半夏秫米汤合栀子豉汤调控PINK1/Parkin信号通路介导线粒体自噬防治失眠伴抑郁的机制OA

Objective:To investigate the effect and mechanism of Banxia Shumitang plus Zhizichi Tang(BZ)on PTEN-induced kinase 1(PINK1)/Parkin signaling pathway in regulating mitochondrial autophagy in preventing insomnia with depression.Methods:Seventy-two 6-week-old male Kunming mice were randomized into a normal group,a model group,low-dose,medium-dose,and high-dose BZ groups(BZ-L,BZ-M,BZ-H,2.92,5.85,11.7 g·kg-1),and an estazolam(0.21 mg·kg-1)group.Insomnia model was established on days 1 to 3.p-Chlorophenylalanine(PCPA)was injected intraperitoneally at a dose of 400 mg·kg-1 into mice of the model group and administration groups for 3 consecutive days.Then,the classic depression model was established with chronic unpredictable mild stress(CUMS)for 28 consecutive days.Administration and modeling were performed simultaneously.After gavage,24 h spontaneous activity test was performed to test sleep state,and sugar preference test and open field test were performed to evaluate anxiety-like state.The morphological changes of the hypothalamus were observed by hematoxylin-eosin(HE)staining,and the number of Nissl bodies was observed by Nissl staining.The ultrastructure of mitochondria was observed by transmission electron microscopy.The mitochondrial membrane potential assay kit(JC-1)was used to examine the mitochondrial function,and a biochemical assay kit was used to measure the ATP content in the hypothalamus.The level of reactive oxygen species(ROS)was determined by the immunofluorescence assay,and the serum levels of 5-hydroxytryptamine(5-HT),noradrenalin(NE),and cortisol(CORT)were determined by enzyme-linked immunosorbent assay(ELISA).Western blot and Real-time quantitative polymerase chain reaction(Real-time PCR)were employed to determine the expression of PINK1,Parkin,microtubule-associated protein 1 light chain 3(LC3),and isolator protein 1(p62)at protein and mRNA levels,respectively.Results:The 24 h spontaneous activity test showed that compared with the normal group,the model group did not exhibit circadian rhythm characteristics in their spontaneous activity time,while the circadian rhythm characteristics were restored in each treatment group(P<0.05).The sucrose preference test indicated that compared with that in the normal group,the sucrose preference rate in the model group reduced(P<0.01),and compared with that in the model group,the sucrose preference rate in each treatment group increased(P<0.01).The open field test revealed that compared with the normal group,the model group exhibited decreased movement distance,average speed,frequency of visits to the central area,and time spent in the central area(P<0.01),and compared with those in the model group,these parameters in each treatment group increased(P<0.05,P<0.01).HE staining showed that compared with the normal group,the model group exhibited disarrangement of neurons in the hypothalamus,enlarged intercellular space,and severe nuclear condensation,while the degree of hypothalamic neuronal damage in each treatment group was alleviated.Nissl staining indicated that compared with the normal group,the model group demonstrated aggravated neuronal damage and increased damaged cells(P<0.01).Compared with that in the model group,the number of damaged cells in each treatment group decreased(P<0.01).Transmission electron microscopy showed that compared with the normal group,the model group exhibited mitochondrial swelling and rupture in the hypothalamus,with visible autophagic lysosomes in the cytoplasm.Compared with those in the model group,only a few autophagic lysosomes were observed in the BZ-L group,and the degree of mitochondrial swelling and rupture was alleviated in the other treatment groups.ROS immunofluorescence assay showed that compared with the normal group,the model group displayed enhanced red fluorescence intensity of mitochondrial ROS(P<0.01),which was attenuated in each treatment group(P<0.01).Mitochondrial membrane potential assay showed that compared with the normal group,the model group had declined mitochondrial membrane potential(P<0.01),which was recovered in each treatment group(P<0.05,P<0.01).The biochemical assay of ATP levels indicated that compared with the normal group,the model group exhibited decreased ATP content(P<0.01),which rebound in each treatment group(P<0.05,P<0.01).ELISA showed that compared with the normal group,the model group exhibited decreased 5-HT content and increased NE and CORT content(P<0.01).Compared with the model group,each treatment group showed increased 5-HT content(P<0.01)and reduced NE content(P<0.01)and CORT content(P<0.05,P<0.01).Western blot of the hypothalamus showed that compared with the normal group,the model group exhibited downregulated protein levels of PINK1,Parkin,and LC3(P<0.01)and upregulated protein level of p62(P<0.01).Compared with the model group,each treatment group showed upregulated protein levels of PINK1,Parkin,and LC3(P<0.05,P<0.01)and downregulated protein level of p62(P<0.05,P<0.01).Real-time PCR showed that compared with the normal group,the model group exhibited downregulated mRNA levels of PINK1,Parkin,and LC3(P<0.01)and upregulated mRNA level of p62(P<0.01).Compared with the model group,each treatment group showed upregulated mRNA levels of PINK1,Parkin,and LC3(P<0.01)and downregulated mRNA level of p62(P<0.01).Conclusion:BZ can ameliorate the insomnia and anxiety state of mice by regulating PINK1/Parkin signaling pathway to improve the mitochondrial function,enhance mitochondrial autophagy,protect neurons,and repair damaged nerve cells.