L-camphor Through The Apparent Regulation Of Stress Particles To Produce Anti-stroke Injury Research

Objective:Clinical treatment of acute ischemic stroke has lacked ineffective treatment.How to inhibit the necrosis of brain tissue in acute ischemic stroke and the level of apoptosis are the key to treatment and recovery.The production of stress granule can inhibit the production of apoptosis and can be regulated by the epigenetic mechanism.In the preliminary study of our team,it was found that L-Camphor,the active component of XingNaoJing injection,was helpful for acute ischemic stroke.Therefore,this paper studies the mechanism of the miR-335 process,which regulated by RNA methylation epigenetic and the effect of L-Camphor to protect brain tissue from acute ischemic stroke.This paper can promote the development of Chinese medicine theory,provide a theoretical basis for clinical prevention and treatment of stroke,and build a foundation for a new generation of new anti-stroke drugs.Methods:1.In the first chapter,the protective mechanism of L-Camphor against acute ischemic stroke injury was studied by using the MCAO model and PC12 serum-free model.(1)The MCAO model was constructed,and miRNA gene chip was used to analyze specifically expressed miRNAs,which were verified by RT-qPCR.(2)tail vein injection of L-camphor was used to intervene in the rat MCAO model,and the expression of cerebral infarction volume,apoptosis level,METTL3 protein and pri-pre-mature miR-335 in each group were detected by TTC staining,TUNEL,western blotting,and rt-qPCR.(3)in PC12 cell OGD/R model,multiple active components of xingnaojing injection were added,and the expression of TIA1 in each group was detected by flow cytometry,so as to test the effect of xingnaojing injection active components on the generation of stress granule.(4)PC12 cell OGD/R model was used to detect the apoptosis level,stress granulation,METTL3 protein expression and pri-pre-mature miR-335 expression of each group by flow cytometry,immunofluorescence double-labeling,western blotting and RT-qPCR.(5)in PC12 cell OGD/R model,multiple effective components of xingnaojing injection were added to detect the expression of RBM3 mRNA in each group by RT-qPCR.(6)in the OGD/R model of PC12 cells,RBM3 expression level and pri-miR-335 methylation level in the nucleus were detected by western blotting and MeRIP experiments.2.In the second chapter,the mechanism of mir-335 promoting the generation of stress particles and protecting brain damage was studied by using the MCAO model and PC12 serum-free model.(1)TTC staining,immunohistochemical method,and TUNEL method were used to detect the cerebral infarction volume,stress granule generation and apoptosis levels in the MCAO model at different time points of reperfusion.(2)with lateral ventricle injection of miR-335 mimic and inhibitor in the MCAO model,we used TTC staining,TUNEL method,immunohistochemistry and western blotting and immune precipitation test different groups(normal group,model group,miR-335 mimic and miR-335 inhibitor groups)to test the cerebral infarction volume,level of apoptosis,stress granule formation,the related protein expression(BCL-2,active caspase 3,ROCK2 and TIA1)and TIA1 phosphorylation level;(3)the target of miR-335 was predicted through the biological information website,and the target protein was verified by the dual luciferase reporter.(4)miR-335 mimics and inhibitors were transfected into PC12 cells by lipo2000,and models were constructed by serum-free stimulation.Immunofluorescence,western blotting and flow cytometry were used to detect the inhibitory effect of miR-335 on the target protein ROCK2 and its influence on the generation of stress granule.(5)the siRNA of ROCK2 was designed and transfected into PC12 cells with lipos2000 to study the target specificity of miR-335 and ROCK2.3.In the third chapter,the molecular mechanism of miR-335 expression decline in acute ischemic stroke was studied by using the MCAO model and PC12 cell OGD/R model.(1)the MCAO model was constructed.TTC staining,TUNEL staining,western blotting,RT-qPCR and immunofluorescence methods were used to detect the cerebral infarction volume,apoptosis level,METTL3 expression,pri-pre-mature-335 expression and the generation of stress granule in the MCAO model at different time points of reperfusion.(2)the OGD/R model of PC12 cells and primary rat cortical neurons was constructed.The generation of stress granule,the expression of METTL3,pri-pre-mature 335 and the level of apoptosis was detected by immunofluorescence double-labeling,western blotting,RT-qPCR and flow cytometry at different glucose and oxygen recovery time points in the cell model.(3)the pri-mir-335 methylation level of PC12 cells stimulated by OGD/R at different glucose and oxygen recovery time points was detected by MeRIP array.(4)CRISPR-Cas9 technology was used to knock out.Changes in pri-mir-335 methylation level and pri-pre-mature-335 expression level after knockout/overexpression were detected by MeRIP method and RT-qPCR.(5)the target of miR-335 was predicted through the biological website,and the target protein was verified by the dual luciferase reporter test.(6)miR-335 mimics and inhibitors were transfected with lipo2000,and the expression of target protein Erfl was detected by western blotting,to verify the targeting.(7)RNAi technology knocked down the expression of Erfl gene in PC12 cells,and the generation of stress granule and the level of apoptosis were detected by immunofluorescence double-labeling and flow cytometry.(8)RBM3 tissue specificity and expression specificity at MCAO were detected by RT-qPCR.(9)the interaction between METTL3 and RBM3 in the nucleus was detected by co-IP experiment,and the pri-mir-335 methylation level was detected by MeRIP experiment.(10)the binding rates of METTL3 and RBM3 to miR-335 promoter regions were detected by ChIP-qPCR.(11)RNA interference experiment of RBM3 was conducted on PC12 cells by siRNA,and the binding rate of METTL3 and miR-335 promoter region was detected by ChIP-qPCR.(12)the expression levels and methylation levels of pri-mir-335 in rbm3-overexpression and RBM3-siRNA were detected by RT-qPCR and MeRIP methods.Results:1.The experimental results in chapter 1 showed that:(1)miRNA gene microarray was used to detect specifically expressed miRNAs in the ischemic cortex of rats and was verified by rt-qPCR.The results showed that mir-335 was decreased in MCAO rats.(2)compared with the model group,the cerebral cortex infarction area and apoptosis level of the MCAO model rats were significantly reduced by L-camphor.(3)compared with the model group,L-camphor significantly reduced the apoptosis level of cerebral cortex in MCAO model rats.(4)compared with the model group,the expression of METTL3 in the l-camphor group increased.(5)compared with the model group,the expression of mir-335 in the cerebral cortex of the L-camphor group was significantly increased,while the expression of pri-mir-335 was decreased.(6)among the effective components of xingnaojing injection,levorotatory camphor has the most obvious promoting effect on cell TIA1 expression.(7)compared with the model group,L-camphor reduced the apoptosis level of OGD/R damaged cells,increased the formation of stress particles in damaged cells,and increased the expression of METTL3.(8)compared with the model group,the expression of miR-335 in the L-camphor group was significantly increased,while the expression of pri-mir-335 was decreased.(9)RT-qPCR results showed that RBM3 mRNA expression level was significantly increased in the L-camphor group compared with the model group.Western blotting results showed that nuclear RBM3 protein expression was significantly higher in the L-camphor group than in the model group.MeRIP experiment showed that compared with the model group,the methylation level of pri-miR-335 in the L-camphor group was increased.2.It was found in the second chapter that:(1)the cerebral infarction area was the highest at 24h after ischemia/reperfusion,and decreased at 36h after ischemia/reperfusion;SG formation was most obvious after ischemia-reperfusion for 6h,while SG formation gradually decreased after 24h and increased after 36h.Apoptosis peaked 24h after reperfusion and decreased 36h after reperfusion.(2)by injecting mir-335 mimics and inhibitors into the lateral ventricle,TTC staining results showed that compared with the model group,infarct volume,and behavior score in the mimics group decreased significantly.(3)compared with the model group,the expressions of ROCK2 and caspase-3 in the miR-335 mimic group were significantly decreased,and the expressions of TIA1 and BCL-2 increased after the detection of western blotting and TUNEL apoptosis.TUNEL staining was used to detect apoptosis in frozen sections.Compared with the model group,mir-335 mimic significantly reduced the level of ischemic cerebral apoptosis.(4)immunohistochemieal results showed that miR-335 mimic significantly increased TIA1 expression compared with the model group.5)TIA1 phosphorylation was detected by immunoprecipitation in rat cerebral cortex.TIA1 phosphorylation levels were increased in the model and inhibitor groups compared with the control group.Also,the miR-335 mimic group significantly inhibited TIA1 phosphorylation.(6)dual luciferase reporter results showed that mir-335 directly targeted the 3' UTR of ROCK2 mRNA in PC12 cells.(7)through the transfection of miR-335 mimic and inhibitor in PC12 cells,it was found that compared with the model group,ROCK2 expression was significantly decreased and TIA1 expression was significantly increased in the miR-335 mimics group.The results of immunofluorescence staining showed that mir-335 mimics significantly promoted the generation of SG.(8)the expression of ROCK2 was inhibited by siRNA,and the results showed that compared with the model group,the expression of ROCK2 in the siRNA-rock2 group was significantly decreased,the expression of TIA1 was significantly increased,and the formation of SG was increased.The results of flow cytometry showed that the apoptosis level of the siRNA-ROCK2 group was significantly lower than that of the model group.(9)siRNA-ROCK2 group significantly inhibited the expression of ROCK2 and increased the expression of TIA1.There was no significant difference in the expression of ROCK2 and TIA1 between the siRNA-ROCK2 group and the co-transfection group.Compared with the model group,SG formation was significantly increased in the co-transfection group and the siRNA-ROCK2 group,but there was no statistically significant difference between the two groups.(10)compared with the model group,the co-transfection and siRNA-ROCK2 groups significantly inhibited the expression of ROCK2,and the expression of TIA1 was increased in the two groups.Cell apoptosis was detected by flow cytometry.Compared with the model group,apoptosis levels were significantly down-regulated in the co-transfection group and the siRNA-ROCK2 group,and there was no statistically significant difference between the two groups.These results indicated that miR-335 specifically targeted 3'-UTR of ROCK2,thereby decreasing ROCK2 expression,promoting SG formation and inhibiting apoptosis.3.According to the experimental study in chapter 3,(1)the cerebral infarction area was the highest at 24h after ischemia-reperfusion and decreased to some extent at 36h after ischemia-reperfusion;(2)the apoptosis rate of ischemic cortex was the highest at 24h after ischemia reperfusion and decreased to some extent at 36h after ischemia reperfusion.After ischemia reperfusion for 6h,the expression of METTL3 was most significantly increased,then gradually decreased,and slightly increased at 36h after ischemia reperfusion.(3)the expression of mature-miR-335 increased at 0h and 6h,and then gradually decreased to the lowest value at 24 h after reperfusion,while the expression of pri-miR-335 and pre-miR-335 was inversely proportional to the expression of mature-miR-335,decreased at 0h and 6h,and then gradually increased.(4)the generation of stress granule was observed at 6h after ischemia reperfusion,while the generation of stress particles was not observed at other time points.(5)the experimental results showed that in the rat MCAO model,the expression level of METTL3 was proportional to the maturity and processing of miR-335.(6)the experimental results showed that the generation level of stress particles in primary cortical neurons and PC12 cells was the highest at OGD/R 0h,and gradually decreased after 0h.(7)western blotting results of METTL3 with different glucose and oxygen recovery time in PC12 cells showed that the expression of METTL3 was the highest at OGD/R 0h,then gradually decreased,and slightly increased at 36h after ischemia/reperfusion.The expression of mature mir-335 increased at 0h and 6h,and then gradually decreased to the lowest value at 24 h after reperfusion,while the expression of pri-miR-335 and pre-miR-335 was opposite to the expression trend of mature miR-335,decreased at 0h and 6h,and then gradually increased.(8)flow cytometry showed that the apoptosis level of PC12 cells was the lowest at OGD/R 0h,and gradually increased after 0h.(9)the results showed that the pri-miR-335 methylation level of normal PC12 cells was the highest at OGD/R 0h,decreased gradually after 0h,and slightly increased at 36h.(10)the expression levels of mature miR-335 and pre-miR-335 in PC12 cells of METTL3 KO were significantly decreased,while the expression levels of pri-miR-335 were increased.The expression levels of mature mir-335 and pre-miR-335 in PC12 cells of METTL3 OE were significantly increased,while the expression levels of pri-miR-335 were decreased.The pri-miR-335 methylation level was increased in PC12 cells of METTL3 OE.(12)dual luciferase reporter gene results confirmed that miR-335 directly targeted the 3' UTR of Erfl mRNA in PC12 cells.(13)the experimental results showed that Erfl protein expression in PC12 cells transfected with miR-335 mimic was significantly decreased compared with the normal control group,which verified the experimental results of mir-335 targeting the regulation of Erfl protein expression.(14)the experimental results showed that compared with the normal PC12 cells,the stress granule production level of PC12 cells with Erfl silenced increased at R0h and R24h.(15)compared with control PC12 cells,the apoptosis rate of PC12 cells which the Erfl protein expression was knocked down was significantly reduced at R0h and R24h.(16)the results showed that the interaction between RBM3 and pri-miR-335 increased after 6h of OGD/R stimulation and the recovery of OGD for 0h in PC12 cells,and decreased after the recovery of OGD/R for 24h.(17)the results showed that the expressions of RBM3 and METTL3 were increased at OGD/R 0h,while the expressions of RBM3 and METTL3 were decreased at OGD/R 24h.Protein inununoprecipitation showed that binding of RBM3 to METTL3 was enhanced at OGD/R 0h,while binding of RBM3 to METTL3 was weakened at OGD/R 24h.(18)rt-qPCR of RBM family proteins in different tissues of rats showed that RBM3 was highly expressed in cerebral cortex of rats(slightly lower than heart and liver tissues).Compared with other RBM family proteins,RBM3 expression in the cerebral cortex of MCAO model rats was significantly decreased.(19)the silencing and overexpression experiments of RBM3 were conducted in PC12 cells,and the results of western blotting showed that the silencing and overexpression were successful.Compared with the normal group,the expression level of RBM3 was significantly different.PC12 cells with RBM3 silencing/overexpression were used for chromatin immunoprecipitation to detect the changes in the interaction between RBM3 silencing/overexpression and miR-335 promoter region.The results showed that the binding between RBM3 silencing group and miR-335 promoter region was significantly reduced compared with the RBM3 overexpression group.The results showed that compared with the RBM3 overexpression group,the expression level of pri-miR-335 in the RBM3 silence group was significantly increased,while the expression level of mature miR-335 was significantly decreased.Meanwhile,this section tested the pri-miR-335 methylation level of each group through the MeRIP experiment.The experimental results showed that,compared with the normal control group,the pri-miR-335 methylation level of the RBM3 overexpression group was significantly increased,while the pri-miR-335 methylation level of the RBM3 silent group was significantly decreased.Conclusion:1.miR-335 regulates the phosphorylation level of TIA1 by regulating the expression of target protein ROCK2,to promote the generation of stress granule and reduce the level of apoptosis,playing a protective role in acute ischemic stroke injury.2.RBM3 and METTL3 interact in the nucleus to promote pri-miR-335 methylation and affect the generation of miR-335.In addition,RBM3 can bind with METTL3 to the promoter region of miR-335 and regulate the pri-miR-335 methylation level.3.L-camphor increased the expression of miR-335,which promoted the generation of stress particles and had the effect of anti-acute ischemic stroke injury.L-camphor promoted the expression of miR-335 by up-regulating the expressions of METTL3 and RBM3 and increasing the methylation level of pri-miR-335.