| Objective:Postoperative cognitive dysfunction(POCD)is often considered as a common complication of general anesthesia in the elderly.Many studies have confirmed that inflammatory responses,especially inflammations of the hippocampus,are closely related to the cognitive impairment.Dual-specificity phosphatase 14(DUSP14,also known as MKP6)has been implicated in the pathogenesis of various inflammatory diseases.However,the exact role and mechanism of DUSP14 in POCD is unclear.Materials and Methods:Part 1: An isoflurane exposure induced aged rat model was successfully constructed: Isoflurane anesthesia group(Iso group)was administered with 1.5%isoflurane for 6 hours and compare with Ctrl group which did not suffer to isoflurane exposure.Evaluation of learning and memory abilities in aged rats was measured using the Morris water maze task test.The pathological changes of hippocampal tissues of the aged rats were detected by Nissl staining.Changes in brain injury markers(S-100β、NSE)were detected by ELISA assay.Part 2: The DUSP14 level in the hippocampal tissues of the aged rats after exposure of isoflurane was detected by IHC assay,q RT-PCR and Western blot,respectively.Part 3: Then we injected AAV9 carrying DUSP14 gene intracerebrally into the hippocampus and finish the transfection for 3 weeks.Evaluation of learning and memory abilities in aged rats was measured using the Morris water maze task test.The DUSP14 expression level was detected by q RT-PCR and WB respectively.The pathological changes of hippocampal tissues of the aged rats were detected by Nissl staining.Changes in brain injury markers(S-100β、NSE)were also detected using ELISA assay.WB assay was used to measure the expression of proteins related to apoptosis(bax、bcl-2 and ccaspase-3).The apoptosis of hippocampal nerve cells was assessed by TUNEL assay.Part 4: In order to find out the effects of overexpression of DUSP14 on isoflurane anesthesia-induced neuronal inflammatory response,we used ELISA and q RT-PCR analysis to measure the expression level of inflammatory cytokines(IL-1β、TNF-α、IL-6)in the hippocampal tissues.IHC were used to detect NLRP3 expression level in the hippocampal tissues.WB assay was used to measure the expression of proteins related to pyroptosis and NLRP3/caspase-1 pathway.Results:Part 1: The representative results in Morris water maze tests were shown that the escape latency time was longer in the Iso group than in the Ctrl group(p<0.001).The number of platform crossing,the percentage of time spent,and the distance covered in the target quadrant were decreased in the Iso group compared with the Ctrl group(p<0.01).By the Nissl staining,rats in the Iso group had significantly fewer neurons than those in the Ctrl group(p<0.001).ELISA assay indicated that the S-100βand NSE levels in the Iso group were remarkably elevated compared to the Ctrl group(p<0.001).Part 2: The expression of DUSP14 in hippocampal tissues from rats in the Iso group was detected using IHC,q RT-PCR and Western blot.The results revealed that the m RNA and protein expression of DUSP14 was significantly decreased in Iso group compared with Ctrl group(p<0.001).Part 3: DUSP14 was successfully overexpressed in isoflurane anesthesia-induced aged rats,which was verified by q RTPCR and Western blot,which show that the expression of DUSP14 was elevated dramatically(p<0.001).The Morris water maze test showed that the overexpression of DUSP14 decreased the escape latency and increase the number of platform crossing,while also enhanced the percentage of time spent and the percentage of distance covered in the target quadrant of the isoflurane exposured aged rats(p<0.01).TUNEL assay results demonstrated that the amount of apoptotic cells in the hippocampus were significantly increased by isoflurane exposure(p<0.001),whereas the overexpression of DUSP14 suppressed its promotive effects on cell apoptosis of aged rats(p<0.01).The Western blot assay showed that Bcl-2 level was significantly decreased,but Bax and cleaved caspase-3 levels were significantly increased by isoflurane exposure(p<0.001),whereas upregulation of DUSP14 reversed the above effects(p<0.001).Nissl staining showed that rats in Iso group evidently displayed decreased number of Nissl staining-positive neuronal cells compared to the Ctrl group(p<0.001),whereas the overexpression of DUSP14 weakened the above results caused by isoflurane treatment(p<0.001).ELISA assay exhibited that the overexpression of DUSP14 inhibited isoflurane-induced levels of S-100β and NSE in aged rats(p<0.001).Part4: The effects of overexpression of DUSP14 on isoflurane anesthesia-induced neuronal inflammatory response were determined in aged rats by ELISA and q RTPCR analysis,the results implied that isoflurane increased the levels of IL-1β,TNF-α and IL-6(p<0.001),whereas the overexpression of DUSP14 reduced the levels of IL-1β,TNF-α and IL-6(p<0.01).IHC showed that the number of NLRP3-positive cells were increased after isoflurane exposure(p<0.001),whereas overexpression of DUSP14 decreased it(p<0.01).Consistently,WB result implied that the protein levels of NLRP3,cleaved caspase-1,IL-1β,IL-18 and GSDMD-NT were upregulated by isoflurane treatment(p<0.001),while the overexpression of DUSP14 partially abolished the above changes and decrease those protein levels(p<0.01).But there were no significant differences in the adaptor protein ASC among four groups(p=0.616).Conclusion:Isoflurane exposure led to brain injury,inflammatory response,cognitive dysfunction in aged rats and decreased the expression of DUSP14.Overexpression of DUSP14 could inhibit apoptosis,inflammation,pyroptosis,brain tissue damage and improve cognitive dysfunction of aged rats after isoflurane anesthesia.Further mechanism studies revealed that DUSP14 may play a neuroprotective effect in POCD through regulating NLRP3 inflammasome-mediated pyroptosis.Overexpression of DUSP14 may effectively protect against isoflurane-induced neuro-inflammation,brain damage and cognitive dysfunction,indicating that DUSP14 may be a potential predictor and therapeutic target for POCD. |
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