Zinc Inhibits LPS-induced Inflammation Response And Its Mechanism In Microglial Cells

BackgroundDepression is a mental emorional disorder caused by many factors,the most important feature is the change of emotion and cognitive function.In recent years,with the rapid development of society,the modern people might be confronted with more and more pressure from the external environment,and under pressure for a long time,will inevitably lead to a series of physical and mental diseases.And frequent suicide cases for depression,make people have to pay more attention to depression.Although depression research for decades,for the pathogenesis of depression are not fully elucidated.But at the moment the proposed hypothesis to the pathogenesis of depression,the inflammation hypothesis of research is increasingly attract the attention of scholars.Clinical studies have found that inflammatory factor levels elevated in patients with depression,and after antidepressant treatment inflammation factor can be restored to normal levels.In addition to treat cancer patients with inflammatory factors found in patients with symptoms of depression,and when stop using the inflammatory factors the symptoms can disappear.Furthermore animal experiments found that anti-inflammatory drugs could be significantly reduced depression behavior induced by Lipopolysaccharides?LPS?,alse can improve cognitive dysfunction caused by depression.The nerve inflammation hypothesis of depression of microglial activation plays a key role in the development of depression.Microglia,as phagocytes in the nervous system,activated can release a large number of inflammatory cytokines,then stimulates neurons,causes a decline in the regeneration of neurons,neurons apoptosis.Glial cells play a key role in maintaining normal brain function,and microglia are closely related to animal behavior.Bacterial infections are associated with a series of depressive symptoms that induce microglial activation and thereby induce secretion of inflammatory factors.In LPS-induced depression models,microglial activation can be blocked by tricyclic antidepressants?TCAs?or selective serotonin reuptake inhibitors?SSRIs?.In addition LPS-induced depression-like symptoms can be achieved by using Microglial activation inhibitor minocycline treatment weakened.Activation of indoleamine 2,3-dioxygenase?IDO?in microglia is essential for the development of depression-like symptoms induced by LPS and other immune stimuli and activation of microglia.And mice with microglial hyperresponsiveness appeared to aggravate LPS-induced depression-like symptoms.This shows that microglia is an important target for the study of neuroinflammation in depression.Zinc is the second most abundant trace element in the human body.It is involved in the formation of multiple structural proteins,regulatory proteins,and enzymatic proteins,and plays an integral role in the regulation of gene expression.Compared with other organs of the human body,the concentration of zinc in the brain is the highest,and the zinc content is highest in the hippocampus and prefrontal cortex.In mammals,appropriate zinc levels are essential for the development of the central nervous system and the differentiation of neural stem cells.In cortical plasticity,zinc regulation plays an important role in brain development,learning and memory processes.Therefore,precise regulation of zinc homeostasis is crucial for the central nervous system and the entire organism.Current studies have found that zinc homeostasis is associated with some neurodegenerative processes,such as Alzheimer's disease,depression,and aging-related loss of cognitive function.Clinical studies have found that serum zinc levels are lower in patients with depression,and zinc levels are inversely related to the severity of depressive symptoms;after successful antidepressant treatment,serum zinc levels may return to normal levels,and depressive symptoms may also be effective improve.Our previous studies have found that stress can make MT,zinc transporter ZnT and ZIP expression change in the stress model,thus reducing the zinc content in the hippocampus.Similarly,in the model of stress-induced depression-like behavior in mice,serum zinc levels were lower in mice with depression-like behaviors.Depression-like behavior of mice after zinc supplementation can be effectively improved,and BDNF,CREB,etc.can also be increased.Animal studies have shown that zinc-deficient rodents can cause depressive-like behavior in rodents,and depression-like behavior can be improved after zinc supplementation.In addition,zinc also has anti-inflammatory and antioxidant effects.Numerous studies have shown that zinc deficiency increases the concentration of inflammatory cytokines and oxidative stress and induces apoptosis and endothelial cell dysfunction,while zinc supplementation can reduce oxidative stress,TNF-alpha and other interleukin pro-inflammatory cytokines.So the regulation of zinc by inflammation is also crucial.zinc finger protein A20?also called tumor necrosis factor alpha-induced protein 3,TNFAIP3?is a kind of C₄ type zinc finger protein.It is the main function of anti-inflammatory and antiapoptotic effect,and zinc finger protein of A20 could play a regulatory function mainly depends on the presence or absence of zinc ions.Population studies have found that depression patients serum zinc levels decline.At the same time documents have also reported lower levels of A20 expression of depression patients,and its expression level increased after antidepressant treatment.After taking SSRI of patients with major depressive disorder A20 expression level significantly increases,and scholars also found that antidepressants fluoxetine can induce the expression of A20 in glial cells.The above studies indicate that A20 may be related to the development of depression,but the specific mechanism remains unclear.In summary,stress-induced imbalance in zinc homeostasis plays an considerable role in the development of depression,and zinc homeostasis also affects the normal function of A20.As a negative feedback regulating factor of inflammation,A20 also has an important role in regulation of inflammation.Therefore,considering the hypothesis of inflammation in depression,explore the links between zinc,A20 and inflammation,and how zinc regulates involvement of A20 in inflammation,thereby providing an experimental basis for the inflammation hypothesis of depression,and new solutions for the treatment of depression.ObjectiveIn this study,LPS induced the establishment of mouse microglial cells?BV2 cells?inflammation model,observed the inhibitory effect of zinc on LPS-induced inflammatory response of BV2 cells and the protection of hippocampal neurons HT-22 activity.And preliminary study of zinc inhibition of inflammatory response mechanism.The research results will help to further improve the mechanism of zinc inhibition of neuroinflammation and provide a new experimental basis for the inflammation hypothesis of depression.Experimental Methods1.Cell cultureBV2 cells,a mouse glial cell line;HT-22 cells,a mouse hippocampal neuronal cell line,were kindly donated by Dr.David Schubert at Salk Institute in Canada.BV2 cells and HT-22 cells were cultured in DMEM high-glucose medium containing 10%fetal bovine serum and 1%double antibody?penicillin/streptomycin?,in a 5%CO₂ humidified atmosphere at 37°C.2.Cell intervention?1?BV2 cells were treated with 100 ng/ml LPS for a time curves?0h,1h,3h,6h,12h,24h?.?2?BV2 cells were treated with different concentrations of gradient zinc sulfate?10?M,15?M,30?M,50?M?for 24h,and BV2 cells were treated with 10?M and 30?M concentrations of zinc sulfate for time curves?0h,1h,3h,6h,12h,24h?respectively.And then collect cells for later use.?3?Treated with 10?M and 30?M zinc sulfate for 1h,then treated with 100 ng/ml LPS for different time?1h,3h,6h?.The experiment was divided into control group,LPS group,10Zn+LPS group,30Zn+LPS group,10Zn group and 30Zn group.3.Co-cultureAfter 10?M and 30?M zinc sulfate was pretreated for 1h,BV2 cells were cultured with 100 ng/ml LPS for 3h,and the supernatant was then used to culture HT-22 cells for24h.4.Cell transfecationWhen the degree of fusion was 80%,BV2 cells were seeded on a multiwell plate and transfected with a ratio of transfection reagent to interfering RNA of 2:1.The A20/TNFAIP3 small interfering RNA and the transfection reagent lipofctamine 3000 were diluted with Opti-MEM serum-free medium,then mixed and incubated at room temperature for 15 min.Each well was pre-incorporated with a medium containing 10%FBS,and an appropriate amount of the above mixture was added according to the specifications of the culture plate.After 24 hours and 36 hours of culture,follow-up experiments were performed and cells was collected for detection.5.Cell viability assayThe activity of BV2 cells and HT-22 cells were detected by CCK-8 kit.6.ROS levels analysisROS assay kit was used to determine ROS levels in BV2 cells.7.Western Blotting analysisThe total protein of BV2 cells was extracted with RIPA lysate and the nuclear and cytoplasmic proteins of BV2 cells were extracted with the nuclear protein cytoplasmic protein extraction kit,followed by heat denaturation for 10 min.SDS polyacrylamide gel electrophoresis 2h,transfer membrane 100min,5%milk closed 2h,primary antibody incubation overnight,rinse 3 times,each time 10min,fluorescent secondary antibody incubation 1h,rinse 3 times,each time 10min,With the use of ImageJ image analysis software gray value analysis.8.Real Time PCRTrizol extracts total RNA from BV2 cells and quantifies the concentration using a microplate reader.The prepared RNA reverse transcription system is then placed on an ordinary PCR machine to obtain cDNA and diluted.Using cDNA as a template,it was mixed with primers,2×SYBR green and DEPC water and then amplified on a StepOne Plus real-time PCR machine.According to the Ct value,take 18S as the internal reference,after the standardization it carries on the statistical analysis.9.ELISA DetectionThe BV2 cell culture supernatants were collected and tested for the change in the concentration of the inflammatory cytokines TNF-?and IL-6 according to the instructions of the ELISA kit.10.Statistical analysisStatistical analysis was performed using statistical software SPSS 16.0,Excel 2007sorting data and graphPad charting.The comparison between each group was performed using t-test and one-way analysis of variance?one-way ANVOA?.Dunnett's test was used between groups.The results were statistically significant at P<0.05.Results1.The influence of LPS stimulation on the expression of CD11b in BV2 cellsBV2 cells were treated with 100 ng/ml LPS for different times.Western Bloting results showed that the expression of CD11b protein at 1h,3h,6h,12h and 24h was significantly higher?P<0.05?,and the difference was statistically significant?P<0.05?.2.The effect of LPS treatment on the activity of BV2 cells at different timesBV2 cells were treated with 100 ng/ml LPS for different times.CCK-8 kit test results showed that:compared with the control group,1h,3h,6h,12h and 24h groups showed no significant difference in cell viability?P>0.05?.3.The effect of LPS treatment on the level of ROS in BV2 cells at different timesThe results showed that ROS levels in BV2 cells were significantly increased at 3h,6h,12h and 24h after LPS treatment compared with control group?P<0.01?,but no significant change at 1h?P>0.05?.4.Effects of LPS treatment on the expression of inflammatory cytokines in BV2 cells at different timesThe results of RT-qPCR showed that the expression levels of TNF-?and IL-6 mRNA were significantly higher in the 1h and 3h groups than in the control group?P<0.05?,while the levels of IL-6 mRNA in 6h group were significantly increased?P<0.01?.ELISA results show that compared with the control group,3h,6h,12h and 24h group TNF-?and IL-6concentrations were significantly increased?P<0.01?.5.Effects of LPS treatment on iNOS and COX2 expression in BV2cells at different timesRT-qPCR results showed that compared with the control group,the expression of iNOS and COX2 mRNA increased significantly in the 3h and 6h groups?P<0.05?;only the expression of COX2 mRNA in 1h group was significantly increased?P<0.01?;The results of protein showed that the expression of iNOS and COX2 in 3h,6h and 12h groups were significantly increased?P<0.05?,and the expressions of iNOS and COX2 in 1h and 24h groups were significantly increased?P<0.01 and P<0.001?,respectively.6.The effect of zinc sulfate on the activity of BV2 cellsBV2 cells were intervened by different concentrations of zinc sulfate.The results showed that the cell viability did not change significantly in 10?M,15?M,30?M and50?M groups compared with the control group?P>0.05?.7.The effect of zinc pretreatment on LPS-induced ROS in BV2CellsThe experiment results showed:compared with the control group,the level of ROS in3hLPS group was significantly increased?P<0.001?,and there was no significant change in the group of 10Zn and 30Zn alone.Compared with 3hLPS group,10Zn+LPS and30Zn+LPS groups ROS levels were significantly lower?P<0.01?.8.The effects of zinc pretreatment on the expression of inflammatory factors in BV2 cells induced by LPSThe results of RT-qPCR and ELISA showed that the expression and concentration of TNF-?and IL-6 mRNA in 3h LPS group,10Zn+LPS group and 30Zn+LPS group were significantly higher than those in control group?P<0.001?.There were no significant changes in both 10Zn group and 30Zn group.Compared with 3h LPS group,the expression of mRNA and concentration of TNF-?and IL-6 in 10Zn+LPS group and 30Zn+LPS group were significantly decreased?P<0.05?.9.The effects of zinc pretreatment on the expression of iNOS and COX2 in BV2 cells induced by LPSThe results of RT-qPCR and Western blotting showed that the expression of iNOS mRNA and protein in the 6h LPS group,10Zn+LPS group,and 30Zn+LPS group significantly increased compared with the control group?P<0.05?.There was no significant change in the zinc sulfate alone group?P>0.05?;Compared with 6h LPS group,the expression of iNOS mRNA and protein in 10Zn+LPS group and 30Zn+LPS group was significantly decreased?P<0.05?.Compared with the control group,COX2 mRNA expression levels in 6h LPS group,10Zn+LPS group and 30Zn+LPS group were significantly increased?P<0.001?,and COX2 protein expression level was significantly increased in 6h LPS group?P<0.001?;Compared with 6h LPS group,COX2 mRNA and protein expression levels in 10Zn+LPS group and 30Zn+LPS group were significantly decreased?P<0.05?.10.Effect of zinc on the activity of HT-22 cells by activated microglia-mediated toxicityAfter co-cultivation,the results of CCK-8 showed that the activity of HT-22 cells in3hLPS group,10Zn+LPS group and 30Zn+LPS group were significantly decreased compared with the control group?P<0.05?,and no significant difference was found between the group of 10Zn and 30Zn alone.Compared with 3hLPS group,the activity of HT-22 cells in 10Zn+LPS group and 30Zn+LPS group was significantly increased?P<0.05?.11.The effect of Zinc on the expression of A20 in BV Cells10?M and 30?M zinc sulfate respectively interfered with BV2 cells at different times.RT-qPCR results showed that compared with the control group,A20 mRNA expression levels of 3h group,6h group,12h group,and 24h group were significantly higher?P<0.01?;In LPS-induced BV2 cells,Western blotting showed that the expression of A20 protein in10Zn+LPS group and 30Zn+LPS group was significantly higher than that in 3h LPS group?P<0.05?.12.Effect of zinc on LPS-induced NF-?B signaling pathway in BV2cellsProtein detection results showed that:1h LPS group phosphorylation I?B?expression levels were significantly higher than the control group?P<0.01?;compared with 1h LPS group,10Zn+LPS group and 30Zn+LPS group phosphorylation I?B?expression levels decreased significantly?P<0.05?.Compared with the Con group,the expression of phosphorylated NF-?B p65 in the nucleus of the 3h LPS group was significantly higher?P<0.01?,while the expression of NF-?B p65 in the cytoplasm was decreased?P<0.01?.Compared with the 3h LPS group,The expression of phosphorylated NF-?B p65 in the10Zn+LPS group and 30Zn+LPS group was significantly decreased?P<0.01?.On the contrary,the expression of NF-?B p65 in the cytoplasm was significantly increased?P<0.001?.13.The effect of zinc on the expression of A20 in BV2 cells after A20interferenceThe results of RT-qPCR and Western blotting showed that the mRNA and protein levels of A20 in the interfering group were significantly lower than those in the NC group?P<0.01?.However,after zinc supplementation in the interference group?ie Si+10Zn group and Si+30Zn group?,the expression level of A20 mRNA and protein was significantly higher than that in the interference group?P<0.05?.Under LPS stimulation,the expression of A20 mRNA in Si+LPS group,Si+10Zn+LPS group and Si+30Zn+LPS group was significantly increased?P<0.05?.14.The effect of zinc on NF-?B in BV2 cells after A20 interferenceWestern blotting results showed that compared with Si+LPS group,the expression of NF-?B p65 protein in the cytoplasm of Zn+group?Si+10Zn+LPS group and Si+30Zn+LPS group?was significantly higher?P<0.05?.The expression of phosphorylated NF-?B p65protein in the nucleus was significantly decreased?P<0.01?.ConclusionIn this study,LPS induced the inflammatory response of BV2 cells,and the effects of zinc pretreatment on oxidative stress and inflammation levels of BV2 cells were observed,and explore the mechanism of action of zinc in inhibiting inflammatory response.According to experimental results,preliminary conclusions are as follows:1.LPS can induce BV2 cells to cause oxidative stress and release inflammatory mediators.2.Zinc can inhibit LPS-induced BV2 cell inflammatory response,and it may have protective effect on the damage of HT-22 cells caused by activation of BV2 cells.3.Zinc may inhibit NF-?B activation by up-regulating A20,thereby inhibiting microglial activation-mediated inflammatory responses.