藏紅花苷緩解血管緊張素Ⅱ誘導(dǎo)的大鼠血管平滑肌細(xì)胞的炎癥反應(yīng)的研究

藏紅花苷緩解血管緊張素Ⅱ誘導(dǎo)的大鼠血管平滑肌細(xì)胞的炎癥反應(yīng)的研究摘要：本研究旨在探究藏紅花苷（Crocin）對血管緊張素Ⅱ（AngⅡ）誘導(dǎo)的大鼠血管平滑肌細(xì)胞（VSMCs）炎癥反應(yīng)的影響。采用CCK-8法評價(jià)Crocin的毒性，并通過Westernblot法檢測其對VSMCs中炎癥相關(guān)蛋白IL-1β、IL-6、TNF-α和NF-κB、信號通路PI3K/AKT、MAPK和JNK表達(dá)的影響。結(jié)果表明，Crocin能夠顯著降低AngⅡ誘導(dǎo)的VSMCs的IL-1β、IL-6和TNF-α表達(dá)水平，并減少NF-κB、PI3K/AKT、MAPK和JNK的激活。同時(shí)，Crocin還可以顯著改善AngⅡ誘導(dǎo)的VSMCs的增殖和遷移能力，提示Crocin具有抗炎和抗增殖遷移的作用。因此，本研究結(jié)果表明Crocin可能是治療血管炎癥反應(yīng)的有前途的新藥。

關(guān)鍵詞：藏紅花苷；血管緊張素Ⅱ；血管平滑肌細(xì)胞；炎癥反應(yīng)；信號通路

Introduction：

血管炎癥反應(yīng)在多種心血管疾病的發(fā)展中起著重要作用。血管平滑肌細(xì)胞是血管的重要組成部分，其在炎癥反應(yīng)中的作用備受關(guān)注。其中，血管緊張素Ⅱ（AngⅡ）是引起血管炎癥反應(yīng)的重要因素之一。研究發(fā)現(xiàn)，AngⅡ能夠刺激VSMCs產(chǎn)生多種炎癥因子，并促進(jìn)細(xì)胞增殖、遷移和纖維化。因此，探究針對AngⅡ誘導(dǎo)的VSMCs炎癥反應(yīng)的藥物具有重要的臨床意義。

藏紅花苷是從藏紅花中提取的活性成分，具有多種生物活性。有研究表明，藏紅花苷能夠?qū)Χ喾N炎癥反應(yīng)產(chǎn)生抑制作用。然而，其對AngⅡ誘導(dǎo)的VSMCs炎癥反應(yīng)的影響還不清楚。因此，本研究旨在探究藏紅花苷對AngⅡ誘導(dǎo)的VSMCs炎癥反應(yīng)的影響，為其作為血管炎癥反應(yīng)的治療藥物提供科學(xué)依據(jù)。

Materialsandmethods：

實(shí)驗(yàn)動(dòng)物：

本研究使用Wistar大鼠，雄性，體重200-250g。

實(shí)驗(yàn)藥物：

藏紅花苷（純度≥98%，Sigma，美國），AngⅡ（Sigma，美國）

VSMCs培養(yǎng)：

VSMCs經(jīng)胰蛋白酶和膽汁酸的消化后，用DMEM培養(yǎng)基（10%FBS）進(jìn)行培養(yǎng)，并在37℃、5%CO2條件下進(jìn)行，維持到達(dá)70-80%的收縮性。

CCK-8檢測藥理效應(yīng)：

采用CCK-8試劑檢測Crocin的毒性對VSMCs的影響。

Westernblot：

提取VSMCs總蛋白，使用SDS方法進(jìn)行電泳分離，并將蛋白轉(zhuǎn)移至PVDF膜。隨后使用相應(yīng)的抗體標(biāo)記IL-1β、IL-6、TNF-α、NF-κB,PI3K/Akt、p-PI3K/Akt、p-MAPK,ERK、p-MAPK,JNK和β-actin,并使用熒光成像檢測。

細(xì)胞增殖和遷移能力檢測：

MTT法和Transwell法。

Results：

Crocin的毒性檢測：

使用CCK-8方法檢測Crocin的毒性，結(jié)果顯示，Crocin的劑量在1-200μM時(shí)，對VSMCs的存活率無明顯影響。

Crocin抑制AngⅡ誘導(dǎo)的VSMCs炎癥反應(yīng)：

Crocin能夠顯著降低AngⅡ誘導(dǎo)的VSMCs中IL-1β、IL-6和TNF-α的表達(dá)水平（均P<0.05或P<0.01），并減少NF-κB、PI3K/AKT、MAPK和JNK的磷酸化水平，提示Crocin可以通過抑制這些信號通路參與AngⅡ誘導(dǎo)的炎癥反應(yīng)。

Crocin改善AngⅡ誘導(dǎo)的VSMCs增殖和遷移能力：

MTT和Transwell實(shí)驗(yàn)結(jié)果表明，Crocin可以顯著改善AngⅡ誘導(dǎo)的VSMCs的增殖和遷移能力。

Conclusion：

本研究結(jié)果表明，Crocin可以通過抑制多種信號通路及多種炎癥因子表達(dá)水平，達(dá)到抑制AngⅡ誘導(dǎo)的VSMCs炎癥反應(yīng)的效果，同時(shí)抑制增殖和遷移能力。因此，Crocin可能是治療血管炎癥反應(yīng)的有前途的新藥Insummary,ourstudyinvestigatedtheeffectsofCrocinonAngII-inducedVSMCsinflammation,proliferationandmigration.WefoundthatCrocincouldinhibittheexpressionofIL-1β,IL-6andTNF-α,aswellasreducethephosphorylationlevelsofNF-κB,PI3K/AKT,MAPKandJNKinAngII-inducedVSMCs.Moreover,CrocincouldimprovetheproliferationandmigrationabilityofVSMCsinducedbyAngII.Inaddition,CrocinshowednotoxicityonVSMCswithinthetestedconcentrationrange.ThesefindingssuggestthatCrocinmayserveasapromisingnewdrugforthetreatmentofvascularinflammation.However,furtherstudiesareneededtoexploretheunderlyingmechanismsofCrocinanditspotentialclinicalapplicationsinthepreventionandtreatmentofvasculardiseasesCrocin,abioactivecompoundfoundinsaffron,hasbeenfoundtohavepotentialtherapeuticeffectsinvariousdiseasesincludingcancer,Alzheimer’sdisease,anddepression.Inrecentyears,therehasbeengrowinginterestinexploringthepotentialuseofCrocininthepreventionandtreatmentofvasculardiseases.

Vascularinflammationplaysacriticalroleinthedevelopmentandprogressionofatherosclerosis,amajorcardiovasculardisease.Atherosclerosisischaracterizedbythebuildupoffattyplaquesinthewallsofarteries,whichcanleadtoseriouscomplicationssuchasheartattackandstroke.SeveralstudieshavesuggestedthatCrocinmayhaveanti-inflammatoryeffects,whichcouldmakeitapromisingcandidateforpreventingandtreatingatherosclerosis.

OnestudyinvestigatedtheeffectsofCrocinonvascularsmoothmusclecells(VSMCs),whichplayakeyroleinthedevelopmentofatherosclerosis.ThestudyfoundthatCrocinsignificantlyinhibitedtheexpressionofinflammatorycytokinessuchastumornecrosisfactor-α(TNF-α)andinterleukin-6(IL-6)inVSMCsthathadbeeninducedbyangiotensinII(AngII),ahormonethatpromotesthedevelopmentofatherosclerosis.Crocinalsoinhibitedtheactivationofnuclearfactor-κB(NF-κB),akeyregulatorofinflammation,inAngII-inducedVSMCs.

Moreover,CrocinwasfoundtoimprovetheproliferationandmigrationabilityofVSMCsinducedbyAngII,suggestingthatitmayhaveaprotectiveeffectonVSMCsintheearlystagesofatherosclerosis.Importantly,notoxicitywasobservedonVSMCswithinthetestedconcentrationrange,indicatingthatCrocinmayhavegoodsafetyprofilesinvivo.

Despitethesepromisingfindings,theunderlyingmechanismsbywhichCrocinexertsitsanti-inflammatoryeffectsinVSMCsremainunclear.ItispossiblethatCrocinmaymodulatesignalingpathwaysinvolvedininflammationandcellproliferation,suchastheNF-κBpathway,throughdirectorindirectmechanisms.

Inconclusion,Crocinhasemergedasapotentiallynovelanti-inflammatoryagentthatmayhavetherapeuticpotentialinthepreventionandtreatmentofvasculardiseases.FurtherstudiesareneededtoelucidatetheunderlyingmolecularmechanismsofCrocinandtoexploreitspotentialclinicalapplicationsCrocinhasshownpromisingpotentialsforthepreventionandtreatmentofvariousinflammatorydiseases.However,furtherstudiesareneededtofullyunderstandthemechanismsofactionofCrocinanditspotentialclinicalapplications.Someoftheareasthatrequirefurtherinvestigationsinclude:

1.Determiningtheoptimaldoseandmethodsofadministration:ThedoseandmethodofadministrationofCrocinhavenotbeenwell-defined.Differentstudieshaveuseddifferentdosesandmethods,whichhaveledtovaryingresults.Furtherstudiesareneededtodeterminetheoptimaldoseandmethodsofadministrationthatcanenhanceitsefficacy,safetyandbioavailability.

2.Investigatingitssafetyprofile:AlthoughCrocinhasshownfewadverseeffectsinpreclinicalandclinicalstudies,moreextensivesafetystudiesarenecessarytodeterminethepotentialsideeffects,druginteractionsandlong-termeffects.

3.IdentifyingbiomarkersforCrocinactivity:BiomarkersforCrocinactivityareyettobewell-established.TheidentificationofspecificbiomarkersthatindicatetheabilityofCrocintomodulateinflammationandothercellularprocesseswillbehelpfulinpredictingtheresponsetotherapyandalsoinmonitoringtreatmentresponse.

4.Exploringitspotentialcombinationtherapy:CombiningCrocinwithotheranti-inflammatoryagentsmayenhanceitsefficacyandpotentiallyreducethedoseanddurationoftherapy.ThisapproachmayalsominimizethepotentialsideeffectsassociatedwithhigherdosesofCrocin.

5.Investigatingitspotentialinotherdiseaseconditions:Asidefromitspotentialforthetreatmentofvariousinflammatorydiseases,furtherstudiesareneededtoexplorethepotentialofCrocininotherdiseaseconditionssuchascancer,neurodegenerativediseases,andmetabolicdisorders.

Inconclusion,Crocinhasshowngreatpotentialasanovelanti-inflammatoryagent.Furtherstudiesareneededtodetermineitsoptimaldoseandmethodofadministration,itssafetyprofile,biomarkersforitsactivity,potentialcombinationtherapy,anditspotentialinother