Name: protection of h9c2 myocardial cells from oxidative stress by crocetin via pink1/parkin pathway-mediated mitophagy
Uploaded: 2023-05-26T15:00:00
Description: Watch this Scientific Journal Video about Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy at JoVE.com

Denne undersøgelse blev støttet af Beijing Natural Science Foundation (nr. 7202119) og National Natural Science Foundation of China (nr. 82274380).

Eksperimenterne blev udført i laboratoriet for fysiologi ved Beijing University of Chinese Medicine, Kina. Alle undersøgelsesmetoder blev udført i overensstemmelse med de relevante retningslinjer og regler fra Beijing Universitet.
1. Cellekultur
<ol><li>Tilsæt 10% føtalt bovint serum og 1% penicillin / streptomycin til Dulbeccos modificerede Eagle medium (DMEM) basisk medium (med 4,5 g / L D-glucose, 4.g.g / L L-glutamin og 110 mg / L natriumpyruvat; se material...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiogenic+shock+after+acute+myocardial+infarction:+a+review.">Cardiogenic shock after acute myocardial infarction: a review.</a> JAMA. 326 (18), 1840-1850 (2021).</li><li>Abbate, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+cardiac+remodeling+benefits+in+patients+undergoing+late+percutaneous+coronary+intervention+of+the+infarct-related+artery:+evidence+from+a+meta-analysis+of+randomized+controlled+trials.">Survival and cardiac remodeling benefits in patients undergoing late percutaneous coronary intervention of the infarct-related artery: evidence from a meta-analysis of randomized controlled trials.</a> Journal of the American College of Cardiology. 51 (9), 956-964 (2008).</li><li>Santoro, G. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+protects+against+common+deletion+of+mitochondrial+DNA-augmented+mitochondrial+oxidative+stress+and+apoptosis.">Melatonin protects against common deletion of mitochondrial DNA-augmented mitochondrial oxidative stress and apoptosis.</a> Journal of Pineal Research. 43 (4), 389-403 (2007).</li><li>La Piana, G., Fransvea, E., Marzulli, D., Lofrumento, N. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+pharmacology+analysis+and+experimental+validation+to+investigate+the+mechanism+of+total+flavonoids+of+rhizoma+drynariae+in+treating+rheumatoid+arthritis.">Network pharmacology analysis and experimental validation to investigate the mechanism of total flavonoids of rhizoma drynariae in treating rheumatoid arthritis.</a> Drug Design, Development, and Therapy. 16, 1743-1766 (2022).</li><li>Wei, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+Chinese+medicine+has+great+potential+as+candidate+drugs+for+lung+cancer:+A+review.">Traditional Chinese medicine has great potential as candidate drugs for lung cancer: A review.</a> Journal of Ethnopharmacology. 300, 115748 (2023).</li><li>Zhi, W., Liu, Y., Wang, X., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+of+traditional+Chinese+medicine+for+the+prevention+and+treatment+of+atherosclerosis.">Recent advances of traditional Chinese medicine for the prevention and treatment of atherosclerosis.</a> Journal of Ethnopharmacology. 301, 115749 (2023).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertensive+heart+disease+and+myocardial+fibrosis:+How+traditional+Chinese+medicine+can+help+addressing+unmet+therapeutical+needs.">Hypertensive heart disease and myocardial fibrosis: How traditional Chinese medicine can help addressing unmet therapeutical needs.</a> Pharmacological Research. 185, 106515 (2022).</li><li>Zhang, X. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+Chinese+medicine+intervenes+ventricular+remodeling+following+acute+myocardial+infarction:+evidence+from+40+random+controlled+trials+with+3,659+subjects.">Traditional Chinese medicine intervenes ventricular remodeling following acute myocardial infarction: evidence from 40 random controlled trials with 3,659 subjects.</a> Frontiers in Pharmacology. 12, 707394 (2021).</li><li>Hao, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+Chinese+medicine+for+cardiovascular+disease:+evidence+and+potential+mechanisms.">Traditional Chinese medicine for cardiovascular disease: evidence and potential mechanisms.</a> Journal of the American College of Cardiology. 69 (24), 2952-2966 (2017).</li><li>Delgado-Montero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+stasis+imaging+predicts+cerebral+microembolism+during+acute+myocardial+infarction.">Blood stasis imaging predicts cerebral microembolism during acute myocardial infarction.</a> Journal of the American Society of Echocardiography. 33 (3), 389-398 (2020).</li><li>Lu, C. 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R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+cell+number+by+hemocytometry+counting.">Estimation of cell number by hemocytometry counting.</a> Cold Spring Harbor Protocols. 2019 (11), (2019).</li><li>Zeng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+potential+value+and+mechanism+of+Kaji-Ichigoside+F1+on+arsenite-induced+skin+cell+senescence.">Assessing the potential value and mechanism of Kaji-Ichigoside F1 on arsenite-induced skin cell senescence.</a> Oxidative Medicine and Cellular Longevity. 2022, 9574473 (2022).</li><li>Chazotte, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+mitochondria+with+JC-1.">Labeling mitochondria with JC-1.</a> Cold Spring Harbor Protocols. 2011 (9), (2011).</li><li>Kyrylkova, K., Kyryachenko, S., Leid, M., Kioussi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+apoptosis+by+TUNEL+assay.">Detection of apoptosis by TUNEL assay.</a> Methods in Molecular Biology. 887, 41-47 (2012).</li><li>Yuan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+impairs+the+autophagic+flux+to+induce+p62-dependent+apoptosis+through+the+upregulation+of+CYLD+in+NRCMs.">Palmitate impairs the autophagic flux to induce p62-dependent apoptosis through the upregulation of CYLD in NRCMs.</a> Toxicology. 465, 153032 (2022).</li><li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting.">Western blotting.</a> Methods. 38 (4), 283-293 (2006).</li><li>Chen, G. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+flavonoids+of+rhizoma+drynariae+restore+the+MMP/TIMP+balance+in+models+of+osteoarthritis+by+inhibiting+the+activation+of+the+NF-&#954;B+and+PI3K/AKT+pathways.">Total flavonoids of rhizoma drynariae restore the MMP/TIMP balance in models of osteoarthritis by inhibiting the activation of the NF-&#954;B and PI3K/AKT pathways.</a> Evidence-Based Complementary and Alternative. 2021, 6634837 (2021).</li><li>Amin, A., Hamza, A. A., Bajbouj, K., Ashraf, S. 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Udforskningen af effektive ingredienser fra komplekse forbindelser af naturlige lægemidler gennem avanceret teknologi har været et hotspot for TCM-forskning29 og kan give laboratoriebevis for fremtidig lægemiddeludvikling efter verifikation. Saflor er et repræsentativt lægemiddel til behandling af "fremme af blodcirkulationen og minimering af blodstasis" og anvendes i vid udstrækning til behandling af myokardieinfarkt30,31. Safran me...

Akut myokardieinfarkt (AMI) er en livstruende myokardienekrose forårsaget af alvorlig og vedvarende iskæmi og hypoxi i koronararterierne 1,2. Perkutan koronar intervention (PCI) er en af de første linje terapeutiske strategier for AMI og beskytter normalt kardiomyocytter mod iskæmisk skade 3,4. Det distale myokardium mangler blod- og iltforsyning, hvis det ikke behandles hurtigt og effektivt efter AMI, hvilket fører til iskæmisk nekrose og yderligere kardiovaskulære komplikationer 5,6. Fremme af kardiomyocytgendannelse og minimering af irreversibel myokardieskade efter at have savnet PCI-kirurgisk mulighed har været et forskningshotspot. Efter AMI er kardiomyocytter i en tilstand af iskæmi og hypoxi, hvilket resulterer i hæmning af mitokondriel oxidativ phosphorylering, reduktion af NAD+ til NADPH og øget enkeltelektronreduktion7. Som følge heraf genererer den ufuldstændige reduktionsreaktion af ilt et overskud af reaktive iltarter (ROS) og fører i sidste ende til oxidativ stressskade på kardiomyocytter8. En overdreven ophobning af ROS udløser lipidperoxidation, hvilket yderligere forstyrrer mitokondriemembranernes struktur og funktion. Resultatet er en kontinuerlig åbning af mitokondriepermeabilitetsovergangsporer og et fald i mitokondriemembranpotentiale, hvilket inducerer apoptose og nekrose.
Angiotensinkonverterende enzym (ACE) hæmmere, angiotensinreceptorblokkere (ARB'er), hæmmere af β-adrenoceptorer, aldosteronantagonister og andre standardlægemidler i AMI kan hjælpe med at forbedre hjertefunktionen efter myokardieinfarkt og forhindre forekomsten af ondartede hændelser, såsom arytmier og venstre ventrikulær remodellering9. Imidlertid påvirkes overlevelse og prognose efter infarkt stærkt af infarktstørrelse, og der er ikke opnået tilfredsstillende resultater for reduktion af kardiomyocytapoptose10,11. Således er udviklingen af lægemidler til fremme af kardiomyocytgendannelse efter myokardieinfarkt blevet et presserende problem.
Traditionel medicin har været en inspirationskilde for moderne farmaceutisk forskning i mange år12,13,14,15. Traditionel kinesisk medicin (TCM) har en lang historie i behandlingen af AMI, og en række randomiserede kontrolforsøg i de senere år har bekræftet, at TCM faktisk kan forbedre prognosen for patienter16,17. Ifølge TCM-teorien er AMI forårsaget af blodstasis18,19, så lægemidler til fremme af blodcirkulationen bruges normalt til behandling af AMI i den akutte fase20. Blandt dem menes safran at have en kraftig effekt på blodaktivering og stasis, og bruges ofte til akut behandling af AMI. Crocetin, en vigtig bestanddel af safran, kan spille en central rolle i beskyttelsen af kardiomyocytter21.
I denne undersøgelse blev H9c2 myokardceller induceret afH2O2for at simulere myokardieiskæmi/reperfusion, hvilket forårsager en kardiomyocytskade på AMI, og crocetin blev anvendt som en intervention for at undersøge dets beskyttende virkning mod oxidativ stressinduceret myokardieskade. Mekanismen for crocetin, der beskytter kardiomyocytter, blev yderligere undersøgt gennem mitofagi. Endnu vigtigere er det, at denne artikel giver en reference til den tekniske tilgang til undersøgelsen af mitofagi, og beskriver hele eksperimentproceduren i detaljer.

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	<tbody>
		<tr>
			<td>0.25% trypsin</td>
			<td>Gibco</td>
			<td>2323363</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>V900929</td>
			<td></td>
		</tr>
		<tr>
			<td>5x protein loading buffer</td>
			<td>Beijing Pulilai Gene Technology</td>
			<td>B1030-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ad-mCherry GFP-LC3B adenovirus</td>
			<td>Beyotime</td>
			<td>C3011</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZF-0514</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L)</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZF-0513</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-free blocking solution</td>
			<td>CST</td>
			<td>15019s</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Parkin antibody</td>
			<td>Santa Cruz</td>
			<td>sc-32282</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PINK1 antibody</td>
			<td>ABclonal</td>
			<td>A11435</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TOM20 antibody</td>
			<td>ABclonal</td>
			<td>A19403</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;-actin&#160; antibody</td>
			<td>ABclonal</td>
			<td>AC026</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>KeyGEN Biotech</td>
			<td>KGP902</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood cell counting plate</td>
			<td>Servicebio</td>
			<td>WG607</td>
			<td></td>
		</tr>
		<tr>
			<td>CAT assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A007-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescence detection system</td>
			<td>Shanghai Qinxiang Scientific Instrument Factory</td>
			<td>ChemiScope 6100</td>
			<td></td>
		</tr>
		<tr>
			<td>CK assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A032-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme Q10 (CoQ 10)</td>
			<td>Macklin</td>
			<td>C6129</td>
			<td></td>
		</tr>
		<tr>
			<td>Crocetin</td>
			<td>Chengdu Ruifensi Biotechnology Co., Ltd.</td>
			<td>RFS-Z01802006012</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI-containing antifluorescence quenching tablets</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZLI-9557</td>
			<td></td>
		</tr>
		<tr>
			<td>DCFH-DA</td>
			<td>Beyotime</td>
			<td>S0033S</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Solarbio</td>
			<td>D8371</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Gibco</td>
			<td>8122091</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced Chemiluminescence (ECL) solution</td>
			<td>NCM Biotech</td>
			<td>P10100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Corning-Cellgro</td>
			<td>35-081-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 7.0&#160;</td>
			<td>https://www.graphpad.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-Px assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A005-1-2</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 myocardial cells</td>
			<td>Beijing Dingguochangsheng Biotech Co., Ltd.</td>
			<td>CS0062</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated goat anti-goat IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZB-2305</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZB-2301</td>
			<td></td>
		</tr>
		<tr>
			<td>JC-1 mitochondrial membrane potential assay kit</td>
			<td>LABLEAD</td>
			<td>J22202</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A020-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A003-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Aladdin</td>
			<td>A2114057</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS assay</td>
			<td>Promega</td>
			<td>G3581</td>
			<td></td>
		</tr>
		<tr>
			<td>Perhydrol</td>
			<td>G-clone</td>
			<td>CS7730</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase inhibitor</td>
			<td>CWBIO</td>
			<td>CW2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Beyotime</td>
			<td>C0351</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylidene difluoride (PVDF) membranes</td>
			<td>Millipore</td>
			<td>ISEQ00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Radioimmunoprecipitation assay (RIPA) lysis buffer</td>
			<td>Solarbio</td>
			<td>R0010</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE gels</td>
			<td>Shanghai Epizyme Biomedical Technology</td>
			<td>PG112</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE running buffer powder</td>
			<td>Servicebio</td>
			<td>G2018-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE transfer buffer powder</td>
			<td>Servicebio</td>
			<td>G2017-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>SOD assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A001-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline powder</td>
			<td>Servicebio</td>
			<td>G0001-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>SLCC9172</td>
			<td></td>
		</tr>
		<tr>
			<td>TUNEL apoptosis assay kit</td>
			<td>Beyotime</td>
			<td>C1086</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Solarbio</td>
			<td>T8220</td>
			<td></td>
		</tr>
	</tbody>
</table>

protection of h9c2 myocardial cells from oxidative stress by crocetin via pink1/parkin pathway-mediated mitophagy

Denne undersøgelse havde til formål at undersøge den oxidative stressbeskyttende virkning af crocetin på H2O2-medierede H9c2-myokardceller gennem in vitro-eksperimenter og yderligere undersøge, om dens mekanisme er relateret til virkningen af mitofagi. Denne undersøgelse havde også til formål at demonstrere den terapeutiske virkning af saflorsyre på oxidativ stress i kardiomyocytter og undersøge, om dens mekanisme er relateret til effekten af mitofagi. Her blev en H 2 O2-baseretoxidativ stressmodel konstrueret og vurderet graden af oxidativ stressskade af kardiomyocytter ved at detektere niveauerne af lactat dehydrogenase (LDH), kreatinkinase (CK), malondialdehyd (MDA), superoxiddismutase (SOD), katalase (CAT) og glutathionperoxidase (GSH Px). Reaktive iltarter (ROS)-detekterende fluorescerende farvestof DCFH-DA, JC-1 farvestof og TUNEL farvestof blev anvendt til at vurdere mitokondrieskader og apoptose. Autofagisk flux blev målt ved transfektering af Ad-mCherry-GFP-LC3B adenovirus. Mitofagy-relaterede proteiner blev derefter påvist via western blotting og immunofluorescens. Crocetin (0,1-10 μM) kunne imidlertid forbedre cellelevedygtigheden betydeligt og reducere apoptose og oxidativ stressskade forårsaget afH2O2. I celler med overdreven autofagisk aktivering kunne crocetin også reducere autofagistrømmen og ekspressionen af mitofagirelaterede proteiner PINK1 og Parkin og vende overførslen af Parkin til mitokondrier. Crocetin kunne reducere H2O2-medieret oxidativ stressskade og apoptose af H9c2-celler, og dets mekanisme var tæt forbundet med mitofagi.

Virkninger af crocetin på celle levedygtighed Crocetin ved 0,1 μM, 0,5 μM, 1 μM, 5 μM, 10 μM, 50 μM og 100 μM havde en signifikant proliferativ virkning på celler, mens crocetin i koncentrationer over 200 μM signifikant hæmmede proliferation af H9c2-celler (figur 1A). Efter 4 timers behandling med 400 μMH2O2blev cellelevedygtigheden reduceret betydeligt, og crocetin kunne til en vis grad vende denne ændring (figur 1B...

Watch this Scientific Journal Video about Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy at JoVE.com

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Baseret på in vitro-eksperimenter afslørede denne undersøgelse crocetins mekanisme til reparation af oxidativ stressskade på kardiomyocytter ved at påvirke mitofagi, hvor PINK1 / Parkin-signalvejen spiller en vigtig rolle.

Beskyttelse af H9c2 myokardceller mod oxidativ stress af Crocetin via PINK1 / Parkin Pathway-medieret mitofagi;

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and ...

Denne protokol giver en reference til tekniktilgangen til studiet af autofag, og den beskriver hele eksperimentelle procedurer i detaljer. Den største fordel ved denne teknik er, at den tillader længdeobservation af ændringen i autofagisk flux. Før du starter eksperimentet, skal du bestemme den effektive infektionskoncentration og træffe de nødvendige sikkerhedsforanstaltninger.Begynd med at centrifugere neutraliserede H9C2 myokardceller ved 179G i fem minutter ved stuetemperatur. Supernatanten kasseres, og cellerne blandes i DMEM-fuldmedium ved forsigtigt at blæse. Opdel cellerne i ni lige store proportioner.Der tilsættes dimethylsulfoxid til kontrolgruppen. Derefter tilsættes varierende koncentrationer af crocetin til de resterende grupper. Frø 100 mikroliter celler pr. Brønd i en 96-brøndplade.Når supernatanten er inkuberet i 24 timer, kasseres den. Derefter vaskes cellerne tre gange med PBS. Tilsæt 100 mikroliter DMEM basalmedium til hver brønd og inkuber cellerne i fire timer ved 37 grader Celsius.Tilsæt nu 20 mikroliter MTS og inkuber i yderligere to timer som demonstreret. Endelig måles absorbansen ved 490 nanometer og beregnes cellelevedygtigheden. For at bestemme ROS fortyndes DCFH-DA i forholdet 1:1000 med det serumfrie medium.Kassér cellens supernatant fra en plade med 48 brønde, der tidligere er podet med H9C2-celler. Vask derefter cellerne to gange med et serumfrit medium. Tilsæt 150 mikroliter fortyndet DCFH-DA til hver brønd og inkuber den ved 37 grader Celsius i 20 minutter.Vask derefter cellerne tre gange med serumfrit medium. Endelig tag billeder ved hjælp af et fluorescensmikroskop. For at analysere mitokondriemembranpotentiale skal du tilføje JC-1-arbejdsløsning til de tidligere dyrkede celler og blande godt.Inkuber dem ved 37 grader Celsius i 20 minutter. Vask derefter cellerne to gange med JC-1 farvningsbuffer. Tilføj komplet medium til hver brønd og tag fotografier ved hjælp af et fluorescensmikroskop.Fastgør cellerne med cellefikseringsopløsning i 30 minutter ved stuetemperatur. Vask dem derefter med PBS én gang. Tilsæt 0,3% Triton-100 til hver brønd og inkuber ved stuetemperatur i fem minutter.Efter to PBS-vaske tilsættes TUNEL-detektionsarbejdsløsning til hver brønd og inkuberes cellerne ved 37 grader Celsius i mørke i 60 minutter. Tilføj DAPI, der indeholder en tablet til dæmpning af fluorescens, og tag billederne som vist. Udskift halvdelen af mediet fra de tidligere dyrkede celler med frisk medium i hver brønd på en 48-brøndplade.Tilsæt mCherry GFP-LC3B adenovirus efterfulgt af polybren for at forbedre infektionseffektiviteten. Udskift mediet hver 24. time. Samtidig med at du bruger et konfokalmikroskop, skal du observere fluorescerende proteinekspression for at bekræfte infektion.Når de er inficeret, skal du dyrke og fange cellerne som beskrevet i manuskriptet. Begynd med at fiksere tidligere dyrkede celler med 4% paraformaldehyd i 10 minutter ved stuetemperatur og tilsæt 0,1% Triton-100. Bloker derefter cellerne med en dyrefri blokopløsning i en time.Derefter inkuberes dem med et primært antistof ved fire grader Celsius natten over. Den næste dag tilsættes et fluorescerende sekundært antistof til cellerne og inkuberer dem i mørke i en time. Til sidst skal du tilføje DAPI, der indeholder anti-fluorescensdæmpende tabletter, før du tager billederne.I denne undersøgelse havde crocetin en signifikant proliferativ virkning på celler. Mens en koncentration over 200 mikromolær hæmmede proliferation af H9C2-celler. Cellelevedygtigheden blev reduceret betydeligt efter hydrogenperoxidbehandling.Crocetin vendte imidlertid denne ændring til en vis grad. Det forbedrede ROS-niveau efter hydrogenperoxidbehandling blev til en vis grad vendt af crocetin. Fluorescensresultaterne bekræftede også reduceret ROS med crocetin.Endvidere genoprettede crocetin desuden mitokondriemembranpotentialet kollapser forårsaget af hydrogenperoxidbehandling. Tilsvarende blev apoptose også vendt af crocetin. Derudover vendte crocetinforbehandlingen autofagistrømmen af H9C2-kardiomyocytter, induceret på grund af hydrogenperoxidbehandling.Crocetin-forbehandling reducerede også translokationen af Parkin-mitokondrier induceret af hydrogenperoxidbehandling. Efter denne procedure kan elektronmikroskopisk også bruges til direkte at observere en mikrofag, hvilket giver mulighed for et mere intuitivt billede af mitokondrier, der opsluges af autofagosomer.

Research

JoVE Journal

Medicine

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

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