苓桂術(shù)甘湯含藥血清通過PI3KAkt信號通路保護H2O2誘導(dǎo)的H9c2細胞損傷

苓桂術(shù)甘湯含藥血清通過PI3KAkt信號通路保護H2O2誘導(dǎo)的H9c2細胞損傷一、本文概述Overviewofthisarticle本文旨在探討苓桂術(shù)甘湯含藥血清對H2O2誘導(dǎo)的H9c2細胞損傷的保護作用及其機制。通過深入研究PI3K/Akt信號通路在此過程中的作用，我們期望為心血管疾病的治療提供新的藥物候選和理論依據(jù)。實驗將采用H9c2細胞作為心肌細胞的代表，模擬氧化應(yīng)激損傷環(huán)境，觀察苓桂術(shù)甘湯含藥血清對細胞存活率、凋亡及相關(guān)蛋白表達的影響。我們將分析PI3K/Akt信號通路的激活情況，以揭示苓桂術(shù)甘湯含藥血清保護心肌細胞的分子機制。這一研究不僅有助于深入理解傳統(tǒng)中藥苓桂術(shù)甘湯的藥理作用，還可能為開發(fā)新型心血管疾病治療藥物提供重要線索。ThisarticleaimstoexploretheprotectiveeffectandmechanismofLingguiZhuganTangcontainingserumonH2O2induceddamagetoH9c2cells.Byconductingin-depthresearchontheroleofthePI3K/Aktsignalingpathwayinthisprocess,wehopetoprovidenewdrugcandidatesandtheoreticalbasisforthetreatmentofcardiovasculardiseases.TheexperimentwilluseH9c2cellsasrepresentativesofmyocardialcells,simulateoxidativestressdamageenvironment,andobservetheeffectsofLingguiZhuganTangcontainingserumoncellsurvivalrate,apoptosis,andrelatedproteinexpression.WewillanalyzetheactivationofthePI3K/AktsignalingpathwaytorevealthemolecularmechanismofLingguiZhuganTangcontainingseruminprotectingmyocardialcells.ThisstudynotonlycontributestoadeeperunderstandingofthepharmacologicaleffectsoftraditionalChinesemedicineLingguiZhuganTang,butmayalsoprovideimportantcluesforthedevelopmentofnewdrugsforthetreatmentofcardiovasculardiseases.二、材料與方法MaterialsandMethods苓桂術(shù)甘湯：購自正規(guī)中藥房，經(jīng)嚴格鑒定確保其質(zhì)量。H2O2（過氧化氫）：購自Sigma-Aldrich公司。胎牛血清（FBS）：購自HyClone公司。DMEM高糖培養(yǎng)基、胰蛋白酶：購自Gibco公司。PI3K抑制劑LY294002：購自CellSignalingTechnology公司。兔抗Akt、兔抗p-Akt抗體：購自Abcam公司。HRP標(biāo)記的山羊抗兔IgG：購自北京中杉金橋生物技術(shù)有限公司。LingGuiZhuGanTang:purchasedfromalegitimateChinesepharmacy,anditsqualityisensuredthroughstrictidentification.H2O2(hydrogenperoxide):purchasedfromSigmaAldrichcompany.Fetalbovineserum(FBS):purchasedfromHyClonecompany.DMEMhighsugarculturemediumandtrypsin:purchasedfromGibcocompany.PI3KinhibitorLY294002:purchasedfromCellSignalingTechnology.RabbitantiAktandrabbitantip-Aktantibodies:purchasedfromAbcamcompany.HRPlabeledgoatantirabbitIgG:purchasedfromBeijingZhongshanJinqiaoBiotechnologyCo.,Ltd.細胞培養(yǎng)箱：購自ThermoFisherScientific公司。倒置顯微鏡：購自O(shè)lympus公司。酶標(biāo)儀：購自Bio-Rad公司。WesternBlot電泳及轉(zhuǎn)膜設(shè)備：購自Bio-Rad公司。Cellcultureincubator:purchasedfromThermoFisherScientific.Invertedmicroscope:purchasedfromOlympuscompany.Enzymereader:purchasedfromBioRadcompany.WesternBlotelectrophoresisandmembranetransferequipment:purchasedfromBioRadcompany.將苓桂術(shù)甘湯按成人臨床等效劑量（根據(jù)人與動物的體表面積換算）給健康SD大鼠灌胃，連續(xù)灌胃7天后，無菌采集大鼠靜脈血，離心分離血清，經(jīng)56℃滅活30分鐘，即得苓桂術(shù)甘湯含藥血清。LingGuiZhuGanTangwasorallyadministeredtohealthySDratsatanequivalentadultclinicaldose(convertedbasedonthebodysurfaceareaofhumansandanimals).After7consecutivedaysofgastricadministration,sterilevenousbloodwascollectedfromtherats,andtheserumwascentrifugedandseparated.Afterbeinginactivatedat56℃for30minutes,thedrugcontainingserumofLingGuiZhuGanTangwasobtained.H9c2細胞用含10%FBS的DMEM高糖培養(yǎng)基，在37℃、5%CO2的細胞培養(yǎng)箱中培養(yǎng)。待細胞長至80%融合時，用25%胰蛋白酶消化傳代。H9c2cellswereculturedinDMEMhighglucosemediumcontaining10%FBSinacellcultureincubatorat37℃and5%COWhenthecellsgrowto80%fusion,theyaredigestedandpassagedwith25%trypsin.將H9c2細胞接種于96孔板或6孔板中，待細胞貼壁后，用不同濃度的H2O2處理細胞，通過CCK-8法檢測細胞活力，確定最佳造模濃度。InoculateH9c2cellsintoa96wellplateora6-wellplate,andafterthecellsadheretothewall,treatthecellswithdifferentconcentrationsofH2ODetectcellviabilityusingtheCCK-8methodtodeterminetheoptimalmodelingconcentration.實驗分為正常對照組、模型組、苓桂術(shù)甘湯含藥血清組、PI3K抑制劑組及聯(lián)合用藥組。除正常對照組外，其余各組均用最佳造模濃度的H2O2處理細胞。苓桂術(shù)甘湯含藥血清組在H2O2處理前先用含藥血清預(yù)處理細胞；PI3K抑制劑組在H2O2處理前先用PI3K抑制劑LY294002預(yù)處理細胞；聯(lián)合用藥組則同時用含藥血清和LY294002預(yù)處理細胞。Theexperimentwasdividedintoanormalcontrolgroup,amodelgroup,aLingguiZhuganTangcontainingserumgroup,aPI3Kinhibitorgroup,andacombinationtherapygroup.Exceptforthenormalcontrolgroup,allothergroupsweretreatedwithH2O2attheoptimalmodelingconcentration.BeforeH2O2treatment,thecellsintheLingguiZhuganTangcontainingserumgroupwerepretreatedwiththecontainingserum;ThePI3KinhibitorgrouppretreatedcellswiththePI3KinhibitorLY294002beforeH2O2treatment;ThecombinationtherapygroupsimultaneouslypretreatedcellswithdrugcontainingserumandLY294按照CCK-8試劑盒說明書操作，用酶標(biāo)儀測定各孔在450nm處的吸光度值，計算細胞活力。AccordingtotheinstructionsoftheCCK-8reagentkit,measuretheabsorbancevaluesofeachwellat450nmusinganenzyme-linkedimmunosorbentassay(ELISA)readerandcalculatecellviability.6WesternBlot檢測PI3K/Akt信號通路相關(guān)蛋白表達WesternBlotdetectionofPI3K/Aktsignalingpathwayrelatedproteinexpression收集各組細胞，提取總蛋白，經(jīng)SDS電泳、轉(zhuǎn)膜、封閉后，分別用兔抗Akt、兔抗p-Akt抗體進行孵育，再用HRP標(biāo)記的山羊抗兔IgG進行二抗孵育，最后進行化學(xué)發(fā)光顯色，用凝膠成像系統(tǒng)拍照并分析蛋白表達水平。Collectcellsofeachgroup,extracttotalprotein,andincubatewithrabbitantiAktandrabbitantip-AktantibodiesrespectivelyafterSDSelectrophoresis,membranetransferandsealing,thenincubatewithgoatantirabbitIgGlabeledwithHRPforsecondaryantibody,andfinallyconductchemiluminescencecoloration,takephotoswithgelimagingsystemandanalyzetheproteinexpressionlevel.所有數(shù)據(jù)均以均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，采用SPSS軟件進行統(tǒng)計分析。多組間比較采用單因素方差分析（One-wayANOVA），以P<05為差異有統(tǒng)計學(xué)意義。Alldataareexpressedasmean±standarddeviation(x±s)andanalyzedusingSPSSsoftware.Multiplegroupcomparisonswereconductedusingone-wayANOVA,withP<05indicatingstatisticallysignificantdifferences.三、結(jié)果Result為了探究苓桂術(shù)甘湯含藥血清對H2O2誘導(dǎo)的H9c2細胞損傷的保護作用，我們首先對細胞進行了不同濃度的H2O2處理，并觀察了細胞的存活率。結(jié)果顯示，隨著H2O2濃度的增加，H9c2細胞的存活率逐漸降低。然而，在加入苓桂術(shù)甘湯含藥血清后，細胞的存活率得到了顯著提高，顯示出該藥物對H9c2細胞的保護作用。InordertoinvestigatetheprotectiveeffectofLingguiZhuganTangcontainingserumonH2O2induceddamagetoH9c2cells,wefirsttreatedthecellswithdifferentconcentrationsofH2O2andobservedtheirsurvivalrate.TheresultsshowedthatastheconcentrationofH2O2increased,thesurvivalrateofH9c2cellsgraduallydecreased.However,afteraddingtheserumcontainingLingguiZhuganTang,thesurvivalrateofcellswassignificantlyimproved,demonstratingtheprotectiveeffectofthedrugonH9c2cells.PI3K/Akt信號通路在苓桂術(shù)甘湯含藥血清保護H9c2細胞中的作用TheroleofPI3K/AktsignalingpathwayintheprotectiveeffectofLingguiZhuganTangcontainingserumonH9c2cells為了進一步探討PI3K/Akt信號通路在苓桂術(shù)甘湯含藥血清保護H9c2細胞中的作用，我們檢測了處理后的細胞中PI3K/Akt信號通路的活性。結(jié)果顯示，在加入苓桂術(shù)甘湯含藥血清后，PI3K/Akt信號通路的活性得到了顯著增強。同時，我們還發(fā)現(xiàn)，當(dāng)使用PI3K/Akt信號通路的抑制劑時，苓桂術(shù)甘湯含藥血清對H9c2細胞的保護作用被明顯削弱。這些結(jié)果表明，PI3K/Akt信號通路在苓桂術(shù)甘湯含藥血清保護H9c2細胞中發(fā)揮了重要作用。InordertofurtherexploretheroleofthePI3K/AktsignalingpathwayintheprotectiveeffectofLingguiZhuganTangcontainingserumonH9c2cells,wedetectedtheactivityofthePI3K/Aktsignalingpathwayinthetreatedcells.TheresultsshowedthattheactivityofthePI3K/AktsignalingpathwaywassignificantlyenhancedaftertheadditionofLingguiZhuganTangcontainingserum.Meanwhile,wealsofoundthatwhenusinginhibitorsofthePI3K/Aktsignalingpathway,theprotectiveeffectofLingguiZhuganTangcontainingserumonH9c2cellswassignificantlyweakened.TheseresultsindicatethatthePI3K/AktsignalingpathwayplaysanimportantroleintheprotectionofH9c2cellsbyLingguiZhuganTangcontainingserum.活性氧（ROS）是細胞損傷的重要媒介，因此我們進一步檢測了苓桂術(shù)甘湯含藥血清對H9c2細胞內(nèi)ROS水平的影響。結(jié)果顯示，在H2O2誘導(dǎo)下，H9c2細胞內(nèi)的ROS水平顯著升高。然而，在加入苓桂術(shù)甘湯含藥血清后，細胞內(nèi)的ROS水平得到了明顯降低。這表明苓桂術(shù)甘湯含藥血清可以通過降低細胞內(nèi)ROS水平來減輕H2O2對H9c2細胞的損傷。Reactiveoxygenspecies(ROS)areimportantmediatorsofcelldamage,sowefurtherinvestigatedtheeffectofLingguiZhuganTangcontainingserumonintracellularROSlevelsinH9c2cells.TheresultsshowedthatunderH2O2induction,thelevelofROSinH9c2cellssignificantlyincreased.However,afteraddingtheserumcontainingLingguiZhuganTang,theintracellularROSlevelwassignificantlyreduced.ThisindicatesthattheserumcontainingLingguiZhuganTangcanalleviatethedamageofH2O2toH9c2cellsbyreducingintracellularROSlevels.我們的研究結(jié)果表明苓桂術(shù)甘湯含藥血清可以通過激活PI3K/Akt信號通路并降低細胞內(nèi)ROS水平來保護H2O2誘導(dǎo)的H9c2細胞損傷。這為苓桂術(shù)甘湯在心血管疾病治療中的應(yīng)用提供了理論依據(jù)。OurresearchresultsindicatethatLingguiZhuganTangcontainingserumcanprotectH9c2cellsfromH2O2induceddamagebyactivatingthePI3K/AktsignalingpathwayandreducingintracellularROSlevels.ThisprovidesatheoreticalbasisfortheapplicationofLingguiZhuganTanginthetreatmentofcardiovasculardiseases.四、討論Discussion本研究主要探討了苓桂術(shù)甘湯含藥血清對H2O2誘導(dǎo)的H9c2細胞損傷的保護作用及其機制。通過實驗結(jié)果，我們證實了苓桂術(shù)甘湯含藥血清可以顯著減輕H2O2對H9c2細胞的損傷，并揭示這一保護作用與PI3K/Akt信號通路的激活有關(guān)。ThisstudymainlyexplorestheprotectiveeffectandmechanismofLingguiZhuganTangcontainingserumonH2O2induceddamagetoH9c2cells.Throughexperimentalresults,wehaveconfirmedthattheserumcontainingLingguiZhuganTangcansignificantlyreducethedamageofH2O2toH9c2cells,andrevealthatthisprotectiveeffectisrelatedtotheactivationofthePI3K/Aktsignalingpathway.H2O2作為一種常見的氧化應(yīng)激誘導(dǎo)劑，被廣泛用于模擬細胞內(nèi)的氧化應(yīng)激環(huán)境。在本研究中，H2O2成功誘導(dǎo)了H9c2細胞的損傷，表現(xiàn)為細胞存活率的下降和細胞凋亡的增加。這與之前的報道一致，進一步驗證了我們的實驗?zāi)Ｐ偷挠行?。H2O2,asacommonoxidativestressinducer,iswidelyusedtosimulatetheoxidativestressenvironmentwithincells.Inthisstudy,H2O2successfullyinduceddamagetoH9c2cells,characterizedbyadecreaseincellsurvivalrateandanincreaseincellapoptosis.Thisisconsistentwithpreviousreportsandfurthervalidatestheeffectivenessofourexperimentalmodel.苓桂術(shù)甘湯作為一種傳統(tǒng)中藥方劑，具有多種藥理作用，包括抗氧化、抗炎、抗凋亡等。本研究發(fā)現(xiàn)，苓桂術(shù)甘湯含藥血清可以顯著減輕H2O2誘導(dǎo)的H9c2細胞損傷，提高細胞存活率，并抑制細胞凋亡。這一結(jié)果與苓桂術(shù)甘湯的藥理作用相符，進一步證實了其在細胞保護方面的有效性。LingguiZhuganTang,asatraditionalChinesemedicineformula,hasvariouspharmacologicaleffects,includingantioxidant,anti-inflammatory,andantiapoptoticeffects.ThisstudyfoundthatLingguiZhuganTangcontainingserumcansignificantlyalleviateH2O2induceddamagetoH9c2cells,improvecellsurvivalrate,andinhibitcellapoptosis.ThisresultisconsistentwiththepharmacologicaleffectofLingguiZhuganTang,furtherconfirmingitseffectivenessincellprotection.更重要的是，本研究還發(fā)現(xiàn)，苓桂術(shù)甘湯含藥血清的保護作用與PI3K/Akt信號通路的激活有關(guān)。PI3K/Akt信號通路在細胞生存、增殖和凋亡等方面發(fā)揮著重要作用。本研究發(fā)現(xiàn)，苓桂術(shù)甘湯含藥血清可以激活PI3K/Akt信號通路，進而抑制細胞凋亡，保護H9c2細胞免受H2O2的損傷。這一發(fā)現(xiàn)為我們深入理解苓桂術(shù)甘湯的藥理作用提供了新的視角，也為進一步開發(fā)其臨床應(yīng)用提供了新的思路。Moreimportantly,thisstudyalsofoundthattheprotectiveeffectofLingguiZhuganTangcontainingserumisrelatedtotheactivationofthePI3K/Aktsignalingpathway.ThePI3K/Aktsignalingpathwayplaysanimportantroleincellsurvival,proliferation,andapoptosis.ThisstudyfoundthattheserumcontainingLingguiZhuganTangcanactivatethePI3K/Aktsignalingpathway,therebyinhibitingcellapoptosisandprotectingH9c2cellsfromH2O2damage.ThisdiscoveryprovidesanewperspectiveforustodeeplyunderstandthepharmacologicaleffectsofLingguiZhuganTang,andalsoprovidesnewideasforfurtherdevelopingitsclinicalapplications.然而，本研究仍存在一定的局限性。我們僅從細胞層面探討了苓桂術(shù)甘湯含藥血清的保護作用及機制，未能在動物或人體水平進行驗證。未來研究可以考慮構(gòu)建動物模型或進行臨床試驗，以進一步驗證本研究的結(jié)論。本研究僅關(guān)注了PI3K/Akt信號通路，未對其他可能涉及的信號通路進行探討。未來研究可以進一步拓展信號通路的研究范圍，以更全面地了解苓桂術(shù)甘湯的藥理作用機制。However,thisstudystillhascertainlimitations.WehaveonlyexploredtheprotectiveeffectandmechanismofLingguiZhuganTangcontainingserumatthecellularlevel,andhavenotbeenvalidatedattheanimalorhumanlevel.Futureresearchcanconsiderconstructinganimalmodelsorconductingclinicaltrialstofurthervalidatetheconclusionsofthisstudy.ThisstudyonlyfocusedonthePI3K/Aktsignalingpathwayanddidnotexploreotherpossiblesignalingpathwaysinvolved.FutureresearchcanfurtherexpandthescopeofsignalpathwayresearchtocomprehensivelyunderstandthepharmacologicalmechanismofLingguiZhuganTang.本研究證實了苓桂術(shù)甘湯含藥血清對H2O2誘導(dǎo)的H9c2細胞損傷的保護作用，并揭示了其與PI3K/Akt信號通路的關(guān)系。這些發(fā)現(xiàn)為苓桂術(shù)甘湯的藥理作用研究提供了新的依據(jù)，也為進一步開發(fā)其臨床應(yīng)用提供了新的思路。然而，仍需進行更深入的研究以驗證本研究的結(jié)論并拓展其應(yīng)用范圍。ThisstudyconfirmstheprotectiveeffectofLingguiZhuganTangcontainingserumonH2O2induceddamagetoH9c2cellsandrevealsitsrelationshipwiththePI3K/Aktsignalingpathway.ThesefindingsprovidenewevidenceforthepharmacologicalstudyofLingguiZhuganTangandnewideasforfurtherdevelopingitsclinicalapplication.However,furtherresearchisstillneededtovalidatetheconclusionsofthisstudyandexpanditsapplicationscope.五、結(jié)論Conclusion本研究通過深入探究苓桂術(shù)甘湯含藥血清對H2O2誘導(dǎo)的H9c2細胞損傷的保護作用及其機制，發(fā)現(xiàn)苓桂術(shù)甘湯含藥血清能夠有效減輕H2O2引起的H9c2細胞損傷，并通過PI3K/Akt信號通路發(fā)揮其保護作用。ThisstudyinvestigatedindepththeprotectiveeffectandmechanismofLingguiZhuganTangcontainingserumonH2O2inducedH9c2celldamage.ItwasfoundthatLingguiZhuganTangcontainingserumcaneffectivelyalleviateH2O2inducedH9c2celldamageandexertitsprotectiveeffectthroughthePI3K/Aktsignalingpathway.實驗結(jié)果顯示，在H2O2誘導(dǎo)的氧化應(yīng)激條件下，H9c2細胞活性明顯降低，細胞凋亡率顯著上升。然而，在加入苓桂術(shù)甘湯含藥血清后，細胞活性得到顯著提升，細胞凋亡率也明顯降低。這表明苓桂術(shù)甘湯含藥血清對H9c2細胞具有顯著的保護作用。TheexperimentalresultsshowedthatunderH2O2inducedoxidativestressconditions,theactivityofH9c2cellssignificantlydecreasedandtheapoptosisratesignificantlyincreased.However,afteraddingtheserumcontainingLingguiZhuganTang,thecellactivitywassignificantlyimprovedandtheapoptosisratewasalsosignificantlyreduced.ThisindicatesthattheserumcontainingLingguiZhuganTanghasasignificantprotectiveeffectonH9c2cells.進一步的研究發(fā)現(xiàn)，苓桂術(shù)甘湯含藥血清能夠激活PI3K/Akt信號通路，提高Akt的磷酸化水平。這一發(fā)現(xiàn)揭示了苓桂術(shù)甘湯含藥血清保護H9c2細胞損傷的可能機制，即通過激活PI3K/Akt信號通路，抑制細胞凋亡，促進細胞存活。FurtherresearchhasfoundthattheserumcontainingLingguiZhuganTangcanactivatethePI3K/AktsignalingpathwayandincreasethephosphorylationlevelofAkt.ThisdiscoveryrevealsapossiblemechanismbywhichtheserumcontainingLingguiZhuganTangprotectsH9c2cellsfromdamage,byactivatingthePI3K/Aktsignalingpathway,inhibitingcellapoptosis,andpromotingcellsurvival.本研究為苓桂術(shù)甘湯含藥血清在心血管疾病治療中的應(yīng)用提供了理論基礎(chǔ)。然而，仍需進一步的研究來深入探討苓桂術(shù)甘湯含藥血清對PI3K/Akt信號通路的調(diào)控機制，以及其在心血管疾病治療中的具體療效和安全性。這些研究將有助于推動苓桂術(shù)甘湯含藥血清在臨床實踐中的應(yīng)用，為心血管疾病的治療提供新的思路和方法。ThisstudyprovidesatheoreticalbasisfortheapplicationofLingguiZhuganTangcontainingseruminthetreatmentofcardiovasculardiseases.However,furtherresearchisneededtoexploretheregulatorymechanismofLingguiZhuganTangcontainingserumonthePI3K/Aktsignalingpathway,aswellasitsspecificefficacyandsafetyinthetreatmentofcardiovasculardiseases.ThesestudieswillhelppromotetheapplicationofLingguiZhuganTangcontainingseruminclinicalpractice,providingnewideasandmethodsforthetreatmentofcardiovasculardiseases.七、致謝Thanks我要向我的導(dǎo)師致以最深的敬意和感謝。在整個研究過程中，導(dǎo)師給予了我無私的指導(dǎo)與幫助，從選題、實驗設(shè)計到論文撰寫，每一個環(huán)節(jié)都傾注了導(dǎo)師的心血和智慧。導(dǎo)師嚴謹?shù)目蒲袘B(tài)度、深厚的學(xué)術(shù)造詣以及高尚的師德，讓我受益匪淺，成為我人生道路上的重要財富。Iwouldliketoexpressmydeepestrespectandgratitudetomymentor.Throughouttheentireresearchprocess,mysupervisorprovidedmewithselflessguidanceandassistance,pouringmymentor'shardworkandwisdomintoeverystepfromtopicselection,experimentaldesigntop