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Ferroptosis is a new type of programmed cell death， characterized by iron overload and lipid peroxidation. Cardiovascular disease （CVD） is an ischemic or hemorrhagic disease of the heart caused by various factors， mainly including myocardial infarction， heart failure， etc. Ferroptosis is involved in the process of myocardial cell damage and plays a driving role in the progression of various CVDs. Its main mechanisms include the destruction of iron homeostasis， the production of reactive oxygen species， the disorder of the antioxidant system， mitochondrial membrane damage， endoplasmic reticulum stress， tumor suppressor gene p53， transcription factor Nrf2 pathway， etc. Myocardial injury is one of the causes of death in many patients with heart disease. Monomers or compounds of traditional Chinese medicine have shown good effects in the treatment of myocardial cell injury caused by ferroptosis， including baicalin protecting cardiac microvascular endothelial cells of myocardial ischemia-reperfusion （I/R） rats through intracellular phosphatidylinositol kinase/phosphokinase B/endothelial nitric oxide synthase （PI3K/Akt/eNOS） pathway， Aralia elata saponin inhibiting myocardial cell ferroptosis through glucocorticoid receptor/p53/solute carrier family 7 members 11 （NR3C1/p53/SLC7A11） pathway， Xinyang tablets improving oxidative stress by regulating phosphorylated serine/threonine protein kinase/stress-activated protein kinase/p53 （MLK3/JNK/p53） signaling pathway. It is of great significance to explore the mechanism of ferroptosis and the protective effect of related traditional Chinese medicine after myocardial cell injury. This article reviews the mechanism of ferroptosis and its relationship with myocardial cells， as well as traditional Chinese medicine monomers and formulas for treating CVDs through the ferroptosis pathway. The article focuses on the pathways and effects of traditional Chinese medicine treatment， so as to provide a reference for the treatment of CVDs with traditional Chinese medicine.
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This study aimed to investigate the effect of Wenyang Zhenshuai Granules(WYZSG) on autophagy and apoptosis of myocardial cells in rats with sepsis via regulating the expression of microRNA-132-3p(miR-132-3p)/uncoupling protein 2(UCP2). Sixty SD rats were randomly divided into modeling group(n=50) and sham operation group(n=10). The sepsis rat model was constructed by cecal ligation and perforation in the modeling group. The successfully modeled rats were randomly divided into WYZSG low-, medium-and high-dose groups, model group and positive control group. Rats in the sham operation group underwent opening and cecum division but without perforation and ligation. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of rat myocardial tissue. Myocardial cell apoptosis was detected by TdT-mediated dUTP nick end labeling(TUNEL) assay. Real-time quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression of miR-132-3p and the mRNA expressions of UCP2, microtubule-associated protein light chain 3(LC3-Ⅱ/LC3-Ⅰ), Beclin-1 and caspase-3 in rat myocardial tissue. The protein expressions of UCP2, LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3 in myocardial tissue were detected by Western blot. Dual luciferase reporter assay was used to verify the regulatory relationship between miR-132-3p and UCP2. The myocardial fibers of sepsis model rats were disordered, and there were obvious inflammatory cell infiltration as well as myocardial cell edema and necrosis. With the increase of the WYZSG dose, the histopathological changes of myocardium were improved to varying degrees. Compared with the conditions in the sham operation group, the survival rate and left ventricular ejection fraction(LVEF) of rats in the model group, positive control group and WYZSG low-, medium-and high-dose groups were decreased, and the myocardial injury score and apoptosis rate were increased. Compared with the model group, the positive control group and WYZSG low-, medium-and high-dose groups had elevated survival rate and LVEF, and lowered myocardial injury score and apoptosis rate. The expression of miR-132-3p and the mRNA and protein expressions of UCP2 in myocardial tissue in the model group, positive control group and WYZSG low-, medium-and high-dose groups were lower, while the mRNA and protein expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3 were higher than those in the sham operation group. Compared with model group, the positive control group and the WYZSG low-, medium-and high-dose groups had an up-regulation in the expression of miR-132-3p and the mRNA and protein expressions of UCP2, while a down-regulation in the mRNA and protein expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3. WYZSG inhibited excessive autophagy and apoptosis of myocardial cells in septic rats and improved myocardial injury, possibly by regulating the expression of miR-132-3p/UCP2.
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Animals , Rats , Rats, Sprague-Dawley , Caspase 3 , Beclin-1/genetics , Stroke Volume , Ventricular Function, Left , Apoptosis/genetics , Autophagy/genetics , Heart Injuries , MicroRNAs/geneticsABSTRACT
OBJECTIVE：To study spectrum-effect relationship of 11 different solvent extracts from Trollius chinensis against hypoxia/reoxygenation injury of cardiomyocyte . METHODS ：HPLC-MS/MS method was used to establish the fingerprints of 11 different solvent extracts from T. chinensis ，the compounds corresponding to the common peaks were identified by comparing with the substance control and literature information. MTT assay was used to detect the effects of 11 different solvent extracts from T. chinensis on the survival rate of rat myocardial H 9c2 cells injured by hypoxia/reoxygenation. The MDA content ，ROS level in cells and LDH content in the supernatant were detected by ELISA. GRA and PLS method were used to analyze the spectrum-effect relationship between the compounds corresponding to common peak and anti-hypoxia/reoxygenation injury of cardiomyocyte （drug effect）. RESULTS ：There were 22 common peaks in 11 different solvent extracts from T. chinensis ，and 22 compounds were identified. Compared with hypoxia/reoxygenation injury group ，survival rate of hypoxia/reoxygenation injury+S 1-S6，S9 and S 10 groups were increased significantly ，while MDA content ，ROS level and LDH content were decreased significantly （P＜0.05）； ROS level and LDH content of hypoxia/reoxygenation injury+S 8 group w ere decreased significantly （P＜0.05）. The r of GRA analysis of 22 compounds with drug effects were all higher than 0.8. Except for peaks 1，2，7，13，14 and 21，r of PLS analysis of rest peaks with drug effects were higher than 0 发。电话：0431-86058683。E-mial：nml2000@163.com （being positive correlation ）. Top 9 common peaks in the list of contribution rate were peak 6＞11＞4＞5＞8＞9＞12＞10＞15. CONCLUSIONS ：Orientin（peak 6），vitexin（peak 11）， orientin-2″-O-β-L-galacto- pyranosl （peak 4），orientin-2″-O-β-D-Pyrine xylosides （peak 5），quercetin-3-O-glucopyranoside（peak 8），vitexin-2″-O-β-L-galactoside（peak 9），hyperoside（peak 12），vitexin-2″-O-β-D-pyrine xylosides （peak 10），2″-O-（2″′- methylbutyry-loxy）-orientin（peak 15）may be the main components of anti-hypoxia/reoxygenation injury of cardiomyocytes.
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Objective To investigate the protective effect of Jiawei Danshen Yin combined with microRNA-21 on IRI myocardial cells,and to study its protective mechanism. Methods H9C2 cardiomyocytes were cultured and divided into four groups: Blank group, IRI model group of cardiomyocytes,miR-21 group,miR-21 combined with Jiawei Danshen Yin group (JWDSY group). Apoptotic rate, apoptosis-related protein and PTEN signaling pathway expression of cardiomyocytes were detected. Results Electron microscopy showed that mitochondria in model group were damaged significantly , and ultrastructural damage in JWDSY group was alleviated; ELISA demonstrated that cTnl was elevated in model group and decreased in JWDSY group with statistical significance (P < 0. 01); flow cytometry re vealed that the apoptotic rate was significantly increased in model group (36. 79 ±2. 12) ,but markedly decreased in JWDSY group (14. 65 ± 0. 94), and the difference was statistically significant ( P < 0. 05 ). Western blot results showed that PTEN expression was uP-regulated and P-Akt expression down-regulated in model group,while PTEN expression decreased and P-Akt expression increased in JWDSY group, and the difference was statistically significant ( P < 0. 05 ). Conclusions miR-21 combined with Jiawei Danshen Yin can inhibit cardiomyocyte apoptosis and protect IRI cardiomyocytes by reducing PTEN expression and activating PI3K/Akt signaling pathway,.
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OBJECTIVE： To study the protective effects of Pericarpium Trichosanthis extract（TPE） on H9c2 myocardial cells injured by hypoxia/reoxygenation（H/R）. METHODS： H/R injury model of H9c2 myocardial cells was established by using sodium dithionite （Na2S2O4） as chemical hypoxia agent. The modeling conditions of different concentrations of Na2S2O4 （0.625-10 mmol/L） and different time of hypoxia （10-60 min） and reoxygenation （2-8 h）， as well as the concentration of TPE （12.5-400 μg/mL） were screened. The cultured H9c2 myocardial cells were randomly divided into normal group， model group， TPE different dose groups （TPE low-dose， medium-dose and high-dose groups， 25， 50， 100 μg/mL） and positive control group （quercetin， 25 μmol/L）. They were pre-treated with relevant medicine for 24 h， and then H/R injury model was established. Cell viability was measured by MTT assay. The levels of LDH， CK-MB， SOD and MDA in cells were tested by ELISA. Apoptosis of H9c2 myocardial cells were observed by flow cytometry. Western blotting was used to detect the protein expression of Bax and Bcl-2 in cells. RESULTS： The condition of H/R injury modeling included modeling concentration of Na2S2O4 2.5 mmol/L， hypoxia time 30 min， reoxygenation time 4 h. 12.5-400 μg/mL TPE showed no toxicity to H9c2 myocardial cells. After treatment， compared with blank group， survival rate and apoptotic rate of H9c2 myocardial cells in model group were increased significantly； the levels of CK-MB， LDH and MDA were increased significantly， while SOD level was decreased significantly； protein expression of Bax and Bax/Bcl-2 ratio were increased significantly， while that of Bcl-2 was decreased significantly （P＜0.05 or P＜0.01）. Compared with model group， above changes of H9c2 myocardial cells were reversed in all dose groups of TPE （P＜0.05 or P＜0.01）. CONCLUSIONS： TPE can protect H9c2 myocardial cells against H/R injury. Its mechanism may be associated with inhibiting the increase of lipid peroxide， improving the ability of scavenging reactive oxygen free radicals， up-regulating the protein expression of Bcl-2 or down-regulating protein expression of Bax， so as to inhibit the cell apoptosis.
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Aim: To establish the standard hypoxia/reoxygenation (H/R) injury model of H9c2 myocardial cells and evaluate the intervention effect of notoginsenoside R, based on RTCA technology. Methods: H9c2 myocardial cells were inoculated into the E-Plate board, 3 wells by a group, then the growth curve and cell index of different hypoxia time, different reoxygenation time and notoginsenoside R, intervention were determined respectively, with the growth status of H9c2 myocardial cells reflected by cell index. Results: Cell survival rate was (48.82 ±5.32)% after hypoxia treatment for 4h and reoxygenation treatment for 16h with replacement of serum-free, glucose-free DMEM medium before hypoxia, and no significant difference was found between the results measured with MTT method. Notoginsenoside R, could protect the hypoxia/reoxygenation injury of H9c2 myocardial cells without time dependence. Conclusions: RTCA technology is an effective means to establish the standard H/R injury model of H9c2 myocardial cells, which also has some guiding significance in discovering new drug targets and mechanisms.
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Aim To establish an in vitro arrhythmia detection technique based on real-time cell analysis (RTCA) Cardio sys-tem with aconitine as a tool drug, and to provide a reliable method for the development of antiarrhythmic drugs. Methods The effects of aconitine on rat cardiac rhythm were detected by eight-channel physiological recorder at the level of whole animal. In vitro cultured cardiac myocytes, the inoculation density of cardiac myocytes was investigated by HTCA method, the effect of aconitine on cardiac beating was monitored by RTCA Cardio system, and the CI value, beating rate, amplitude and irregular rhythm of cardiac myocytes were analyzed. Results Eight-channel physiological recorder was used to detect the effects of aconitine on whole animals. The results showed that aconitine(50 mg • g"1) could induce arrhythmias such as ventricular tachycardia, ventricular fibrillation, shortened RR interval and increased heart rate. RTCA Cardio system showed that aconitine (2-8 p,M) could induce arrhythmias such as increased cardiac cell beating frequency, decreased beating amplitude and abnormal beating state in a dose-dependent manner. Conclusions RTCA Cardio system can rapidly, sensitively and accurately detect the arrhythmia induced by aconitine in cardiac myocytes, which provides methodological reference for the development of antiarrhythmic drugs.
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Objective To investigate the total flavones of rhododendra(TFR) on contractility of rat myocardial cells and its possible mechanism. Methods The contraction amplitude and contraction frequency of primary cultured rat myocardial cells were observed by image analysis system. The intracellular free Ca2 + content was measured by calcium ion imaging system. Results 10 and 100 nmol /L hU Ⅱ significantly accelerated the contraction frequency of myocardial cells,and 10 nmol /L hU Ⅱ increased the contraction amplitude of myocardial cells,but 100 nmol /L hU Ⅱ reduced the contraction amplitude of myocardial cells. TFR 300 mg /L significantly slowed the contraction frequency of rat myocardial cells and increased the contraction amplitude. TFR in the range of 33. 3 ～ 300 mg /L could significantly inhibit the increase of contraction frequency,the decrease of contraction amplitude and the increase of intracellular free Ca2 + content induced by 100 nmol /L hU Ⅱ. Conclusion TFR can slow down the contraction frequency of myocardial cells and increase its contractility,which may be related to the decrease of free Ca2 + content in myocardial cells.
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Objective: To study the injury effect and molecular mechanism of high glucose on myocardial cells. Methods: Myocardial cells H9c2 were cultured and divided into the control group treated with DMEM containing 5.5 mmol/L glucose, the high glucose group treated with DMEM containing 35 mmol/L glucose, and the N-acetylcysteine (NAC) group pre-treated with 1000 ?mol/L NAC and treated with DMEM containing 1000 ?mol/L NAC and 35 mmol/L glucose. The production of ROS and the expression of mitochondria pathway apoptosis molecules in cells as well as the contents of collagen and collagen metabolism molecules were measured. Results: After 8 h, 16 h and 24 h of treatment, ROS RFU as well as Bax, CytC, Caspase-3 and Caspase-9 protein expression in cells and Col-I, Col-III, PINP and PIIINP protein levels in culture medium of high glucose group were higher than those of control group, Bcl-2 protein expression were lower than those of control group, but CTX-I protein levels in culture medium were not significantly different from those of control group; after 24 h of treatment, Bax, CytC, Caspase-3 and Caspase-9 protein expression in cells as well as Col-I, Col-III, PINP and PIIINP protein levels in culture medium of NAC group were lower than those of high glucose group whereas Bcl-2 protein expression was higher than that of high glucose group. Conclusions: High glucose can induce myocardial cell apoptosis, increase collagen synthesis and accelerate interstitial fibrosis by increasing the production of reactive oxygen species.
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Objective:To study the injury effect and molecular mechanism of high glucose on myocardial cells.Methods:Myocardial cells H9c2 were cultured and divided into the control group treated with DMEM containing 5.5 mmol/L glucose, the high glucose group treated with DMEM containing 35 mmol/L glucose, and the N-acetylcysteine (NAC) group pre-treated with 1 000 μmol/L NAC and treated with DMEM containing 1 000 μmol/L NAC and 35 mmol/L glucose. The production of ROS and the expression of mitochondria pathway apoptosis molecules in cells as well as the contents of collagen and collagen metabolism molecules were measured.Results:After 8 h, 16 h and 24 h of treatment, ROS RFU as well as Bax, CytC, Caspase-3 and Caspase-9 protein expression in cells and Col-I, Col-III, PINP and PIIINP protein levels in culture medium of high glucose group were higher than those of control group, Bcl-2 protein expression were lower than those of control group, but CTX-I protein levels in culture medium were not significantly different from those of control group; after 24 h of treatment, Bax, CytC, Caspase-3 and Caspase-9 protein expression in cells as well as Col-I, Col-III, PINP and PIIINP protein levels in culture medium of NAC group were lower than those of high glucose group whereas Bcl-2 protein expression was higher than that of high glucose group.Conclusions:High glucose can induce myocardial cell apoptosis, increase collagen synthesis and accelerate interstitial fibrosis by increasing the production of reactive oxygen species.
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OBJECTIVE: To explore the protective effect of ginkgo-diyidamolum injection on rat myocardial cells injured by oxygen- glucose deprivation. METHODS: The model of oxygen-glucose deprivation in rat myocardial cells was established. The cells were randomly divided into five groups: normal group, oxygen-glucose deprivation group, different ginkgo-diyidamolum injection dosage groups(0.28, 0.84, 1.40 mg•mL-1). The effects of ginkgo-diyidamolum injection on NO level, malondialdehyde (MDA) level, changes in the concentrations of lactate dehydrogenase (LDH), superoxide dismutase (SOD) activity were evaluated. Cells apoptosis rate was determined by flow cytometry. The gene transcription level of myocardial cell gap junction protein 43 (Cx43) was monitored by RT-PCR. RESULTS: Compared with oxygen-glucose deprivation group, ginkgo-diyidamolum injection could reduce the content of intracellular NO, MDA, LDH and enhance the activity of SOD. Ginkgo-diyidamolum injection high dose and middle dose groups obviously improved the myocardial cell morphology, inhibited cell apoptosis and Cx43 gene in myocardial cells. CONCLUSION: Ginkgodiyidamolum injection has a strong protective effect when myocardial cells injured by oxygen-glucose deprivation, which is related to the antioxidant effect, inhibition of cell apoptosis, and maintaining the role of myocardial gap junction channel.
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OBJECTIVE To study the effect of total flavonoids of Dracocephalum heterophyllum (TFDH),a Uygur medicine,on cardiomyocyte hypertrophy induced by norepinephrine(NE),and to provide insights into the mechanism.METHODS Cardiomyocytes of primarily cultured neonatal rats were used as models. Myocardial hypertrophy was induced by NE 2 μmol·L-1. The cells were divided into cell control group,NE 2 μmol·L-1model group,and TFDH 10,25 and 50 μmol·L-1+NE 2 umol·L-1groups.CCK-8 method was used to observe the activity of myocardial cells,while RT-PCR technique was used to detect the expression of mRNA of cardiac hypertrophy gene atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC). The internal factors were glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Confocal laser scanning was used to detect the surface area of myocardial cells and[Ca2+]i.The activity of Ca2+-ATP was measured with enzymatic reaction of fragmentation cells. The concentration of NO and the activity of NOS were determined with colorimetry. RESULTS Compared with the cell control group, cardiomyocytes were stimulated at 48 h by NE 2 μmol·L-1, which could decrease the survival rate of cardiomyocytes from(95±1)% to(78+5)%,surface area increased from (178±29)μm2to(274± 38)μm2(P<0.05),the expression of mRNA of ANP and β-MHC increased from(1.00±0.01)and(1.00± 0.02)to(2.76±0.55)and(2.69±0.31),respectively(P<0.05).The concentration of[Ca2+]iincreased from 1.00±0.12 to(1.52 ± 0.41)μmol·L-1,while the activity of Ca2+-ATP decreased from 1.01±0.14 to(0.41± 0.06)μmol·L-1(P<0.05).The concentration of NO decreased from 1.50±0.14 to(1.12±0.05)μmol·L-1,and the activity of NOS decreased from 0.86±0.06 to(0.52±0.10)μmol·L-1(P<0.05).TFDH 10,25 and 50 μmol·L-1could inhibit the decline of the survival rate,increase of the surface area and the increased expressions of mRNA of ANP and β-MHC that were induced by NE(P<0.05).At the same time,it also could inhibit the increase of the concentration of[Ca2+]i,the decreased of activity of Ca2+-ATP,and the decline of the concentration of NO and the activity of NOS (P<0.05). CONCLUSION TFDH can improve the activity of cardiomyocyte hypertrophy induced by NE, down-regutate mRNA expressions of proto oncogene ANP and β-MHC,and reduce the surface area of cardiomyocytes induced by NE.The mecha-nism may be related to promoting the release of NO and regulating the concentration of [Ca2+]iand the activity of Ca2+-ATP.
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Aim To assess the regulatory effects of Po-lygonatum sibiricum polysaccharides(PSP)on Toll-like receptor 4 (TLR4 )-myeloid differentiation factor 88 (MyD88)-nuclear factor κB(NF-κB)signaling path-way in anoxia /reoxygenation-H9c2 myocardial cells. Methods The H9c2 myocardial cells cultured in vitro were randomly divided into groups:control group (C group),hypoxia /reoxygenation group (H/R group), PSP group,TLR4 inhibitor group(TAK-242 group)and PSP +TLR4 inhibitor group(PSP +TAK-242 group). The cells were cultured in normal condition for 27 h in C group.The cells were subjected to 21 h hypoxia fol-lowed by 6 h reoxygenation in H/R.Definitely,the cells in TAK-242,PSP and PSP +TAK-242 groups were treated with PSP and TAK-242 with the final con-centration of 1 .5 g·L -1 and 1 μmol·L -1 for 1 2 h before 21 h hypoxia,then the cells were exposed to the normal culture condition for another 6 h.After the treatment,cell survival rate was tested by MTT method. The contents of tumor necrosis factor-α(TNF-α)and interleukin-1 β(IL-1 β)were determined by enzyme-linked immunosorbent assay(ELISA).The protein ex-pression levels of NF-κB and inhibitor κBα(IκBα) were detected by Western blot,and the expression lev-els of TLR4 and MyD88 mRNA were detected by fluo-rescence quantitative PCR method.Results Compared with H/R group,the cell survival rates were significant-ly increased,while the inflammatory cytokines contents and NF-κB protein expression were dramatically de-creased in groups PSP,TAK-242 and PSP +TAK-242, whereas the NF-κB expression was significantly down-regulated,and the IκBα protein expression was in-creased.The mRNA expression levels of TLR4 and MyD88 were markedly decreased.Conclusion PSP might protect H9c2 myocardial cells against H/R inju-ry,which may be associated with the inhibition of TLR4-MyD88-NF-κB pathway.
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To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken (Gallus gallus) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.
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Apoptosis , Aspirin , Cell Survival , Chickens , Heat Stress Disorders , Heat-Shock Proteins , Hot Temperature , HSP90 Heat-Shock Proteins , In Vitro Techniques , Proto-Oncogene Proteins c-akt , Reactive Oxygen Species , TransducersABSTRACT
Objective Bone morphogenetic protein 4 ( BMP4) induces rat myocardial hypertrophy in H9c2 cells, but the spe-cific mechanism remains unclear .The study aimed to elucidate the role of autophagy in myocardial hypertrophy and its relationship with extracellular signal regulating kinase ( ERK1/2) signal transduction pathway . Methods H9c2 cardiomyocytes were randomly divided into 4 groups:control group, BMP4 group, BMP4+PD98059 (ERK1/2 signal pathway inhibitor) group and BMP4+3MA (autophagy inhibitor) group.With PD98059(50μmol/L) and 3MA(5mmol/L) blocking for 30min, BMP4 (50μg/L) were added.Expressions of tiny tube related proteins 1 light chain 3 (LC3) and p-ERK1/2 were detec-ted after culturing for 30min.48h later, measurements were made on cell surface area , average protein content and α-smooth muscle actin (α-SMA ) protein expression level .Cell morphology and size were measured respectively by inverted microscope and Image J software .Total protein content was detected by BCA , and western blot was used to measure the expressions of LC3, ERK1/2, p-ERK1/2 and α-SMA. Resul ts 30min later, the expressions of LC3 and p-ERK1/2 were significantly higher in BMP4 group than in control group [(1.54 ±0.05) vs (1.95 ±0.11),(0.94 ±0.04) vs (1.33 ± 0.06),P0.05). Conclusion Autophagy may participate in BMP4-induced rat myo-cardial hypertrophy in H9c2 cells through ERK1/2 signal transduction pathway .The clinical treatment of myocardial hypertrophy may benefit from the blocking of autophagy or ERK 1/2 signal transduction pathway .
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Objective To establishment the model of angiotensin Ⅱ (AngⅡ) inducinged cardiomyocyte hypertrophy in H9c2 myocardial cells, and observinge the the changes of ubiquitin binding protein leveljunction protein ubiquitination level. Methods Myocardial cell isolated and subcultured H9c2 were cultured in vitro andmyocardial cell, they were divided into three groups in different AngⅡ concentration (10-8, 10-7, 10-6 mol/L), with in different hours (0, 6, 12, 24, 48, 72 h), and there is also a control group; measured the total protein contentlevel were measured useby BCA method , measured the number of cardiomyocytes in the same area were measured, and the relative surface area of myocardial cells were calculated. The expression of ubiquitin binding protein cardiomyocyte hypertrophy was time- and concentration-dependently induced by AngⅡ, wheremanifested, as total cellular protein content,relative cell surface area and the expression of ubiquitin binding proteinubiquitinated desmin significantly increasing compared with the control group (P < 0.05), which were most significant in 10-7 mol/L AngⅡtreated group for 48 h. Conclusions Cardiomyocyte hypertrophy model induced by 10-7 mol/L AngⅡ for 48 h , was successfully established , where H9c2 cardiomyocyte hypertrophy was induced by 10-7 mol/L AngⅡ in 48 h, and ubiquitin binding proteinubiquitinated desmin significantly increased in cardiomyocyte hypertrophy.
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Objective The stimulating factors and etiology of myocyte hypertrophy are not yet clear.We aim to investigate the mechanisms of bone morphogenetic protein 4 (BMP4)-induced H9C2 myocyte hypertrophy in rats by activating the extracellular signal regulating kinase ( ERK1/2) signaling pathway. Methods Rat H9C2 myocytes were cultured, the model of BMP4-induced H9C2 myocyte hypertrophy was established, and the cells were randomly di-vided into control A, BMP4 A, and angiotensinⅡ( AngⅡ) groups to be treated for 48 hours.The changes in the expression of ERK1/2 protein phosphorylation were observed at 0, 10, 30, 60, and 120 min after treated with BMP4 at the concentration of 50 μg/L and at 30 min after treated with BMP4 at 0, 10, 50, and 100 μg/L.The changes of the H9C2 myocytes were also observed after blocking the ERK1/2 signaling pathway.The cells were divided into control B, BMP4 B, BMP4+PD98059, and PD98059 groups to be cultured with 50μg/L BMP4 for another 48 hours after 30 min blockage with PD98059 at 50 ×10 -6 mol/L.The size and area of the cells were measured by inverted microscopy and image J Software, their total protein content detected by BCA, and the expressions of p-ERK1/2 and brain natriuretic peptide ( BNP) determined by Western blot. Results Compared with control A, the BMP4 A, and AngⅡ groups showed markedly larger cell areas ([649.74 ±2.03] vs [744.85 ±1.31] and [748.39 ±1.54] μm2), higher protein contents ([243.18 ±3.69] vs [340.09 ±2.14] and [347.43 ± 3.30] pg/cell), and higher expressions of the BNP protein ([0.72 ±0.44] vs [0.96 ±0.55] and [1.07 ±0.55]/GAPDH), with statistically significant differences between any two groups (P>0.05).After BMP4 intervention, the expression of p-ERK1/2 manifes-ted a time-dependent trend of first going up and then coming down, reaching the peak at 30 min, with the ERK1/2 protein phosphoryl-ation significantly higher at 30 min than at 10 min (P>0.05), especially than at 0, 60, and 120 min (P>0.01).The level of p-ERK1/2 protein phosphorylation in the mytocytes was elevated with the increased concentration of BMP4, remarkably higher at 50μg/L than at 0 and 10μg/L, but lower than at 100μg/L ( P>0.01) .The cell area, protein content, and BNP protein expression of the H9C2 myocytes were significantly lower in the BMP4+PD98059 group ([650.49 ±1.28] μm2, [244.06 ±3.48] pg/cell, and [0.55 ±0.15]/GAPDH) than in the BMP4 B group ([746.04 ±1.45] μm2, [343.57 ±4.11] pg/cell, and [0.79 ±0.55]/GAPDH) (P>0.01). Conclusion BMP4 may induce hypertrophy of rat H9C2 myocytes by activating the ERK1/2 signaling path-way.Early blocking of ERK1/2 activation may contribute to the management of myocardial hypertrophy.
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Objective: To study the effects of Compound Granules of Yinxingye (Ginkgo leaf) on H2O2-induced cellular injury in H9c2 myocardial cells of rats. Methods: H9c2 cells were treated with H2O2 to establish a myocardial injury model, and then pretreated by Compound Granules of Yinxingye at the concentration of 25, 50, 100, and 200 μg/mL for 24 h. Cell viability was measured by methyl thiazolyl tetrazolium (MTT). Morphological changes were observed by phase contrast microscope. Lactic dehydrogenase (LDH), creatine kinase-MB (CK-MB), malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO) levels were detected by biochemical kits. Intracellular reactive oxygen species and calcium ion concentration and mitochondrial membrane potential were measured by laser confocal microscopy. Results: The exposure of H9c2 cells to 500 μmol/L H2O2 for 6 h could reduce cell viability significantly and induce oxidative stress injury. The Compound Granules of Yinxingye pretreatment could improve the cell viability, improve the damage cell morphology change, reduce LDH, CK, MDA, and NO levels in the cell supernatant, increase SOD activity. ROS generation and concentration of intracellular calcium could be reduced and the in mitochondrial membrane potential declined. Conclusion: The Compound Granules of Yinxingye has the protective effect on H2O2-induced cellular injury in H9c2 myocardial cells.
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Aim To investigate the effect of Sonic Hedgehog on normal hearts and hypoxic-ischemic myo-cardial cells. Methods A method for left anterior de-scending artery ( LAD ) ligation was employed to con-struct the myocardial infarction model, and ultrasonic cardiogram was used for identification. Western blot and immunofluorescence staining were used to detect expressions of Shh, Ptch-1, Smo and Gli-1 in H9C2 cells and H2 O2-induced H9C2 cells, and that in 12 ca-ses of myocardial infarction tissues and 9 cases of nor-mal myocardium, respectively. Agonist and antagonist of Shh pathway were adminstered in the hypoxic-ische-mic myocardial H2 O2-induced H9C2 cell model, and once again expressions and strength of Shh, Ptch-1, Smo, Gli-1 were detected. Results Shh and Gli-1 were not expressed in normal hearts, but expressed in hearts with myocardial infarction;Ptch-1 and Smo were expressed in both normal hearts and hearts with myo-cardial infarction. Under the action of agonist, expres-sions of Shh and Gli-1 increased in the hypoxic-ische-mic H9C2 cell model. Similarly, Shh and Gli-1 were not expressed in normal H9C2 cells, but in H2 O2-in-duced H9C2 cells, and Ptch-1 and Smo were expressed in both normal H9C2 and in H2 O2-induced H9C2 cells. Conclusion Shh signaling pathway can be acti-vated in the condition of ischemia and oxidative stress, and then it promotes the repairing of myocardial cell damage.
ABSTRACT
This research firstly establishes the oxidative damage model of H9c2 induced by H2O2 and screens the concentration range of intestines absorption liquid of Qi benefiting and blood circulation activating formula which possess external myocardium protection function. Then, the thesis chooses 4 dosages to conduct experiments：examining the protection function of intestines absorption liquid of Qi benefiting and blood circulation activating formula on H9c2 to provide reference for clinical prevention and curing of relative heart diseases of oxidative stress injury; as well as examining the H9c2 cardiac muscle cell vigour, cellular morphology, SOD, MDA and other indexes to primarily evaluate and discuss the functional mechanism of intestines absorption liquid of Qi benefiting and blood circulation activating formula. The results show that the intestines absorption liquid of Qi benefiting and blood circulation activating formula has relatively better protection function toward the H9c2 cardiac muscle cell damage induced by H2O2 and presents concentration dependency to some extent. The intestines absorption liquid of Qi benefiting and blood circulation activating formula can increase SOD vigour, and decrease MDA emission, thus decreasing the formation of abnormal cell and strengthening the oxidation resistance of cardiac muscle cell. The intestines absorption liquid of Qi benefiting and blood circulation activating formula has protection function to some extent.