Exploration Of The Anti-depressant Mechanisms Of Jiaweisinisan Based On Hippocampal Endoplasmic Reticulum Stress

ObjectiveBy establishing a rat model of depression,this study aimed to investigate the anti-depressant mechanisms of Jiaweisinisan based on cerebrospinal fluid proteomics from the perspective of hippocampal endoplasmic reticulum stress.Meanwhile,the possibility of glucose-regulated protein 94(GRP94)content in cerebrospinal fluid(CSF)as a potential biomarker of depression and the intervention effects of JWSNS were also investigated.This study will provide some theoretical basis for the application of liver-regulating treatment formulas to prevent and treat psychiatric disorders.Methods1.Establishment of depression model in ratsThis study proposes to establish a rat depression model by adopting the chronic unpredictable mild stress(CUMS)method.Except for the CON group,the three groups of rats were exposed to various mild stressors and combined with isolated rearing.The stressors included fasting(24 h),water deprivation(24 h),continuous light(12 h),foot shock(1 mA,2 s,10 times),white noise(5 h),stroboscopic illumination(5 h),thermal swimming(45℃,5 min),ice water swimming(4℃,5 min),restraint(12 h),humidified cage(10 h),and housing with another four rats(10 h).Stressors were given randomly one to two daily for 8 weeks without the same stressor for 3 days.2.Effects of JWSNS on depressive symptoms and cognition in CUMS ratsSixty-four young male Wistar rats were randomly and equally divided into normal control group(CON),depression model group(CUMS),JWSNS medication group(JWSNS)and quercetin medication group(QUE).Four groups of rats were administered gavage twice daily for 8 weeks from the time of modeling.Rats in the JWSNS group were given a gavage dose of 16.92 g/kg,rats in the QUE group were given a gavage dose of 50 mg/kg,and rats in the CON and CUMS groups were given equal doses of saline by gavage.After modeling,the rats were tested for depressive symptoms and cognitive function by several behavioral approaches.3.Source analysis of the elevated GRP94 protein in the CSF proteomics of CUMS ratsCerebrospinal fluid samples from CUMS rats were analyzed by tandem mass tag(TMT)labeling quantitative proteomics technique.Proteins with a fold change of more than 1.2-fold(up-regulation greater than 1.2-fold or down-regulation less than 0.83-fold)and p-values less than 0.05 were screened as differentially expressed proteins.Parallel reaction monitoring(PRM)was used for quantitative analysis of target proteins and target peptides.ELISA was performed to detect GRP94 levels in rat cerebrospinal fluid,plasma,choroid plexus,prefrontal cortex,and hippocampus to determine the source of elevated GRP94 protein in cerebrospinal fluid proteomics.Pearson correlation analysis was used to calculate the correlation between GRP94 levels in CSF and behavioral data.Pearson correlation analysis was employed to calculate the correlation between GRP94 content in hippocampus and behavioral data.4.Hippocampal endoplasmic reticulum stress and cell apoptosis in CUMS ratsImmunofluorescence(IF)and Western-blot(WB)were used to detect the expression of chaperone proteins GRP94 and GRP78 in the hippocampus of CUMS rats.IF and WB were performed to detect protein expression on the PERK pathway in rat hippocampus,including p-PERK,EIF2α,p-EIF2α,ATF4,and CHOP.The protein expression of ATF6 and p-IRE1 in rat hippocampus were detected by IF and WB.Cell apoptosis of rat hippocampus was examined by TUNEL method.Molecular docking was used to verify the affinity and binding activity of quercetin(an important active ingredient of JWSNS)with GRP94 and four related proteins on the PERK pathway(PERK,EIF2α,p-EIF2α,and CHOP).5.Establishment of endoplasmic reticulum stress model by localized injection of tunicamycin(TM)in rat hippocampusForty young male Wistar rats were randomly and equally divided into four groups,namely,the control group(Control),low-dose TM group(TM-L),medium-dose TM group(TM-M),and high-dose TM group(TM-H).The three TM model groups received 5 ul of low,medium and high doses of tunicamycin(25 μM,50 μM,75 μM)by hippocampal localization injection,and the Control group received equal doses of DMSO solvent by hippocampal localization injection.Forty-eight hours after localization injection,CSF was collected from each group of rats.ELISA was used to detect GRP94 content in plasma,hippocampus and cerebrospinal fluid of all rats to determine the optimal concentration of tunicamycin injection.6.Behaviors,endoplasmic reticulum stress and cell apoptosis in hippocampus in TM-M injected model ratsSixty young male Wistar rats were randomly and equally divided into four groups,namely the control group(Control),the medium-dose TM group(TM-M),JWSNS medication group(JWSNS)and quercetin medication group(QUE).Rats in the Control group were localized and injected with 5 ul DMSO solvent in the hippocampus,while the remaining three groups were localized and injected with an equal amount of 50μM tunicamycin in the hippocampus.All rats received advanced gavage twice daily for 2 weeks prior to hippocampal stereotaxic injection.Rats in the JWSNS group were given a gavage dose of 16.92 g/kg,rats in the QUE group were given a gavage dose of 50 mg/kg,and rats in the Control and TM-M groups were given equal doses of saline by gavage.Forty-eight hours after positional injection,depressive symptoms and cognitive functions of rats were examined by several behavioral techniques.ELISA was used to detect GRP94 content in plasma,hippocampus and cerebrospinal fluid of rats in each group.The protein expression of GRP94 and p-EIF2α in rat hippocampus were detected by immunofluorescence method.The cell apoptosis of rat hippocampus was detected by TUNEL method.Results1.After 8 weeks of modeling,rats in the CUMS group showed significant depression-like behavior,anxiety-like behavior,and cognitive dysfunction.The main manifestations were a decrease in sucrose preference,an increase in immobility time,a reduction in the time ratio of open arms,and a decline in the number of platform crossings.After JWSNS treatment,the above behaviors were significantly improved in CUMS rats.2.In CSF proteomics data,GRP94 protein and EIF2α protein levels were elevated in the CUMS group of rats compared with the CON group.Both GRP94 and EIF2α protein levels were reduced in the CSF of JWSNS group compared with the CUMS group.The quantitative results of PRM were consistent with the TMT results.ELISA results showed that GRP94 content in CSF of CUMS rats was significantly higher compared with the CON group.The GRP94 content in CSF of JWSNS rats was significantly lower compared with the CUMS group.There were no significant differences of GRP94 content in plasma between CON,CUMS and JWSNS groups.However,GRP94 content was significantly higher in the hippocampus of CUMS rats compared with the CON group.The GRP94 content in the hippocampus of JWSNS rats was significantly decreased compared with the CUMS group.The correlation analysis revealed a significantly positive correlation of GRP94 protein content in CSF and hippocampus.There was no significant correlation between the GRP94 content in CSF or hippocampus and the sucrose preference of rats.There was no significant correlation between the GRP94 content in CSF or hippocampus and the immobility time of forced swimming test in rats.There was a remarkable correlation between the GRP94 content in CSF or hippocampus and the results of elevated plus maze test in rats.There was a significant correlation between the GRP94 content in CSF or hippocampus and the results of social interaction test in rats.There was a statistically significant correlation between GRP94 content in CSF or hippocampus and the results of Morris water maze test in rats.3.IF and WB results indicated that GRP94 and GRP78 protein expression in hippocampus were significantly increased in CUMS rats compared with the CON group.However,JWSNS could significantly reduce GRP94 and GRP78 protein expression in hippocampus compared with the CUMS group.Compared with the CON group,the protein expression of p-PERK and p-EIF2α in hippocampus of CUMS rats were significantly enhanced.While the protein expression of p-PERK and p-EIF2α in hippocampus of JWSNS rats were significantly reduced compared with the CUMS group.There were no significant differences in the protein expression of EIF2α and ATF4 in hippocampus between CON,CUMS and JWSNS groups.Compared with the CON group,CHOP protein expression in hippocampus of CUMS rats was significantly higher.Moreover,CHOP protein expression in hippocampus of JWSNS rats was significantly lower than that of CUMS group.No significant differences were found for ATF6 and p-IRE1 protein expression in hippocampus of CON,CUMS and JWSNS rats.Compared with the CON group,cell apoptosis was significantly increased in DG and CA3 regions of hippocampus of CUMS rats.Compared with the CUMS group,cell apoptosis in DG and CA3 regions of hippocampus was significantly decreased in JWSNS group.Molecular docking results revealed that quercetin(an important active ingredient of JWSNS)showed strong affinity and binding activity to GRP94 and four related proteins on the PERK pathway(PERK,EIF2α,p-EIF2α,and CHOP).4.Forty-eight hours after the localized hippocampal injection of low-dose tunicamycin,there were no statistical differences in GRP94 protein levels in plasma,hippocampus,and CSF.Forty-eight hours after the medium-dose tunicamycin injection,GRP94 content in the hippocampus and CSF were significantly higher in the TM group compared with the Control group.Forty-eight hours after the high-dose tunicamycin injection,hippocampal GRP94 content was significantly higher in the TM group compared with the Control group,and there was no significant difference in GRP94 content in CSF and plasma.Forty-eight hours after the medium-dose TM injection,GRP94 content in hippocampus and CSF in the JWSNS group decreased dramatically compared with the TM-M group,and the GRP94 content in plasma did not show significant changes.Eight days after medium/high-dose TM injection,hippocampal GRP94 levels were significantly elevated,and there were no significant differences for GRP94 levels in CSF and plasma.5.Forty-eight hours after the hippocampal localized injection of medium-dose tunicamycin,TM-M rats showed significant anxiety-like behavior,social impairment and cognitive dysfunction.The main symptoms included decreases in the entry frequency ratio of open arms,the time ratio of left chamber,and the time ratio of platform-located quadrant,etc.JWSNS treatment resulted in significant remission of the above behaviors.Immunofluorescence results showed that the protein expression of GRP94 and p-EIF2α in DG and CA1 areas of hippocampus were significantly increased in TM-M group compared with the Control group.Meanwhile,the protein expression of GRP94 and p-EIF2α in DG and CA1 areas of hippocampus was significantly decreased in JWSNS group compared with TM-M group.Compared with the Control group,cell apoptosis was significantly enhanced in the hippocampal CA1 area of TM-M group,and there was no significant variation of cell apoptosis in the DG area.Compared with TM-M group,cell apoptosis in CA1 area of hippocampus was significantly reduced in JWSNS group,and cell apoptosis in DG area was not significantly changed.Conclusions1.The CUMS rats exhibited depression,anxiety and cognitive impairment,which are in line with the pathological mechanism of "liver dysfunction" in Chinese medicine.The liver-regulating formula JWSNS could effectively alleviate depression-like behaviors and cognitive impairment in CUMS rats.2.Endoplasmic reticulum stress occurred in the hippocampus of CUMS rats,which was accompanied by activation of the PERK pathway.Persistent and excessive endoplasmic reticulum stress may lead to increased cell apoptosis in hippocampus.JWSNS could alleviate endoplasmic reticulum stress and reduce cell apoptosis in hippocampus,thereby improving depressed emotion and cognitive function in CUMS rats.3.Elevated GRP94 levels in cerebrospinal fluid may be a potential biomarker of depression.JWSNS can reduce GRP94 levels in cerebrospinal fluid by alleviating the hippocampal endoplasmic reticulum stress response.Therefore,reduced GRP94 levels in cerebrospinal fluid may be a potential biomarker for the antidepressant effect of JWSNS.