Knockout of farnesoid X receptor gene aggravates cisplatin-induced kidney injury / 中南大学学报(医学版)

OBJECTIVES@#Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand activated transcription factors and belongs to bile acid receptor. Studies have shown that the expression of FXR in renal tissue can reduce renal injury via regulation of glucose and lipid metabolism, inhibition of inflammatory response, reduction of oxidative stress and renal fibrosis. However, it is unclear whether FXR is involved in autophagy in renal diseases. This study aims to investigate the role of FXR in cisplatin-induced acute renal injury and whether its mechanism is related to autophagy regulation.@*METHODS@#Twelve male WT or FXR-KO mice at 12 weeks were randomly divided into a WT group, a WT+cisplatin group, a FXR-KO group, and a FXR-KO+cisplatin group, with 6 mice in each group. The WT+cisplatin group and the FXR-KO+cisplatin group were intraperitoneally injected with cisplatin (20 mg/kg), and the WT group and the FXR-KO group were intraperitoneally injected with equal volume of cisplatin solvent. Seventy-two hours later, the mice were killed and blood and renal tissue samples were collected. The levels of SCr and BUN were detected by immunoturbidimetry. After the staining, the pathological changes of renal tissue were observed under optical microscope. The protein levels of LC3 and p62 were detected by Western blotting and immunohistochemistry. The clearance of damaged mitochondria and the accumulation of lysosomal substrate were observed under electron microscope. The apoptosis of renal tubular epithelial cells was detected by TUNEL.@*RESULTS@#Compared with the WT group or the FXR-KO group, both SCr and BUN levels in the WT+cisplatin group or the FXR-KO+cisplatin group were significantly increased (P<0.01 or P<0.001), and SCr and BUN levels in the FXR-KO+cisplatin group were significantly higher than those in the WT+cisplatin group (both P<0.05). Under the light microscope, there were no obvious pathological changes in the renal tissue of mice in the WT group and the FXR-KO group. Both the WT+cisplatin group and the FXR-KO+cisplatin group had vacuolar or granular degeneration of renal tubular epithelial cells, flat cells, lumen expansion, brush edge falling off, and even exposed basement membrane and tubular formation. The scores of renal tubular injury in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the score in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05). Under the transmission electron microscope, the mitochondria of mouse tubular epithelial cell in the WT+cisplatin group and the FXR-KO+cisplatin group was swollen, round, vacuolated, cristae broken or disappeared; the lysosome was uneven and high-density clumps, and the change was more obvious in the FXR-KO+cisplatin group. Western blotting showed that the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased in the WT+cisplatin group compared with the WT group and the FXR-KO+cisplatin group compared with FXR-KO group (P<0.05 or P<0.01); compared with the FXR-KO group, the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased significantly in the FXR-KO+cisplatin group (both P<0.05). Immunohistochemistry results showed that the expression of total LC3 and p62 in renal cortex of the WT+cisplatin group and the FXR-KO+cisplatin group was increased significantly, especially in the FXR-KO+cisplatin group. TUNEL results showed that the mice in the WT group and the FXR-KO group had negative staining or only a few apoptotic tubular epithelial cells, and the number of apoptotic cells in the WT+cisplatin group and the FXR-KO+cisplatin group were increased. The apoptosis rates of renal tubular epithelial cells in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the apoptosis rate in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05).@*CONCLUSIONS@#Knockout of FXR gene aggravates cisplatin induced acute renal injury, and its mechanism may be related to inhibiting autophagy and promoting apoptosis.

Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis / 法医学杂志

OBJECTIVES@#To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of β-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis.@*METHODS@#SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of β-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins.@*RESULTS@#The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of β-arrestin 2 expression in SH-SY5Y cells.@*CONCLUSIONS@#CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the β-arrestin 2 signal.

Effects of trichostatin A on FHIT and WWOX genes expression, cell growth inhibition and apoptosis induction in hepatocellular carcinoma WCH 17 cell line

Previously, we evaluated the effect of trichostatin A (TSA) on the expression of DNA methyltransferase 1 (DNMT1) in Hepatocellular Carcinoma (HCC). Fragile histidine triad (FHIT) and WW domain-containing oxidoreductase (WWOX) are two of the most common down-regulated genes in many cancers located on chromosome 3p14.2 and 16q23.3-24.1 respectively. The aim of the current study was to assess the effect of TSA on these genes expression, cell growth, and apoptosis in HCC WCH 17 cell. The cells were seeded and treated with TSA at different times. Then, MTT assay, flow cytometry, and qRT-PCR were achieved to determine viability, apoptosis and gene expression respectively. Cell growth was significantly inhibited, 92 to 36% after 24 h, 86 to 28% after 48 h, and 78 to 24% after 72 h. The results of flow cytometry confirmed that TSA increased apoptosis compared to the control group, the apoptosis percentage increased to 12%, 16%, and 18% in comparison to control groups (2%). Significant up-regulation of the genes was observed in all treated groups. We concluded that re-expression of silenced WWOX and FHIT genes could be achieved by TSA resulting in cell growth inhibition and apoptosis induction in WCH 17 cell.

Yin-Yang-1 decreases Fas-induced apoptosis in acute lymphoblastic leukemia under hypoxic conditions: its implications in immune evasion / YY1 induce la disminución de la apoptosis a través de Fas en la leucemia linfoblástica aguda en condiciones hipóxicas: implicaciones en la evasión de la respuesta inmunitaria

Abstract Background: Acute lymphoblastic leukemia (ALL) is an aggressive malignant disease with high prevalence in pediatric patients. It has been shown that the downregulation of Fas expression is correlated with an inadequate response in ALL, although these mechanisms are still not well understood. Several reports demonstrated that hypoxia is involved in dysfunctional apoptosis. Yin-Yang-1 (YY1) transcription factor is involved in resistance to apoptosis, tumor progression, and it is increased in different types of cancer, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood, but it is known that YY1 negatively regulates Fas expression. The study aimed to evaluate the effect of YY1 on Fas expression under hypoxic conditions in ALL. Methods: Leukemia cell line RS4; 11 was cultured under normoxic and hypoxic conditions. YY1, Fas receptor, and hypoxia-inducible factor (HIF-1α) expression were analyzed. After treatment with a Fas agonist (DX2), apoptosis was analyzed through the detection of active caspase 3. Data were analyzed using Pearson’s correlation. Results: Leukemia cells co-expressed both HIF-1α and YY1 under hypoxia, which correlated with a downregulation of Fas expression. During hypoxia, the levels of apoptosis diminished after DX2 treatment. The analysis revealed that patients with high levels of HIF-1α also express high levels of YY1 and low levels of Fas. Conclusions: These results suggest that YY1 negatively regulates the expression of the Fas receptor, which could be involved in the escape of leukemic cells from the immune response contributing to the ALL pathogenesis.

Resumen Introducción: La leucemia linfoblástica aguda (LLA) es una enfermedad con alta prevalencia en la población pediátrica. El mecanismo por el cual el receptor de Fas participa en la regulación inmunitaria en los tumores es desconocido, pero se sabe que está subexpresado en LLA. El factor de transcripción Ying-Yang-1 (YY1) está involucrado en la resistencia a la apoptosis y la progresión tumoral; se encuentra aumentado en diferentes tumores, incluida la LLA. Aunque los mecanismos que regulan la expresión de YY1 en LLA son desconocidos, se sabe que YY1 regula la expresión del receptor de Fas. El objetivo de este trabajo fue evaluar el efecto de YY1 en la expresión de Fas en condiciones de hipoxia en la LLA. Métodos: Se cultivaron células RS4;11 en condiciones de hipoxia y se analizó la expresión de YY1, receptor de Fas y HIF-1α. La apoptosis fue inducida usando un agonista de Fas (DX2) y se analizó con la detección de caspasa 3 activa. Los datos se analizaron mediante correlación de Pearson. Resultados: Las células RS4;11 coexpresaron HIF-1αy YY1 en hipoxia, lo cual correlaciona con una baja expresión de Fas. La apoptosis se encontró disminuida durante condiciones de hipoxia, después del tratamiento con DX2. El análisis bioinformático mostró que los pacientes con altos niveles de HIF-1αpresentan YY1 elevado y bajos niveles del receptor de Fas. Conclusiones: Estos resultados sugieren que YY1 regula negativamente la expresión del receptor de Fas, lo cual podría estar involucrado en el escape de las células leucémicas a la respuesta inmunitaria, contribuyendo a la patogénesis de la LLA.

lncRNA OIP5-AS1 targets ROCK1 to promote cell proliferation and inhibit cell apoptosis through a mechanism involving miR-143-3p in cervical cancer

Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.

CLEC3B protects H9c2 cardiomyocytes from apoptosis caused by hypoxia via the PI3K/Akt pathway

Ischemic heart disease (IHD) is one of the leading causes of death worldwide. C-type lectin domain family 3 member B (CLEC3B) is a C-type lectin superfamily member and is reported to promote tissue remodeling. The serum levels of CLEC3B are downregulated in patients with cardiovascular disease. However, the molecular mechanisms of CLEC3B in IHD is not well-characterized. Therefore, we overexpressed CLEC3B and silenced CLEC3B in H9c2 rat cardiomyocytes for the first time. We then constructed a model of IHD in vitro through culturing H9c2 cardiomyocytes in serum-free medium under oxygen-deficit conditions. Then, Cell Counting Kit-8 (CCK-8), flow cytometry, qRT-PCR, and western blot assays were performed to investigate cell viability, apoptosis, and expression levels of CLEC3B, phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt), and cleaved-caspase 3. We observed that the mRNA expression of CLEC3B was decreased in hypoxic H9c2 cardiomyocytes (P<0.05). Overexpression of CLEC3B increased cell viability (P<0.01), inhibited cell apoptosis (P<0.05), upregulated the levels of p-PI3K/PI3K and p-Akt/Akt (P<0.01 or P<0.05), and downregulated expression of cleaved-caspase 3 (P<0.001) in hypoxic H9c2 cardiomyocytes while silencing of CLEC3B caused the opposite results. Inhibition of the PI3K/Akt pathway reversed the protective effect of CLEC3B on hypoxic H9c2 cardiomyocytes. Our study demonstrated that CLEC3B alleviated the injury of hypoxic H9c2 cardiomyocytes via the PI3K/Akt pathway.

Glycogen synthase kinase 3ß participates in late stages of Dengue virus-2 infection

BACKGROUND Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3β, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3β during Dengue virus-2 (DENV-2) infection was investigated. METHODS GSK3β participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS Phosphorylation of GSK3β-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3β reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3β. CONCLUSIONS The results suggest that GSK3β participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.

LncRNA SNHG7/miR-34a-5p/SYVN1 axis plays a vital role in proliferation, apoptosis and autophagy in osteoarthritis

BACKGROUND: Osteoarthritis (OA) is one of the most common rheumatic diseases of which clinical symptoms includes swelling, synovitis and inflammatory pain, affect patients' daily life. It was reported that non-coding RNAs play vital roles in OA. However, the regulation mechanism of ncRNA in OA pathogenesis has not been fully elucidated. METHODS: The expression of SNHG7, miR-34a-5p and SYVN1 was detected using qRT-PCR in tissues, serum and cells. The protein expression of SYVN1, PCNA, cleavage-caspase 3, beclinl and LC3 were measured using western blot. The RNA immunoprecipitation (RIP), RNA pulldown, and luciferase reporter assays were used to verify the relationship between SNHG7, miR-34a-5p and SYVN1. The MTT and flow cytometry assay was performed to detected cell proliferation and cell apoptosis respectively. RESULTS: In this study, SNHG7 and SYVN1 expression were down-regulated, but miR-34a-5p was up-regulated in OA tissues and IL-1P treated cells compared with normal tissues and chondrocyte. Functional investigation revealed that up-regulated SNHG7 or down-regulated miR-34a-5p could promote cell proliferation and inhibit cell apoptosis and autophagy in OA cells. More than that, RIP, pulldown and luciferase reporter assay was applied to determine that miR-34a-5p was a target miRNA of SNHG7 and SYVN1 was a target mRNA of miR-34-5p. Rescue experiments showed that overexpression of miR-34a reversed high expression of SNHG7-mediated suppression of apoptosis and autophagy as well as promotion of proliferation, while its knockdown inhibited cell apoptosis and autophagy and promoted cell proliferation which could be impaired by silencing SYVN1. In addition, SNHG7 regulated SYVN1 through sponging miR-34a-5p. CONCLUSION: SNHG7 sponged miR-34a-5p to affect cell proliferation, apoptosis and autophagy through targeting SYVN1 which provides a novel sight into the pathogenesis of OA.

Discovery of the ARP2 protein as a determining molecule in tumor cell death

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.

Evaluation of Collagen Fibers, MMP2, MMP9, 8-OHdG and Apoptosis in the Aorta of Ovariectomized LDL Knockout Mice Submitted to Aerobic Exercise / Avaliação de Fibras de Colágeno, MMP2, MMP9, 8-OH-Dg e Apoptose na Aorta de Ratas LDL Nocaute Ovariectomizadas Submetidas a Exercício Aeróbico

Abstract Background: In menopause, there is greater cellular exposure to oxidative stress, related to the decreased antioxidative effects of estrogen. These metabolic changes favor the progression of cardiovascular diseases, such as atherosclerosis. Abnormal function of the aorta - the most important artery - is associated with many cardiovascular diseases. Collagen, especially types I and III, is one of the most important aortic wall components and it can be affected by many factors, including menopause. The 8-OHdG is one of the main markers of DNA oxidative damage induced by reactive oxygen species (ROS). Objective: We aimed to investigate effects of moderate aerobic training on the ascending aorta of LDL-knockout (LDL-KO) and ovariectomized female mice. Methods: A total of 15 C57BL/6 mice and 15 LDL-KO mice were divided into experimental groups. The thickness and volume density of types I and III collagen fibers were performed by morphoquantitative analysis, whereas the MMP-2 and MMP-9 and 8-OHdG were detected by immunohistochemistry and apoptosis was detected by the TUNEL assay. The significance level for all tests was p < 0.05. Results: Exercise causes an increase in the thickness of the aorta in LDL-KO groups, particularly accentuated in the ovariectomized groups. The type I collagen fibers showed an increase in volume density influenced by training in both Control groups and in the LDL-KO group. Type III collagen density decreased in both groups. The MMP-2 showed moderade immunostaining in the tunica media in LDL-KO groups, which did not occur in the control groups and the MMP-9 stained irregularly in all tissues. The marker 8-OhdG was stronger in the exercise training groups. Additionally, the ovariectomy, the exercise training and the LDL-KO treatments increased apoptosis. Conclusion: These results suggest that moderate-intensity aerobic exercise in ovariectomized mice associated to an increase in LDL rate possibly increases oxidative stress and apoptosis induction.

Resumo Fundamento: Na menopausa, há maior exposição celular ao estresse oxidativo, relacionada à diminuição dos efeitos antioxidantes do estrogênio. Essas alterações metabólicas favorecem a progressão das doenças cardiovasculares, como a aterosclerose. A função anormal da aorta - a artéria mais importante - está associada a muitas doenças cardiovasculares. O colágeno, especialmente os tipos I e III, é um dos mais importantes componentes da parede da aorta e pode ser afetado por muitos fatores, incluindo a menopausa. Por sua vez, 8-OHdG é um dos principais marcadores de danos oxidativos do DNA induzidos por espécies reativas de oxigênio (EROS). Objetivo: Investigar os efeitos do treinamento aeróbico moderado na aorta ascendente de camundongos fêmeas, nocaute para LDL (LDL-KO) e ovariectomizadas. Métodos: Um total de 15 animais C57BL/6 e 15 animais LDL-KO foram divididos em grupos experimentais. A espessura e a densidade de volume das fibras de colágeno tipos I e III foram realizadas por análise morfoquantitativa; MMP-2 e MMP-9 e 8-OHdG foram detectadas por imunohistoquímica; e a apoptose foi detectada pelo ensaio TUNEL. O nível de significância adotado para todos os testes realizados foi p < 0,05. Resultados: o exercício causa aumento da espessura da aorta em grupos LDL-KO, particularmente acentuada em grupos ovariectomizados. As fibras de colágeno de tipo I mostraram aumento da densidade de volume influenciado pelo treinamento em animais controle e LDL-KO. A densidade do colágeno tipo III diminuiu em ambos os grupos. A MMP-2 mostrou imunomarcação moderada na túnica média em animais LDL-KO; em grupos controle, a MMP-9 marcou irregularmente em todos os tecidos. O marcador 8-OHdG foi mais forte nos grupos de treinamento de exercícios. Além disso, a ovariectomia, o treinamento físico e os tratamentos de LDL-KO aumentaram a apoptose. Conclusão: Esses resultados sugerem que exercícios aeróbicos de intensidade moderada em camundongos ovariectomizados associados ao aumento da taxa de LDL, possivelmente, aumentam o estresse oxidativo e a indução da apoptose.

The long non-coding RNA H19 induces hypoxia/reoxygenation injury by up-regulating autophagy in the hepatoma carcinoma cells

BACKGROUND: Long non-coding RNA H19 (H19) plays an important role by regulating protein expression in different tissues and organs of the body. However, whether H19 induces hypoxia/reoxygenation (h/R) injury via increase of autophagy in the hepatoma carcinoma cells is unknown. RESULTS: H19 was expressed in the hepatoma carcinoma cells (Hep G2 and HCCLM3 cells) and its expression was most in 8 h/24R. The knockdown of H19 and 3-MA (an autophagy inhibitor) protected against h/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The knockdown of H19 and 3-MA also decreased the autophagic vesicles (AVs) and the expression of Beclin-1 and the ration of LC3-II/LC3-I, and increased cell viability, the expression of Bcl-2 and P62 and the phosphorylation of PI3K, Akt and mTOR. In addition, chloroquine (CQ, an inhibitor of autophagy flux) markedly decreased formation of autophagy flux (the ration of LC3-II/LC3-I). The results of the knockdown of H19 group were similar to those of the 3-MA (or CQ) group. Rapamycin (a mTOR inhibitor, an autophagy activator) further down-regulated h/R-induced decrease of the phosphorylated PI3K, Akt and mTOR. The knockdown of H19 cancelled the effect of rapamycin. The overexpression of H19 further expanded h/R-induced increase of the ration of LC3-II/LC3-I and decrease of the phosphorylated PI3K, Akt and mTOR. CONCLUSIONS: Our results suggest that the long non-coding RNA H19 induces h/R injury by up-regulation of autophagy via activation of PI3K-Akt-mTOR pathway in the hepatoma carcinoma cells.

Effect of miR-134 against myocardial hypoxia/reoxygenation injury by directly targeting NOS3 and regulating PI3K/Akt pathway

Abstract Purpose To reveal the function of miR-134 in myocardial ischemia. Methods Real-time PCR and western blotting were performed to measure the expression of miR-134, nitric oxide synthase 3 (NOS3) and apoptotic-associated proteins. Lactic dehydrogenase (LDH) assay, cell counting kit-8 (CCK-8), Hoechst 33342/PI double staining and flow cytometry assay were implemented in H9c2 cells, respectively. MiR-134 mimic/inhibitor was used to regulate miR-134 expression. Bioinformatic analysis and luciferase reporter assay were utilized to identify the interrelation between miR-134 and NOS3. Rescue experiments exhibited the role of NOS3. The involvement of PI3K/AKT was assessed by western blot analysis. Results MiR-134 was high regulated in the myocardial ischemia model, and miR-134 mimic/inhibitor transfection accelerated/impaired the speed of cell apoptosis and attenuated/exerted the cell proliferative prosperity induced by H/R regulating active status of PI3K/AKT signaling. LDH activity was also changed due to the different treatments. Moreover, miR-134 could target NOS3 directly and simultaneously attenuated the expression of NOS3. Co-transfection miR-134 inhibitor and pcDNA3.1-NOS3 highlighted the inhibitory effects of miR-134 on myocardial H/R injury. Conclusion This present work puts insights into the crucial effects of the miR-134/NOS3 axis in myocardial H/R injury, delivering a potential therapeutic technology in future.

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

Hydrogen sulfide protects H9c2 cardiomyoblasts against H2O2-induced apoptosis

Reactive oxygen species (ROS) are highly reactive chemical species that may cause irreversible tissue damage, and play a critical role in cardiovascular diseases. Hydrogen sulfide (H2S) is a gasotransmitter that acts as a ROS scavenger with cardio-protective effects. In this study, we investigated the cytoprotective effect of H2S against H2O2-induced apoptosis in cardiomyocytes. H9c2 rat cardiomyoblasts were treated with H2S (100 μM) 24 h before challenging with H2O2 (100 μM). Apoptosis was then assessed by annexin V and PI, and mitochondrial membrane potential was measured using a fluorescent probe, JC-1. Our results revealed that H2S improved cell viability, reduced the apoptotic rate, and preserved mitochondrial membrane potential. An increased Bcl-2 to Bax ratio was also seen in myocytes treated with H2S after H2O2-induced stress. Our findings indicated a therapeutic potential for H2S in preventing myocyte death following ischemia/reperfusion.

Ropivacaine promotes apoptosis of hepatocellular carcinoma cells through damaging mitochondria and activating caspase-3 activity

BACKGROUND: Recent evidences indicated that some local anaesthetic agents played a role in inhibiting the proliferation of cancer cells; Whether ropivacaine is able to promote apoptosis of hepatocellular carcinoma (HCC) cells is still unclear. The aim of this study was to investigate the effect of ropivacaine on the apoptosis of HCC cells. METHODS: In the present study, we treated the HCC cell lines, Bel7402 and HLE with ropivacaine. MTT, DAPI stain, trypan blue exclusion dye assay, flow cytometry, electron microscopy, computational simulation, laser confocal microscope, Western blotting, and enzyme activity analysis of caspase-3 were applied to detect the growth and apoptosis of HCC cells and to explore the role mechanism of ropivacaine. RESULTS: Ropivacaine was able to inhibit proliferation and promote apoptosis of HCC cells in a dose- and time-dependent manner. Ropivacaine also has a trait to inhibit the migration of HCC cells; ropivacaine damaged the mitochondria of HCC cells. The results also indicated that ropivacaine was able to interact with caspase-3, promote cytoplasmic caspase-3 migration into the nucleus, stimulate cleavage of caspase-3 and PARP-1, caspase-9 proteins, inhibit the expression of Bcl-2, promote expression of Apaf-1 and mitochondria release cytochrome C, and activate the activity of caspase-3. CONCLUSIONS: Ropivacaine has a novel role in promoting apoptosis of HCC cells; The role mechanism of ropivacaine maybe involve in damaging the function of mitochondria and activating the caspase-3 signalling pathway in HCC cells. Our findings provide novel insights into the local anaesthetic agents in the therapy of HCC patients.

Molecular aspects of the teratogenesis of rubella virus

Rubella or German measles is an infection caused by rubella virus (RV). Infection of children and adults is usually characterized by a mild exanthematous febrile illness. However, RV is a major cause of birth defects and fetal death following infection in pregnant women. RV is a teratogen and is a major cause of public health concern as there are more than 100,000 cases of congenital rubella syndrome (CRS) estimated to occur every year. Several lines of evidence in the field of molecular biology of RV have provided deeper insights into the teratogenesis process. The damage to the growing fetus in infected mothers is multifactorial, arising from a combination of cellular damage, as well as its effect on the dividing cells. This review focuses on the findings in the molecular biology of RV, with special emphasis on the mitochondrial, cytoskeleton and the gene expression changes. Further, the review addresses in detail, the role of apoptosis in the teratogenesis process.

Long non-coding RNA MALAT1 enhances the apoptosis of cardiomyocytes through autophagy inhibition by regulating TSC2-mTOR signaling

BACKGROUND: Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy. METHODS: Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins. RESULTS: H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy. CONCLUSION: These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.

Apoptotic mechanisms in rabbits with blast-induced acute lung injury

Abstract Purpose: To investigate the apoptotic mechanisms in rabbits with blast-induced acute lung injury (ALI). Methods: A total of 40 rabbits were randomly divided into a blank control group (A, n=10) and an experimental group (EXP, n=30). Explosion-induced chest-ALI models were prepared and sampled at different time points (4, 12, and 24h after modeling, T1-T3) to test the lung dry weight/wet weight ratio (W/D) and arterial oxygen pressure (PaO2), apoptosis of lung tissue by the TUNEL assay, and Caspase-3, Bax, and Bcl-2 levels by immunohistochemical analysis. Furthermore, lung tissue was sampled to observe pathological morphology by microscopy. Results: Under a light microscope, Group EXP exhibited obvious edema in the pulmonary interstitial substance and alveoli, a large number of red blood cells, inflammatory cells, and serous exudation in the alveolar cavity, as well as thickening of the pulmonary interstitial fluid. Compared to Group A, the W/D ratio was significantly increased in Group EXP (P<0.01), while PaO2 was significantly reduced (P<0.01). The apoptosis index was significantly increased (P<0.01), and caspase-3 and Bax/Bcl-2 levels were increased (P<0.01). Conclusion: Apoptosis plays an important role in the occurrence and development of acute lung injury in rabbits by participating in lung injury and promoting the progression of ALI.

The protection effect and mechanism of hyperbaric oxygen therapy in rat brain with traumatic injury

Abstract Purpose: To investigate the effect of hyperbaric oxygen therapy (HBOT) on traumatic brain injury (TBI) outcome. Methods: The modified Marmarou's weight drop device was used to generate non-lethal moderate TBI rat model, and further developed in vitro astrocytes culturing system. Then, we analyzed the expression changes of interested genes and protein by quantitative PCR and western blot. Results: Multiple HBO treatments significantly reduced the expression of apoptosis promoting genes, such as c-fos, c-jun, Bax and weakened the activation of Caspase-3 in model rats. On the contrary, HBOT alleviated the decrease of anti-apoptosis gene Bcl-2 and promoted the expression of neurotrophic factors (NTFs), such as NGF, BDNF, GDNF and NT-3 in vivo. As a consequent, the neuropathogenesis was remarkably relied with HBOT. Astrocytes from TBI brain or those cultured with 21% O2 density expressed higher NTFs than that of corresponding controls, from sham brain and cultured with 7% O2, respectively. The NTFs expression was the highest in astrocytes form TBI brain and cultured with 21% O2, suggesting a synergistic effect existed between TBI and the following HBO treatment in astrocytes. Conclusion: Our findings provided evidence for the clinical usage of HBO treating brain damages.