A preliminary study on the effect of arsenic exposure on necroptosis of mouse myocardial cells / 中华地方病学杂志

Objective:To study the effects of arsenic exposure on necroptosis pathway and inflammatory response of mouse myocardial cells.Methods:Sixty male C57BL/6J mice were randomly divided into control group (group C) and low, medium, and high dose arsenic exposure groups (groups L, M, H) based on body weight using a random number table method. Each group had 15 mice, and they drank 0.00, 0.15, 1.50, and 15.00 mg/L arsenic trioxide (As 2O 3) solution prepared with deionized water. The exposure period was 12 weeks. Hematoxylin-eosin (HE) staining and Masson trichrome staining of paraffin-embedded heart tissues were used to observe the histopathology changes of the heart. Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes of myocardial cells. The quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of inflammatory genes [tumor necrosis factor (TNF)-α and interleukin(IL)-6] and the genes involved in necroptosis pathway [receptor-interacting protein (RIP) 1, RIP3 and mixed-lineage kinase domain-like protein (MLKL)]. Protein expressions of RIP1 and RIP3 in the heart were assessed by western blotting. Results:Histopathological examination results showed there were myocardial necrosis, inflammatory cells infiltration and fibroblasts hyperplasia and other changes in groups M and H. TEM analysis revealed marked ultrastructural changes in groups M and H, including fractured myofibril, fractured Z lines of sarcomere, and swollen mitochondria with fractured cristae. Compared with group C (1.00 ± 0.00), the mRNA expression of RIP1 in group H was significantly up-regulated (1.41 ± 0.06, P < 0.05); the mRNA expressions of RIP3 (1.29 ± 0.14, 1.56 ± 0.08), MLKL (1.23 ± 0.05, 1.36 ± 0.07), TNF-α (2.20 ± 0.10, 2.23 ± 0.18) and IL-6 (1.87 ± 0.16, 1.63 ± 0.15) were significantly up-regulated in groups M and H ( P < 0.05). The protein expressions of RIP1 (0.43 ± 0.04, 0.50 ± 0.04) and RIP3 (0.68 ± 0.02, 0.84 ± 0.05) in groups M and H were higher than those in group C (0.25 ± 0.01, 0.45 ± 0.04, P < 0.05). Conclusion:Subchronic arsenic exposure induces histopathological changes such as myocardial necrosis and fibrosis in mice, inducing necroptosis and inflammatory reactions in myocardial cells.

Effect and mechanism of Danggui Buxue Decoction-containing serum in mitigating H9c2 cell injury caused by exposure to intermittent low oxygen / 中国中药杂志

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.

Role of PNPT1 in cardiomyocyte apoptosis induced by oxygen-glucose deprivation / 南方医科大学学报

OBJECTIVE@#To explore the effect of inhibiting polyribonucleotide nucleotidyl-transferase 1 (PNPT1) on oxygen-glucose deprivation (OGD)-induced apoptosis of mouse atrial myocytes.@*METHODS@#Cultured mouse atrial myocytes (HL-1 cells) with or without OGD were transfected with PNPT1-siRNA or a negative control siRNA (NC-siRNA group), and the cell survival rate was detected using CCK-8 assay. The expression levels of ACTB and TUBA mRNA were detected with qPCR, and the protein expression of PNPT1 was detected with Western blotting. The apoptosis rate of the treated cells was determined with flow cytometry, the mitochondrial membrane potential was detected using JC-1 kit, and the mitochondrial morphology was observed using transmission electron microscope.@*RESULTS@#With the extension of OGD time, the protein expression levels of PNPT1 increased progressively in the cytoplasm of HL-1 cells (P < 0.05). Transfection with PNPT1-siRNA significantly reduced PNPT1 expression in HL-1 cells (P < 0.05). Exposure to OGD significantly enhanced degradation of ACTB and TUBA mRNA (P < 0.05) and markedly increased the apoptosis rate of HL-1 cells (P < 0.05), and these changes were significantly inhibited by transfection with PNPT1-siRNA (P < 0.05), which obviously increased mitochondrial membrane potential and improved mitochondrial morphology of HL-1 cells exposed to OGD.@*CONCLUSION@#Inhibition of PNPT1 improves mitochondrial damage and reduces degradation of apoptotic-associated mRNAs to alleviate OGD-induced apoptosis of mouse atrial myocyte.

Improvement Effect and Its Mechanism of Active Components of Averrhoa carambola on High Glucose-induced Injury of H 9c2 Myocardial Cell / 中国药房

OBJECTIVE：To study the imp rovement effects of 2-dodecyl-6-methoxycyclohexa-2，5-diene-1，4-dione（DMDD） from Averrhoa carambola on H 9c2 myocardial cell injury induced by high glucose and its mechanism. METHODS ：H9c2 myocardial cells were divided into normal group ，high glucose group ，osmotic pressure control group ，DMDD high ，medium and low concentration groups （8，4，2 μmol/L）. Normal group and high glucose group were treated with low glucose DMEM medium （containing 5.5 mmol/L glucose ，similarly hereinafter ） and high glucose DMEM medium （containing 33.3 mmol/L glucose ， similarly hereinafter ）for 48 h，respectively. The cells in osmotic pressure control group were cultured in low glucose DMEM medium containing 27.5 mmol/L mannitol for 48 h. In DMDD groups ，cells were cultured in high glucose DMEM medium for 24 h， and then in high glucose DMEM medium containing corresponding concentration of DMDD for 24 h. At the end of cell culture ，MTT metho d was used to detect the cell survival rate. The activities of ROS ， GSH-Px and LDH in cellsupernatant were detected by using related kits. ELISA assay was used to detect the levels of TNF-α and IL-6 in cell supernatant. Cell apoptosis was d etected by acridine orange/ethidium bromide （AO/EB）staining. Western blot assay was used to detect the expression of apoptosis related proteins （cleaved caspase- 3，Bcl-2，Bax）and the phosphorylation level of nuclear factor κB （NF-κB）/NF-κB suppressor protein α（IκBα）signaling pathway related proteins （NF-κB p65，IκBα）. RESULTS ：Compared with the normal group ，survival rate ，the activity of GSH-Px and protein expression of Bcl- 2 in high glucose groups were decreased significantly（P＜0.01）；the activities of ROS and LDH ，the levels of TNF-α and IL-6，the protein expression of Bax and cleaved caspase-3，and the phosphorylation level of NF-κB p65 and IκBα were increased significantly（P＜0.01）；the cells showed orange yellow fluorescence ，and the number of cells with fuzzy morphology increased significantly ，showing an obvious apoptotic state. There was no statistical significance in above indexes of osmotic pressure control group compared with normal group. Compared with high glucose group ，the activities or levels of above indexes （except for cell survival rate an LDH activity in low concentration group ）were reversed significantly in DMDD groups （P＜0.05 or P＜0.01）；the orange yellow fluorescence in the cells decreased significantly ，and the cell morphology was relatively complete. CONCLUSIONS ：DMDD can significantly improve H9c2 myocardial cell injury induced by high glucose ；the mechanism of which may be associated with suppressing oxidative stress and inflammatory response ，regulating the expression of apoptosis related protein and NF-κB/IκBα pathway related protein.

Research progress on in vitro models of cardiomyocyte injury / 中国中药杂志

Cardiovascular diseases seriously endanger human health and life. The accompanying myocardial injury has been a focus of attention in society. Chinese medicine,serving as a natural and precious reservoir for the research and development of new drugs,is advantageous in resisting myocardial injury due to its multi-component,multi-pathway,and multi-target characteristics. In recent years,with the extensive application of culture method for isolated cardiomyocytes,a cost-effective,controllable in vitro model of cardiomyocyte injury with uniform samples is becoming a key tool for mechanism research on cardiomyocyte injury and drug development.A good in vitro model can reduce experimental and manpower cost,and also accurately stimulate clinical changes to reveal the mechanism. Therefore,the selection and establishment of in vitro model are crucial for the in-depth research. This study summarized the modeling principles,evaluation indicators,and application of more than ten models reflecting different clinical conditions,such as injuries induced by hypoxia-reoxygenation,hypertrophy,oxidative stress,inflammation,internal environmental disturbance,and toxicity. Furthermore,we analyzed advantages and technical difficulties,aiming to provide a reference for in-depth research on myocardial injury mechanism and drug development.

Cyclovirobuxine D ameliorates aldosterone-induced primary neonatal rat cardiac myocyte injury via Ca / 中国药理学通报

Aim To investigate the protective effect of cyclovirobuxine D(CVB-D) on aldosterone (ALD)-induced primary neonatal rat cardiac myocytes (PNRC-Ms) injury and the possible mechanism. Methods PNRCMs were extracted by trypsin, and the PNRCMs injury model was established by ALD (10 μmol · L

Sal b attenuates cardiomyocyte injury by regulating priming of nlrp3 inflammasomes / 中国药理学通报

Aim To investigate whether Sal B alleviates hypoxic-induced cardiomyocyte injury by regulating the priming of NLRP3 inflammasomes. Methods The effects of Sal B on growth of H9C2 cells were examined by CCK8 assay,then the appropriate concen tration of Sal B was selected. The expression level of LDH was detected by Microliter assay. The expression levels of cTn/IL-lp were measured by Elisa assay. The protein and mRNA levels of TLR4/Myd88/I-RAK1/NF-kB/NLRP3 were detected by Western blot and qPCR. Results Hypoxia intervention notably reduced the viability of H9C2 cells and increased the expression levels of cTn/IL-IP. Besides,the protein and mRNA expression levels of TLR4/Myd88/IRAK1/NF-kB/NLRP3 were significant uP-regulated after hypoxia intervention. However, the viability of H9C2 cells increased, the secretion levels of LDH/cTn/IL-1 p were reduced,and the protein and mRNA levels of TLR4/Myd88/IRAK1/NF-KB/NLRP3 were significant inhibited after pretreated with Sal B. Conclusion Sal B attenuates cardiomyocyte injury by regulating the priming of NLRP3 inflammasome.I.

Study on Regulation Effects of Volatile Oil from Angelica sinensis on Autophagy of Myocardial Cell H 9C2 in Rats with Hypoxia/reoxygenation Injury / 中国药房

OBJECTIVE：To investigate the regulation effects of volatile oil from Angelica sinensis on autophagy of myocardial cell H 9C2 in rats with hypoxia and reoxygenation （H/R）injury. METHODS ：Using myocardial cell H 9C2 as subject ，CCK-8 method was used to screen the optimal concentration and administration time of volatile oil from A. sinensis. The acitivity of LDH in cell supernatant was determined after treated with volatile oil from A. sinensis by ELISA. Using autophagy inhibitors （3-methyladenine，5 mmol/L） as positive control ，MDC method and Western blotting assay were used to detect average fluorescence intensity of MDC and the expression of autophagy related proteins [Beclin- 1，microtubule associated protein light chain 3Ⅱ（LC3Ⅱ），LC3Ⅰ] in H 9C2 cells after treated with medicines. RESULTS ：After treated with 0.6 μmol/L violate oil from A. sinensis for 6 h，compared with blank group ，LDH activity in cell supernatant ，average fluorescence intensity of MDC ，the expression of Beclin- 1，LC3Ⅱ/LC3Ⅰ ratio in cells were increased significantly in H/R group ，while the expression of p 62 was decreased significantly （P＜0.05 or P＜0.01）. Compared with H/R group ，the activity of LDH in cell supernatant of H/R+drug group as well as average fluorescence intensity of MDC ，the expression of Beclin- 1，LC3Ⅱ/LC3Ⅰratio in cells in H/R+drug group and H/R+autophagy inhibitor group were decreased significantly ，while the expression of p 62 were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：The volatile oil from A. sinensis can reduce the autophagy level of H/R injury myocardial cells by regulating the expression of autophagy related proteins.

Xuefu Zhuyu Oral Liquid () Prevents Apoptosis of Ischemic Myocardium Cells in Rats by Regulating SIRT1 and Its Pathway-Related Genes / 中国结合医学杂志

OBJECTIVE@#To observe the changes of ischemic myocardial cells apoptosis in rats following intervention with Xuefu Zhuyu Oral Liquid (, XFZY), as well as changes of protein expression of silent information regulator 1 (SIRT1) and SIRT1 pathway-related genes.@*METHODS@#H9c2 rat myocardial cells were divided into 6 groups: control group, oxygen glucose deprivation (OGD) group, SIRT1 siRNA group, OGD+SIRT1 siRNA group, OGD+XFZY group, and OGD+SIRT1 siRNA+XFZY group. Quantitative fluorescent polymerase chain reaction (PCR) and Western blot were used to detect the concentration variations of SIRT1 and its pathway-related genes and corresponding protein expression after XFZY intervention and SIRT1 transfection.@*RESULTS@#Compared with the control group, the mRNA and protein expressions of SIRT1 were decreased obviously, while the mRNA and protein levels of P53, FoxO1, FoxO3, FoxO4 and nuclear factor kappa B (NF-ΚB) were increased in the OGD group, SIRT1 siRNA group, and OGD+SIRT1 siRNA group (P<0.01). Compared with the OGD group and OGD+SIRT1 siRNA group, the treatment of XFZY inhibited the decline in SIRT1 mRNA and protein expressions (P<0.01), and down-regulated the mRNA and protein levels of P53, FoxO1, FoxO3, FoxO4 and NF-ΚB, respectively (P<0.05 or P<0.01).@*CONCLUSION@#XFZY could prevent myocardial cells apoptosis probably by increasing the mRNA and protein expressions of SIRT1 and inhibiting the mRNA and protein expressions of P53, NF- K B, FoxO1, FoxO3 and FoxO4.

Intrauterine infection and fetal cardiovascular system development / 国际儿科学杂志

When the pathogen infects the fetus,the pathogenic microorganism and the infection product are recognized by the corresponding receptor.The fetus innate immune system is passively activated,which produces proinflammatory cytokines,induces cascade reaction of cytokines,and releases a large number of inflammatory factors secreted by the body.Its toxic effect can cause damage to the brain,lung,small intestine and heart and other important organs of the whole body,which seriously threatens the life of the perinatal infants and their subsequent survival quality.It has been found that fetal cardiovascular system is one of the important target organs of intrauterine infection.Cytokines produced by cardiac inflammation and induced by intrauterine infection can damage myocardial cells,affect the proliferation of myocardial cells,and cause damage to cardiac function.Moreover,the persistent influence of infection on fetus leads to fetal vascular remodeling and changes in fetal cardiopulmonary hemodynamics.This article reviews the effects of pathogens of intrauterine infection and fetal cardiac inflammation,cardiac hemodynamics,cardiomyocyte development,gene program of cardiomyocyte and cardiac structure development on fetal cardiovascular system.

Naringin pretreatment alleviating myocardial cell hypoxia/reoxygenation injury by suppressing endoplasmic reticulum stress-induced apoptosis / 中国药理学通报

Aim: To study the effect of naringin (Nar) on endoplasmic reticulum stress (ERS) -induced apoptosis in H9c2 myocardial cell hypoxia/reoxygenation (H/R) injury and its molecular mechanism. Methods: H9c2 cells were cultured in vitro and randomly classified into five groups: normal control (group C), H/R group, H/R with Nar 10 mg · L-1 (group L), H/R with Nar20 mg · L-1 (group M) and H/R with Nar40 mg · L-1 (group H). Myocardial cells were normally cultured until the end of the experiment in group C. The myocardial cells were treated by hypoxia for 4 h before reoxygenation for 24 h in group H/R. The myocardial cells were cultured with Nar(10, 20, 40 mg · L-1) respectively 6 h before hypoxia, and after 6 h they were treated by hypoxia for 4 h before reoxygenation for 24 h in group L, group M and group H. The survival rate of cells was determined by MTT method after experiment. The apoptosis of cardiomyocytes was detected by TUNEL. CCAAT/enhancer-binding protein-homologous protein(CHOP), activating transcription factor-4(ATF4), eukaryotic initiation factor 2α (eIF2α), p-eIF2α, double-stranded RNA like endoplasmic reticulum kinase (PERK) and p-PERK were all assessed by Western blot. Results: Compared with group C, H9c2 cell viability decreased, induced apoptosis and the protein expression of CHOP, ATF4, peIF2α/eIF2α and p-PERK/PERK in group H/R were significantly increased (P < 0. 05). Compared with group H/R, H9c2 cell viability increased, the apoptosis and the protein expression of CHOP, ATF4, peIF2α/eIF2α and p-PERK/PERK were reduced in group L, group M and group H. Of the three groups, group M and group H showed the most significant effect (P < 0. 05). Conclusions: The Nar pretreatment can reduce myocardial cell apoptosis caused by H/R injury, suggesting that Nar can help to relieve the ERS-associated apoptosis through the PERK-eIF2α-ATF4- CHOP pathway.

Moxibustion improves cardiac function by up-regulating autophagy-related proteins of cardiomyocytes in rats with chronic heart failure / 针刺研究

OBJECTIVE: To observe the effect of moxibustion on cardiac function and the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and selective autophagy receptor signaling adaptor sequestosome 1 (SQSTM1/p62) in rats with chronic heart failure (CHF), so as to explore its underlying mechanisms in preventing and treating CHF. METHODS: Sixty male SD rats were randomly divided into normal, model, moxibustion, autophagy inhibitor 3-methyladenine (3-MA) and autophagy agonist rapamycin (RAPA) groups (n＝12 rats/group). The CHF model was established by intrape-ritoneal injection of adriamycin (ADR, 2 mg/kg, once every week for 12 weeks). Mild moxibustion was applied to bilateral "Feishu" (BL13) and "Xinshu" (BL15) for 20 min every time. Rats of the 3-MA group were treated by intraperitoneal injection of 3-MA suspension (15 mg/kg), and those of the RAPA group treated by gavage of RAPA suspension (2 mg/kg). All the treatments were given once a day for 3 weeks. The heart rate (HR), cardiac output (CO), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and maximum rising and lowering rates of left ventricular pressure (±dp/dtmax) were measured for assessing the cardiac performance. Histopathological changes of the left ventricular myocardium were observed by HE staining. The expression levels of LC3-Ⅰ， LC3-Ⅱ and p62 proteins of the left ventricle myocardium tissue were detected by Western blot. RESULTS: After modeling, the pathological changes of myocardium (as myocardial cell swelling with vacuoles, myocardial fibre breakage, etc.) were obvious, and the HR, LVEDP, LC3-Ⅱ and LC3-Ⅱ/Ⅰ protein expression levels were significantly increased in the model group compared with the normal group (P<0.01), while the CO, LVSP, ±dp/dtmax, and the expression of p62 protein were significantly down-regulated (P<0.01). Following the interventions, the myocardial injury was reduced, the HR, LVEDP, LC3-Ⅱ and LC3-Ⅱ/Ⅰ levels in both moxibustion and 3-MA groups were significantly decreased (P<0.05, P<0.01), while the CO, LVSP, ±dp/dtmax and p62 expression level were significantly increased relevant to the model group (P<0.05, P<0.01). In addition, the ratio of LC3-Ⅱ/Ⅰ was significantly increased, and the expression level of p62 significantly down-regulated in the RAPA group compared with the model group (P<0.01). CONCLUSION: Moxibustion can improve cardiac function in CHF rats, which may be related to its effects in down-regulating the ratio of LC3-Ⅱ/Ⅰ and up-regulating the expression of p62 protein to inhibit cardiomyocyte autophagy.

Effect and molecular mechanism of serum exosomes of diabetic mice on regulating H9C2 myocardial cell injury / 解放军医学杂志

[Abstract] Objective To investigate the effect of exosome in cultured in vitro H9C2 myocardial cells injury of diabetic mice and its mechanism. Methods The mouse model of diabetic myocardial injury was established by using db/db mice (n=10) and their mate mice db/+ (n=5). Serum exosomes were isolated and quantitated using the exosome isolation reagent and EXOCET Quantitation kit. The serum exosomes were labeled with PKH26 (red fluorescent cell linker) to detect the endocytosis in H9C2 cells. The expressions of exocrine associated protein and inflammatory cytokines in H9C2 cells with or without exosome stimulation were detected by Western blotting. TUNEL was used to detect apoptosis. A neutralizing antibody of Rab1a was used for blocking experiment. Results Db/db mice produced more exosomes than db/+ mice (30.95×109/ml vs. 10.45×109/ml, P<0.01). Moreover, H9C2 cells cultured in vitro could swallow more serum-exosomes derived from db/db mice. Meanwhile, serum exosome from db/ db mice, as used to interfere H9C2 cells, significantly increased the expression of inflammatory cytokines, such as 6.2 folds to IL-6 and 2.6 folds to IL-1β (P<0.01). Furthermore, the apoptosis in H9C2 cells increased compared to those from db/+ mice. Mechanism studies announced that the increased expression of Rab1a in exosomes-derived from db/db mice, and blocking the expression of Rab1a in exocrine of db/db mice with Rab1a neutralizing antibody could significantly inhibit the endocytosis and apoptosis of H9C2 cells. Conclusions Serum exosomes isolated from db/db mice may trigger inflammation and apoptosis of cardiomyocytes cultured in vitro, which may be involved in the evolution of diabetic myocardial injury. Inhibition of exosome secretion or intervention of its regulatory molecules may become a new research target for the treatment of diabetic myocardial injury.

Vital role of RIP3 in the mechanism of myocardial cell necroptosis / 中华危重病急救医学

Myocardial ischemia/reperfusion (I/R) injury seriously endangers human health and is a potential hidden danger in the treatment of cardiovascular diseases, among which myocardial necrosis is one of the mechanisms of myocardial I/R injury. Numerous studies have shown that necrosis factor is widely involved in the regulation of myocardial cell necrosis, but its specific mechanism is not fully understood. Receptor interacting protein 3/receptor interacting protein kinase 3 (RIP3/RIPK3) is an essential protein in necroptosis pathways, and activated RIP3 can cause irreversible necrosis of myocardial cells. On the basis of introducing RIP3 molecule, this paper focused on the role and mechanism of RIP3 mediated necroptosis pathway in myocardial cell necroptosis, with a view to providenew ideas and insights for the treatment of myocardial I/R injury.

Study on Protective Effect of Atorvastatin-induced Increase of EPC-MVs on Myocardial Cells in STEMI Patients / 中国药房

OBJECTIVE： To observe the protective effect of atorvastatin-induced increase of EPC-MVs on myocardial cells in ST-segment elevation myocardial infarction （STEMI） patients， and to investigate its potential mechanism. METHODS： Totally 168 STEMI patients was collected from our hospital during Feb. 2015-Feb. 2018， and then divided into group A （88 cases） and group B （94 cases） according to the dose of atorvastatin. All patients received percutaneous coronary intervention， and then given Bivaleridine for injection， Clopidogrel bisulfate tablets and Atorvastatin calcium tablets. Group A was given Atorvastatin calcium tablets 20 mg， once a day. Group B was given Atorvastatin calcium tablets 20 mg， twice a day. A treatment course lasted for 30 d， and two groups were treated for 3 courses at least. The levels of blood lipid （TC， LDL-C， HDL-C） （before treatment and 30th， 60th， 90th day after treatment） and the number of EPCs positive cells （30th， 60th day after treatment） were observed in 2 groups. The expression of miRNA of EPC-MVs （60th day after treatment） was detected， and the expression difference of miRNA were validated. Target gene and KEGG pathway enrichment of miRNA with most significant expression difference were analyzed， and the effects of it on the proliferation of cardiac HCM-a cells were evaluated. The occurrence of ADR was recorded in 2 groups. RESULTS： Totally 8 patients withdrew from the study in group A， and 6 patients in group B. There was no statistical significance in the levels of TC， LDL-C and HDL-C or the number of EPCs positive cells in peripheral blood between 2 groups before treatment or 30th day after treatment （P＞0.05）. After treatment， the level of HDL-C in 2 group （60th and 90th day after treatment） and the number of EPCs positive cells in peripheral blood in group B （60th day after treatment） were increased significantly， and group B was significantly higher or more than group A at the same time point （P＜0.05）. Microarray analysis showed that compared with group A， 16 miRNAs expressed more than 1.5 times differentially in EPC-MVs of group B， 7 of which were up-regulated and 9 down-regulated. Top five differentially expressed genes were hsa-miR-126 （up-regulated）， hsa-miR-1275 （up-regulated）， hsa-miR-7704 （down-regulated）， hsa-miR-105-5p （down-regulated）， and hsa-miR-3180 （down-regulated）. Fluorescence quantitative polymerase chain reaction results showed that compared with group A， relative expression of hsa-miR-126 and hsa-miR-1275 in group B were increased significantly； and relative expression of hsa-miR-7704， hsa-miR-105-5p and hsa-miR-3108 were decreased significantly （P＜0.05）. The expression difference of hsa-miR-126 was the most significant， and its target genes included Ang-1， PDGF， p38 MAPK， Smad2/3， HIF-1， TGF-β， etc. The signaling pathways involved in regulation mainly included angiogenesis signaling pathway， chronic myelogenous leukemia related pathway， renal epithelial cell carcinoma related pathway and so on. CCK-8 test showed that the optical density （OD） of cells in hsa-miR-126 specific interfering substance group was decreased significantly， and the OD value of cells in simulated substance group was increased significantly， compared with blank group （P＜0.05）. There was no statistical significance in the incidence of ADR as diarrhea， nausea and vomiting， rash， etc. （P＞0.05）. CONCLUSIONS： Different doses of atorvastatin can regulate the level of HDL-C， and large dose of atorvastatin can increase the number of EPCs significantly， but dose not influence the safety of drug use. This effect may be associated with up-regulating the expression of hsa-miR-126 in EPC-MVs so as to promoting the proliferation of myocardial cells.

Effect of Dracocephalum Heterophyllum Benth Flavonoid on Myocardial Cell Hypertrophy Induced by Noradrenaline in Rats / 医药导报

Objective To probe into effect of Dracocephalum heterophyllum Benth flavonoids(DHBF) on noradrenaline (NE) -induced myocardial cell hypertrophy, and to study the mechanism. Methods SD neonatal rat myocardial cells were cultured in vitro. The experiment was divided into normal control group, model control group (NE 2 μmol·L-1) , prazosin group (prazosin 50 μmol·L-1) ,low-,medium-and high-dose DHBF group (DHBF 10,25,50 μmol·L-1) .Cardiomyocyte hypertrophy were induced by NE(2 μmol·L-1) . DHBF and prazosin were intervened respectively. CCK-8 method was used to observe the activity of myocardial cells. RT-PCR technique was used to detect the expression of mRNA of cardiac hypertrophy c-jun and brain natriuretic peptide (BNP) ,and Western blotting was used to detect the protein expression of CaN and NFAT-3 in myocardial cells. Confocal laser scanning was used to detect the surface area of myocardial cell. Results Compared with the normal control group, survival rate of cardiomyocytes was significantly decreased, expression of mRNA of c-jun and BNP significantly upregulated, protein expression of CaN and NFAT-3 decreased, and surface area increased in model control group (P<0.05) . Compared with the cell of model control group, low-,medium-and high-dose DHBF significantly reversed NE-induced decrease of the survival rate, increase of surface area, increase of c-jun and BNP mRNA, and increase of CaN and NFAT-3 protein expression (P<0.05) . Conclusion DHBF can improve the survival rate of cardiac hypertrophy patients, down-regulate c-jun and BNP mRNA expression, decrease CaN and NFAT-3 protein expression, and decrease NE-induced surface area of cardiomyocytes.

Effect of Dracocephalum heterophyllum Benth Flavonoid on Rat Myocardial Cell Hypertrophy Induced by Angiotensin Ⅱ / 中国药学杂志

OBJECTIVE: To observe the effect of Dracocephalum heterophyllum flavonoids(DHBF) of Uygur Medicine on cardiomyocyte hypertrophy, which were induced by angiotensin Ⅱ(AngⅡ), and it could provide a basis for further study to the mechanism. METHODS: SD rats, 0 - 3 d of age, neonatal rat myocardial cells cultured in vitro, the experiment was divided into control group, AngⅡ(1 μmol•L-1) group, different concentrations of DHBF(10, 25, 50 μmol•L-1) + AngⅡ(1 μmol•L-1) groups, cardiomyocyte hypertrophy were induced by AngⅡ 1 μmol•L-1 and was intervened using DHBF respectively, CCK-8 method was used to observe the activity of myocardial cells, RT-PCR technique was used to detect the expression of m RNA of cardiac hypertrophy gene atrial natriuretic peptide(ANP) and brain natriuretic peptide(BNP), the internal factor was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Confocal laser scanning was used to detect the surface area of myocardial cell was [Ca2+]i; the activity of Ca2+-ATP was measured by the enzymatic reaction of fragmentation cell; the concentration of NO and the activity of NOS were determined by colorimetry. RESULTS: Compared with the control group, the activity of myocardial cell was(85% ± 5%) in the AngⅡgroup, which was increased significantly after it was dealed with DHBF and AngⅡ(P < 0.05); RT-PCR results showed the expression of mRNA of ANP and BNP were increased by using AngⅡ, which were lower by using DHBF and AngⅡ. The surface area of myocardial cell were increased by using AngⅡ, which could be reversed by using DHBF and AngⅡ. Confocal laser scanning showed the concentration of [Ca2+]i was increased by using AngⅡ, but which was lower significantly by using DHBF and AngⅡ(P < 0.05). The enzymatic reaction of fragmentation cell results showed the activity of Ca2+-ATP was decreased by using AngⅡ, but which was increased significantly by using DHBF and AngⅡ(P < 0.05). Colorimetry results showed the concentration of NO and the activity of NOS were decreased by using AngⅡ, but which was increased significantly by using DHBF and AngⅡ(P < 0.05). CONCLUSION: DHBF can improve the activity of hypertrophy cardiomyocytes which were induced by angⅡ, downregulate expression of mRNA of ANP and BNP, reduce surface area of cardiomyocytes induced by AngⅡ, the mechanism of action may be related to promoting the release of NO, regulating the concentration of[Ca2+]i and the activity of Ca2+-ATP.

Protective effects of oridonin on adriamycin-induced myocardial apoptosis through inhibiting autophagy / 局解手术学杂志

Objective To investigate the effects and mechanisms of oridonin on adrimycin-induced myocardial apoptosis. Methods Cells were divided into H9C2,adriamycin,oridonin(5 μM),oridonin(10 μM) and oridonin(20 μM) group.Cells were treated with adriamy-cin except H9C2 group and cells in oridonin(5,10,20 μM)group were treated with oridonin at the same time.The concentrations of superox-ide dismutase(SOD) and malondialdehyde(MDA) were detected and the cell proliferation was detected by CCK8 assay.Cell apoptosis was determined by flow cytometry.The expressions of autophagy-related proteins(Beclin1,P62 and LC3) were measured by western blot.And the expression of LC3 also detected by immunofluoresence.Results Compared with H9C2 group,the concentration of SOD decreased and MDA increased in adriamycin group;compared with adriamycin group,SOD increased but MDA decreased in oridonin(5,10,20 μM)group signifi-cantly.Meanwhile,cell proliferation was inhibited and apoptosis was induced in adriamycin group compared with H9C2 group;but cell prolif-eration rate was increased and apoptosis rate was decreased in oridonin(5,10,20 μM)group compared with adriamycin group.In addition,ad-riamycin up-regulated the protein level of Beclin1 and ratio of LC3Ⅱ/LC3Ⅰ,and inhibited the expression of P62;oridonin(5,10,20 μM) attenuated the effects of adriamycin on Beclin1,ratio of LC3Ⅱ/LC3Ⅰand P62 notably.Conclusion Oridonin alleviated adriamycin-indued myocardial apoptosis by inhibiting autophagy.

Overexpression of human telomerase reverse transcriptase inhibits myocardial apoptosis induced by hypoxia/reoxigenation in rats / 基础医学与临床

Objective To investigate the effects of human telomerase reverse transcriptase (TERT) on myocardial apoptosis induced by hypoxia/reoxigenation (Hypo/Ro) in rats. Methods Adenovirus mediated transfection of TERT gene into myocardial cells of neonatal rats, lentivirus-mediated siRNA silencing the TERT. To investigate the protective effects of human telomerase reverse transcriptase on myocardial hypoxia/reoxigenation injury in rats. Results The apoptosis rate of Hypo/Ro group was significantly higher than that of the control group. Compared with the Hypo/Ro group, TERT over-expression group significantly reduced the rate of apoptosis, and over-expression of TERT decreased the expression of CytC gene and increased the expression of P21 gene in cardiomyo-cytes (P＜0.05).Conclusions Over expression of telomerase reverse transcriptase inhibits myocardial apoptosis induced by Hypo/Ro. The mechanism may be explained by the regulation to the expression of P21 and CytC genes.

Protective Effect of Compatibilities of Ginsenosides Rg1 and Aconitine on Myocardial Cells of in vitro Cul-tured Heart Failure Model / 中国药房

OBJECTIVE:To investigate the protective effect of the compatibilities of ginsenosides Rg1 and aconitine on myocar-dial cell of in vitro cultured heart failure model. METHODS:The myocardial cells of neonate rat were grouped into normal control group,model group,positive control group(Deslanoside injection,1×10-7 mol/L),ginsenosides Rg1 group(1×10-8 mol/L),acon-itine group (1 × 10-9 mol/L) or their compatibilities groups (1∶1,2∶1,1∶2,V/V). Except for normal control group,other groups were given 0.8%pentobarbital sodium to induce heart failure model of myocardial cells. After modeling,each group was given rele-vant medicine for 1 h,and then the activities of T-ATPase,Ca2+-Mg2+-ATPase,Na+-K+-ATPase in cells were all detected. The activi-ties of acyl carrier protein(ACP)and lactate dehydrogenase(LDH),and the contents of brain natriuretic party(BNP),TNF-α and total glycogen were measured in cell culture fluid. RESULTS:Compared with normal control group, T-ATPase and Ca2+-Mg2+-ATPase activities were decreased significantly in model group;meanwhile,Na+-K+-ATPase activity was increased signifi-cantly,and ACP,LDH activities and BNP content in cell culture fluid were increased significantly(P0.05). CONCLUSIONS:Compatibility of ginsenosides Rg1 and aconitine can improve ATPase activities and membranous permeability,regulate BNP secretion and protect myocardial cell of heart failure model,especially the compatibility of ginsenosides Rg1 to aconitine of 2∶1 ratio.