| The manifestations of cognitive dysfunction such as memory loss,attention impairment,and unfavorable speech after chemotherapy for breast cancer patients are receiving increasing attention.This not only brings mental suffering to the patients,but also has a serious impact on their daily living ability and quality of life.However,the onset of chemotherapy-induced cognitive dysfunction is insidious and not easily recognized,and the specific mechanism of action has not yet been elucidated.It is important to explore and clarify the effects and mechanism of action of chemotherapy on cognitive function of breast cancer patients for the prevention and treatment of the disease.On the one hand,this study was conducted to clarify the relationship between chemotherapy and cognitive function and its influencing factors from the clinical perspective,and on the other hand,the molecular mechanism of cognitive dysfunction caused by chemotherapy drugs was further explored by constructing a chemotherapy cognitive impairment model in mice,which provides new ideas and theoretical basis for clinical treatment.Objective:1.To clarify the effects of chemotherapy on subjective and objective cognitive dysfunction in breast cancer patients.2.To observe the effects of chemotherapy on the learning and memory ability and the tissue structure of hippocampal region in mice.3.To explore the molecular mechanism of cognitive function changes in mice caused by chemotherapy.Methods:1.Patients with breast cancer who were newly diagnosed to receive chemotherapy for the first time were selected for the study,and the Mo CA Neuropsychological Scale test and the FACT-Cog,SDS,and SAS subjective questionnaires were administered at four time periods: before chemotherapy(T0),during chemotherapy(T1),within 1 week after the end of chemotherapy(T2),and 6 months later(T3).1.1 The effect of chemotherapy on patients’ cognitive function was clarified by comparing the changes in subjective and objective cognitive function between the healthy control group and the chemotherapy group at T2.1.2 The basic information of the participants in the study was collected,and the factors influencing chemotherapy-related changes in cognitive function in breast cancer patients were analyzed by using pearson correlation analysis and regression analysis.1.3 The cognitive function of breast cancer patients at different time points before and after chemotherapy was analyzed by single factor repeated measurement method.2.C57BL/6J male mice were randomly divided into control group(sham group),epirubicin group(EPI group,intraperitoneal injection of epirubicin 4mg/kg),paclitaxel group(PTX group,intraperitoneal injection of paclitaxel 20mg/kg),and cyclophosphamide group(CTX group,intraperitoneal injection of cyclophosphamide 50mg/kg).A total of 8injections were given every 3 days.2.1 The learning memory ability of mice was detected by Morris water maze.2.2 The changes of hippocampal tissue structure of mice were observed by HE staining method and Nissl staining method.2.3 The effect of chemotherapy on the density of dendritic spines in the hippocampal region was observed by Golgi staining.2.4 The expression of Iba-1 in the hippocampal region of mice was detected by immunofluorescence technique to observe the effect of chemotherapy on microglia in the hippocampal region.3.C57BL/6J male mice were randomly divided into control group(sham group),epirubicin group(EPI group,4mg/kg),paclitaxel group(PTX group,20mg/kg),and cyclophosphamide group(CTX group,50mg/kg).A total of 8 intraperitoneal injections were given every 3 days.3.1 Apoptosis detection.Detection of apoptosis in mouse hippocampal neurons by TUNEL method.To detect the expression of apoptosis-related proteins Bax,Bcl2 and Caspase-3 in the hippocampal region using Western blot technique.To observe the co-localization and expression of apoptosis protein Caspase-3 with neuronal cells Neu N in mouse hippocampal region using immunofluorescence double-labeling technique.3.2 To detect the expression of SOD and MDA,indicators of oxidative stress in mouse hippocampus,using kits.3.3 To detect the inflammatory mediators TNF-α,IL-1β,IL-6 and IL-18 in mouse serum and hippocampus by ELISA technique;to observe the co-localization and expression of the inflammatory mediator IL-1β with microglia Iba-1 in mouse hippocampus by immunofluorescence double-labeling technique.3.4 The levels of NLPR3,caspase-1,IL-1β and IL-18 m RNA in mouse hippocampal tissue were detected by q RT-PCR.3.5 The expression levels of NLPR3,caspase-1,IL-1β and IL-18 in mouse hippocampal tissues were detected by Western blot.Results:1.Results of the effect of chemotherapy on subjective and objective cognitive function in breast cancer patients.1.1(1)In the T0 stage before chemotherapy,there was no significant difference in Mo CA and SDS scores in the chemotherapy group compared with the control group(P>0.05),but the FACT-Cog scores(P=0.012)in the chemotherapy group already showed a decrease and SAS scores(P=0.030)showed an increase compared with the control group.(2)At the T2 stage after the start of chemotherapy,the total Mo CA scale score and each dimensional index,the total FACT-Cog score and each dimensional index,SAS and SDS scores of patients in the chemotherapy group were significantly decreased compared with the control group(P<0.01).1.2(1)Patient age,education level,and chemotherapy regimen were the influencing factors of objective cognitive dysfunction in breast cancer patients after chemotherapy.Among them,patients aged ≥60 years,with low education level and using an anthracycline-containing chemotherapy regimen had low Mo CA total scores as well as low scores of each dimensional index and more severe objective cognitive dysfunction.(2)Patient age,education level,chemotherapy regimen,and SAS and SDS scores were all influential factors for chemotherapy-related cognitive impairment in breast cancer patients(P<0.05).Among them,patients aged ≥60 years,with low education level,using anthracycline-containing chemotherapy regimen,with anxiety and depression had lower FACT-Cog total score and each dimension score,and more severe subjective cognitive dysfunction.1.3(1)Mo CA scores of breast cancer chemotherapy patients were significantly different at different time points before and after chemotherapy,T0,T1,T2 and T3.Compared with the pre-chemotherapy T0 stage,the total Mo CA scores as well as all dimensions were lower in the post-chemotherapy T1 and T2 stages(P<0.05);only the total Mo CA scores,visuospatial and executive ability and verbal ability were lower in the T3stage(P<0.05).The differences in abstraction ability and orientation were not significant in T2 compared to T1 stage,but the Mo CA scale total score and scores of all other remaining dimensions decreased(P<0.05).Mo CA scale score as well as all dimensions rebounded in T3 compared to T2 stage(P<0.05).(2)FACT-Cog scores of breast cancer chemotherapy patients were significantly different at different time points of T0,T1,T2,and T3(P<0.01).Compared with the pre-chemotherapy T0 stage,the subjective cognitive function FACT-Cog total score and each dimension score decreased in T1 and T2 stages after chemotherapy(P<0.05);the FACT-Cog total score and Cog PCI,Cog PCA and Cog QOL dimensions decreased in T3 stage(P<0.05),and the Cog Oth dimension was not significantly different.The total FACT-Cog score and the scores of each dimension decreased significantly(P<0.05)in T2 compared with the T1 stage.The total FACT-Cog score and the indicators of each dimension rebounded to different degrees(P<0.05)in T3 stage compared with T2 stage.2.Results of the effects of chemotherapeutic drugs on learning memory function and hippocampal tissue structure in mice.2.1 The results of Morris water maze showed that:(1)in the positioning navigation experiment,the mean shortening of escape latency was not significantly different between groups of mice in the first 3 days of the experiment(P>0.05).From the 4th and 5th days,the escape latency of mice in the paclitaxel,epirubicin and cyclophosphamide groups was significantly prolonged compared with that in the control group(P<0.05).Therefore,chemotherapy reduced the ability of mice to recognize spatial markers,leading to their impaired learning ability.(2)In the space exploration experiment,The cumulative duration of mice in the target quadrant of chemotherapy group was less than that in the control group(P<0.05),and The Times of crossing the quadrant were less than that in the control group(P<0.05).2.2 HE staining results: The cone cells in the hippocampal CA1 area of mice in the control group were structurally dense and intact,with uniform and normal morphology and neat arrangement.In the chemotherapy group,the number of cone cells in the CA1 area of hippocampus was reduced,the structure was loose,the morphology was abnormal,and the arrangement was disordered,among which the neuronal cells in the epirubicin group had the most serious degeneration and necrosis.2.3 Nissl staining: Nisin bodies were abundant in the hippocampal neurons of control mice,with dark coloring.In contrast,in the chemotherapy group,the number of neuronal nisomes in hippocampal neurons was reduced and the staining was light.In the epirubicin group,the neuronal cells were obviously swollen and the Nisin bodies were lysed in translucent areas.2.4 Golgi staining showed that the number and density of hippocampal CA1 dendritic spines in chemotherapy group were lower than those in control group(P<0.05),and the density and number of dendritic spines in the epirubicin group were more significantly reduced compared with those in the paclitaxel and cyclophosphamide groups,and the difference was statistically significant(P<0.05).2.5 The immunofluorescence results showed that in the control group,the expression of Iba-1 was low,the cell membrane was small,and the morphology was normal.In the chemotherapy group,the expression of Iba-1 increased and the cell membrane became larger.In the epirubicin and cyclophosphamide groups,the expression of Iba-1 was significantly increased,the cell membrane was significantly larger,and the cell morphology was round.3.Results of the effects of chemotherapeutic agents on apoptosis,inflammation and oxidative stress in the hippocampal region of mice.3.1(1)The results of TUNEL staining showed that the number of TUNEL-positive cells in mice in the chemotherapy group was higher when compared with control group(P<0.05).The number of TUNEL positive cells in mice in the paclitaxel group increased more significantly(P<0.05).(2)Western blot showed that the Bax/Bcl-2 ratio and cleaved-caspase-3 protein expression in the brain tissues of mice in the chemotherapy group were significantly higher than those in the control group(P<0.05).The increase was most significant in the paclitaxel group(P<0.05).(3)The results of immunofluorescence double-labeling method showed that the expression level of caspase-3 and immunofluorescence intensity were significantly increased in the hippocampal tissues of mice in the chemotherapy group,while the expression level and immunofluorescence intensity of Neu N were significantly decreased.Among them,compared with the epirubicin and cyclophosphamide groups,the caspase-3 positive cells and immunofluorescence intensity increased more significantly in the paclitaxel group,while the Neu N positive cells and immunofluorescence intensity decreased more significantly.And from the staining results,the apoptotic protein caspase-3 expression was concentrated in neuronal cell locations;therefore,there was co-localized expression of Neu N and caspase-3,and Neu N diminished with the enhancement of caspase-3 fluorescence intensity.3.2 The results of oxidative stress index measurement in the hippocampus showed that the SOD activity decreased(P<0.05)and the MDA content increased(P<0.05)in all groups of mice after chemotherapy,indicating that the antioxidant capacity of mice in the chemotherapy group was reduced and the oxidative stress response was obvious.Among them,the differences in SOD activity and MDA content between the paclitaxel,epirubicin and cyclophosphamide groups were not significant when compared among the chemotherapy groups,indicating that the antioxidant capacity was close between the groups.3.3(1)The results of ELISA technique to detect inflammation levels in mice serum and hippocampus showed that the levels of TNF-α,IL-1β,IL-6,IL-18 in mice serum and hippocampal tissues in the chemotherapy group were higher than those in control group(P<0.05),with the most significant increase in the epirubicin group(P<0.01).(2)The results of immunofluorescence double-labeling method showed that the number of IL-1βand Iba-1 positive cells and immunofluorescence intensity in the hippocampal tissue of chemotherapy group were higher than those in the control group(P<0.05).Among them,the increase of IL-β and Iba-1 positive cells and immunofluorescence intensity were more obvious in the epirubicin group compared with the paclitaxel and cyclophosphamide groups(P<0.05).And from the staining results,the expression of inflammatory factor IL-βwas concentrated in the location of microglia expressing Iba-1;therefore,there was co-localized expression of Iba-1 and IL-β,and IL-β was enhanced with the enhancement of Iba-1 fluorescence intensity.3.4 The results of q RT-PCR showed that the m RNA levels of NLRP3,caspase-1,IL-1β and L-18 in hippocampal tissues of mice in the chemotherapy group were all increased to different degrees(P<0.05).Among the chemotherapy groups,the most significant increase was observed in the epirubicin group(P<0.05).3.5 Western blot results showed that the expression levels of NLRP3,caspase-1,IL-1βand L-18 in hippocampal tissues of mice in the chemotherapy group were all increased to different degrees compared with the control group,among which the increase in the epirubicin group was obvious and the difference was statistically significant(P<0.05).Conclusion:1.Chemotherapy can cause subjective and objective cognitive dysfunction in breast cancer patients,and over time,both subjective and objective cognitive functions recovered to varying degrees six months after chemotherapy,but executive and language functions recovered more slowly.2.Advanced age,low education level,and anthracycline-containing chemotherapy regimen are common risk factors affecting subjective and objective cognitive dysfunction;anxiety and depression are risk factors affecting subjective cognitive dysfunction,not related to objective cognitive dysfunction.3.Chemotherapy can lead to decreased learning memory function,pathological damage to hippocampal histological structures,decreased density of dendritic spines,and microglia activation in mice.4.Chemotherapy can lead to apoptosis and increased levels of oxidative stress in neuronal cells in the hippocampus of mice.5.Chemotherapy can lead to increased levels of inflammatory mediators in the serum and hippocampus of mice and activation of the NLRP3 inflammasome-caspase-1-IL-1β/IL-18 pathway. |
