Activation of the wnt/β-catenin/CYP1B1 pathway alleviates oxidative stress and protects the blood-brain barrier under cerebral ischemia/reperfusion conditions

Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier.However,the potential links between them following ischemic stroke remain largely unknown.The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway.Meanwhile,Wnt/β-catenin pathway activation by the pharmacological inhibito r,TWS119,relieved oxidative stress,increased the levels of cytochrome P4501B1(CYP1B1)and tight junction-associated proteins(zonula occludens-1[ZO-1],occludin and claudin-5),as well as brain microvascular density in cerebral ischemia rats.Moreove r,rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress,suppression of the Wnt/β-catenin pathway,aggravated cell apoptosis,downregulated CYP1B1and tight junction protein levels,and inhibited cell prolife ration and migration.Overexpression ofβ-catenin or knockdown ofβ-catenin and CYP1B1 genes in rat brain mic rovascular endothelial cells at least partly ameliorated or exacerbated these effects,respectively.In addition,small interfering RNA-mediatedβ-catenin silencing decreased CYP1B1 expression,whereas CYP1B1 knoc kdown did not change the levels of glycogen synthase kinase 3β,Wnt-3a,andβ-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivatio n/reoxygenation.Thus,the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling,and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress,increased tight junction levels,and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

Inhibition of the immunoproteasome LMP_(2) ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway

Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2(LMP2)in the pathophysiology of ischemia stroke.However,the relationship between the immunoproteasome LMP2 and the BBB remains unclear.Methods:Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion(MCAO/R).Three days before MCAO,the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region.The rat brain microvascular endothelial cell(RBMVEC)line was exposed to oxygen–glucose deprivation/reperfusion(OGD/R)to mimic ischemic conditions in vitro.The RNA interference-mediated knockdown of LMP2 orβ-catenin was analysed in vivo and in vitro.Analysis of the quantity of extravasated Evans blue(EB)and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB.Immunofluorescence and Western blotting were employed to detect the expression of target proteins.Cell migration was evaluated using a scratch migration assay.The results of immunofluorescence,Western blotting and cell migration were quantified using the software ImageJ(Version 1.53).Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference(LSD)test.Results:Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins[occludin,claudin-1 and zonula occludens(ZO-1)]in the MCAO/R group compared with the sham group(P<0.001).However,inhibition of the immunoproteasome LMP2 restored the expression of these proteins,resulting in higher levels of occludin,claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group(P<0.001).In addition,inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability[e.g.,the quantity of extravasated EB:LMP2-shRNA group(58.54±7.37)μg/g vs.control-shRNA group(103.74±4.32)μg/g,P<0.001],and promoted the upregulation of Wnt-3a andβ-catenin proteins in rats following MCAO/R.In vitro experiments,OGD/R induced marked upregulation of LMP2,proapoptotic protein Bax and cleaved caspase-3,and downregulation of occludin,claudin-1,ZO-1 and Bcl-2,as well as inhibition of the Wnt/β-catenin pathway Wnt-3a andβ-catenin proteins in RBMVECs,compared with the control group under normal culture conditions(P<0.001).However,silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R.Silencing ofβ-catenin by transfection of RBMVECs withβ-catenin-si RNA aggravated the downregulation of tight junction proteins,and reduced the proliferation and migration of RBMVECs following OGD/R,compared with the control-siRNA group(P<0.001).LMP2-si RNA andβ-catenin-si RNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.Conclusions:This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury,and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

Hypolipidemic effects of Alismatis Rhizoma decoction on the lipid profile in hyperlipidemia rats by RNA-sequencing

As a disorder of lipid metabolism,hyperlipidemia(HLP)is characterized by elevated levels of lipids in the blood circulation.It is consistently related to the development of cardiovascular events and diseases associated with metabolic syndrome.Alismatis Rhizoma decoction(ARD),a well-known traditional Chinese medicine prescription,has long been used for treating vertigo,which is a symptom experienced by HLP patients.In this study,we aimed to investigate the hyperlipidemic activity and the potential molecular mechanisms of ARD in HLP rats at the transcriptional level.RNA sequencing and transcriptome analysis were performed collaboratively,including analysis of differentially expressed genes(DEGs),GO functions,and KEGG pathway analysis.The results showed that 1981 DEGs(1370 upregulated and 611 downregulated)were identified in the HFD group compared with the CON group.Moreover,474 DEGs(350 upregulated and 124 downregulated)were detected in the ARD group compared with the HFD group.Furthermore,GO analysis revealed that DEGs were mainly involved in the following functions:developmental process,response to an external stimulus,ion transport,alcohol binding,and plasma membrane part.Pathway analysis suggested that these DEGs were significantly enriched in bile secretion,malaria,cell adhesion molecules,retinol metabolism,the sphingolipid signaling pathway,chemical carcinogenesis,and the T cell receptor signaling pathway.In conclusion,our study demonstrated that ARD alleviated the lipid metabolism disorder caused by HLP through multiple mechanisms,which provided vital scientific evidence for further pharmacological studies of ARD.

Upper limb arthromyodynia can be treated with Notopterygium by down-regulating P2X3 to mediate PKC-induced inflammatory response

Rheumatoid arthritis(RA)is one of the most common refractory diseases in the world,and traditional Chinese medicine Notopterygium(NE)has been used in the treatment of upper limb pain for a long time.NE can significantly reduce the expression of inflammatory pain target P2X3 receptor in rats with upper-limb arthritis.To verify the relationship between the mechanism of NE for“upper limb paralysis”and the P2X3 receptor-mediated PKC inflammatory response pathway,UPLC was taken to measure the exact medicinal substance of ethyl acetate from NE.Sprague Dawley rats were randomly divided into a blank group,a model group,a live-action group,and a positive group.The joint cavity was removed after 21 d.Moreover,a model group,a live group,and a positive group were also set up with RA-FLS cells in our in vitro study.The expressions of P2X3 and PKC inflammation pathway indicators were detected by Western blotting analysis.A P2X3 inhibitor(A-317491)acted on RA-FLS cells,and a model group and a positive group were set.Then the protein expression of PKC was detected.NE reduced the expressions of P2X3,Rab7,PKC,and NF-κB at the protein level in both systems.NE and P2X3 receptor antagonists reduced the expressions of key proteins in the PKC pathway in RA-FLS cells to similar extents,and their effects were not additive.NE could effectively improve the“forelimb pain”of RA rats,with a mechanism closely related to the P2X3/Rab7/PKC/NF-κB pathway.