| Objective:This study aimed to observe the effects of lipopolysaccharide(LPS)intraperitoneal(i.p.)injection on rats and determine the underlying mechanism.Thus,a rat model with depression in Parkinson’s disease(dPD)was established,and the curative effect and mechanism of Xifeng-Jieyu prescription(XJP)in the treatment of dPD rats were discussed.Methods:Part 1:The Sprague-Dawley rats were administered LPS(0.5 mg/kg,i.p.)for either 1,2,or 4 consecutive days to establish a rat model with dPD.The sucrose preference test(SPT),the open field test(OFT),and the rotarod test evaluated depression-like and motor behaviors.Resting-state functional magnetic resonance imaging(R-fMRI)and echo-planar diffusion tensor imaging(DTI)were used to detect the intrinsic activities and the integrity of white matter fibers in the brain.Hematoxylin-eosin(HE)staining was used to observe the morphological structure of the prefrontal cortex(PFC)and hippocampus(HIP).The expression of c-Fos,ionized calcium-binding adapter molecule-1(Iba-1),and tyrosine hydroxylase(TH)was evaluated using immunohistochemistry.Part 2:To investigate the therapeutic effect and mechanism of XJP on dPD rats.The Sprague-Dawley rats were injected with LPS(0.5 mg/kg,i.p.)for 4 days to establish the dPD rat model.Then,for 14 days,the traditional Chinese medicine group was intragastric administration of XJP,the western medicine group was intragastric administration of fluoxetine hydrochloride,and the control group was intragastric administration of corresponding volume of distilled water.The SPT,OFT,and rotarod test evaluated depression-like and motor behaviors.R-fMRI and DTI were used to detect the intrinsic activities and the integrity of white matter fibers in the brain.The expression of c-Fos,Iba-1,and TH was evaluated using immunohistochemistry.The concentration of interleukin-1α(IL-1α),interleukin-6(IL-6),tumor necrosis factor(TNF-α),interferon-y(IFN-γ),and vascular endothelial growth factor(VEGF)was measured using Luminex technology.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the mRNA expression of the key molecules(Notch1,Jagged1,Hes1,and Hes5)of the Notch signaling pathway.The protein expression of the key molecules(Notch1,Jagged1,Hes1,and Hes5)of Notch signaling pathway were detected by western blot.Results:Part 1:1.Behavioral tests results:In the SPT,compared with the control group,the sucrose preference decreased significantly in the LPS-4 d group(P<0.0001);After 2 days of LPS injection,sucrose preference decreased(P=0.0023).The sucrose preference was also decreased in the LPS-1 d group(P=0.0001).In the OFT,the total horizontal distance(P<0.0001)and the center distance(P=0.0008)were significantly reduced in the LPS-4 d group compared with the control group.After 2 days of LPS injection,the total horizontal distance decreased(P<0.0001),but the center distance had no statistical significance.The total horizontal distance was decreased(P=0.0018)in the LPS-1 d group,but the center distance was not statistically significant.In the rotarod test,the standing time was significantly reduced in the LPS-4 d group compared with the control group(P<0.0001).The standing time was decreased after 2 days of LPS injection(P=0.0109).The standing time was not statistically significant in the LPS-1 d group.2.MRI results:The R-fMRI results showed that,compared with the control group,the ALFF value of the LPS-4 d group in HIP was significantly lower than that of the control group(P<0.005),and the intrinsic activities in the HIP were significantly reduced in the LPS-4 d group.The DTI results showed that the FA values of the bilateral HIP,bilateral PFC,bilateral VTA,and DRN significantly decreased(F=285,P<0.0001;Left HIP,P=0.0009;Right HIP,P=0.002;Left PFC,right PFC,left VTA,right VTA and DRN,P<0.0001),the MD values increased significantly(F=147.2,P<0.0001;Left HIP,P=0.0077;Right HIP,P=0.0026;Left PFC,P=0.0009;Right PFC,P=0.0014;Left VTA,P=0.0003;Right VTA and DRN,P<0.0001).The integrity of white matter fibers was greatly destroyed within 4 days of LPS treatment.3.HE staining:HE staining results of brain tissue showed that the neurons of rats in the control group had complete structure,regular cell morphology,orderly and dense arrangement,uniform distribution of chromatin,complete nuclear membrane,and clear nucleoli.In the LPS group,the neurons were atrophied and loosely arranged,some cells showed vacuolar degeneration,nuclear shrinkage,and deep cytoplasmic staining,and some neurons were degraded and necrotic.4.Immunohistochemical results:Compared with the control group,there were no statistical differences in the expression of c-Fos,Iba-1,and TH after 1 day of LPS injection.In the LPS-2 d group,the expression of c-Fos in the PFC(P=0.0153)and SN(P=0.0316)was significantly increased,while the expression of c-Fos in the HIP was not significantly different,and the expression of Iba-1 in the HIP(P=0.0003)and SN(P=0.0142)was significantly increased.The expression of Iba-1 in the PFC was not statistically significant,and the TH expression in the SN was reduced by 23%(P=0.0062).The expression of c-Fos in PFC(P=0.0013),HIP(P=0.0069),and SN(P=0.0001)were significantly increased in the LPS-4 d group.The expression of Iba-1 in the PFC(P=0.0084),HIP(P=0.002),and SN(P=0.0311)was significantly increased,and the expression of TH in the SN was decreased by 28%(P=0.0018).Part 2:1.Behavioral tests results:In the SPT,compared with the control group,rats in the LPS group had significantly lower sucrose preference(P<0.0001);Compared with the LPS group,the sucrose preference of rats in the FXT,XJPM,and XJPH groups was significantly increased(P<0.0001),but there was no statistical significance in XJPL group.In the OFT,compared with the control group,the total horizontal distance(P<0.0001)and the center distance(P=0.0149)of rats in the LPS group were significantly reduced.Compared with the LPS group,the total horizontal distance of rats in the FXT,XJPM,and XJPH groups was significantly increased(FXT,P=0.001;XJPM,P=0.0008;XJPH,P<0.0001),the center distance of rats in the XJPM and XJPH groups were increased to different degrees(XJPM,P=0.0004;XJPH,P=0.0046),but there was no statistical significance in the XJPL group.In the rotarod test,the standing time of rats in the LPS group was significantly reduced compared with the control group(P<0.0001);Compared with the LPS group,the standing time of rats in the FXT,XJPM,and XJPH groups was significantly increased(P<0.0001),but there was no statistical significance in the XJPL group.2.MRI results:The R-fMRI results showed that compared with the LPS group,the fALFF values of olfactory bulb glomeruli,olfactory plexus,and accessory olfactory bulb glomeruli of rats in the FXT group were significantly increased,the intrinsic activities were significantly increased(P<0.005).The fALFF values of the olfactory globular layer,olfactory plexus layer,accessory olfactory globular layer,and HIP of rats in the XJPM and XJPH groups were significantly increased(P<0.005),the intrinsic activities were significantly increased.The DTI results showed that compared with the LPS group,the FA values of bilateral PFC,bilateral VTA,and DRN in rats of the FXT group were significantly increased,the MD value of bilateral PFC and DRN brain region decreased significantly.The FA values of the bilateral HIP,bilateral PFC,bilateral VTA,and DRN in the XJPM group were significantly increased,the MD values of the bilateral HIP,bilateral PFC,and DRN were significantly decreased.The FA values of the bilateral HIP,bilateral PFC,bilateral VTA,and DRN in the XJPH group were significantly increased,the MD values of the bilateral HIP,bilateral PFC,and DRN were decreased significantly.3.HE staining results:HE staining results of brain tissue showed that the neurons in the HIP region of rats in the control group had complete structure,regular cell morphology,orderly and dense arrangement,uniform chromatin distribution,complete nuclear membranes,and clear nucleoli.In the LPS group,neurons atrophied and were arranged loosely,some cells showed vacuolar degeneration,nuclear pyknosis,and deep cytoplasm staining,and some neurons degenerated and necrotic.The neuron injury in the FXT,XJPM,and XJPH groups were significantly lower than in the LPS group.4.Immunohistochemical results:Compared with the control group,the expression of c-Fos in the PFC(P=0.0164),HIP(P=0.0293),and SN(P=0.0284)of rats in the LPS group was significantly increased;the Iba-1 expression in the PFC(P<0.0001),HIP(P=0.0135),and SN(P=0.0161)were significantly increased;and the TH expression in the SN was decreased by 32.57%(P=0.0014).Compared with the LPS group,the c-Fos expression in the PFC(P=0.0064),HIP(P=0.0031),and SN(P<0.0001)in the XJPM group was significantly increased;the Iba-1 expression in the PFC(P=0.032),HIP(P=0.0229),and SN(P=0.0438)were significantly decreased;while the expression of TH in the SN was significantly increased(P=0.0006).Compared with the LPS group,the expression of c-Fos in the PFC(P<0.0001),HIP(P=0.011),and SN(P=0.0121)of rats in the XJPH group were significantly increased;the expression of Iba-1 in the PFC(P=0.0118),HIP(P=0.0211),and SN(P=0.0213)were significantly decreased;and the TH expression in the SN was also significantly increased(P=0.0244).5.The level of inflammatory cytokines:Compared with the control group,the inflammatory cytokines in the LPS group were significantly increased(IL-1α,P=0.0223;IL-6;P=0.0024;TNF-α,P=0.0191;IFN-γ,P=0.0027;VEGF,P=0.0066);Compared with the LPS group,the level of inflammatory cytokines in the FXT group was significantly decreased(IL-1α,P=0.0142;IL-6,P=0.0028;IFN-γ,P=0.0037;VEGF,P=0.01),the level of the inflammatory cytokines in the XJPM group was significantly decreased(IL-1α,P=0.0106;IL-6,P=0.0022;TNF-α,P=0.0452;IFN-γ,P=0.0021;VEGF,P=0.0066)and inflammatory cytokines in the XJPH group was also decreased(IL-1α,P=0.0167;IL-6,P=0.0033;IFN-γ,P=0.0053;VEGF,P=0.0101).6.The expression of Notch signaling pathway key molecules:Compared with the control group,the mRNA expression of and in the LPS group was significantly increased(P<0.0001),the protein expression was also significantly increased(Notch1,P=0.0166;Jagged1,P=0.005;Hes1,P=0.0033;Hes5,P=0.0018);Compared with the LPS group,the mRNA expression of Notch1,Jagged1,Hes1,and Hes5 of rats in the FXT group were significantly decreased(P<0.0001),the protein expression was significantly decreased(Notch1,P=0.0331;Jaggedl,P=0.0007;Hes1,P=0.0002;Hes5,P=0.0078);Compared with the LPS group,the gene expression of Notch1(P<0.0001),Jagged1(P<0.0001),and Hes5(P<0.0001)in the XJPM group were significantly decreased,and the protein expression of Notch1,Jagged1,Hes1,and Hes5 were significantly decreased(Notch1,P=0.0217;Jaggedl,P=0.0011;Hes1,P=0.001;Hes5,P=0.0227);Compared with the LPS group,the mRNA expression of Notch 1(P<0.0001),Jagged1(P<0.0001),and Hes5(P=0.01)in the XJPH group were significantly decreased,and the protein expression of Jagged1,Hes1,and Hes5 were significantly decreased(Jagged1,P=0.0054;Hes1,P=0.0375;Hes5,P=0.0483).Conclusion:1.LPS injection(0.5 mg/kg,i.p.)for 4 consecutive days could be used to successfully create a rat model with dPD.Our modeling method is easier to perform,takes less time,induces behavioral,histopathological,and white matter integrity changes,and simulates the pathogenetic process of dPD in this rat model with dPD.2.Xifeng-Jieyu prescription relieved LPS-induced depression-like and motor behaviors.The underlying mechanisms may involve inhibiting the mRNA and protein expression of the key molecules(Notchl,Jagged1,Hes1,and Hes5)of Notch signaling pathway,thus inhibiting the microglial activation,reducing the secretion of inflammatory factors,and alleviating the inflammatory response,reducing the damage of neurons in the brain,improving the intrinsic activities of brain regions,protecting the integrity of white matter fibers,playing anti-inflammatory and neuroprotective effects,and finally achieving the purpose of treating dPD. |
