| Chemotherapy leaves a growing number of cancer patients alive,but patients receiving chemotherapeutic agents are often plagued with some side effects.Cancer survivors have developed slow processing speed,memory impairment,inability to concentrate,and language difficulty,which is often attributed to the receipt of chemotherapy,and called chemotherapy induced cognitive impairment(also known as “chemobrain”).This phenomenon greatly deteriorates the survivors’ quality of life and prevents them from resuming their pre-cancer lives.Breast cancer patients were found to be the most reported patients with impaired cognitive function after chemotherapy.Paclitaxel and doxorubicin(DOX)are still the most commonly used chemotherapeutic agents to manage breast cancer.Despite DOX is known not to target the central nervous system or even across the blood–brain barrier(BBB),it could still impair cognitive functions and induces chemobrain in rodents and patients.Therefore,prevention or alleviation of chemobrain is important for the treatment and rehabilitation of cancer patients,especially breast cancer patients.Several agents(such as cholinesterase inhibitors,modafinil,and anti-inflammatory agents)have been tried clinically to treat chemobrain,but little effect has been achieved.So it is necessary to find new methods to combat chemotherapy-induced cognitive impairment so as to improve the quality of life in cancer survivors.Polydatin(PLD)is a glycoside of resveratrol(Res)extracted from the root of Polygonum cuspidatum.Although PLD and Res could transform each other in vivo,PLD is the main substance in serum after administration of PLD or Res,suggesting that PLD has better oral absorption and metabolic stability than Res.Furthermore,PLD exhibits diverse biological functions,including antiinflammatory,antioxidant and immunoregulation.Studies have shown that PLD can alleviate chemotherapy-induced oxidative stress and lipid peroxidation to protect organisms.More importantly,PLD can cross the BBB to prevent Parkinson’s disease,reduce brain edema following cerebral infarction,protect against cerebral nerve injury caused by ischemia/reperfusion injury and alleviate cognitive impairment caused by chronic drinking.Nevertheless,there have been no reports on the effects of PLD on chemotherapy-induced cognitive impairment.In the present study,we aimed to study the effects of PLD on symptoms of chemobrain and to evaluate the protection of Polydatin on oxidative stress,inflammatory response,and apoptosis in the hippocampus of doxorubicin-induced “chemobrain” rats and its possible mechanisms and pathway.Objective: Doxorubicin can impair cognitive functions and induce chemobrain in cancer survivors.Polydatin(PLD)is known to have many biological activities,including anti-inflammatory,antioxidant and immunoregulation.Nevertheless,there have been no reports on the effect of PLD on chemotherapy-induced cognitive impairment.This study aimed to determine whether symptoms of chemobrain can be prevented or relieved by PLD in rats with doxorubicin-induced cognitive impairment.To evaluate the protection of Polydatin on oxidative stress,inflammatory response,and apoptosis in the hippocampus of doxorubicin-induced “chemobrain” rats and its possible mechanisms and pathway.Methods:1 Animal model: Six-week-old male Sprague–Dawley rats(Beijing HFK Bioscience Co.Ltd.,China),weighting 210–230 g,were used for the experiments.Rats were kept in a regulated environment(temperature,22 ± 1°C;relative humidity,45–55%;12-h light/dark cycle).Through general observation,the rats without abnormal behavior were selected for underwater swimming observation,and the rats with abnormal movement were excluded.Rats were randomly divided into four groups: shamoperated group(Sham,n=6),doxorubicin-treated group(DOX,n = 6),polydatin administered group(PLD,n = 6),and doxorubicin-treated and polydatin administered group(DOX+PLD,n = 6).To generate the rat model of chemotherapy,2 mg/kg of DOX(Melone Pharmaceutical Co.,Ltd.,China)was intraperitoneally(IP)injected for the DOX group once a week for 4 weeks.For PLD groups,50 mg/kg of PLD(Aladdin,China)was taken orally every day for 3 weeks.In the DOX+PLD group,2 mg/kg of DOX was IP injected once a week for 4 weeks,and the PLD was taken orally at 50 mg/kg per day for 4 weeks.Rats in the sham group were administered an equal volume of solvent.Behavioral analyses of each group were observed after 28 days.2 HE staining and toluidine blue staining were used to observe the pathological changes of the hippocampus between the four groups.Morris water-maze task was used to assess spatial learning and memory in rats.Step-down avoidance task was used to assess short-term memory in rats.3 The levels of malondialdehyde(MDA)and glutathione(GSH)were measured using maleic dialdehyde assay Kit and Glutathione(GSH)Assay Kit.The levels of tumor necrosis factor alpha(TNF-α),prostaglandin E2(PEG-2)and cyclooxygenase-2(COX-2)were assessed using ELISA Kit for tumor necrosis factor alpha,ELISA Kit for prostaglandin E2 and ELISA Kit for cyclooxygenase.Western blotting was used to detect the expression of Nrf-2,p-IκBα,IκBα,p-p65,p65,cleaved caspase-3,cleaved caspase-9 in the hippocampal tissues.The TUNEL Apoptosis Assay Kit was used to label apoptotic cells.DAPI was added for 5 min to stain the nuclei.All the statistical analyses were performed using the Graph Pad Prism 8.0(Graph Pad Inc.,San Diego,CA,USA).Student’s t-test was used to compare the differences between the two groups,and multiple group comparisons were analyzed with one-way analysis of variance(ANOVA).Each experiment was repeated at least three times.Data were presented as mean ± SD(standard deviation).A value of P< 0.05 was considered statistically significant.Results:1.We employed the Morris water-maze task to assess spatial learning and memory in rats(n = 6).The DOX group observed a decrease in spatial memory ability relative to levels exhibited in the sham group,whereas the DOX +PLD group exhibited an increase in spatial memory relative to that observed in the DOX group.The escape latency in the DOX group was significantly longer than that in the sham group,and this difference began on the second day.The latency difference between the DOX+PLD group and the sham group began on the third day.Over time,the latency in the DOX +PLD group was significantly shorter than that in the DOX group.Besides,rats in the DOX+PLD group spent more time in the target quadrant than rats in the DOX group.Moreover,there was no difference between the sham group and the PLD group.These results demonstrate that PLD did not show neurotoxicity.DOX leads to decreased spatial learning/memory,while PLD improves rat’s spatial learning/memory abilities in the DOX induced chemobrain models.2.Short-term memory was assessed by the step-down avoidance task.The latency time of the step-down in the DOX group was 38.1 ± 9.0 s,which was significantly less than the sham group(123.5 ±24.8 s).Co-administration of PLD significantly increased the step-through latency time(76.2 ± 20.2 s)in the DOX +PLD group.However,there was no difference between the sham group and the PLD group.These results demonstrate that PLD did not show neurotoxicity.DOX leads to decreased short-term memory,while PLD improves rat’s short-term memory ability in the DOX induced chemobrain models.3.Hematoxylin-Eosin(HE)staining: The sham group and PLD group had normal histological structures of the hippocampus.DOX resulted in nuclear pyknosis and degeneration of neuronal cells in the hippocampus.Co-administration of PLD and DOX partially restored the histological features of the hippocampus.4.The extent of neurodegeneration in the hippocampus was further assessed using toluidine blue staining.DOX resulted in complete loss of histological architecture of hippocampal neurons.Conversely,co-administration of PLD and DOX partially restored neuron features manifested by the rounded neuronal build and defined nuclei.Sham group and PLD group showed no difference.5.In order to assess the effect on hippocampal oxidative stress,we detected the levels of GSH and MDA.DOX caused a significant increase in the content of MDA(3.2 ± 0.7 nmol/mgprot),while coadministration of PLD reduced this elevation(1.9 ± 0.4 nmol/mgprot).DOX reduced GSH levels(5.8 ± 1.6 μmol/gprot),while co-administration of PLD prominently restored GSH to normal levels(9.7 ± 2.1 μmol/gprot).We further studied the effect of PLD on DOX-induced pro-inflammatory response.TNF-α levels were significantly increased by DOX(45.3 ± 9.1 pg/mgprot),while the administration of PLD reduced this elevation(16.5 ± 7.2 pg/mgprot).Besides,PGE-2 levels were increased in the DOX group(85.9 ± 22.9 pg/mgprot),and restored to normal levels in the DOX+PLD group(55.0 ± 15.2 pg/mgprot).DOX caused an increase in COX-2 levels(0.3 ± 0.1 ng/mgprot),while PLD treatment could attenuate DOX induced elevation(0.1 ± 0.03 ng/mgprot).In addition,there was no statistical difference between the PLD group and the sham group.These results indicated that DOX triggered oxidative stress and inflammatory response in the hippocampal tissues.PLD treatment significantly attenuated DOX induced oxidative stress and inflammatory response.6.The number of TUNEL positive cells in the DOX group was higher than that in the sham group,while coadministration of PLD could reduce the number of TUNEL positive cells.The levels of cleaved caspase-3 and cleaved caspase-9 were higher in the DOX group than the sham group.Conversely,coadministration of PLD decreased the expression of cleaved caspase-3 and cleaved caspase-9.These results indicated that PLD inhibited neuronal apoptosis in the hippocampus of rats.7.Nuclear factor E2-related factor 2(Nrf2)is a major transcription factor of the antioxidant stress.We found that PLD could significantly increase the expression of Nrf2.Nrf2 levels in the DOX+PLD group was much higher than that in the DOX group.Nuclear factor kappa B(NF-κB)is associated with inflammatory responses.So we assayed the expression of NF-κB pathway-associated proteins.DOX treatment enhanced the expression of p-IκB and p-p65,indicating that DOX activated the NF-κB pathway.In contrast,co-administration of PLD could inhibit the activation of IκB and p65.These results demonstrated that PLD prevented DOX-induced oxidative stress and proinflammatory response through Nrf2 and NF-κB pathway.Conclusion: The chemobrain models were established by intraperitoneal injection of doxorubicin(DOX,2 mg/kg)in rats once a week for 4 weeks(DOX group and DOX+PLD group).We found that PLD treatment significantly protected against DOX induced learning and memory impairment,restored hippocampal histopathological architecture.We indicated that PLD presents a promising neuroprotective agent which could protect against DOX induced cognitive impairment.DOX treatment caused cognitive impairment in SD rats,and PLD could prevent these deficits by its antioxidant,antiinflammatory,and anti-apoptotic activities through Nrf2 and NF-κB pathway. |
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