| Objective: This study established POCD model using lipopolysaccharide(LPS)induced BV2 cells in vitro to mimic neuroinflammation and performed tibial fracture surgery in vivo to observe the activation of microglia,and then explored the therapeutic effect of dexmedetomidine on POCD,and initially explored the role of SALL1/JARID2 in the improvement of POCD by dexmedetomidine.It is expected to provide novel molecular mechanism for the clinical application of dexmedetomidine and experimental basis for the follow-up in-depth study of the POCD mechanism.Methods: 1.Using 0.1μg/ml and 1μg/ml LPS to induce BV2 cells,MTT was used to detect cell viability after induction 6h,12 h,24h,36 h and 48 h,which explored the best LPS induction concentration and time for POCD model building in vitro.Afterwards,BV2 cells were pretreated with 0.1 μM,1 μM and10 μM dexmedetomidine(Dex)before LPS induction,and MTT was used to detect cell viability and explore the most effective Dex concentration.2.BV2 cells were randomly divided into Control group,LPS group and Dex group.Immunofluorescence of CD68 was applied to observe the activation of microglia,quantitative real-time polymerase chain reaction(q RT-PCR)was applied to determine microglia M1/M2 type marker changes,q RT-PCR and Western blot was used to detect the expression of SALL1 and its downstream genes.3.A count of 60 C57BL/6 male mice were randomly divided into 3groups,including Sham group,Model group and Dex group.Mice in the Sham group were received only anesthesia without tibial fracture surgery.Mice in the Model group underwent tibial fracture surgery under inhalation anesthesia.Mice in the Dex group received intraperitoneal injection of 25μg/kg 0.5 h before anesthesia.After the operation,the three groups of mice were subjected to Morris water maze experiment to evaluate cognitive function.After the behavioral test,peripheral blood was collected,and brain tissue was taken after cardiac perfusion;immunofluorescence of Iba-1 was used to observe the activation of microglia in the hippocampus,q RT-PCR was used to detect the expression of M1/M2 type markers in microglia,the content of inflammatory factors including TNF-α,IL-6 and IL-10 in serum and hippocampus were determined by Enzyme-linked immunosorbent assay(ELISA),q RT-PCR and western blot were performed to determine the expression of SALL1 and JARID2 in hippocampus.Results: 1.After treating BV2 cells with 0.1μg/ml and 1μg/ml LPS for 6h,12 h,24h,36 h and 48 h,the cell viability results showed that 0.1μg/ml LPS significantly increased the cell viability of BV2 cells after 24 h induction(P<0.05);0.1μM,1μM and 10μM Dex pretreated BV2 cells,and the results showed that 0.1μM Dex could significantly inhibit the activation of BV2 cells(P<0.05).2.The results of experiments in vitro showed that compared with the Control group,the microglia activation marker(CD68)was significantly increased,which means BV2 cells in the LPS group were significantly activated,while the microglia activation marker(CD68)was significantly reduced in the Dex group.In addition,the m RNA levels of microglia M1 type markers(i NOS and CD32)in the LPS group were significantly higher than those in the Control group.Compared with the LPS group,the m RNA levels of i NOS and CD32 in the Dex group were significantly lower;and compared with the Control group,the m RNA levels of microglia M2 markers(CD206 and Arg1)were significantly reduced in the LPS group.Compared with the LPS group,the m RNA levels of CD206 and Arg1 in the Dex group were significantly increased(P<0.05).The results of in vivo experiments showed that the escape latency of the Model group was longer than that of the Sham group,and the escape latency of the Dex group was shorter than that of the Model group(P<0.05);compared with the Sham group,the number of crossings in the Model group decreased significantly,the number of crossing in the Dex group was higher than that in the Model group(P<0.05).Immunofluorescence showed that the expression of Iba-1 in the hippocampus of the Model group was significantly increased compared with the Sham group,and the expression of Iba-1 in the hippocampus of the Dex group was less than that in the Model group.The m RNA levels of i NOS and CD32 in the Model group were significantly higher than those in the Sham group,while the Dex group was significantly lower than that in the Model group(P<0.05);compared with the Sham group,the m RNA levels of CD206 and Arg1 were significantly reduced in the Model group and significantly increased in the Dex group(P<0.05).The ELISA test showed that the levels of TNF-α and IL-6 in the serum and hippocampus of the Model group were significantly higher than those in the Sham group,and the content of TNF-α and IL-6 in the Dex group was significantly lower than that in the Model group(P<0.05).In addition,the IL-10 content in the serum and hippocampus tissue of the Model group mice was significantly lower than that in the Sham group,and the IL-10 content in the Dex group was significantly higher than that in the Model group(P<0.05).3.In vitro experiments showed that the m RNA levels of SALL1 and JARID2 in the LPS group were significantly lower than those in the Control group,the expressions of SALL1 and JARID2 in the Dex group were significantly increased(P<0.05),while the m RNA levels of Dux,Mreg and Phc1 were no significant difference among the three groups(P>0.05).The protein expression of SALL1 and JARID2 in the LPS group was significantly lower than that in the Control group,and the protein expression of SALL1 and JARID2 in the Dex group was significantly increased(P<0.05);in addition,in vivo experiments showed that compared with the Sham group,the m RNA and protein level of SALL1 and JARID2 in the Model group were decreased,the m RNA and protein expressions of SALL1 and JARID2 in the Dex group were significantly higher than those in the Model group(P<0.05).Conclusion: 1.0.1μg/ml LPS with 24 h induction can significantly activate BV2 cells,and 0.1μM Dex can significantly inhibit the activation of BV2 cells.2.After establishment of POCD,microglia were significantly activated,M1 type markers and pro-inflammatory cytokines of microglia cells were significantly increased,and the expression of M2 type markers and anti-inflammatory cytokines were decreased;dexmedetomidine can markedly improve cognitive dysfunction of mice after surgery by inhibiting the activation of microglia and reversing the expression of M1/M2 type markers and inflammatory factors in microglia.3.SALL1 decreased when microglia cells were activated,and the expression of its downstream gene JARID2 also decreased,and dexmedetomidine can increase the expression of SALL1 and JARID2. |
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