基于MCT1调控的髓鞘轴突能量代谢探究加减薯蓣丸改善血管性痴呆伴抑郁小鼠抑郁行为和髓鞘再生

Modified Shuyuwan Ameliorates Depressive Behaviors and Promotes Myelin Regeneration in Mouse Model of Vascular Dementia Complicated with Depression by Regulating Energy Metabolism of Myelin Axons via MCT1

目的:探究加减薯蓣丸对血管性痴呆(VaD)伴抑郁小鼠的作用机制。方法:3月龄雄性C57/BL6小鼠7只,采用双侧颈动脉狭窄(BCAS)构建VaD模型,激光散斑成像测量BCAS小鼠术前、术后局部脑血流量(rCBF);随后采用BCAS法结合慢性不可预测刺激(CUMS),构建VaD伴抑郁小鼠模型,将BCAS/CUMS小鼠分为模型组、氟西汀组(0.01 g·kg^(-1))、加减薯蓣丸高、中、低剂量组(20、10、5 g·kg^(-1)),对照组为假手术且无慢性不可预测刺激(sham)组,每组10只。悬尾实验、糖水偏好实验检测小鼠抑郁行为,免疫荧光法检测小鼠胼胝体(CC)髓鞘相关糖蛋白(MAG)、髓鞘碱性蛋白(MBP)、神经丝蛋白重链(NF200)、抗非磷酸化神经细丝蛋白抗体(SMI32)的分布及表达变化;蛋白免疫印迹法(Western blot)检测小鼠胼胝体中单羧酸转运蛋白1(MCT1)、MBP、MAG、少突胶质细胞糖蛋白(MOG)、淀粉样蛋白前体(APP)、NF200、朗飞氏结标记物(Caspr)、电压门控性钠通道(Nav1.6)蛋白表达,乳酸(LD)试剂盒检测各组小鼠血清中LD含量的表达,超微透射电镜观察髓鞘超微结构。结果:激光散斑成像结果显示,BCAS术后10 min,局部脑血流量即刻出现下降(P<0.01),直至术后2周,局部脑血流量仍然呈脑低灌注状态且无法恢复至术前水平;行为学检测结果显示,与sham组比较,模型组小鼠糖水偏好百分比下降(P<0.01),悬尾实验中不动时间显著增加(P<0.01);加减薯蓣丸组及氟西汀组干预后糖水偏好百分比升高(P<0.01),悬尾实验中不动时间显著减少(P<0.01);LD检测结果显示,BCAS/CUMS小鼠LD含量最高(P<0.01),加减薯蓣丸干预后可显著降低LD含量(P<0.01);免疫荧光结果显示,与sham组比较,BCAS/CUMS小鼠胼胝体MAG、MBP、NF200的荧光强度表达降低,SMI32荧光强度增加,加减薯蓣丸高、中、低剂量组及氟西汀组均可不同程度增加MAG、MBP、NF200荧光强度,降低SMI32荧光强度;Western blot结果显示,与sham组比较,BCAS/CUMS小鼠胼胝体中MCT1、MBP、MOG、MAG、NF200、Caspr蛋白表达水平下降(P<0.05,P<0.01),APP、Nav1.6蛋白相对表达水平升高,加减薯蓣丸干预后可以不同程度改善上述趋势(P<0.05,P<0.01);超微透射电镜结果表明,与sham组比较,BCAS/CUMS造模后髓鞘超微结构破坏,轴突萎缩,加减薯蓣丸干预后,髓鞘结构受损程度减轻。结论:加减薯蓣丸可能通过上调MCT1,改善VaD小鼠LD在髓鞘与轴突中的转运障碍,促进胼胝体髓鞘再生并增加髓鞘结构完整性,改善抑郁表型。

Objective:To explore the mechanism of modified Shuyuwan in treating vascular dementia(VaD)complicated with depression in mice.Method:The VaD model was established by bilateral carotid artery stenosis(BCAS)in seven 3-month-old male C57/BL6 mice.The regional cerebral blood flow(rCBF)of mice was measured by laser speckle imaging before and after BCAS surgery.Then,the BCAS method was combined with chronic unpredictable mild stress(CUMS)to establish a mouse model of VaD complicated with depression.BCAS/CUMS mice were assigned into BCAS/CUMS,fluoxetine(0.01 g·kg^(-1)),and high-,medium-,and low-dose(20,10,5 g·kg^(-1),respectively)modified Shuyuwan groups.The shame group underwent sham operation without CUMS(n=10).The tail suspension test and sucrose preference test were carried out to examine the depressive behaviors of mice.The distribution and expression of myelin-associated glycoprotein(MAG),myelin basic protein(MBP),neurofilament heavy polypeptide(NF200),and anti-nonphosphorylated neurofilament epitope antibody(SMI32)in the corpus callosum(CC)were detected by the immunofluorescence assay.Western blot was employed to determine the protein levels of monocarboxylate transporter 1(MCT1),MBP,MAG,oligodendrocyte glycoprotein(MOG),amyloid precursor protein(APP),NF200,contactin-associated protein(Caspr),and voltage-gated sodium channel(Nav1.6)in the corpus callosum.The level of lactic acid in the serum was measured by the lactic acid assay kit,and the ultrastructure of myelin was observed by ultraprojective electron microscope.Result:Laser speckle imaging showed that rCBF decreased immediately 10 min after BCAS surgery(P<0.01),and the rCBF was still cerebral hypoperfusion and did not return to the preoperative level 2 weeks after surgery.Behavioral test results showed that compared with the sham group,the BCAS/CUMS group presented decreased percentage of sucrose preference(P<0.01)and prolonged immobile time in the tail suspension test(P<0.01).Compared with the BCAS/CUMS group,fluoxetine and modified Shuyuwan increased the percentage of sucrose preference(P<0.01)and shortened the immobile time in the tail suspension test(P<0.01).The level of lactic acid was the highest in the BCAS/CUMS mice(P<0.01),and modified Shuyuwan lowered the lactic acid level(P<0.01).Immunofluorescence results showed that compared with the sham group,the BCAS/CUMS group presented decreased fluorescence intensity of MAG,MBP and NF200 and increased fluorescence intensity of SMI32 in the corpus callosum,and such changes were reversed by modified Shuyuwan at different doses and fluoxetine.Western blot results showed that compared with the sham group,the BCAS/CUMS modeling down-regulated the protein levels of MCT1,MBP,MOG,MAG,NF20,and Caspr(P<0.05,P<0.01)and up-regulated the protein levels of APP and Nav1.6 in the corpus callosum,and the above trends were reversed by modified Shuyuwan(P<0.05,P<0.01).Compared with the sham group,BCAS/CUMS modeling led to myelin ultrastructure damage and axon atrophy,which were alleviated by modified Shuyuwan.Conclusion:Modified Shuyuwan can ameliorate the transport disorder of lactic acid between myelin sheath and axon by upregulating the expressin of MCT1,promote the regeneration of myelin sheath in the corpus callosum,and improve the integrity of myelin sheath structure,thereby alleviating depression in VaD mice.