Study On Liver-regulating Formula Inhibiting Stress Injury Based On Promoting Recruitment Of CD4~+ T Lymphocytes To The Choroid Plexus

ObjectiveThe aim of this study is to investigate the effects of liver-regulating formula JWSNS on stress resilience by establishing a chronic unpredictable mild stress(CUMS)rat model from the perspective of choroid plexus(CP)CD4+T-lymphocyte recruitment and transport.Methods1.CUMS model establishingThe study was conducted on 100 Wistar male adult rats of 180-220g.The rats were moulded in CUMS for 8 weeks combined with solitary housing,except for the Control group,which was housed in 4 rats/cage.Stressors included white noise(85 dB,5 hours),food deprivation(12 hours),water deprivation(12 hours),stroboscopic illumination(300 flashes/minute,5 h),plantar electric shock(1 mA,2 s/time,every 30 s interval,10 electric shocks in total),soiled cage(10 h),continuous lighting at night(20:00-8:00 the next day,12 h),restraint(12 h),paired with four other stressed animals(10 h),food and water deprivation(24 hours),thermal swimming(45℃,5 min),1-2 stressors are randomly applied daily and the same stressors are not repeated for 3 days.2.Drug interventionsAll animals were randomly divided into four groups based on scurose preference:Control group,CUMS group,JWSNS group,and Quercetin group.Gavage was given to each group of rats from the day the stress was applied for 8 weeks and was terminated at the same time when the stress was over.The rats in the JWSNS group were given twice daily by gavage at a dose of 16.9 g·kg·d-1(twice the equivalent adult dose of 60 kg body weight)and the Quercetin group was given twice daily by gavage at the following dose:Quercetin 50mg·kg·d-1.The Control and CUMS groups were given equal amounts of saline gavage(4mL·kg-1 body weight)at fixed time points each day.In addition,the stressed rats were divided into resilient and susceptible groups according to behavioural Z-normalisation by behavioural observations.3.Indicator testing(1)Behavioural observations and analysis of stress resilienceAfter modelling,the rats were tested for pleasure deficit in the sugar water preference test,despair in the forced swim test,anxiety in the elevated cross maze test,and social competence in the three-box social test.Z-normalisation of the behaviour was carried out to determine the resilience and susceptibility of the rats.(2)A study of CD4+T lymphocyte recruitment to the choroid plexus(CP)Flow cytometry was used to detect the number of CD4+ T lymphocytes in CP tissues between groups;immunofluorescence and Western-blot were used to detect the expression of CD4+ T in CP tissues,and the above indicators were analysed for resilient ity and susceptibility.The correlation between behavioural and CD4 fluorescence intensity was assessed by correlation analysis.(3)CP tight junctions and the expression of chemokines and adhesion moleculesThe expression of tight junction scaffold proteins zonula occludens-1(ZO-1),C-C chemokine ligand 2(CCL2),C-C chemokine ligand 5(CCL5),C-C chemokine ligand 11(CCL11),Chemokine(C-X-C Motif)Ligand 9(CXCL9),Chemokine(C-X-C Motif)Ligand 10(CXCL10),intercellular cell adhesion molecule-1(ICAM-1),and Vascular cell adhesion Protein 1(VCAM-1)in CP tissues was measured by immunofluorescence.Western-blot was used to detect the expression of CCL5,CCL1 and ICAM1 proteins in CP tissues,and the above indicators were analysed for resilient ity and susceptibility.The correlation between CD4 fluorescence intensity and chemokines and adhesion molecules was observed by correlation analysis.(4)Cytokine assays in peripheral,CP,cerebrospinal fluid,hippocampusThe levels of interleukin-1(IL-1 β),interleukin-6(IL-6),interleukin-4(IL-4)and interleukin-10(IL-10)in plasma,cerebrospinal fluid(CSF),CP and hippocampal tissues were measured by ELISA in all groups of rats,and the above indicators were analysed for resilient ity and susceptibility.The correlation between CD4 fluorescence intensity and cytokines was observed by correlation analysis.(5)Polarization of hippocampal microgliaThe polarization of microglia in the DG,CA1 and CA3 regions of the hippocampus was detected by immunofluorescence.The expression of ionized calcium binding adapter molecule 1(iba-1),inducible nitric oxide synthase(iNOS)and Arginase-1 protein was examined by western blot,and the above indicators were analysed for resilient ity and susceptibility.Correlation analysis was performed to observe the correlation between iba-1,iNOS and Arginase-1 protein expression and CD4 protein expression and cytokines.(6)Culture of HAPI microglia cell lines and analysis of the effect of CSF on HAPI microglia in each group of ratsThe toxicity of different concentrations of lipopolysaccharide(LPS)and CSF on HAPI microglia was measured by CCK-8.HAPI microglia were stimulated with LPS and treated with CSF and divided into Control,LPS,LPS+Con-CSF,LPS+CUMS-CSF,LPS+JWSNS-CSF groups.The survival rate of HAPI microglia in each group was detected by CCK-8,and the expression of iba-1,iNOS and Arginase-1 in HAPI microglia was detected by immunofluorescence.Results1.By behavioural Z-normalisation analysis,the results showed that the proportion of stress resilient rats was significantly higher in the JWSNS group(22/24)compared to the CUMS group(7/24).Significant anxiety and depression-like symptoms(P<0.01,P<0.05),as well as social impairment(P<0.01),were observed in the susceptible rats compared to the resilient rats.CUMS rats showed significant depressive symptoms such as pleasure deficit(P<0.01)and despair(P<0.01),as well as anxiety-like emotions(P<0.01),accompanied by a decrease in social skills(P<0.01),and treatment with JWSNS effectively alleviated these abnormal behaviours(P<0.05,P<0.05,P<0.01,P<0.01)in stress rats.2.CD4+ T lymphocyte recruitment was reduced in CP of susceptible rats compared to resilient rats(P<0.05).JWSNS significantly promoted CD4+T lymphocyte recruitment to CP in CUMS rats(P<0.01).Correlation analysis showed that CD4 expression correlated with the percentage of elevated open arm entries(r=0.779,P<0.01),percentage of time(r=0.753,P<0.01),percentage of distance travelled(r=0.644,P<0.01),and social time(r=0.755,P<0.01),but not with sugar depletion(r=0.432,P=0.057).3.Compared with resilient rats,susceptible rats decreased the expression of(P<0.05),CCL11(P<0.01),CXCL10(P<0.01)and ICAM-1(P<0.01)in CP JWSNS effectively maintained the tight junctions of CP in CUMS rats and increased the expression of ZO-1(P=0.028<0.05),increased the expression of CP chemokines CCL5(P<0.01).CCL11(P<0.05),CXCL10(P<0.01)and adhesion molecule ICAM-1(P<0.01)in CUMS rats.Correlation analysis showed that the expression of CD4 correlated with ZO-1(r=0.54,P<0.05),CCL5(r=0.659,P<0.01),CCL11(r=0.621,P<0.01),CXCL10(r=0.659,P<0.01)and ICAM-1(r=0.632,P<0.01).4.Compared to the resilient rats,the susceptible rats had significantly higher CP,CSF and hippocampal pro-inflammatory cytokines IL-1β and IL-6(P<0.05,P<0.05,P<0.05,P<0.05,P<0.05,P<0.05)and significantly lower IL-10(P<0.01,P<0.05,P<0.05).No statistically significant differences in plasma IL-1β,IL-6,IL-4 and IL-10(P=0.067-1.000)between the groups compared to the CUMS group.Compared with the Control group,CP,CSF and hippocampal pro-inflammatory cytokines IL-1β and IL-6(P<0.01,P<0.05,P<0.05,P<0.01,P<0.05,P<0.01)were significantly higher,and IL-10 in CSF was significantly lower in the CUMS group(P<0.01)in CSF.Compared with the CUMS group,JWSNS significantly decreased pro-inflammatory cytokines IL-1β(P<0.01,P<0.05)and IL-6(P<0.05,P<0.01)in CP and hippocampus;decreased pro-inflammatory cytokine IL-1β in CSF(P<0.05)and increased anti-inflammatory cytokine IL-10 in CSF(P<0.01).No significant difference in plasma IL-1 β(F=2.444,P=0.122)between the groups.The correlation analysis showed that IL-1β in CP was positively correlated with IL-1β in CSF(r=0.603,P<0.01).IL-6 in CP was positively correlated with IL-6 in CSF(r=0.454,P<0.05)IL-4 in CP was positively correlated with IL-4 in CSF(r-=0.550,P<0.01);IL-6 in CSF was positively correlated with IL-6 in hippocampus(r=0.714,P<0.01).CD4 protein expression was negatively correlated with IL-6 in CP(r=-0.475,P<0.05)and positively correlated with IL-10 in CP(r=0.493,P<0.05).5.The number of positive cells in DG region,CA1 region,CA3 region iba-1(P<0.01.P<0.01,P<0.01)and iNOS(P<0.01,P<0.05,P<0.05)was significantly increased in susceptible rats compared with resilient rats.JWSNS effectively inhibited the abnormal activation of microglia in the DG,CA1 and CA3 regions of the hippocampus of CUMS rats,reduced the number of M1 phenotypes and decreased the expression of iba-1(P<0.01,P<0.01,P<0.01)and iNOS(P<0.01,P<0.01,P<0.01).Correlation analysis showed that iba-1 and iNOS protein expression were positively correlated with hippocampal IL-1 content(r=0.635,P<0.01,r=0.690,P<0.01),and with hippocampal IL-6(r= 0.643,P<0.01,r=0.693,P<0.01),and negatively correlated with CD4 protein expression(r=-0.564,P<0.01,r=-0.564,P<0.01).6.There was no significant difference in the survival rate of HAPI microglia after 24 and 36 h(P=0.058-0.938)of LPS stimulation at different concentrations(1ng·ml·-1-100ug·ml-1).Both 5%and 10%CSF could inhibited the proliferation of microglia(P<0.05);immune-fluorescence showed a significant decrease in iba-1(P<0.05,P<0.05,P<0.01),and iNOS(P<0.01,P<0.05,P<0.01)expression in the LPS+5%JWSNS-CSF group compared to the LPS+5%CUMS-CSF group.Conclusion1.Susceptible rats showed marked depression-like and anxiety-like symptoms and social impairment,with reduced recruitment of CP to CD4.JWSNS effectively improves depression-like and anxiety-like behavior,enhances socialization,promotes stress resilience ratio and increases CD4+T lymphocyte recruitment to CP in CUMS rats.2.Reduced expression of chemokines and adhesion molecules,increased levels of pro-inflammatory cytokines and reduced levels of anti-inflammatory cytokines in susceptible rats.JWSNS increased the expression of chemokines,adhesion molecules and altered cytokine levels in CUMS rats,this alteration correlated with CD4 protein expression.3.M1-type microglia expression was significantly elevated in susceptible rats,suggesting that polarization of microglia affects stress susceptibility.JWSNS effectively reduced hippocampal microglia density and inhibited Ml-type microglia polarization;CSF in the JWSNS group of rats effectively alleviated the abnormal activation of HAPI microglia in vitro,which may be related to altered CSF cytokine levels.