Therapeutic Effect And Mechanism Of Trilobatin On Oxygen-Glucose Deprivation And Reoxygenation-Induced Neuronal Injury

Objective: To investigate therapeutic effect and mechanism of trilobatin(TLB)on oxygenglucose deprivation and reoxygenation(OGD/R)-induced neuronal injury the effect of trilobatin.Methods:1.The cerebral ischemic/reperfusion model was induced by OGD/R in primary rat astrocytes and cortical neurons.Primary cortical neurons were randomly divided into 6 groups after OGD 2 h/R 24 h: control,control + TLB 25 μM,OGD/R,OGD/R + TLB 6.25 μM,OGD/R+ TLB 12.5 μM,OGD/R + TLB 25 μM.Primary rat astrocytes were randomly divided into6 groups after OGD 1.5 h/R 48 h: control,control + TLB 50 μM,OGD/R,OGD/R + TLB12.5 μM,OGD/R + TLB 25 μM,OGD/R + TLB 50 μM.Cell viability was detected by MTT assay,and cell damage was detected by LDH release assay;Cell morphology was observed under inverted microscope;The levels of IL-1β,IL-6,TNF-α,ROS,MDA and SOD and Sirt3 activities were detected by ELISA assay,and the Sirt3 expression was detected by Western blot.2.Sirt3-KO primary rat astrocytes and cortical neurons were generated by a CRISPR/Cas9 system,and simulate the damage of rat primary cortical neurons and astrocytes by OGD/R.Primary rat cortical neurons were randomly divided into 8 groups: WT + control,WT +control + TLB 25 μM,WT + OGD/R,WT + OGD/R + TLB 25 μM,Sirt3-KO + control,Sirt3-KO + control + TLB 25 μM,Sirt3-KO + OGD/R,Sirt3-KO + OGD/R + TLB 25 μM,and the primary astrocytes were randomly divided into 8 groups: WT + control,WT + control+ TLB 50 μM,WT + OGD/R,WT + OGD/R + TLB 50 μM,Sirt3-KO + control,Sirt3-KO+ control + TLB 50 μM,Sirt3-KO + OGD/R,Sirt3-KO + OGD/R + TLB 50 μM.The expression level of intracellular Sirt3 m RNA was detected by RT-PCR;Cell viability were determined using MTT assay,and cytotoxicity was determined by the LDH release assay.3.Loss of Sirt3 in CRISPR/Cas9-dependent knockout rat,and Sirt3 m RNA level was detected by RT-PCR method.Cerebral ischemia-reperfusion injury(CIRI)model was established by middle cerebral artery occlusion(MCAO)method.SD rats were divided into8 groups: WT + sham,WT + sham + TLB 20 mg/kg,MCAO,WT + MCAO + TLB 20mg/kg,Sirt3-KO + sham,Sirt3-KO + sham + TLB 20 mg/kg,Sirt3-KO + MCAO,Sirt3-KO+ MCAO + TLB 20 mg/kg.The administration group was given TLB 20 mg/kg by gavage,the sham and MCAO groups were given the same volume of normal saline by gavage for three consecutive days,twice a day.Then,neurological deficits were measured using Longa5 method,TTC staining was used to detect the volume of cerebral infarction.Results:1.The results showed that OGD/R reduced the cell viability and increased LDH release in primary rat cortical neurons and astrocytes than those of control,and the neurons shrink,reduction in cell number,even death;Whereas,TLB reversed these changes after OGD/R.Besides,the results of ELISA assay showed that the SOD and Sirt3 activities was decreased,while,IL-1β,IL-6,TNF-α,ROS and MDA levels were obviously increased in OGD/R group than those of control;However,TLB significantly reversed these changes.Notably,TLB not only down-regulated the levels of IL-1β,IL-6,TNF-α,ROS and MDA,upregulated SOD and Sirt3 activities than those of OGD/R group,but also increased Sirt3 expression and its activity.2.Sirt3-KO neurons or astrocytes showed more severe injury after OGD/R insult than those of WT neurons or astrocytes.However,the protective effects of TLB on OGD/R-induced injury were partially occluded in Sirt3-KO astrocytes and Sirt3-KO neurons than those of WT astrocytes or neurons.3.The results showed that the neurological function scores and cerebral infarct volume were significantly increased after MCAO insult in Sirt3 KO rats than that of WT rats.Whereas,TLB treatment partly lost its ability to reduction of MCAO-induced injury in Sirt3-KO rats.Conclusion: TLB protects against OGD/R-induced neuroinflammation and oxidative injury via activation of Sirt3.