Biological Activity Study Of SAC-Gallic Acid Conjugates On Ischemic Stroke

Research purposes:Ischemic Stroke?IS?is an acute cerebrovascular disease.It is ischemic necrosis of localized brain tissue caused by blood circulation disorder,ischemia and hypoxia in the brain and the second leading cause of death and disability in the world.Therefore,it is particularly urgent to find new drugs to prevent and treat ischemic stroke.?1?In this study,a series of cysteine analogs and SAC-Gallic acid conjugates were synthesized,and the best protective effect methyl S-?4-fluorobenzyl?-N-?3,4,5-trimethoxybenzoyl?-L-cysteinate?MTC?was selected by establishing an in vitro ischemia-reperfusion induced PC12 cells damage model;?2?Through the model of PC12 cells ischemia reperfusion,the protective mechanism and molecular mechanism were discussed;?3?Established a rat middle artery occlusion?MCAO?model in vivo to further verify its specific action pathway and mechanism of protective and anti-inflammatory effects on stroke;?4?Combined with previous work in the laboratory:FGFR1 improved pain perception by regulating T-type Ca²⁺channels that played an important role in pain transmission,and pain research induced by the proteasome inhibitor bortezomib,prompting us to further design and develop FGFR1antagonists,proteasome agonist and MTC conjugates,as new anti-inflammatory,analgesic and neuroprotective multi-target drugs.Methods:The cysteine analogs and SAC-Gallic acid conjugates was designed and synthesized using the principle of medicinal chemical hybridization.Rat PC12 cells and Kunming suckling mouse neurons were cultured in vitro to simulate nerve cell ischemia model.There were blank control group,ischemic group and ischemic post-dosing group?0.1,0.3,1,3?M?.The best compound MTC is selected by the CCK-8cell activity assay;PC12 cells were injured by ischemia-reperfusion,the changes of cell karyotype after MTC treatment were detected by Hoechst 33258 method and the apoptosis was detected by FITC/PI method;Detected the corresponding changes of LDH,SOD,CAT and GPx in cells by LDH,SOD,CAT and GPx kits and detected MTC effect on PC12 cells apoptosis and antioxidant enzymes;Western blot analysis was performed to analyze the expression levels of PI3K,p-Akt,cleaved caspase-9,cleaved caspase-3,bax,bcl-2,p-erk and other proteins after ischemia-reperfusion induced PC12cell injury by MTC,as well as the effect of PI3K/AKT inhibitor LY294002 pretreatment on the expression levels of PI3K,p-Akt;TREK1 plasmid was transfected into COS7cells,and the effect of TREK1 current density was observed after MTC was added;A model of middle artery occlusion was established in rats,and SD rats were randomly divided into three groups:sham operation group?sham?,model group?vehicle?,and MTC treatment group.HE staining was used to detect the effect of MTC on the hippocampus of rat brain;Detected the anti-inflammatory activity of MTC in Raw264.7 cells by Griess method.Results:It was revealed that MTC is the most effective compound.In the comparison of similar compounds,seven types of hydrogen sulfide donor compounds can play the role of protecting ischemic nerve cells.Among them,MTC showed good effect at low concentration?1?M?;Hoechst 33258 results showed no obvious shrinkage and rupture of the nuclei after MTC treatment,and Annexin V-FITC/PI further confirmed the anti-apoptotic effect of MTC;Ischemia-reperfusion increased the number of PC12 cells apoptosis,LDH levels were significantly increased,LDH was significantly reduced in the MTC post-treatment group;MTC post-treatment increased SOD and decreased ROS activity in PC12 cells after ischemia-reperfusion induced injury,and played an antioxidant role,the activity of CAT and GPx increased to clear the peroxides in PC12 cells;MTC post-treatment increased the expression of PI3K,p-AKT,p-ERK protein levels,and PI3K inhibitor LY294002 would inhibit the effect of MTC;MTC post-treatment reduced the expression of cleaved caspase-3 protein level,inhibited the activation of apoptotic cascade to protect a large number of PC12 cells from ischemia-reperfusion injury;MTC post-treatment increased the expression of bcl-2,decreased the expression of bax,and reduced the apoptosis of PC12 cells;Meanwhile,MTC can significantly reduce the current density of the heterologously expressed TREK1 channel;HE staining results in the hippocampus of rats can be seen,compared with the blank control group,the gap between the cells widened,the characteristic of apoptosis was obvious,and the number of neurons decreased;Compared with ischemia,the intercellular space was shortened and the number of neurons increased in the dosing group.;Griess method revealed that the anti-inflammatory activity of MTC was much better than SAC?IC50>150?M?and GA?69.25±4.22?.Conclusions:1.We synthesized and screened the best compound:SAC-Gallic acid conjugates methyl S-?4-fluorobenzyl?-N-?3,4,5-trimethoxybenzoyl?-L-cysteinate?MTC?,compared with previous H₂S donors,our results showed that low-dose MTC has a better protective effect on ischemic neurons;2.MTC had neuroprotective effect,and its mechanism was related to antioxidant and activation of PI3K/AKT and MEK/ERK pathways,inhibition of mitochondrial apoptosis signaling pathways,and reduction of endoplasmic reticulum stress and increased cell survival.3.MTC played a protective role in ischemic neurons by regulating TREK1 double-hole potassium channels.4.At the same time,our laboratory revealed that MTC has anti-inflammatory effects.Although the effect of MTC on inflammatory factors and labor pain in cerebral ischemic rats needs to be further studied,the above results indicated that MTC has both ischemic neuron protection and ischemic anti-inflammatory effects.