After completion of the acclimatization period, the animals were divided into four groups (n = 6 rats/group) as follows: normal control group (NCA), cisplatin control group (CCA), low dose CCNE + cisplatin group (CCNE-L) and high dose CCNE + cisplatin group (CCNE-H). The rats in the CCNE-L and CCNE-H groups were orally treated with CCNE at doses of 100 and 400 mg/kg, respectively for five weeks, while the animals in the NCA and CCA groups received normal saline for five weeks. Except for the rats in the NCA group, all the other groups were administered with intraperitoneal injection of cisplatin (2.5 mg/kg) once a week for four weeks starting from the second week of CCNE administration (Fig. 1). Cisplatin as well as CCNE were dissolved in normal saline before administering to the animals. The selection of doses of cisplatin and CCNE, as well as the duration of administration was based on previous studies (Wang et al., 2020; Arafa and Atteia, 2020; Liu et al., 2021). The weight of the animals was measured on a weekly basis throughout the duration of the treatment. After the last treatment, the animals were subjected to series of behavioural test.

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Fig. 1 Time course of the experimental protocol of the whole study.

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2.4 Forced swimming test (FST)

The FST is used for evaluation depressive-like behavior in animals. The procedure used in the FST was based on a previously reported protocol (Porsolt et al., 1977). In brief, the animals were initially subjected to a pre-test session for 15 mins, each rat was placed in a cylindrical transparent tank (30 × 40 cm) containing 30 cm of water. Twenty-four hours after the pre training test, the animals were subjected to FST for 5 min and the total swimming, climbing and immobility time was recorded.

2.5 Open field test (OFT)

The OFT assesses the exploratory behavior and anxiety in models of neuroprotection. A rectangular open field divided into six rectangular subunits was used for the experiment. Each rat was positioned at the middle of the open field and the exploratory behaviours of the rats including total number of line crossing made with all the four paws and the rearing time were evaluated with a 10 min period.

2.6 Morris water maze test (MWMT)

The MWMT is used for evaluating spatial learning in animals and uses distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform. The method of Chen et al. (2021) was adopted for the MWMT. Briefly, a circular water pool (180 cm in diameter) was filled with water (25 ± 1 °C) to a height of 40 cm. The animals were initially trained to locate the position of a hidden platform in a target quadrant submerged 1 cm below the surface of the water. The first four days was for acquisition trial, and the rats were randomly place inside the pool wall from four different quadrant positions and allowed to adapt to the water surface for 2 s before been gently released to swim and locate the hidden platform within 60 s. The time taken for the animals to locate the hidden escape platform was recorded within 60 s. The animals that were unable to locate the hidden platform within 60 s were gently guided to the location of the hidden platform and allowed to stay on the platform for 10 s. For the exploratory test, the hidden platform was removed on the fifth day of the experiment and the time spent by each rat in the target quadrant where the hidden platform was previously positioned was recorded within 60 s.

2.7 Y maze test

The Y maze test is widely used for evaluating behavioral task relating to spatial learning and memory using spontaneous alternation and recognition memory test. The protocol used for the Y maze test was according to previously reported method (Olasehinde et al., 2020). Briefly, the maze consisted of three identical arms A, B and C (35 cm × 30 cm × 15 cm). The animals were placed at one end of maze arm and were allowed to freely navigate through the maze for 5 min. Spontaneous alternation was accessed by the pattern of complete entry into each arm (the rat’s hind paws goes entirely into the arm). The frequency of spontaneous alternation into the arms was recorded based on successive entries into the three arms on overlapping triplet sets (ABC, BCA, CAB).

2.8 Rotarod test

After the treatment period, the motor coordination of the rats was evaluated using a rotarod apparatus. The test evaluates balance and coordination based on the length of time the animals stays on the rotating rod. The animals were subjected to pre-test sessions which was performed four times daily for three days in a row. The rats were positioned on the rotarod accelerating from 0 to 20 rpm for 600 s. The test was terminated when rats fell off the rotating rod or at the end of the allotted trial time. The time spent by each rat to dismount from the rotating rod was recorded.

2.9 Assessment of thermal hyperalgesia

The hotplate and tail withdrawal test was used for evaluating thermal hyperalgesia in the rats. The hotplate was set at 52 ± 1.0 °C and each animal was placed individually on the hot plate apparatus. The time it takes for the first display of latency of pain including behaviours such as paw licking, jumping or shaking was recorded and taken as an index of pain threshold. A threshold time of 40 s was fixed to circumvent paw injury. For the tail withdrawal test, the tail of each rat was dipped into hot water bath at 50°± 1.0 °C until the initial display of signs of pain including tail flicking, withdrawal or struggle. A threshold period of 12 s was fixed to circumvent injury.

2.10 Assessment of mechanical hyperalgesia

Mechanical hyperalgesia was evaluated by estimating the threshold limit of paw withdrawal to von Frey filaments. The animals were individually placed in plexiglass boxes and von Frey filaments was perpendicularly applied on the hind paw for 6 s. Paw flinching/rapid withdrawal was adjudged as a nociceptive response. The next filament with lesser or greater force was applied in the presence or absence of a nociceptive response, respectively.

2.11 Animal sacrifice

The rats were put under sodium thiopental anaesthesia (100 mg/kg) and sacrificed by cervical dislocation. The whole brain tissue were dissected, washed with normal saline and weighed. The brain tissues were further homogenized in phosphate buffer saline, centrifuged at 6000 rmp for 15 min, and the supernatant obtained after centrifugation was utilised for the estimation of biochemical parameters.

2.12 Assessment of antioxidant enzymes and malonaldehyde

Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and malonaldehyde (MDA) in the brain tissue homogenate was determined with the aid of biochemical assay kits obtained Abbkine (Wuhan, China) according to specifications in the instruction manual of the manufacturer.

2.13 Determination of proinflammatory cytokines level

The following inflammatory related parameters were analysed in the brain tissues homogenate: IL-6, IL-1β, TNF-α and NF-κB using ELISA kits from Abcam (UK) and Cusabio (China). The procedures followed were in accordance with the specified instructions from the manufacturers guide.

2.14 Determination of AChE activity

The assessment of AChE activity was performed based on the reported protocol of Ellman et al. (1961). The hydrolysis of acetylthiocholine iodide by AChE to produce thiocholine was measured spectrophotometrically at 412 nm. Briefly, the brain tissue supernatant (0.1 mL), 2.5 mL of phosphate buffer saline solution and 0.1 mL of 10 mM DTNB (5,5-dithio-bis-2-nitrobenzoic acid) were thoroughly mixed together for 5 min at room temperature and the absorbance of the mixture was read at 412 nm. Thereafter, 5 µL of acetylthiocholine iodide (50 mM) was added and the absorbance was recorded every 2 min for 10 min.

2.15 Histopathological examination

For the histology, a small portion of the brain tissues was taken and fixed in 10 % buffered formalin solution. The fixed brain tissues were dehydrated in serial dilutions of alcohol solution and further subjected to embedding using paraffin blocks (5 μm thickness). The sectioned brain tissues were stained with hematoxylin & eosin and the slides visualized under light microscope.

2.16 Statistical analysis

GraphPad Prism Version 5 software was employed for statistical analysis and the results were conveyed as means ± SD (n = 6). Significant differences between treated and untreated groups were examined using one-way ANOVA or two-way ANOVA coupled with Newman-Keuls comparison or Bonferroni’s comparison test. Values were adjudged statistically significant at p < 0.05.

3.1 CCNE improved body weight (BW) loss and food intake in cisplatin treated rats

As shown in Fig. 2A, the final BW of the CCA group was significantly reduced in comparison to the NCA group after 5 weeks of treatment. Whereas, the significant BW loss observed in the CCA rats was prevented in the groups administered with CCNE (100 and 400 mg/kg) in a dose dependent manner (p < 0.001). The food consumption in CCA group was also markedly decreased following cisplatin administration (Fig. 2B). Nevertheless, CCNE dose dependently improved food intake compared to the CCA group (Fig. 2B; p < 0.001).

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Fig. 2 Effect of treatment with CCNE (100 and 400 mg/kg) on Body weight (A) and food consumption (B). Results were expressed as mean ± SD (n = 6) and comparison between groups was analysed using one way ANOVA with Newman-Keuls multiple comparison test. **p < 0.001 vs NCA group; ##p < 0.001 vs CCA group; $$p < 0.05 vs CCA group.

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3.2 CCNE alleviated depressive-like behaviours in cisplatin treated rats in the FST

Fig. 3A-C illustrates the effect of treatment with CCNE in the FST. The results indicated that relative to NCA rats, the swimming and climbing time of the CCA group were significantly lower (Fig. 3A-B; p < 0.001), while the immobility time was markedly increased when juxtaposed to the NCA group (Fig. 3C; p < 0.001). The administration of CCNE at both doses led to notable decline in immobility time in comparison to the CCA rats. Additionally, marked accrual in climbing behavioural time and the time spent swimming was observed in the CCNE administered groups in comparison to the CCA rats (Fig. 3A-C; p < 0.001).

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Fig. 3 Effect of treatment with CCNE (100 and 400 mg/kg) on depressive-like behavioural parameters. Climbing (A), swimming (B), immobility time (C), crossing (D) and rearing (E) parameters in the FST and OFT. Results were expressed as mean ± SD, n = 6. Results were expressed as mean ± SD (n = 6) and comparison between groups was analysed using one way ANOVA with Newman-Keuls multiple comparison test. **p < 0.001 vs NCA group; ##p < 0.001 vs CCA group; $$p < 0.05 vs CCA group.

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Additionally, the CCA rats showed decrease in the number of crossing (Fig. 3D) and rearing (Fig. 3E) in the OFT compared to the NCA rats. Interestingly, CCNE treated animals showed significant and dose dependent increases in the crossing and rearing behaviours when compared to the untreated CCA group (Fig. 3D-E).

3.3 CCNE improved memory impairment in cisplatin treated rats in the MWMT

As indicated in Fig. 4A, the escape latency of the NCA animals was progressively and markedly reduced over the four days test period suggesting normal ability of the NCA rats to learn. Whereas, the CCA rats displayed significantly higher escape latency time throughout the four days of the test when compared to the NCA animals, suggesting impaired learning ability (p < 0.001). Treatment with CCNE (100 and 400 mg/kg) markedly suppressed the escape latency time compared to the CCA group (Fig. 4A; p < 0.001 and p < 0.05). Furthermore, the CCA rats showed marked reduction in the time spent in the target quadrant in comparison to the NCA group, while CCNE treatment dose dependently improved the time spent in the target quadrant when juxtaposed to the CCA group (Fig. 4B; p < 0.001 and p < 0.05).

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Fig. 4 Effect of treatment with CCNE (100 and 400 mg/kg) on spatial memory and learning. Escape latency time (A), quadrant time (B), and % spontaneous alternation (C) in the MWM and Y maze tests. Results were expressed as mean ± SD, n = 6. Results were expressed as mean ± SD (n = 6) and comparison between groups was analysed using Two way ANOVA with Bonferroni’s multiple comparison test. **p < 0.001 vs NCA group; ##p < 0.001 vs CCA group; $$p < 0.05 vs CCA group.

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3.4 CCNE improved memory impairment in cisplatin treated rats in the Y maze test

The results from the Y maze test is displayed in Fig. 4C. Significant decrease in percentage of spontaneous alternation was observed in the CCA rats compared to NCA rats (p < 0.01), suggesting impediment in spatial learning and memory. The effect of cisplatin was significantly reversed by CCNE treatment. Supplementation with CCNE (100 and 400 mg/kg) dose dependently increased the percentage of spontaneous alternation in the treated rats compared to the CCA group (Fig. 4C).

3.5 CCNE prevented thermal and mechanical hyperalgesia in cisplatin treated rats

The results shown in Fig. 5A-B indicated that the CCA group displayed significant decrease in pain threshold as observed by significant decline in pain latency reaction in the hot plate and tail withdrawal tests. Whereas, treatment with CCNE produced significant and concentration related improvement in pain threshold response when juxtaposed with the CCA rats. The rats in the treated groups had higher paw and tail withdrawal thresholds (Fig. 5A-B; p < 0.001 and p < 0.05). In addition, the paw withdrawal threshold in the von Frey filament test was notably lower for the animals in the CCA group when compared to NCA group (Fig. 5C; p < 0.05). On the other hand, the paw withdrawal threshold was significantly increased in the 400 mg/kg CCNE administered group when juxtaposed to the CCA group (Fig. 5C; p < 0.05).

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Fig. 5 Effect of treatment with CCNE (100 and 400 mg/kg) on thermal and mechanical hyperalgesia. Paw withdrawal latency (A), tail withdrawal latency (B), paw withdrawal threshold (C) and latency to fall (D) Results were expressed as mean ± SD (n = 6) and comparison between groups was analysed using one way ANOVA with Newman-Keuls multiple comparison test. **p < 0.001 vs NCA group; ##p < 0.001 vs CCA group; $$p < 0.05 vs CCA group.

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3.6 CCNE prevented impairment in motor coordination in cisplatin treated rats

The results from the rotarod test is presented in Fig. 5D, the CCA group showed reduced performance as observed by their significantly reduced latency to fall when compared to the NCA group, suggesting impaired motor coordination. However, the performance of the CCNE (100 and 400 mg/kg) treated rats was greatly improved in comparison to the CCA group. The groups treated with CCNE displayed longer latency to fall, suggesting improvement in motor coordination (Fig. 5C; p < 0.001).

3.7 CCNE decreased AChE activity and lipid peroxidation in cisplatin treated rats

The administration of cisplatin induced significant increase in AChE activity and MDA levels in the CCA rats compared to normal control rats. CCNE significantly prevented these changes in a concentration dependent manner (Fig. 6A-B; p < 0.001).

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Fig. 6 Effect of treatment with CCNE (100 and 400 mg/kg) on AChE (A), MDA level (B), SOD (C), CAT (D) and GPx (E). Results were expressed as mean ± SD (n = 6) and comparison between groups was analysed using one way ANOVA with Newman-Keuls multiple comparison test. **p < 0.001 vs NCA group; ##p < 0.001 vs CCA group; $$p < 0.05 vs CCA group.

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3.8 CCNE improved enzymatic antioxidants in cisplatin treated rats

The brain activities of SOD, CAT and GPx in the cisplatin control group (CCA) were notably decreased compared to the normal control (Fig. 6C-E; p < 0.001). Nevertheless, cisplatin injected rats that were treated with CCNE resulted in a dose dependent and significantly higher activities of SOD, CAT and GPx when juxtaposed to the untreated cisplatin control rats (p < 0.001)

3.9 CCNE alleviated inflammatory mediators in cisplatin treated rats

Fig. 7A-D shows the effects of treatment with CCNE on inflammatory mediators in cisplatin administered rats. In the NCA rats, the levels of proinflammatory cytokines including IL-6, IL-1β and TNF-α were markedly lower than in the CCA rats. Whereas, in the CCNE treated groups, the concentration of these proinflammatory cytokines were markedly abated in the brain when compared to the CCA group (Fig. 7A–C; p < 0.001). Furthermore, NF-κB level in the CCNE treated groups was also significantly abated compared to the CCA control group (Fig. 7D; p < 0.001).

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Fig. 7 Effect of treatment with CCNE (100 and 400 mg/kg) on inflammatory mediators. IL-6 (A), IL-1β (B), TNFα (C) and NF-кB (D). Results were expressed as mean ± SD (n = 6) and comparison between groups was analysed using one way ANOVA with Newman-Keuls multiple comparison test. **p < 0.001 vs NCA group; ##p < 0.001 vs CCA group; $$p < 0.05 vs CCA group.

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3.10 CCNE attenuated histopathological alterations in cisplatin treated rats

The histopathological examination of the CCA treated group showed distinctive pathological alterations as evident by congested blood vessels, degenerated and swollen neurons as well as infiltration of inflammatory cells compared to the NCA group that showed relatively normal brain histology (Fig. 8A-B). Interestingly, the marked improvement were noticed in the histopathology of the brain tissues of the CCNE pre-treated groups compared to CCA control group (Fig. 8C-D).

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Fig. 8 Effects of treatment with CCNE on brain histological changes (H & E stain) in normal and cisplatin treated- rats. (A) NCA group, (B):CCA group, (C) CCNE-L group, (D) CCNE-H group. Black arrow indicate congested blood vessels; red arrow indicate swollen neurons; purple arrow indicate infiltration of inflammatory cells.

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