Biochanin A,as the Lrg1/TGF-β/Smad2 pathway blockade,attenuates blood-brain barrier damage after cerebral ischemiareperfusion by modulating leukocyte migration patterns

Background:Biochanin A is an excellent dietary isoflavone that has the concomitant function of both medicine and foodstuff.The attenuation function of biochanin A on blood-brain barrier(BBB)damage induced by cerebral ischemia-reperfusion remains unclear.Methods:C57BL/6 mice were subjected to 1 h middle cerebral artery occlusion(MCAO)followed by 24 h reperfusion.The infarct volume of the brain was stained by TTC,while leakage of the brain was quantitatively stained by Evans blue,and the neurologic deficit score was measured.Microglial-induced morphologic changes were observed via immunofluorescence staining,and rolling and adhering leukocytes in venules were observed via two-photon imaging,while the inner fluorescein isothiocyanate-albumin of venules were compared with those of surrounding interstitial area through venular albumin leakage.Results:The attenuation effect of biochanin A on tight junction injury was compared in ischemia-reperfusion mice or conventional knockdown of leucine-richα2-glycoprotein 1(Lrg1)mice.Biochanin A could ameliorate BBB injury in mice with cerebral ischemiareperfusion in a dose-dependent manner by strengthening the immunostaining volume of occludin,claudin-5,and zonula occludens-1.The amoeba morphologic changes of microglial combined with the elevated expression of Lrg1 could be relieved under the treatment of biochanin A.Biochanin A played a countervailing role on the rolling leukocytes in the vessel,while the leakage of blood vessels was reduced.Biochanin A diminished its functions to further improved attenuation for tight junction injury on conventional Lrg1-knockout mice,as well as the inhibition effects on TGF-β1,and the phosphorylation of suppressor of mothers against decapentaplegic 2(Smad2)/Smad2 via western blot assay.Conclusion:Biochanin A could alleviate tight junction injury induced by cerebral ischemiareperfusion and blocked the Lrg1/TGF-β/Smad2 pathway to modulate leukocyte migration patterns.

Exploring the attenuation mechanisms of Dalbergia odorifera leaves extract on cerebral ischemia-reperfusion based on weighted gene co-expression network analysis

The attenuation function of Dalbergia odorifera leaves on cerebral ischemia-reperfusion(I/R)is little known.The candidate targets for the Chinese herb were extracted from brain tissues through the high-affinity chromatography.The molecular mechanism of D.odorifera leaves on cerebral I/R was investigated.Methods:Serial affinity chromatography based on D.odorifera leaves extract(DLE)affinity matrices were applied to find specific binding proteins in the brain tissues implemented on C57BL/6 mice by intraluminal middle cerebral artery occlusion for 1 h and reperfusion for 24 h.Specific binding proteins were subjected to mass-spectrometry to search for the differentially expressed proteins between control and DLE-affinity matrices.The hub genes were screened based on weighted gene co-expression network analysis(WGCNA).Then,predictive biology and potential experimental verification were performed for the candidate genes.The protective role of DLE in blood-brain barrier damage in cerebral I/R mice was evaluated by the leakage of Evans blue,western blotting,immunohistochemistry,and immunofluorescent staining.Results:952 differentially expressed proteins were classified into seven modules based on WGCNA under soft threshold 6.Based on WGCNA,AKT1,PIK3CA,NOS3,SMAD3,SMAD1,IL6,MAPK1,TGFBR2,TGFBR1,MAPK3,IGF1R,LRG1,mTOR,ROCK1,TGFB1,IL1B,SMAD2,and SMAD518 candidate hub proteins were involved in turquoise module.TGF-β,MAPK,focal adhesion,and adherens junction signaling pathway were associated with candidate hub proteins.Gene ontology analysis demonstrated that candidate hub proteins were related to the TGF-βreceptor signaling pathway,common-partner SMAD protein phosphorylation,etc.DLE could significantly reduce the leakage of Evans blue in mice with cerebral I/R,while attenuating the expression of occludin,claudin-5,and zonula occludens-1.Western blotting demonstrated that regulation of TGF-β/SMAD signaling pathway played an essential role in the protective effect of DLE.Conclusion:Thus,a number of candidate hub proteins were identified based on DLE affinity chromatography through WGCNA.DLE could attenuate the dysfunction of bloodbrain barrier in the TGF-β/SMAD signaling pathway induced by cerebral I/R.

Study on screening potential allergenic proteins from infant milk powders based on human mast cell membrane chromatography and histamine release assays

Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic(CMC)method based on human mast cells(HMC-1) for screening potential allergens in infant formula milk powders(IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns(10 mm ? 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic(RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.