氯马斯汀对慢性疼痛模型小鼠抑郁样行为改善作用及神经机制研究

Improvement and neural mechanism of clemastine on depressive-like behavior in chronic pain model mice

目的:探讨氯马斯汀(clemastine,CLE)对完全弗氏佐剂(complete Freund's adjuvant,CFA)诱导的慢性疼痛模型小鼠抑郁样行为的影响和可能的机制。方法:30只雄性C57BL/6J小鼠按照随机数字表法分为生理盐水对照组(NS组)、疼痛模型组(CFA组)、疼痛模型+CLE干预组(CFA-CLE组),每组10只。CFA组、CFA-CLE组小鼠在右后足足底中部皮下注射10μL CFA建立慢性炎性疼痛模型,CFA-CLE组小鼠在CFA注射第7天腹腔注射CLE(10 mg/kg),1次/d,持续14 d,CFA组和NS组小鼠注射等体积0.9%氯化钠溶液。采用von Frey纤维丝检测3组小鼠机械缩足阈值(paw withdrawal mechanical threshold,PWMT);采用旷场实验检测小鼠的活动度,采用糖水偏爱实验、悬尾实验、强迫游泳实验检测小鼠抑郁样行为;采用免疫组织化学染色方法检测小鼠海马区少突胶质细胞标志物髓鞘碱性蛋白(myelin basic protein,MBP)、新生神经元标志物双皮质素(doublecortin,DCX)和成熟神经元标志物微管相关蛋白2(microtubule-associated protein 2,MAP2)的表达情况。采用GraphPad Prism 8.0软件进行统计分析,3组间比较采用单因素方差分析,PWMT结果采用重复测量方差分析。结果:痛阈检测结果显示,时间和组别对PWMT影响的交互效应是显著的(F=4.390,P<0.001),并且时间主效应(F=13.44,P<0.001)与组别主效应(F=25.38,P<0.001)均有显著性。在1 d、7 d、14 d、21 d,CFA组小鼠的PWMT均低于NS组(均P<0.001);CFA组与CFA-CLE组小鼠在1 d、7 d、14 d、21 d的PWMT均显著低于0 d(均P<0.001);而在不同时间点CFA-CLE组小鼠的PWMT与CFA组相比均差异无统计学意义(均P>0.05)。3组小鼠旷场实验中的运动总距离差异无统计学意义(F=1.15,P>0.05)。3组小鼠的糖水偏爱率、悬尾不动时间和强迫游泳不动时间均差异有统计学意义(F=5.46,13.44,14.29,均P<0.05);CFA组小鼠的糖水偏爱率[(65.78±11.61)%]低于NS组[(80.55±6.41)%],悬尾不动时间和强迫游泳不动时间[(124.60±18.16)s,(82.63±13.40)s]均高于NS组[(88.11±12.83)s,(48.09±9.78)s](均P<0.05);CFA-CLE组小鼠的糖水偏爱率[(78.04±9.10)%]高于CFA组,悬尾不动时间和强迫游泳不动时间[(89.96±11.93)s,(56.37±12.82)s]均低于CFA组(均P<0.05)。3组小鼠海马区MBP、DCX和MAP2阳性细胞的吸光度均差异有统计学意义(F=11.92,6.55,9.31,均P<0.01);CFA组小鼠海马MBP、DCX和MAP2阳性细胞的吸光度均低于NS组(均P<0.01);而CFA-CLE组小鼠海马MBP、DCX和MAP2阳性细胞的吸光度均高于CFA组(均P<0.05)。结论:CLE可能通过促进海马少突胶质细胞再髓鞘化和提高神经元可塑性改善CFA慢性疼痛模型小鼠的抑郁样行为。

Objective To study the effect of clemastine(CLE)on pain-related depressive-like behavior induced by complete Freund's adjuvant(CFA)and its possible regulatory mechanism.Methods Thirty C57BL/6J male mice were randomly divided into normal saline control group(NS group),CFA induced pain model group(CFA group)and CFA+CLE intervention group(CFA-CLE group),with 10 mice in each group.The mice in CFA group and CFA-CLE group were subcutaneously injected with 10μL CFA in the middle of the right hind foot to establish the chronic inflammatory pain model.Then 7 days later,mice in CFA-CLE group were intraperitoneally injected with CLE(10 mg/kg)once a day for 14 days,meanwhile mice in CFA group and NS group were injected with an equal volume of 0.9%sodium chloride solution.The von Frey fiber was used to evaluate the paw withdrawal mechanical threshold(PWMT)of mice in the three groups.Open field test was used to detect the activity of mice.Sucrose preference test,tail suspension test(TST)and forced swimming test(FST)were used to detect the depressive-like behavior of mice.Immunohistochemical staining was used to detect the expression of myelin basic protein(MBP)(a marker for oligodendrocytes),doublecortin(DCX)(a marker for newly formed neurons),and microtubule-associated protein 2(MAP2)(a marker for mature neurons)in the hippocampus of mice.GraphPad Prism 8.0 software was used for statistical analysis.Multiple group comparisons were conducted by one-way ANOVA.The pain threshold data was analyzed by repeated ANOVA.Results The results of pain threshold showed that the interaction effect of time and group on PWMT was significant(F=4.390,P<0.001),and both the time main effect(F=13.44,P<0.001)and group main effect(F=25.38,P<0.001)were significant.At 1 d,7 d,14 d,21 d,the PWMT of mice in CFA group were significantly lower than those of NS mice(all P<0.001).The PWMT of mice in CFA group and CFA-CLE group at 1 d,7 d,14 d and 21 d were significantly lower than those at 0 d(all P<0.001).However,there were no significant differences in PWMT between CFA-CLE group and CFA group at different time points(all P>0.05).There was no significant difference in the total distance among the 3 groups(F=1.15,P>0.05).The differences of the sucrose preference percentage and the immobility time in TST,FST were significant among the 3 groups(F=5.46,13.44,14.29,all P<0.05).The sucrose preference percentage of mice in CFA group were lower than that of NS group((65.78±11.61)%,(80.55±6.41)%)(P<0.05).In TST and FST,the immobility time in CFA group((124.60±18.16)s,(82.63±13.40)s)were both longer than those of NS group((88.11±12.83)s,(48.09±9.78)s)(both P<0.05).The sucrose preference percentage of CFA-CLE group((78.04±9.10)%)was higher than that of CFA group(P<0.05).In TST,FST,the immobility time in CFA-CLE group((89.96±11.93)s,(56.37±12.82)s)were both shorter than those of CFA group(both P<0.05).Immunohistochemical results showed that the differences of the average absorbance of MBP,DCX,MAP2 in hippocampus were significant among the 3 groups(F=11.92,6.55,9.31,all P<0.01).The absorbance of MBP,DCX,MAP2 in hippocampus in CFA group were lower than those of NS group(all P<0.01).The absorbance of MBP,DCX,MAP2 in hippocampus of mice in CFA-CLE group were higher than those of CFA group(all P<0.05).Conclusion Clemastine can relieve CFA-induced depressive-like behaviors in mice by strengthening hippocampal oligodendrocytes remyelinate and enhancing neuronal plasticity.