| With its high prevalence,recurrence risk,depression is a severe psychiatric disease with a heavy health burden.Patients with major depressive disorder have elevated risk of suicide.The pathogenesis of depression itself remains unknown and there is relative lack of clinical antidepressant with safety and effectiveness.Thus,it is crucial to explore the pathophysiological mechanisms of depression and to find novel antidepressants.Patients with depression are usually detected neuroinflammation in the central nervous system(CNS).Post-mortem studies have revealed the microglia activation,and the reduced density and size and dysfunction of astrocytes in the prefrontal cortex and hippocampus of depressive patients.These results provide the information of physiopathological changes in end-stage of depression,but could not well explain the neuroinflammatory and immune inflammatory changes in early-stage or developmental process of depression.Astrocytes generate and secrete neuroinflammatory cytokine and chemokines,which regulate immune defense in CNS.Reactive astrocytes increased the burden of neuroinflammation in Alzheimer's disease,but the role astrocytes playing in depression-related neuroinflammation is still unknown and need to be explored.Chronic stress is one of the important factors to induce depression,which activates the hypothalamic-pituitary-adrenal(HPA)axis and causes sustained higher level of glucocorticoid.Previously reported by us,increased interleukin-1?(IL-1?)maturation and IL-1?-related CNS inflammation were detected in chronic prefrontal cortex(PFC)of unpredictable mild stress(CUMS)-exposed rats,with decreased expression of astrocytes marker protein GFAP.To explore the immune regulatory changes in early stage of chronic stress,stress level of corticosterone is to be used to stimulates primary astrocytes cultures obtained from Sprague-Dawley(SD)rat,with the aim to find out whether the immune-inflammatory changes characterized by IL-1? maturation happened in astrocytes and what is the possible mechanism underlying.Based on these,furthermore evaluation is to be done for the intervene of traditional Chinese medicine components,to find antidepressants with noval pathway.Physical concentrations of human serum cortisol vary from 137 to 283 nanomolar per liter(nM),and free hippocampal corticosterone concentrations by microdialysis in rat brain are dozens to hundreds nM,for example 27 ng/mL(about 80 nM),under stress conditions.Therefore,100 nM corticosterone stimulation astrocytes for 24 h,48 h,72 h are applied in the present study,and the mRNA and protein expression levels of pro-inflammatory cytokines IL-1? in primary astrocyte cultures of SD rat.Compared with normal group,the mRNA and protein levels of total IL-1? were unchanged,while the maturation of IL-1? was significantly increased compared to their corresponding control groups(p<0.05).And the protein levels of NOD-like receptor pyrin domain containing 3(NLRP-3)(p<0.01)and mature Caspase-1(p<0.001)were significantly increased,suggesting corticosterone exposure inducing NLRP3 inflammasome activation and IL-1? maturation.Our previous work confirmed thioredoxin-interacting protein(TXNIP)was involved in the promotion of NLRP3 inflammasome activation and IL-1? maturation.We have analyzed protein expression following 100 nM corticosterone treatment of SD rat primary astrocyte cultures.TXNIP was suggested to be an endogenous inhibitor of thioredoxin(TRX),an important regulator for cellular redox homeostasis.The high expression of TXNIP inhibit activity of TRX-1,causing the accumulation of ROS,activating NLRP3inflammasome and cleavage IL-1? to mature.The present study showed that 100 nM corticosterone exposure for 72 h significantly decreased the expression of TRX-1(p<0.001)and increased the ROS levels(p<0.001)in primary astrocyte cultures of rat.Glucocorticoid is a potential transcriptional regulator for a set of mRNAs,including TXNIP.We have characterized that 100 nM corticosterone treatment significantly increased TXNIP mRNA levels(p<0.001),twice of normal control group,in primary astrocyte cultures of SD rat.These results support the hypothesis that stress level corticosterone exposure induce gene and protein expression of TXNIP in astrocytes,downregulated TRX-1 protein and accumulate ROS,resulting in immuno-inflammatory changes in astrocytes characterized by NLRP3 inflammasome activation and IL-1? maturation.Further studies in mouse C 8-D1A astrocytes were done.And the similar results were observed that the expression level of total IL-1? was unchanged,the mature IL-1? was significantly increased compared to their corresponding control groups(p<0.001),the protein levels of NLRP3(p<0.01),mature Caspase-1(p<0.001)were significantly increased,TXNIP protein was highly expressed(p<0.01),the TRX-1 was significantly decreased(p<0.01),the ROS levels was significantly increased(p<0.001),TXNIP mRNA was highly expressed(p<0.001).These results in mouse C8-D1A astrocytes were highly consistent with that obtained in primary astrocytes culture of SD rat,supporting stress level corticosterone increased the TXNIP expression,decreased the TRX-1 protein levels,accumulated the ROS and resulted in astrocytic immuno-inflammatory changes characterized by NLRP3 inflammasome activation and IL-1? maturation.The in vivo study was done in C57BL/6 mice.Corticosterone stimulated mice were injected corticosterone at 20 mg/kg/d(s.c.),while antidepressant treated mice were injected fluoxetine hydrochloride at 10 mg/kg/d(i.g.)for 21 days.The TXNIP protein levels was significantly higher(p<0.001)in mice cortex of corticosterone groups and TRX-1 was significantly decreased(p<0.01),and the protein of NLRP3(p<0.001),Caspase-1(p<0.001)and mature IL-1?(p<0.01)were significantly increased.And these changes could be reversed by the antidepressant fluoxetine,as the TXNIP protein levels significantly decreased(p<0.001),TRX-1 protein increased(p<0.001)and NLRP3 inflammasome activation and IL-1? maturation decreased(p<0.01).Active astrocytes can generate and secrete neuroinflammatory cytokine and chemokines.Long-term corticosterone exposure may decrease the expression of GFAP,the marker of astrocytes.We found that the expression of GFAP levels were increased by corticosterone in primary astrocyte cultures of SD rat,suggesting activation of astrocytes.Emerging evidence suggests that the end product of glycolysis lactate of astrocytes can transport to neurons for energy support and TXNIP is one of the negative regulator of glucose metabolism.Is the glycolysis influenced by stress or glucocorticoid?We determined the extracellular levels of lactate and intracellular mRNA levels of 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase-3(PFKFB3),a key enzyme of glycolysis,in corticosterone-exposured primary astrocyte cultures of SD rat.The results showed that 100 nM corticosterone exposure for 72 h significantly decreased the extracellular lactate(p<0.01)and the intracellular PFKFB3 mRNA levels(p<0.01)in primary rat astrocytes,indicating stress level corticosterone exposure induced glycolysis disturbances in astrocytes.Epimedium flavonoids,stilbene compounds and curcumin show the property of anti-inflammatory,antioxidant effects and neuroprotective potential.It is unknown whether these traditional Chinese medicine compounds could reverse the astrocytic changes induced by stress level corticosterone.Thus,we screened the intervention activities of these components of(Resveratrol,Pterostilbene,Curcumin,Polydatin,Baohuoside I,Icariin,Icaritin,EpimedinA,Epimedin B,Epimedin C,Mulberroside A)in the corticosterone treated astrocytes models with immuno-inflammatory changes characterized by NLRP3 inflammasome activation and IL-1? maturation.As we found,the Icariin(2.5,5,10 and 20 ?M)and Baohuoside I(2.5,5,10 and 20 ?M)reversed the mRNA levels of TXNIP in model astrocytes with excellent dose-response.TXNIP protein expression levels were significantly decreased,TRX-1 protein expression levels were increased,ROS levels were decreased,NLRP3 inflammasome activation and IL-1? maturation were suppressed by Icariin(5,10 and 20 ?M)and Baohuoside I(5,10 and 20 ?M)treatments.Our data revealed that Resveratrol(10,20 and 40 ?M),Pterostilbene(2.5,5 and 10 ?M),Mulberroside A(5,10 and 20 ?M)significantly decreased ROS levels and reversed the immune-inflammatory changes induced by corticosterone,although showing non-significant effect on TXNIP mRNA.Our results indicated that these traditional Chinese medicine components could reverse corticosterone-induced astrocytic immuno-inflarmmatory changes characterized by NLRP3 inflammasome activation and IL-1? maturation through different pathways like the TXNIP mRNA and protein inhibition by Icariin and Baohuoside I and the ROS elimination by Resveratrol,Pterostilbene,and Mulberroside A.Thus,these components may be astrocytes protective against stress-induced immuno-inflammatory changes.Summary:The stress level corticosterone increased IL-1? maturation by activated NLRP3 inflammasome,causing immune-inflammatory changes in primary astrocyte cultures of SD rat and C8-D1A mouse astrocytes.In consistent with these,activated NLRP3 inflammasome and mature IL-1? were observed in mice cortex after chronic corticosterone exposure.Corticosterone exposure decreased extracellular lactate and PFKFB3 mRNA levels of astrocytes,inducing astrocytic activation marker expression and glycolysis disturbance.In addition,we evaluated and found the reverse activities of Icariin and Baohuoside I through TXNIP inhibition pathway and of Resveratrol,Pterostilbene,Mulberroside A through ROS elimination pathway.These results indicate novel pathological mechanism of stress induced astrocytic immuno-inflammatory changes and provide experiment basis for new antidepressants discovery by mechanism of reverse of astrocytes immune-inflammatory changes. |
PDF Full Text Request