| Objective:To study a novel brain-targeted rutin derivative for the treatment of ischemic stroke through ACE2/Ang1-7 signal transduction through anti-inflammation.Methods:(1)The lipopolysaccharide(Lipopolysaccharide,LPS)solution with the final concentration of1,25,50,100 ng/m L,and 1 μg/m L was used to activate BV2 and HMC3 microglia by immunoblotting,and the expression of NF-κb-p65 and i NOS were detected by Western blotting.The optimal drug dose for microglia modeling was 1 μg/m L.1 μg/m L LPS was selected to incubate cells for 2,4,6,8,12,and 24 h to obtain the optimal drug action time.The detection method was the same as above.The effect of LPS on cell viability at different concentrations and durations of action,the effect of rutin derivative SS-31-HA-RT(SHR)on the survival rate of BV2 and HMC3 cells and the improvement of LPS-induced survival rate of BV2 and HMC3 cells by SHR were examined by MTT.A nitric oxide(NO)detection kit was used to detect the changes in NO release before and after administration.Flow cytometry and immunofluorescence were used to detect the changes in the reactive oxygen family(Reactive oxygen species,ROS)and CD11 b,and to detect the changes in M1 and M2 types of microglia.Changes in i NOS,TNF-α,HIF-1α,NF-κb-p65,ARG1,and IL-10 at different concentrations were detected by immunoblotting.(2)We then used the transient cerebral ischemia(t MCAO)model to explore the ameliorative effect of SHR on inflammation in the t MCAO model.A transient cerebral ischemia(t MCAO)model was prepared in male SD rats weighing approximately 250 g using a modified Longa wire bolus method.After 2 h of ischemia,each group of drugs was injected into the abdominal cavity and the vessel was reperfused after 1 h.The volume of cerebral infarction was measured 24 h later using 2,3,5-triphenyl tetrazolium chloride(TTC)staining.The expression of TNF-α and HIF-1α in the cerebral cortex of rats after ischemia was detected by immunohistochemistry.The expression of microglia marker in the cerebral cortex of rats after ischemia was detected by immunofluorescence.Immunofluorescence was used to detect the transformation of microglia in the cerebral cortex after ischemia.Immunoblotting was used to detect the changes of pro-inflammatory factors(TNF-α,HIF-1α,IL-6)and anti-inflammatory factors(IL-10,ARG1)in brain tissue homogenates after ischemia.(3)To investigate the mechanism of the anti-inflammatory effect of SHR,we used an ACE2inhibitor(MLN-4760)and a TFEB inhibitor(CCI-779)to explore the changes in inflammatory factors in each group.Immunofluorescence and Western blotting were used to detect the interaction between ACE2 and TFEB in microglia.Primary rat brain microvascular endothelial cells(r BMECs)were co-cultured with BV2 microglia,and the transendothelial resistance(TEER)was detected daily to obtain the blood-brain barrier(Blood-brain-barrier,BBB)model in vitro.Elisa kit was used to detect the effects of SHR on the contents of ACE2,ANG2,and Ang1-7 in separately cultured r BMECs and BV2 cells,as well as in the co-culture system,to sort out how SHR regulates inflammation by affecting intercellular ACE2 signal transduction.After LPS modeling the apparent permeability coefficient(Pa PP)of free diffusion of Na F from the inner chamber to the outer chamber was detected to study the protective effect of SHR on the inflammatory the injury BBB.The MTT method was used to detect the protective effect of SHR on SH-SY5 Y neurons under inflammatory modeling.Flow cytometry was used to detect the effects of drugs on ROS content,mitochondrial membrane potential,and apoptosis rate of SH-SY5 Y cells after the inflammatory injury.The inverted fluorescence microscopy was used to detect the changes of Ki67 expression of SHR after inflammatory injury to verify the protective effect in SHR on inflammatory injured neurons.Results:(1)The results of Western blotting showed that when the concentration of LPS was 1 μg/m L,the expression of NF-κb-p65 and i NOS protein in microglia was higher than that of other concentrations,and the cells were damaged.At the same concentration,the longer the incubation time of LPS,the higher the expression of NF-κb-p65 and i NOS protein and the same obvious damage to cells.Therefore,LPS incubation of 1 μg/m L for 24 h was selected as the model concentration of this experiment.The MTT results showed that SHR was not toxic to BV2 and HMC3 cells and could repair the cell damage caused by LPS modeling.Compared with the model group,the NO release of BV2 cells treated with 25 mmol/L SHR decreased significantly,and the NO release decreased significantly after 50 and 100 mmol/L SHR treatment.In HMC3 cells,compared with the LPS model group,there was no significant change in NO release after25 mmol/L SHR treatment,but NO release decreased significantly after 50 and 100 mmol/L SHR treatment.Flow cytometry and immunofluorescence results showed that compared with the model group,the ROS level of BV2 and HMC3 cells in the treatment group decreased.The expression of CD11 b decreased significantly,indicating that the number of activated microglia decreased.The M1 phenotype of microglia decreased while the M2 phenotype increased after drug incubation,indicating that SHR can promote the polarization transformation of microglia to M2.The changes in inflammatory factors detected by immunoblotting can also prove the good anti-inflammatory effect of SHR.(2)In the ischemic model group,the cerebral infarction volume was 23.91%.While in the SHR treatment group,the cerebral infarction volume was significantly reduced to 6.72%.The results of immunohistochemistry and Western blotting showed that the expression of inflammatory factors in the cerebral cortex increased after ischemia and decreased significantly after SHR administration.The immunofluorescence results showed that SHR could also stimulate the polarization transformation of microglia in the cerebral cortex and reduce the M1 activation rate of microglia.(3)The results of western blotting showed that ACE2 and TFEB could affect the expression of inflammatory factors to some extent,and there might be some relationship between them.Western blotting and immunofluorescence showed that SHR enhanced the fluorescence intensity of ACE2 and promoted the nuclear translocation of TFEB.When the expression of TFEB was inhibited by its inhibitor CCI-779 or si RNA,the expression of ACE2 was decreased correspondingly,and the expression and nuclear translocation of TFEB were significantly decreased by ACE2 inhibitor MLN-4760.The Elisa method also verified the same results in BV2 and r BMECs cells cultured alone.Interestingly,in the co-culture system,SHR can promote r BMECs to secrete more ACE2.ACE2 degrades ANG2 produced by BV2 cells,while the degradation product Ang1-7 can act on r BMECs again,providing more protection for r BMECs cells.It is verified that SHR plays a neuroprotective role in the co-operation of r BMECs and microglia through ACE2 and TFEB signals.In the blood-brain barrier model in vitro,according to the determination of Pa PP,we can see that SHR reduced the permeability of BBB and maintained its integrity.In the in vitro cell experiment of SH-SY5 Y,SHR can improve the changes in apoptosis rate,ROS,and facilitate cell proliferation,which further verified the protective effect of SHR on neurons.Conclusions:(1)SHR can significantly reduce the expression of inflammatory factors in microglia and regulate the polarization process and phenotypic changes.(2)SHR significantly reduced the volume of cerebral infarction and neurological damage in t MCAO rats,reduced the inflammation caused by ischemic stroke,and could change the microglia in the cerebral cortex from pro-inflammatory M1 type to anti-inflammatory M2 type.(3)SHR can promote r BMECs cells to secrete more ACE2 to act on BV2 cells,and its degradation product Ang1-7 can provide favorable conditions for r BMECs cells.The blood-brain barrier could be strengthened by SHR treatment,which lays a good foundation for SHR to repair the inflammatory injury and plays a great neuroprotective role in ischemic stroke. |
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