黄连解毒汤调控FBXL5/IRP2改善VaD小鼠脑铁代谢紊乱、髓鞘损伤及激越攻击行为

Effect of Huanglian Jiedutang in Improving Brain Iron Metabolism Disorders， Myelin Damage， and Aggressive Behavior in VaD Mice via Regulation of FBXL5/IRP2

摘要:

目的 探讨黄连解毒汤通过调控F盒富含亮氨酸重复序列蛋白5（FBXL5）/铁调节蛋白2（IRP2）通路对血管性痴呆（VaD）小鼠脑铁代谢紊乱、髓鞘损伤及攻击行为的影响。方法 将60只C57BL/6J小鼠随机分为假手术组、模型组、利培酮组（2 mg·kg^-1·d^-1）和黄连解毒汤低、中、高剂量组（0.25、0.5、1 g·kg^-1·d^-1），每组10只。采用双侧颈总动脉狭窄（BCAS）法建立VaD模型。从第7周开始给予药物干预2周。在第9周进行行为学评估，包括触摸逃避实验和居民入侵者测试。行为学测试结束后，收集下丘脑腹外侧腹内侧（VMHvl）组织样本。蛋白免疫印迹法（Western blot）检测髓鞘相关糖蛋白（MAG）、髓鞘少突胶质细胞糖蛋白（MOG）、髓鞘碱性蛋白（MBP）、4-羟基-2-壬烯醛（4-HNE）、谷胱甘肽过氧化物酶4（GPX4）、转铁蛋白受体1（TFR1）、铁蛋白轻链（Ft-L）、膜铁转运蛋白1（FPN1）、FBXL5和IRP2表达；免疫荧光检测MBP荧光强度；透射电镜观察髓鞘超微结构改变；Perl's染色检测组织铁沉积；酶联免疫吸附测定法（ELISA）检测丙二醛（MDA）和超氧化物歧化酶（SOD）水平。结果 与假手术组比较，模型组小鼠啃咬次数、攻击行为、易激惹评分明显升高，攻击潜伏期显著缩短（P<0.01）；MAG、MOG和MBP的表达水平及MBP的荧光强度均显著降低（P<0.01），髓鞘超微结构紊乱。Ft-L、TFR1表达升高，FPN1表达降低，铁沉积增加（P<0.01）。抗氧化剂GPX4和SOD降低，4-HNE和脂质过氧化产物MDA升高（P<0.01）。FBXL5蛋白表达降低，IRP2蛋白表达升高（P<0.01）。与模型组比较，黄连解毒汤中、高剂量组和利培酮组小鼠啃咬次数、攻击行为和激惹评分降低（P<0.05，P<0.01），攻击潜伏期增加（P<0.01）；黄连解毒汤中、高剂量组MAG、MOG、MBP表达水平及MBP免疫荧光均升高（P<0.01）。超微板层结构排列有序；Ft-L、TFR1表达降低，FPN1表达升高，铁沉积减少（P<0.01）；GPX4、SOD表达水平升高，4-HNE、MDA表达水平降低（P<0.01）；FBXL5蛋白表达升高，IRP2蛋白表达降低（P<0.01）。结论 黄连解毒汤可能通过调控VMHvl区FBXL5/IRP2表达，改善VaD小鼠脑铁代谢紊乱、髓鞘损伤及激越攻击行为。

Abstract:

Objective This study aims to investigate the effect of Huanglian Jiedutang on brain iron metabolism disorders， myelin damage， and aggressive behavior in vascular dementia （VaD） mice by regulating the F-box leucine-rich repeat protein 5 （FBXL5）/iron regulatory protein 2 （IRP2） pathway.Method Sixty C57BL/6J mice were randomly divided into six groups： sham operation group， model group， risperidone group （2 mg·kg^-1·d^-1）， and low-dose， medium-dose， and high-dose groups of Huanglian Jiedutang （0.25， 0.5， 1 g·kg^-1·d^-1）， with 10 mice in each group. The VaD model was established by bilateral carotid artery stenosis （BCAS）. Drug intervention was administered for two weeks starting from the seventh week. Behavioral assessments， including the touch escape and resident-intruder tests， were conducted in the ninth week. After the behavioral tests， ventromedial hypothalamus ventrolateral （VMHvl） tissue samples were collected. Western blot was used to detect the expression of myelin-associated glycoprotein （MAG）， myelin oligodendrocyte glycoprotein （MOG）， myelin basic protein （MBP）， 4-hydroxynonenal （4-HNE）， glutathione peroxidase 4 （GPX4）， transferrin receptor 1 （TFR1）， ferritin light chain （Ft-L）， ferroportin 1 （FPN1）， FBXL5， and IRP2. Immunofluorescence was used to measure MBP fluorescence intensity. Transmission electron microscopy was employed to observe ultrastructural changes in the myelin sheath. Perl's staining was used to detect tissue iron deposition. Malondialdehyde （MDA） and superoxide dismutase （SOD） levels were measured by enzyme linked immunosorbent assay （ELISA）.Result Compared to the sham operation group， the model group exhibited a significant increase in biting， aggression， and irritability scores， along with a reduced latency of the attack （P<0.01）. The expression levels of MAG， MOG， and MBP， as well as the fluorescent density of MBP， were significantly decreased （P<0.01）. Disordered myelin ultrastructure， increased Ft-L and TFR1 expression， decreased FPN1 expression， and elevated iron deposition were observed （P<0.01）. Antioxidants GPX4 and SOD were reduced， while 4-HNE and lipid peroxidation product MDA were increased （P<0.01）. FBXL5 protein expression decreased， and IRP2 protein expression increased （P<0.01）. Compared with the model group， in the middle-dose and high-dose groups of Huanglian Jiedutang and the risperidone group， the number of bites， aggressive behaviors， and irritability scores were reduced （P<0.05， P<0.01）， while the latency of the attack increased （P<0.01）. In the middle-dose and high-dose groups of Huanglian Jiedutang， the expression levels of MAG， MOG， and MBP， as well as MBP immunofluorescence increased （P<0.01）. The ultrastructure was orderly arranged. Ft-L and TFR1 expression decreased； FPN1 expression increased， and iron deposition was reduced （P<0.01）. GPX4 and SOD expression levels increased， and 4-HNE and MDA expression levels decreased （P<0.01）. FBXL5 protein expression increased， and IRP2 protein expression decreased （P<0.01）.Conclusion Huanglian Jiedutang may alleviate brain iron metabolism disorders， myelin damage， and aggressive behavior in VaD mice by regulating FBXL5/IRP2 expression in the VMHvl region.