Effect of sodium fluoride on myocardial damage by regulating apoptosis through JNK signaling pathway / 环境与职业医学

Background It has been found that fluoride may cause cardiomyocyte damage. c-Jun N-terminal kinases (JNK) signaling pathway plays an important role in apoptosis, but its role in fluorosis-induced cardiomyocyte damage is still unknown yet. Objective To explore the toxic effect of sodium fluoride (NaF) on H9c2 cardiomyocytes of rats and whether NaF affects cardiomyocyte apoptosis through the JNK signaling pathway. Methods According to the concentrations of sodium fluoride and whether sp600125 (JNK inhibitor) was added, cardiomyocytes of rats were divided into six groups, including control group, SP600125 group (SP group), 0.24, 0.48, and 0.96 mmol·L−1 NaF groups, and 0.96 mmol·L−1 NaF+SP600125 group (NaF+SP group). Cardiomyocytes exposed to NaF for 24 h were observed using a fluorescence inverted microscope. The changes of cell viability at 24, 48, and 72 h after the treatment were detected by CCK-8 method. The levels of reactive oxygen species (ROS) at 24 h after the treatment in H9c2 cardiomyocytes were determined by fluorescent probe method. The expression levels of Bcl-2, Bax, Caspase-3, and JNK mRNA at 24 h after the treatment were detected by real-time PCR. The protein expression levels of Bcl-2, Bax, Caspase-3, and p-JNK at 24 h after the treatment were detected by Western blotting. Results Compared with the control group, after being exposed to 0.48 and 0.96 mmol·L−1 NaF for 24 h, the cell growth density decreased. With the increase of NaF concentration, rounded cells and some suspended dead cells appeared. At 24h after exposure to NaF, the cell viability of the 0.48 and 0.96 mmol·L−1 NaF groups decreased compared with the control group (P<0.05). At 48h and 72h after exposure to NaF, the cell viability levels of the NaF treated groups were significantly lower than that of the control group (P<0.05). After NaF exposure for 24 h, compared with the control group, the intracellular ROS levels were increased (P<0.05); the mRNA expression levels of Bcl-2 were decreased to varying degrees, especially in the 0.48 and 0.96 mmol·L−1 NaF groups (P<0.05); the mRNA expression levels of Bax, Caspase-3, and JNK were increased (P<0.05); the protein expression levels of Bcl-2 were reduced (P<0.05); the protein expression levels of Bax, Caspase-3, and p-JNK were elevated (P<0.05). Compared with the 0.96 mmol·L−1 NaF group, the cell viability of the NaF+SP group was increased, the intracellular ROS level was decreased, the mRNA expression levels of Bax, Caspase-3, and JNK were decreased, the protein expression level of Bcl-2 was increased, and the protein expression levels of Bax, Caspase-3, and p-JNK were decreased (P<0.05); the expression level of Bcl-2 mRNA had a rising trend but showed no statistical significance (P>0.05). Conclusion Cardiomyocyte damage after excessive fluoride exposure may result from fluoride inducing excessive ROS production in cardiomyocytes, which may activate the JNK signaling pathway and induce cardiomyocyte apoptosis.

Effects of acteoside on hypoxia/reoxygenation-induced cardiomyocyte damage by regulating the Rnd 3/NF-κB pathway / 中国药房

OBJECTIVE To explore the effects of acteoside on hypoxia/reoxygena tion（H/R）-induced cardiomyocyte damage by regulating Rho family GTPase 3（Rnd3）/nuclear factor κB（NF-κB）pathway. METHODS The H 9c2 cardiomyocyte were divided into control group （no administration ，no modeling ），H/R group （only modeling ），H/R+AS-L group ，H/R+AS-M group ， H/R+AS-H group （10，30，90 μmol/L acteoside for above 3 groups firstly ，and then modeling ），H/R+pcDNA group [transfecting pcDNA （empty vector ） firstly，and then modeling] ，H/R + pcDNA-Rnd 3 group [overexpression of Rnd 3 by transfecting pcDNA-Rnd3（Rnd3 overexpression vector ）firstly，and then modeling] ，H/R+AS-H+si-NC group [transfecting si-NC （negative control）firstly，and then giving 90 μmol/L acteoside and modeling]，H/R+AS-H+si-Rnd3 group [inhibiting overexpression of Rnd 3 by transfecting si-Rnd 3 （Rnd3 small interfering RNA ） firstly，and then giving 90 μ mol/L acteoside and modeling]. After corresponding treatment ，the apoptotic rate ，release of lactate dehydrogenase （LDH），malondialdehyde（MDA）level，the activity of superoxide dismutase （SOD），the level of tumor necrosis factor α（TNF-α），interleukin 1β（IL-1β）and interleukin- 6（IL-6）， mRNA and protein expression of Rnd 3 and NF-κB subunit p65（NF-κB p65），the expression of aspartate proteolytic enzyme 3 （Cleaved Caspase- 3）protein and Cleaved Caspase- 9 protein were detected. RESULTS Different concentrations of acteoside could reduce the apoptotic rate of H/R-induced H 9c2 cardiomyocyte，the protein expressions of Cleaved Caspase- 3 and Cleaved Caspase-9，mRNA and protein expressions of NF-κB p65，the levels of LDH release and MDA ，TNF-α，IL-1β and IL-6，while increase the activity of SOD and mRNA and protein expressions of Rnd 3（P＜0.05），in a dose-dependent manner. Overexpression of Rnd 3 could decrease the apoptotic rate of H 9c2 cardiomyocyte，protein expressions of NF-κB p65，Cleaved Caspase- 3 and Cleaved Caspase- 9， the levels of LDH release ， MDA， TNF-α，IL-1β and IL-6，while increase the protein expression of Rnd 3 and the activity of SOD （P＜0.05）. The inhibition overexpression of Rnd 3 could weaken the inhibitory effects of acteoside on H/R-induced apoptosis of H 9c2 cardiomyocyte， oxidative stress and inflammatory reaction （P＜0.05）. CONCLUSIONS Acteoside could regulate Rnd 3/NF-κ B pathway by promoting the expression of Rnd 3 and inhibiting the expression of NF-κB p65，inhibit cardiomyocyte apoptosis ，oxidative stress and inflammation reaction so as to relieve the H/R-induced cardiomyocyte damage.

Effect of Trans-oleic Acid on Proliferation Inhibition and Apoptosis Induction in H9c2 Cardiomyocyte / 中国药学杂志

OBJECTIVE: To investigate the effects and possible mechanism of trans-oleic acid (9t-C18:1) on proliferation inhibition and induction apoptosis in H9c2 cardiomyocyte. METHODS: H9c2 rat cardiomyocytes were cultured in vitro. High, medium and low (600, 300, 150 μmol•L-1) dose of 9t-C18:1 groups and the negative control (NC) group were administered to H9c2 cardiomyocytes. The effect of 9t-C18:1 on cell proliferation was tested using cell counting kit-8 (CCK-8) assay. Morphological changes of cells were observed by AO-EB staining. Intracellular reactive oxygen species (ROS) and apoptosis were detected by flow cytometry. The expression of Bcl-2 and Bax genes was detected by quantitative real time- polymerase chain reaction (QRT-PCR). The expression of Bcl-2 and Bax protein was determined by flow cytometry after immunofluorescence staining. RESULTS: The typical morphological characteristics of apoptosis were observed by fluorescence microscope. The result of CCK-8 assay indicated that 9t-C18:1 have an certainly inhibitory effect on the proliferation of H9c2 cells. ROS level and apoptosis rate were significantly increased. Bcl-2 gene and protein expression were down-regulated, and Bax gene and protein expression were up-regulated, compared with NC group(P<0.05, P<0.01). CONCLUSION: 9t-C18:1 can inhibit the proliferation and induce the apoptosis of H9c2 cardiomyocyte, and its mechanism may be related to promoting the mitochondrial apoptotic pathway.

Effect of genipin on cardiomyocyte apoptosis and autophagy in rat after hypoxia / reoxygenation injury / 解剖学报

Objective To investigate the effect of genipin on cardiomyocyte apoptosis and autophagy in rat after hypoxia/ reoxygenation (H/ R) injury. Methods The method with hypoxia treatment of H9c2 cells for 12 hours and then reoxygenation treatment for 4 hours was used in the present study in order to establish H/ R model. The H9c2 cells were divided into control group (Con), genipin group (GE), model group (H/ R), model + genipin group (H/ R+ GE). Cell viability was detected by cell counting kit-8(CCK-8). Cell apoptosis was determined by flow cytometry. Transmission electron microscopy was used to observe autophagosomes. The expression of Bax, Bcl-2, P62, Beclin1, LC3-Ⅱ, protein kinase B(Akt), p-Akt, mammalian target of rapamycin(mTOR), p-mTOR proteins were assessed by Western blotting. Results Genipin pretreatment enhanced the cell viability, prevented cell apoptosis and autophagosome accumulation, and reduced the ratio of the cross-sectional areas of the autophagic structures to that of the cytoplasm after H/ R injury in H9c2 cells. Western blotting showed that genipin pretreatment decreased the expression of Bax, LC3-Ⅱ, Beclin1 proteins and increased the expression of Bcl-2, p62, p-Akt, p-mTOR proteins after H/ R injury. Conclusion Genipin can inhibit H/ R injury-induced apoptosis and autophagy, which may be through activating Akt/ mTOR signaling pathway.

Study on Preparation ，Characterization and Cytotoxicity of Baicalin PEG-PE Nanomicelles / 中国药房

OBJECTIVE： To prepare Baicalin-loaded Polyethylene glycol-derivatized phosphatidylethanolamine （BAI@PEG-PE） nanomicelles， and to characterize it and study its cytotoxicity. METHODS： BAI@PEG-PE nanomicelles were prepared by film hydration method and their appearance characteristics were observed. The particle size， polydispersity index， Zeta potential， drug-loading amount and encapsulation efficiency of the nanomicelles were detected. Drug release of BAI raw material and BAI@PEG-PE nanomicelles in pH 7.4 phosphate buffer were compared within 1-84 h. Using coumarin 6 as fluorescent probe， the distribution of PEG-PE nanomicelles in H9c2 cardiomyocytes were observed. H9c2 cardiomyocytes were divided into model group， BAI raw material group and BAI@PEG-PE nanomicelles group. After treated with serum-free DMEM medium containing no or corresponding drugs for 0.5 h， isoproterenol was used to induce cardiomyocyte apoptosis. Nuclear morphology， cell apoptosis rate and protein expression of Bcl-2 and Bax were compared with among 3 groups. RESULTS： Prepared BAI@PEG-PE nanomicelles were uniform globular shape. The particle size was （16.7±0.8） nm， PDI was 0.11±0.01 and Zeta-potential was （－18.4±0.6） mV； drug-loading amount was （7.84±0.65）％， encapsulation efficiency was （85.7±4.9）％ （n＝3）. Accumulative release rate was 76.5％ within 84 h. BAI raw material was released completely within 24 h. PEG-PE nanomicelles could strengthen the intake of coumarin 6 in H9c2 cardiomyocytes， mainly gathering around mitochondria. Compared with model group， the apoptosis morphology of cardiomyocytes were improved significantly in BAI raw material group and BAI@PEG-PE nanomicelles group； apoptosis rate was decreased significantly； protein expression of Bcl-2 was increased significantly； protein expression of Bax was decreased significantly with statistical significance （P＜0.05 or P＜0.01）. Above effects of BAI@PEG-PE nanomicelles group were more significant （P＜0.05 or P＜0.01）. CONCLUSIONS： BAI@PEG-PE nanomicelles are prepared successfully， and show significant sustained-release effect and myocardial targeting， and can prevent cardiomyocyte apoptosis.

Astragaloside Ⅳ regulates Nrf2/Bach1/HO-1 signaling pathway and inhibits H9c2 cardiomyocyte injury induced by hypoxia-reoxygenation / 中国中药杂志

Astragaloside Ⅳ(AS-Ⅳ) has protective effects against ischemia-reperfusion injury(IRI), but its mechanism of action has not yet been determined. This study aims to investigate the protective effects and mechanism of AS-Ⅳ on H9c2 cardiomyocyte injury induced by hypoxia-reoxygenation(H/R). The H/R model of myocardial cells was established by hypoxic culture for 12 hours and then reoxygenation culture for 8 hours. After AS-Ⅳ treatment, cell viability, the reactive oxygen species(ROS) levels, as well as the content or activity of superoxide dismutase(SOD), malondialdehyde(MDA), interleukin 6(IL-6), and tumor necrosis factor alpha(TNF-α), were measured to evaluate the effect of AS-Ⅳ treatment. The effect of AS-Ⅳ on HO-1 protein expression and nuclear Nrf2 and Bach1 protein expression was determined by Western blot. Finally, siRNA was used to knock down HO-1 gene expression to observe its reversal effect on AS-Ⅳ intervention. The results showed that as compared with the H/R model group, the cell viability was significantly increased(P<0.01), ROS level in the cells, MDA, hs-CRP and TNF-α in cell supernatant and nuclear protein Bach1 expression in the cells were significantly decreased(P<0.01), while SOD content, HO-1 protein expression in cells and expression of nuclear protein Nrf2 were significantly increased(P<0.01) in H/R+AS-Ⅳ group. However, pre-transfection of HO-1 siRNA into H9c2 cells by liposome could partly reverse the above effects of AS-Ⅳ after knocking down the expression of HO-1. This study suggests that AS-Ⅳ has significant protective effect on H/R injury of H9c2 cardiomyocytes, and Nrf2/Bach1/HO-1 signaling pathway may be a key signaling pathway for the effect.

Effect of C3G overexpression on proliferation and apoptosis of high glucose-induced H9C2 cardiomyocytes / 重庆医学

Objective To observe the effects of Crk-SH3 domain guanine exchange factor(C3G)overexpression on the pro-liferation and apoptosis of high glucose-induced H9C2 cardiomyocytes.Methods H9C2 cardiomyocytes were transiently transfected with pCXN2-Flag(empty plasmid)and pCXN2-Flag-hC3G(human C3G mRNA)plasmids,then conducted high glucose(HG)in-tervention.The experiment was divided into the blank group,empty vector group,C3G overexpression group,blank + HG group, empty vector + HG group and C3G overexpression+ HG group.The C3G protein expressions,apoptosis and proliferation rate were respectively detected in each H9C2 cardiomyocytes groups.Results The proliferation rate in the blank+ HG group and empty vec-tor+ HG group were significantly decreased compared with the blank group and empty vector group,while the apoptosis rate was significantly increased.Compared with blank group and empty vector group,blank + HG and empty vector + HG group,the C3G protein expression and proliferation rate in the C3G overexpression group and C3G overexpression+ HG group were increased sig-nificantly,while the apoptosis rate was decreased significantly.Conclusion High glucose inhibits H9C2 myocardial cell proliferation and promots its apoptosis;furthermore,C3G overexpression can reversed the decrease of high glucose-induced H9C2 cardiomyocyte prolifer-ation rate and apoptosis increase.C3G overexpression can promote the survivability of high glucose-induced H9C2 cardiomyocytes.