| Objective:Depression is a common type of psychiatric disorder in the world,characterized by depressed mood,loss of self,decreased interest,irritability,sleep disturbance,and severe suicide.About 350 million people worldwide suffer from depression,and depression is expected to be the leading cause of disability in the world over the next20 years.According to statistics,the prevalence rate of depression in China is about3% to 6%,and it is increasing year by year.The pathogenesis of depression is complex that pathogenesis include the monoamine neurotransmitter hypothesis,the brain-derived neurotrophic factor hypothesis,the hypothalamic-pituitary-adrenal(HPA)hypothesis,and the immunoinflammatory hypothesis.In recent years,with the deep research on depression,the immunoinflammatory hypothesis has attracted more and more researchers' attention which is that the occurrence of depression may be due to the activation of the immune system and cause the excessive secretion of cytokines,which provides a new research direction for the prevention and treatment of depression.Although there have been many advances in the development and measures of new drugs for the treatment of depression,there is still no satisfactory method.Studies have suggested that exercise can relieve depressive symptoms,showing that anti-depression in sports has received increasing attention from researchers.Although there are many studies on anti-depression in sports,for example,exercise can adjust the structure and function of synapses,promote the regeneration of hippocampal neurons,and increase the expression of brain-derived neurotrophic factors,but the use of exercise as a treatment for depression is still in the initial phase,the detailed mechanism of exercise antidepression has not been fully elucidated.It is well-knownthat exercise generates a large amount of lactate,which can shuttle through different cells and organs.The lactate in the brain is mainly derived from neurons and glial cells,which can provide energy for neurons.Not only that,lactate also acts as a signaling molecule in the central nervous system.With the in-depth study of lactate,peripheral injection of lactate has been found to have anti-inflammatory and anti-depressant effects,but the specific mechanism of action has not yet been elucidated.Microglia are immune cells of the central nervous system,and its activity is associated with a variety of pathological conditions,such as psychological stress,pathological aging,and chronic infection,regulating the response of the nervous and immune systems to different immune,physiological,and psychological responses.Studies have shown that microglia play an important role in neuroplasticity,impaired neuroprotection,and worsening of depression during pathological processes.More and more research shows that the occurrence of depression is related to the inflammatory reaction caused by excessive activation of microglia and the deterioration of hippocampal tissue.Our previous study found that lactate improve the depression-like behavior of mice induced by lipopolysaccharide(LPS)and inhibit LPS-induced inflammatory responses,but the specific molecular mechanism has not yet been elucidated.Therefore,this subject explores the specific anti-inflammatory effects of lactate from the cellular level.Methods:1.Culture primary hippocampal microglia in vitro.C57BL/6 neonatal rat hippocampus cultured mixed glial cells were allowed to grow to 10-12 days,the separation of microglia using lidocaine,purification after the third to fifth generation of microglia cells for experimental studies.2.Microglia were treated with LPS as the stimulator for 3 h,6 h,12 h,and 24 h respectively.ELISA was used to detect the expression of proinflammatory cytokines TNF-? and IL-1? in each group.3.CCK-8 was used to detect the effect of 10 mM,20 mM,and 30 mM lactate treatment on the activity of microglia for 24 h.4.Treated microglial cells with three concentrations of 10 mM,20 mM,30 mM lactate and LPS for 3 h,6 h,and 12 h.ELISA was used to detect the expression of proinflammatory cytokines TNF-? and IL-1? in each group.5.After treated with LPS for 3 h,6 h,12 h and 24 h,the expression of NF-?B phosphorylation and total protein were detected by Western blot.6.The microglia were treated with 20 mM lactate and LPS for 3 h,6 h,and 12 h.Western blot was used to detect the expression of NF-?B phosphorylation and total protein.7.The microglial cells were treated with LPS for 3 h,6 h,12 h,and 24 h respectively.The expression of NF-?B phosphorylation protein was detected by immunocytochemistry.8.When LPS was used to treat microglia for 6 h,the effect of lactate on the level of NF-?B phosphorylation protein in microglia was observed by immunocytochemical staining.9.After treatment with LPS for 3 h,6 h,12 h,and 24 h,the expression of NLRP3inflammasome-related proteins was detected by Western blot.10.After treated with 20 mM lactate and LPS microglia for 3 h,6 h and 12 h,the expression of NLRP3 inflammasome-related protein was detected by Western blot.11.The microglia cells were treated with LPS for 3 h,6 h,12 h,and 24 h respectively.The expression of NLRP3 inflammasome-associated protein was observed by immunocytochemical staining.12.After treatment of LPS with microglia for 3 or 6 hours,the effect of lactate on NLRP3 inflammasome-associated protein levels in microglia was observed by immunocytochemical staining.Results:1.Compared with the control,LPS treatment microglia cells 3 h,6 h,12 h,the expression of TNF-? was significantly increased(p <0.05);LPS treatment microglia 3h,6 h and 12 h,the expression of IL-1? was significantly increased(p<0.05).2.Treatment of microglia with three different concentrations of 10 mM,20 mM,and30 mM lactate for 24 h did not affect the activity of microglia.3.When 3 h,6 h microglia were treated with 10 mM,20 mM,30 mM lactate and LPS,three concentrations of lactate down-regulated the expression of TNF-?compared with the LPS group(p< 0.05);When 20 mM,30 mM lactate and LPS were added to treat microglia cells for 3,6,and 12 hours,20 mM and 30 mM lactate could down-regulate the expression of IL-1? compared with LPS group.(p<0.05).4.Western blot results showed that compared with the control group,LPS treated microglia cells significantly increased the level of NF-?B phosphorylation protein in microglia after 3 h,6 h and 12 h(p<0.05).There was no significant change in total NF-?B protein.5.Western blot results showed that the expression of NF-?B phosphorylation protein was significantly decreased after 6 h and 12 h of lactate treatment in LPS group compared with LPS group at each time point(p<0.05).6.The results of immunocytochemical staining showed that the level of NF-?B phosphorylation protein in microglia was increased 6 h and 12 h after LPS treatment compared with the control group,and the fluorescence intensity of LPS treatment 6 h was the strongest;LPS induced LPS-induced downregulation of P-NF-?B expression compared to the LPS group.7.Western blot results showed that LPS treatment microglia cells 3 h,6 h,12 h,compared to the control group,microglia NLRP3 protein levels were significantly increased(p <0.05),and after LPS stimulation 6 h The level of NLRP3 protein reached the peak(p<0.001).After LPS treatment of microglia for 3 h,6 h,and 12 h,the ASC protein level was significantly higher than that of the control group(p<0.01)and stimulated by LPS.After 3 h,the expression of ASC protein was the highest(p<0.001).After LPS treated microglia for 6 h and 12 h,the level of Caspase-1protein was significantly higher than that of the control group(p<0.05).At 6 h,the expression of Caspase-1 protein was the highest(p<0.01).8.Western blot results showed that compared with the LPS group at each time point,NLRP3 protein expression was significantly decreased after 6 h of lactate treatment of microglia(p<0.001);when lactate treated microglia 3 h,6 h,12 h,the level of ASC protein decreased significantly compared with the LPS group at each time point;when the microglia were treated with lactate for 6 h,the Caspase-1 protein level decreased significantly compared with the LPS group at each time point(p<0.01).9.The results of immunocytochemical staining showed that compared with the control group,the microglia NLRP3 protein expression increased at 3 h,6 h,and 12 h after LPS treatment,and the fluorescence intensity was the strongest at 6 h after LPS treatment;The expression of ASC protein in glial cells was increased at 3 h,6 h and12 h after LPS treatment,and the fluorescence intensity was the strongest at 3 h after LPS treatment.The microglia Caspase-1 protein was treated with LPS for 3 h and 6 h.After 12 h,the expression increased,and the fluorescence intensity was the strongest at 6 h after LPS treatment.10.The results of immunocytochemical staining showed that the expression of NLRP3 in LPS group was increased compared with the control group at 6 h after LPS and lactic acid treatment of microglia.Compared with LPS group,the expression of NLRP3 induced by LPS was significantly lower than that of LPS group.LPS and Lactate treated microglial cells for 3 h compared with the control group,LPS group ASC expression increased;compared with the LPS group,lactate LPS-induced ASC expression increased significantly decreased;LPS and lactate treatment microglia 6 h compared with the control group,the expression of Caspase-1 in the LPS group increased.Compared with the LPS group,the LPS-induced increase in Caspase-1expression was significantly downregulated by lactate.Conclusion:1.LPS induces inflammatory reaction in mouse primary hippocampal microglia.2.Lactate inhibit LPS-induced inflammatory reaction of primary hippocampal microglia in mice.3.Lactate may exert anti-inflammatory effects by inhibiting the production of pro-inflammatory factors TNF-?,IL-1?,activation of NF-?B,and activation ofNLRP3 inflammasome. |
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