The Mechanism Of MiR-182-5p Delivered By Plasma Exosomes On Perioperative Neurocognitive Disorders By Activating NF-κB Pathway

Objective: The objective of this study was to explore miRNAs differentially expression in plasma of elderly patients with perioperative neurocognitive disorders(PNDs)which were screened and verified by high-throughput sequencing,and the molecular pathways enriched by differentially expressed genes were analyzed by bioinformation.The expression of miR-182-5p in plasma,hippocampal neurons and related inflammatory factors in PNDs model rats was verified by animal experiments.The Animal and cell experiments were further used to explore the possible mechanism and effect of miR-182-5p delivered by plasma exosomes on surgical operations-induced neuroinflammation and cognitive disorder in aged rats with PNDs.Methods: In the first part of the clinical experiment,120 elderly patients undergoing abdominal surgery with general anesthesia were selected and divided into observation and control groups by 1SD method,using the Mini Mental State Examination Scale(MMSE)and the Montreal Mental Inventory(Mo CA)respectively on the first day before surgery and the seventh day after surgery,when the difference in the seventh day and before surgery MMSE or Mo CA of a patient decreases by more than or equal to 1 standard deviation,the patient is considered as PNDs and included in the observation group.The significantly differential expression miRNAs in the peripheral plasma of elderly patients with PNDs were found by high throughput sequencing.The differential expression of miRNAs in the peripheral plasma was verified by q RT-PCR.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to analyze the main molecular pathways enriched by differential genes.In the second part of the experiment,18 month old SD rats were selected and divided into three groups: control group,sham-operated group,and group of surgical operation,6 cases in each group.Morris water maze test and conditional fear test were used to detect changes in cognitive function,q RT-PCR was used to detect the expression of miR-182-5p in rat plasma and hippocampal tissue,H&E staining was used to observe morphological changes in rat hippocampal neurons,immunofluorescence was used to detect the expression of p-P65 protein in rat hippocampus,and ELISA was used to detect TNF-α and IL-6 in rat hippocampal tissue after anesthesia and surgery.The third part of study is animal and cell experiments.Aged PNDs rat models were induced by anesthesia and exploratory laparotomy.Subsequently,exosomes and miR-182-5p were inhibited by injection of GW4869 and miR-182-5p-sponge respectively.Then,exosomes were extracted from the plasma of rats in each group,followed by the determination of the morphology and diameters of exosomes as well as the expression of exosome markers CD63 and CD81 by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA),and Western blot.Besides,the Morris water maze(MWM)and fear condition test were used to evaluate the learning and memory of rats.Western blot to detect the expression levels of neurotrophic factors(brain-derived neurotrophic factor(BDNF)and nerve growth factor(NGF))as well as NF-κB pathway-related proteins(p65 and p-P65)in rat hippocampal tissues or PC-12 cells;q RT-PCR to assess the expression levels of miR-182-5p and BDNF in rat plasma,plasma exosomes,hippocampal tissues,and PC-12 cells;ELISA to evaluate the levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6,and IL-1β in rat hippocampal tissues;H&E staining was used to observe morphological changes in rat hippocampal neurons and dual-luciferase reporter assay to verify the targeting relationship between miR-182-5p and BDNF.Results:(1)It was found that 16 miRNAs such as miR-182-5p were significantly higher in the plasma of observation group than those in the control group,and 26 miRNAs such as miR-106-5p were notably lower in the plasma of observation group than those in the control group,and 9 miRNAs were identified as new miRNAs in the plasma of observation group by high throughput sequencing.The results of q RT-PCR of miR-96-5p,miR-182-5p,miR-1260 a and miR-106b-5p is consistent with the above sequencing results.(2)According to GO analysis,in terms of cell function,differential miRNAs are concentrated in the cytoplasm,while in terms of molecular function,they are concentrated in protein binding.The main molecular pathways enriched by differential genes in KEGG analysis are tumor related pathways,MAPK signaling pathways,and neurotrophic factor signaling pathways.(3)The second part of the study showed that the cognitive function was significantly reduced and successfully induced a rat model of PNDs in the operation group compared with the control group and sham-operated group,The escape latency was significantly prolonged,the number of crossing the platform quadrant and the target quadrant residence time were reduced,and the damage of hippocampal neurons is more serious in the operation group compared with the control group and anesthesia group.Compared with the control group and the sham-operated group,the expression of miR-182-5p in the plasma and hippocampus of the aged rats in the operation group was significantly increased,Anesthesia and surgery activated the expression of p-P65,TNF-α,IL-6 and other inflammatory factors in the hippocampus of aged rats.(4)miR-182-5p expression was up-regulated in PNDs rats and could be delivered by plasma exosomes.Inhibition of plasma exosomes or miR-182-5p could significantly ameliorate learning and memory disorders in GW4869 group and miR-182-5p-sponge group,inhibit the expression p-P65 protein in the hippocampal tissue of PNDs rats,decrease the levels of inflammatory cytokines TNF-α,IL-6,and IL-1β;increase the expression of BDNF and NGF;In addition,miR-182-5p could also target and inhibit BDNF.Conclusion:(1)There are significantly up-regulated in the expression of miR-182-5p and other miRNAs in the plasma of elderly PNDs patients,The main molecular pathways enriched by differential miRNAs are MAPK signaling pathway and neurotrophic signaling pathway.(2)Surgical operation induced the occurrence of PNDs,promoted the expression of miR-182-5p and activated the NF-κB signaling pathway in aging rats.(3)miR-182-5p delivered by plasma exosomes promotes surgical operationinduced neuroinflammation and cognitive dysfunction in aged PNDs rats by activating the NF-κB pathway and targeting BDNF.