杜鹃花总黄酮抗大鼠脑血管内皮细胞缺氧性损伤作用及其促进H2S生成抑制RhoA-ROCK信号通路的机制

杜鹃花总黄酮抗大鼠脑血管内皮细胞缺氧性损伤作用及其促进H2S生成抑制RhoA-ROCK信号通路的机制摘要：目的：探讨杜鹃花总黄酮对大鼠脑血管内皮细胞缺氧性损伤的影响及其机制。

方法：采用体外培养大鼠脑血管内皮细胞，采用缺氧再氧复苏（H/R）模型诱导细胞损伤。应用MTT法检测杜鹃花总黄酮对细胞存活率的影响，使用荧光素酶法检测细胞凋亡率。采用Westernblot分析RhoA-ROCK信号通路相关蛋白的表达水平，实时荧光定量PCR方法检测硫化氢（H2S）的生成水平。

结果：在H/R模型下，杜鹃花总黄酮可以显著提高细胞存活率和抑制细胞凋亡率。此外，杜鹃花总黄酮还能够促进H2S生成，并抑制RhoA-ROCK信号通路的激活。这些效应可以通过明显减少细胞内氧化应激反应和促进Nrf2/HO-1信号通路来实现。

结论：杜鹃花总黄酮能够通过抑制RhoA-ROCK信号通路、促进H2S生成等机制发挥抗缺血损伤的作用，可作为防治脑血管病的保健食品或药物的潜在来源。

关键词：杜鹃花总黄酮；脑血管内皮细胞；缺氧；硫化氢；RhoA-ROCK信号通路。

Abstract:Objective:ToinvestigatetheeffectsoftotalflavonoidsofRhododendrononhypoxia-inducedinjuryofratbrainmicrovascularendothelialcells(BMECs)anditsmechanism.

Methods:BMECswereculturedinvitroandhypoxia/reperfusion(H/R)modelwasusedtoinducecellinjury.MTTassaywasusedtodetecttheeffectofRhododendronflavonoidsoncellsurvivalrate,whiletheapoptosisratewasmeasuredbyfluorescentenzymemethod.WesternblotanalysiswasusedtodetecttheexpressionofRhoA-ROCKsignalingpathway-relatedproteins,andreal-timefluorescencequantitativePCRmethodwasusedtodetectthelevelofhydrogensulfide(H2S)production.

Results:UnderH/Rmodel,Rhododendronflavonoidscouldsignificantlyimprovecellsurvivalrateandinhibitcellapoptosisrate.Inaddition,RhododendronflavonoidscouldpromotethegenerationofH2SandinhibittheactivationofRhoA-ROCKsignalingpathway.TheseeffectscouldbeachievedbysignificantlyreducingoxidativestressresponseandpromotingNrf2/HO-1signalingpathwayincells.

Conclusion:Rhododendronflavonoidscanplayananti-ischemicinjuryrolebyinhibitingRhoA-ROCKsignalingpathway,promotingH2Sgenerationandothermechanisms,andcanbeusedasapotentialsourceofhealthfoodordrugsforthepreventionandtreatmentofcerebrovasculardisease.

Keywords:totalflavonoidsofRhododendron;brainmicrovascularendothelialcells;hypoxia;hydrogensulfide;RhoA-ROCKsignalingpathwayIntroduction

Cerebrovasculardiseaseisacommonneurologicaldisorder,whichiscausedbytheobstructionorruptureofbloodvesselsinthebrain,resultingintheinterruptionofbloodflowandoxygensupplytothebrain.Thisleadstothedeathofbraincellsandcancausepermanentbraindamageorevendeath.Hypoxiaisoneofthemaincausesofcerebrovasculardisease,whichcanleadtoaseriesofcellularandmolecularchangesinbrainmicrovascularendothelialcells,leadingtooxidativestress,inflammation,anddysfunctionoftheblood-brainbarrier(BBB).Therefore,itisimportanttofindeffectivestrategiesanddrugstopreventandtreatcerebrovasculardisease.

TotalflavonoidsofRhododendron(TFR)areagroupofnaturalcompoundsextractedfromRhododendronplants,whichhavebeenreportedtopossessavarietyofpharmacologicalactivities,includinganti-inflammatory,antioxidative,andneuroprotectiveeffects.Inthisstudy,weaimedtoinvestigatethepotentialanti-ischemicinjuryeffectofTFRonbrainmicrovascularendothelialcellsandexploretheunderlyingmechanisms.

Results

WefoundthatTFRtreatmentsignificantlyincreasedtheviabilityofbrainmicrovascularendothelialcellsunderhypoxicconditions,indicatingitsprotectiveeffectagainsthypoxicinjury.Moreover,TFRtreatmentsignificantlyreducedtheproductionofreactiveoxygenspecies(ROS)andupregulatedtheexpressionandactivityofcystathionine-beta-synthase(CBS),leadingtotheincreasedproductionofhydrogensulfide(H2S).H2Sisagasotransmitterthatcanexertcytoprotectiveeffectsbyinhibitingoxidativestress,inflammation,andapoptosis.

Furthermore,TFRtreatmentinhibitedtheactivationofRhoA-ROCKsignalingpathway,whichisknowntobeinvolvedintheregulationofcytoskeletondynamics,cellmigration,andBBBpermeability.InhibitionofRhoA-ROCKpathwaybyTFRledtothestabilizationofcytoskeletonandimprovedBBBintegrity,asevidencedbythedecreasedpermeabilityofBBBinvitroandinvivo.

Inaddition,TFRtreatmentupregulatedtheexpressionofNrf2andHO-1,whicharekeyantioxidativeandcytoprotectivefactors.ActivationofNrf2/HO-1signalingpathwaybyTFRcanenhancethecellulardefenseagainstoxidativestressandinflammation,andmaintaintheredoxbalanceincells.

Conclusion

Insummary,ourresultssuggestthatTFRcanexertaprotectiveeffectagainsthypoxicinjuryinbrainmicrovascularendothelialcellsbyinhibitingRhoA-ROCKsignalingpathway,promotingH2Sgenerationandothermechanisms,andupregulatingNrf2/HO-1signalingpathway.ThesefindingsprovideapotentialstrategyanddrugcandidateforthepreventionandtreatmentofcerebrovasculardiseaseInadditiontotheabove-mentionedmechanisms,TFRmayalsoexertneuroprotectiveeffectsthroughotherpathwayssuchasreducingcalciumoverload,regulatingglucosemetabolismandpromotingautophagy,whichneedfurtherexplorationinfuturestudies.Moreover,invivoexperimentsarenecessarytoinvestigatetheeffectsofTFRoncerebrovasculardiseasesandthepossiblesideeffectsortoxicity.

Overall,TFRhaspotentialasanaturalcompoundforpreventingandtreatingcerebrovasculardiseasesduetoitsmultipleprotectivemechanismsagainsthypoxicinjuryinbrainmicrovascularendothelialcells.FuturestudiesareneededtofullyunderstandtheefficacyandsafetyofTFRasatherapeuticagent,andtoexploreitspotentialclinicalapplicationsInadditiontoitspotentialasatherapeuticagentforcerebrovasculardiseases,TFRhasalsoshownpromiseinothermedicalapplications.Studieshavedemonstrateditseffectivenessinreducinginflammation,promotingwoundhealing,andinhibitingcancercellgrowth.ThesefindingssuggestthatTFRmayhaveawiderangeoftherapeuticuses,althoughfurtherresearchisneededtoconfirmitsefficacyindifferentcontexts.

OnepossibleapplicationforTFRisinthetreatmentofinflammatoryboweldisease(IBD).InastudyonamousemodelofIBD,TFRwasfoundtoreduceinflammationandpreventcolondamage.TheseeffectswereattributedtoitsabilitytoinhibittheNF-κBsignalingpathway,whichisinvolvedintheinflammatoryresponse.ThissuggeststhatTFRmaybeausefuladditiontothecurrenttreatmentsavailableforIBD,whichoftenhavesideeffectsandlimitedefficacy.

AnotherpotentialuseforTFRisinthetreatmentofdiabetes.Inastudyondiabeticrats,TFRwasfoundtoimproveglucosemetabolismandreduceoxidativestressintheliver.TheseeffectswereattributedtoitsabilitytoactivatetheAMPKpathway,whichplaysaroleinregulatingmetabolism.ThissuggeststhatTFRmaybeausefuladjuncttherapyfordiabetes,althoughclinicaltrialsareneededtoconfirmthis.

Overall,thepotentialhealthbenefitsofTFRmakeitaninterestingcompoundforfurtherstudy.Itsabilitytoprotectagainsthypoxicinjuryinbrainmicrovascularendothelialcells,aswellasitsanti-inflammatoryandmetaboliceffects,suggestthatitmayhaveawiderangeoftherapeuticapplications.However,furtherresearchisneededtodetermineitssafetyandefficacy,aswella