| Studies have shown that sepsis survivors may experience long-term cognitive impairment,manifested as decreased memory ability,visualisation perception and executive difficulties,which make the quality of life of patients decline and bring a great burden to society and families.Therefore,it is of great clinical significance to draw attention to the prevention and treatment of cognitive dysfunction caused by sepsis.However,the pathogenesis of septic cognitive impairment has not yet been completely clarified.It has been found that miR-125a-5p may play an important role in the inflammatory response,so this study first verified the effect of miR-125a-5p targeting HIF1AN on BV-2 microglial activation and causing microglial inflammation by negatively regulating HIF-1α expression levels through cellular experiments.The inflammatory activation phenotype of BV-2 microglia was induced by LPS and IFN-γ,and the expression of miR-125a-5p,HIF1 AN and HIF-1α in BV-2 microglia was detected under the inflammatory phenotype,demonstrating that the miR-125a-5p/HIF1AN/HIF-1α axis is indeed associated with the inflammatory response,in which miR-125a-5p agonists promote microglia-mediated inflammatory response,while antagonizing miR-125a-5p attenuates the inflammatory response.Animal experiments were used to establish a mouse model of sepsis-related cognitive dysfunction by LPS to further verify the mechanism by which miR-125a-5p is involved in the production of sepsis-related cognitive impairment in mice,and the learning and memory ability of septic cognitive impairment mice was improved by regulating miR-125a-5p and reducing the expression of neuroinflammatory factors.The aim of this study was to investigate the mechanism by which miR-125a-5p/HIF1AN/HIF-1α axis activates microglia and produces inflammatory factors to mediate neuroinflammation and then participate in sepsis-related cognitive impairment by establishing a cellular inflammatory injury model and an animal model of cognitive impairment in sepsis,and to improve the learning and memory ability of septic cognitive impairment mice by regulating miR-125a-5p,providing possible coping strategies and new intervention targets for the treatment of sepsis-induced cognitive dysfunction.Part Ⅰ miR-125a-5p targets HIF1AN to negatively regulate HIF-1α involved in microglial activationObjective:To investigate the expression of miR-125a-5p/HIF1AN/HIF-1α in BV-2 microglia induced by LPS and IFN-y.Methods:First,the inflammatory model of BV-2 microglia was established:BV-2 microglia were divided into two groups:control group(CON group)and model group(LPS+IFN-γ group).Fresh medium was added to PBS in the control group and LPS(150 ng/ml)and IFN-γ(20 ng/ml)in the model group.After 12 h of continuous culture in the cell incubator after administration,the changes in the expression of miR-125a-5p gene and inflammatory cytokines IL-1β mRNA and TNF-α mRNA were detected by real-time PCR;the changes in the expression of HIF1AN mRNA and protein and HIF-1α mRNA and protein were detected by real-time PCR and Western blot;and the changes in iNOS expression were detected by real-time PCR,Western blot,and immunofluorescence staining.Secondly,a miRNA transfection model under inflammatory conditions in BV-2 microglia was established:BV-2 microglia were divided into six groups:CON group,LPS+IFN-γ group,agonist negative control group(NC agomir+LPS+IFN-γ group),miR-125a-5p agonist group(miR agomir+LPS+IFN-γ group);antagonist negative control group(NC antagomir+LPS+IFN-γ group)and miR-125a-5p antagonist group(miR antagomir+LPS+IFNy).BV-2 microglia were transfected using miR-125a-5p agonists,antagonists or their respective negative controls and treated with LPS+IFN-γ for 12 h.Real-time PCR was used to detect the changes of miR-125a-5p gene and inflammatory cytokines IL-1β mRNA and TNF-α mRNA expression;real-time PCR and Western blot were used to detect the changes of HIF1AN mRNA and protein and HIF-1α mRNA and protein expression;realtime PCR,Western blot and immunofluorescence staining were used to detect the changes of iNOS expression.Finally,a HIF-1α small interfering RNA transfection model was established in BV-2 microglia under inflammatory conditions:BV-2 microglia were divided into four groups:CON group,LPS+IFN-γ group,small interfering RNA negative control group(NC siRNA+LPS+IFN-γ group)and HIF-1α small interfering RNA group(HIF-1βsiRNA+LPS+IFN-γ group);HIF-1α small interfering RNA and its negative control were used and treated with LPS+IFN-γ for 12 hours.Real-time PCR was used to detect the changes of HIF-1α mRNA and inflammatory cytokines IL-1β mRNA and TNF-α mRNA expression;real-time PCR and Western blot were used to detect the changes of HIF-1αprotein expression;real-time PCR,Western blot and immunofluorescence staining were used to detect the changes of iNOS expression.Results:First,in the BV-2 microglial inflammation model,compared with the CON group,the expression of IL-1β mRNA and TNF-α mRNA in the LPS+IFN-γ group was increased(P<0.05);the expression of iNOS mRNA and protein was increased(P<0.05);the red fluorescence intensity of iNOS positive microglia was increased;the expression of miR125a-5p gene was increased(P<0.05);the expression of HIF1AN mRNA and protein was decreased(P<0.05);and the expression of HIF-1α mRNA and protein was increased(P<0.05).Secondly,in the miRNA transfection model under inflammatory conditions in BV-2 microglia,miR-125a-5p gene expression was increased in the miR agomir+LPS+IFN-y group compared with the NC agomir+LPS+IFN-γ group(P<0.05);inflammatory cytokines IL-1β mRNA and TNF-α mRNA expression were increased(P<0.05);iNOS mRNA and protein expression were increased(P<0.05);iNOS positive microglia red fluorescence intensity was increased;HIF1AN mRNA and protein expression was decreased(P<0.05);HIF-1α mRNA and protein expression was increased(P<0.05).Compared with the LPS+IFN-γ group,there were no significant differences in the expression of inflammatory cytokines IL-1β mRNA and TNF-α mRNA in the NC agomir+LPS+IFN-γ group(P>0.05);there were no significant differences in iNOS,HIF1AN and HIF-1α mRNA and their protein expression(P>0.05).Compared with the antagomir+LPS+IFN-γ group,miR-125a-5p gene expression decreased in the miR antagomir+LPS+IFNγ group(P<0.05);inflammatory cytokines IL-1β mRNA and TNF-α mRNA expression decreased(P<0.05);iNOS mRNA and protein expression decreased(P<0.05);iNOS positive microglia red fluorescence intensity decreased;HIF1AN mRNA and protein expression increased(P<0.05);HIF-1α mRNA and protein expression decreased(P<0.05);compared with the LPS+IFN-γ group,inflammatory cytokines IL-1β mRNA and TNF-αmRNA levels were not significantly different in the NC antagomir+LPS+IFN-γ group(P>0.05),and iNOS,HIF1AN and HIF-1α mRNA and protein levels were not significantly different(P>0.05).Compared with the NC siRNA+LPS+IFN-γ group,HIF-1α mRNA and protein expression decreased in the HIF-1α siRNA+LPS+IFN-γ group(P<0.05);inflammatory cytokines IL-1β mRNA and TNF-α mRNA expression decreased(P<0.05);iNOS mRNA and protein expression decreased(P<0.05);iNOS positive microglia red fluorescence intensity decreased;compared with the LPS+IFN-γ group,inflammatory cytokines IL-1β mRNA and TNF-α mRNA expression were not significantly different in the NC siRNA+LPS+IFN-γ group(P>0.05),and iNOS and HIF-1α mRNA and protein levels were not significantly different(P>0.05).Conclusion:In the presence of LPS+IFN-y,the expression of miR-125a-5p is increased,the expression of inflammatory cytokines IL-1β mRNA and TNF-α mRNA and microglial activation marker iNOS is increased,the expression of HIF1AN is decreased,and the expression of HIF-1α is increased in microglia.By regulating the expression levels of miR125a-5p and HIF-1α,it is found that miR-125a-5p negatively regulates HIF-1α by targeting HIF1AN to promote the expression of inflammatory cytokines IL-1β and TNF-α,and plays a regulatory role in microglial activation,thus demonstrating that the miR-125a5p/HIF1AN/HIF-1α axis is associated with inflammatory regulation.Part Ⅱ Role of miR-125a-5p in cognitive dysfunction in sepsis in adult male C57BL/6 miceObjective:To investigate the role of miR-125a-5p in cognitive dysfunction in male C57BL/6 mice with sepsis.Methods:A model of cognitive dysfunction in sepsis was established:8-week-old male C57BL/6 mice were randomly divided into two groups:control group(CON group)and model group(LPS group).CON group received a single intrapulmonary injection of normal saline and LPS group received a single intrapulmonary injection of 10 mg/kg LPS.After intrapulmonary injection of LPS 10 mg/kg for 12 h,the two groups of mice were trained in the conditioned fear test,and after 12 h of training,the conditioned fear association environment test was performed,and the auditory conditioning stimulation test was performed 2 h after the completion of the association environment test to record the coagulation time of the two groups of mice,and the hippocampus-dependent memory(related to the association environment test)and hippocampus-independent memory ability(related to the auditory conditioning stimulation test)of the two groups of mice were analyzed;followed by the Y-maze spontaneous alternation test to record the number of spontaneous alternations of the two groups of mice and analyze the spatial memory ability of the two groups of mice.Subsequently,hippocampus tissues were immediately sampled to detect changes in the expression of inflammatory cytokines TNF-α mRNA and IL-1βmRNA and miR-125a-5p gene by real-time PCR;changes in the expression of HIF1AN mRNA and its protein,HIF-1α mRNA and its protein,and iNOS mRNA and its protein were detected by real-time PCR and Western blot.Secondly,a miRNA transfection model of cognitive dysfunction in sepsis was established:8-week-old male C57BL/6 mice were randomly divided into four groups:control group(CON group),model group(LPS group),antagonist negative control group(NC+LPS group)and miR-125a-5p antagonist group(antagomir+LPS group).To maintain the transfection effect,miR antagomir antagonist transfection complex or negative control transfection complex was prepared 1 day before fear conditioning training,and bilateral hippocampus CA1 intracerebral injection pretreatment was immediately performed using a brain stereotypic device,and 12 h after the injection,mice in the LPS,NC+LPS,and antagomir+LPS groups were intraperitoneally injected with a single dose of LPS 10 mg/kg;mice in the CON group were intraperitoneally injected with a single dose of saline.Twenty-four hours after intrapulmonary injection of LPS,conditioned fear test training was performed,24 h after training,conditioned fear test association environment test was performed,and 2 h after association environment test,auditory conditioned stimulation test was performed to record the coagulation time of the four groups of mice and assess the hippocampus-dependent memory ability and hippocampus-independent memory ability of the four groups of mice;then Y-maze spontaneous alternation test was performed to record the number of spontaneous alternations of the four groups of mice and assess the spatial working memory ability of the four groups of mice.Subsequently,hippocampus tissues were immediately sampled to detect changes in the expression of inflammatory cytokines TNF-α mRNA and IL-1β mRNA and miR-125a-5p gene by real-time PCR;changes in the expression of HIF1AN mRNA and its protein,HIF-1α mRNA and its protein,and iNOS mRNA and its protein were detected by real-time PCR and Western blot.Results:Compared with CON group,the percentage of coagulation time in association environment test was shortened(P<0.05),hippocampus-dependent memory was impaired in LPS group;there was no significant difference in the percentage of coagulation time in auditory conditioning stimulation test(P>0.05),hippocampus-independent memory was not impaired in LPS group;Y-maze spontaneous alternation rate was decreased(P<0.05),spatial working memory ability was impaired in mice;miR-125a-5p gene expression was increased(P<0.05);inflammatory cytokines IL-1β mRNA and TNF-α mRNA expression were increased(P<0.05);iNOS mRNA and protein expression were increased(P<0.05);HIF1AN mRNA and protein expression were decreased(P<0.05);HIF-1α mRNA and protein expression were increased(P<0.05).Compared with the NC+LPS group,the antagomir+LPS group had an increased percentage of coagulation time in the association environment test(P<0.05)and alleviated hippocampus-dependent memory impairment in mice;there was no significant difference in the percentage of coagulation time in the auditory conditioning stimulation test(P>0.05),and hippocampus-independent memory was not impaired in mice;the Y-maze spontaneous alternation rate was increased(P<0.05)and alleviated spatial working memory impairment in mice;the expression of inflammatory cytokines IL-1β mRNA and TNF-α mRNA was decreased(P<0.05);the expression of iNOS mRNA and protein was decreased(P<0.05);the expression of HIF-1α mRNA and protein was decreased(P<0.05);compared with the LPS group,the levels of inflammatory cytokines IL-1β mRNA and TNF-α mRNA in the NC+LPS group were not significantly different(P>0.05);the levels of iNOS,HIF1AN and HIF-1α mRNA and protein were not significantly different(P>0.05).Conclusion:After intrapulmonary injection of LPS in mice,behavioral experiments including conditioned fear test and Y-maze spontaneous alternation test showed that compared with CON group,hippocampus-dependent memory decreased and spatial working memory ability decreased in LPS group,iNOS and inflammatory cytokines IL-1βand TNF-α expression increased and miR-125a-5p expression increased in LPS group.After pretreatment with miR-125a-5p antagonist,conditioned fear test and Y-maze spontaneous alternation test showed that hippocampus-dependent memory and spatial working memory ability were correspondingly improved in LPS group,and iNOS and inflammatory cytokines IL-1β and TNF-α expression decreased in hippocampus compared with negative control group.This result is in agreement with in vitro assays,therefore,this experiment provides a new approach to improve sepsis-related cognitive dysfunction by regulating miR-125a-5p expression and then the regulation of neuroinflammatory factors. |
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