β-Caryophyllene Protect Brain Ischemic Stroke By Regulating MiR-218/HMGB1 Signal Pathway

MicroRNA(miRNA)is a class of endogenous and non-coding single-stranded small molecules consisting of 21–23 bases that regulate target genes through post-transcriptional levels of degradation and inhibition,and miR-218 is a newly identified important factor with anti-inflammatory effects.The high mobility group B1(HMGB1)is a nuclear protein widely expressed in eukaryotic cells.HMGB1 can be released to the outside of the cell under various conditions stimulation.One way of HMGB1 release is passive release by dead,necrotic or damaged cells.These stimuli include inflammatory response,immune response,cell migration,invasion,amplification,differentiation,resistance to pathogenic microorganisms and tissue regeneration.β-caryophyllene(BCP)is a bicyclic sesquiterpene with potent anti-inflammatory activity that inhibits inflammatory cascade reactions.Studies have shown that BCP can reduce the volume of cerebral infarction after CIR injury in rats.ObjectiveThis study aimed to explore the expression of miR-218 and HMGB1 in mouse models of ischemic stroke,and the effects of their expression changes on the series of injuries caused by ischemic stroke,and their expression relationship.Based on this,further explore BCP regulation of this pathway.MethodsIn vivo1.in vivo experiments:The first part: about 20 g C57B/L male mice were randomly divided into different groups: sham operation group(sham);injury group(I/R).Mice in sham group underwent only surgery without ischemia;mice in injured group underwent middle cerebral artery embolization(MCAO),ischemia 1 h reperfusion 24 h..The second part: randomly divided about 20 g C57B/L male mice into different groups: sham;I/R;miR-218agomir;miR-218antagomir;GV-HMGB1;RNAi-HMGB1;miR-218--agomir+GV-HMGB1;miR-218antagomir+RNAi-HMGB1.The mice in the sham group underwent only surgery without ischemia;the injured mice underwent middle cerebral artery embolization(MCAO),ischemia 1 h and reperfusion 24 h;and the other groups were injected with miR-218agomir、miR-218antagomir、GV-HMGB1 、 RNAi-HMGB1 、 miR-218 agomir GV-HMGB1 and miR-218 antagomir RNAi-HMGB1 at the lateral ventricle one week before the middle cerebral artery embolizationThe third part: about 20 g C57B/L male mice were randomly divided into different groups: sham operation group(sham);injury group(I/R);administration group(BCP I/R).The mice in the sham group underwent only surgery without ischemia;the injured group underwent middle cerebral artery embolization(MCAO),ischemia 1 h reperfusion 24 h;The administration group was given intragastric administration once a day five days before the middle cerebral artery embolization operation,re-gastric administration before the sixth day,and 24 h.after ischemia 1 h.After 24 hours of reperfusion,the dead mice were removed,and the viable mice were scored and statistically.Mice were perfused with paraformaldehyde in the heart and sliced,respectively,for fish detection,fluorescence test,histochemical test,HE staining test and Nissl staining test.mice were isolated from cortical hippocampal tissue after brain extraction.cortical tissue was tested for WB,qPCR,ELISA and double luciferase respectively.In vitroPrimary neuronal cells were cultured in vitro.Part 1: Randomly divided the inoculated cells into different groups: normal group(normal);injury group(OGD).Normal group did not do treatment,normal culture;injury group on the sixth day of half-change solution added sugar-free medium,12 hours after hypoxia 1 h reoxygenation 24 h..Part 2: The inoculated cells were randomly divided into different groups: sham operation group(normal);injury group(OGD);miR-218 overexpression group(miR-218mimic);miR-218 silencing group(miR-218inhibitor);HMGB1 overexpression group(GV-HMGB1);HMGB1 silencing group(RNAi-HMGB1);double overexpression group(miR-218 mimic GV-HMGB1);double silencing group(miR-218 inhibitor RNAi-HMGB1).Normal group did not do treatment,normal culture;injury group on the sixth day of half-change solution added sugar-free medium,12 hours after hypoxia 1 h re-oxygenation 24 h.When the other groups were cultured for three days and half,the sugar-free medium was added to the miR-218mimic、miR-218inhibitor、GV-HMGB1、RNAi-HMGB1 、 miR-218 mimic GV-HMGB1 and miR-218 inhibitor RNAi-HMGB1,for the sixth day,respectively.After 12 hours,hypoxia h re-oxygenated for 24 h.Part 3: Randomly divided the inoculated cells into different groups: normal group(normal);injury group(OGD);administration group(BCP OGD).Normal group did not do treatment,normal culture;injury group in the culture of the sixth day half-change solution added sugar-free medium,hypoxia 1 h re-oxygenation 24 h;the group in the culture of the sixth day half-change solution added sugar-free medium at the same time,12 hours after hypoxia 1 h re-oxygenation 24 h.The cells were detected by fish,fluorescence,WB,qPCR and ELISA after 24 hours of re-oxygenation.ResultsThe experimental results show that:1.After ischemic stroke,the neuropathic disorder occurred in mice,the area of cerebral infarction increased,the survival rate of brain cells decreased,and the number of niposomes decreased.2.In vitro and in vivo showed a significant decrease in miR-218 expression and a significant increase in HMGB1 expression.3.Luciferase assay showed that miR-218 and HMGB1 could bind directly.4.MR-218 overexpression protects against brain injury after stroke,HMGB1 overexpression exacerbates brain injury.MiR-218 overexpression can reverse such damage exacerbation to some extent,suggesting that miR-218 may be HMGB1 upstream factor.5.The expression of TLR4 and RAGE proteins and mRNA is highly correlated with HMGB1,and the TNF-α 、 IL-1 β and IL-6 of inflammatory factors are also associated with the expression of miR-218 and HMGB1.6.BCP can reverse miR-218 reduction to some extent after ischemic stroke,suggesting that BCP can play a brain protective role by acting on miR-218/HMGB1/RAGE/TLR4 signaling pathways.ConclusionThe results confirmed the protective effect of miR-218 inhibition of HMGB1/TLR4/RAGE signaling,which in turn inhibited inflammation,oxidative stress and apoptosis.Moreover,BCP potentially attenuates ischemic brain injury by activating miR-218 and inhibiting HMGB1/TLR4/RAGE signaling,reducing IL-1β/IL-6/TN F-α expression,inhibiting neuronal death and inflammatory responses.These data suggest that BCP protects against inflammatory injury in ischemic stroke by regulating miR-218/HMGB1/TLR4/RAGE signaling pathways,providing new insights into the therapeutic mechanisms of this treatment protocol.