小檗碱通过修复MAMs结构调控钙信号通路改善CUMS大鼠抑郁样行为OA

OBJECTIVE To investigate whether berberine ameliorates depression-like behavior in chronic unpredictable mild stress(CUMS)rats by restoring the structure of mitochondria-associated endoplasmic reticulum membranes(MAMs)and regulating the calcium signaling pathway.METHODS The physicochemical properties of berberine were characterized by scanning electron microscopy,ultraviolet-visible spectroscopy,Fourier transform infrared spectroscopy,fluorescence spectroscopy and nuclear magnetic resonance spectroscopy.The in vivo antidepressant effect of berberine was evaluated using a CUMS rat model.Rats were randomly assigned to four groups:a control group,model group,model+sertraline 5.25 mg·kg-1 group,and model+berberine 50 mg·kg-1 group.The treatment groups received daily intragastric administration for 4 consecutive weeks while the control and model groups received an equal volume of normal saline.The body mass of the rats was measured before the experiment(day 0)and at the end of the experiment(day 28).Depression-like behavior was assessed using the sucrose preference rate and immobility time in the forced swimming test.Anxiety-like behavior and locomotor activity were evaluated by residence time in the central area,number of crossing,and number of rearing in the open field test.Spatial learning and memory ability were assessed via escape latency and the number of platform crossing in the Morris water maze test.Meanwhile,the small intestinal propul-sion rate was measured to evaluate gastrointestinal motility.HE staining and Nissl staining were performed to observe hippocampal neuronal damage.RNA sequencing was performed to screen the key signaling pathways regulated by berberine.An in vitro injury model was established using corticosterone(CORT)-induced rat adrenal pheochromocytoma cell line(PC12)that were divided into a control group,CORT 400 μmol·L-1 group and CORT+berberine 40 μmol·L-1 group.Transmission electron microscopy was used to observe MAMs ultrastructure.Mitochondrial calcium and reactive oxygen species(ROS)levels were detected using Rhod-2 AM and DCFH-DA fluorescent probes,respectively.The fluo-rescence intensity of the mitochondrial calcium uniporter(MCU)and the co-localization of inositol 1,4,5-trisphosphate receptor type 1(IP3R1)and voltage-dependent anion channel 1(VDAC1)proteins were detected by immunofluorescence staining.RESULTS The results of physical and chemical character-ization confirmed that berberine had typical needle-like crystal morphology and characteristic spectrum,clear molecular structure and typical properties,which could be used for subsequent in vivo and in vitro experiments.The in vivo results showed that,compared with the control group,the mass gain of rats in the model group was significantly reduced,the sucrose preference rate was significantly decreased,the immobility time of forced swimming was significantly prolonged,the latency to leave the central area of the open field was significantly increased,the number of crossing and rearing was significantly reduced,the escape latency of Morris water maze was significantly prolonged,the number of platform crossing was significantly reduced,and the intestinal propulsion rate was significantly lowered.HE and Nissl staining showed that hippocampal neurons in the model group were loosely arranged,nuclear condensation was obvious,and the number of Nissl bodies was significantly decreased.Compared with the model group,berberine intervention significantly increased mass gain and the sucrose preference rate,significantly shortened the immobility time in the forced swimming test,shortened the latency to leave the central area,increased the number of crossing and rearing in the open field test,shortened the escape latency and increased the number of platform crossing in the Morris water maze test,and significantly elevated the small intestinal propulsion rate.HE and Nissl stain-ing of the hippocampus showed densely arranged neurons,reduced intercellular spaces,decreased pyknotic nuclei,and markedly increased Nissl bodies.Transcriptome sequencing analysis found that the calcium signaling pathway was a key regulatory pathway for its antidepressant efficacy.The in vitro results showed that berberine effectively repaired CORT-induced MAMs ultrastructural damage in PC12 cells,significantly inhibited mitochondrial calcium overload and oxidative stress,reduced MCU fluorescence intensity,and decreased the abnormal co-localization of IP3R1/VDAC1.CONCLUSION Berberine can significantly improve the depression-like behavior of CUMS rats by repairing MAMs structure and regulating the calcium signaling pathway.