Expression Levels Of Caspase-3 Research Articles

Insulin Ameliorates Folliculogenesis in An Experimental Model of PCOS Mice.

Insulin is an essential factor that controls female reproductive system. Insulin signaling via Foxo1 and Akt1 can improve steroidogenesis, cell proliferation, and protein synthesis. We aimed to determine the effect of insulin on possible changes in gene expression, hormonal status, and histological aspects of the ovary following the induction of the animal model of polycystic ovary syndrome (PCOS). In this experimental study, 24 adult female NMRI mice weighing 25-30 g were randomly placed in three groups: control, PCOS (60 mg/kg dehydroepiandrosterone (DHEA) for 20 days, and PCOS+insulin (60 mg/kg DHEA for 20 days+100 μL insulin diluted in water twice a week for 30 consecutive days). Blood specimens were obtained from the heart and the serum levels of testosterone, progesterone, and estradiol were measured. Right, and left ovaries were removed for real-time polymerase chain reaction (PCR) and stereological study. DHEA injection significantly amplified the concentration of testosterone, progesterone, and estradiol. While insulin treatment amended the level of reproductive hormones. DHEA injection significantly reduced the expression levels of Irs1-4, Pdk1, Pi3k, and Akt1-3 and raised the expression level of Caspase-3. However, insulin administration amplified expression levels of Irs1-4, Pdk1, Pi3k, and Akt1-3, and reduced Caspase-3. The total volume of ovarian tissue in mice receiving DHEA significantly declined compared to the control group. Besides, a substantial decrease was detected in the number of ovarian antral, Graafian, and primordial follicles and also in the total number of corpus luteum following DHEA administration. Comparison of structural alterations in ovarian tissue between the PCOS+insulin and the PCOS groups displayed that insulin administration improved the total number of Graafian, primordial, and antral follicles and also corpus luteum. In general, short-term insulin treatment showed improvement in hormonal balance, folliculogenesis, and insulin resistance in the ovaries of the PCOS mice model.

Роль цитокинов и регуляторных молекул клеточного цикла в прогнозировании достижения ответа на терапию у больных хроническим миелолейкозом

Introduction. Despite significant advances in therapy of patients with chronic myeloid leukemia (CML), improved survival rates, development of resistance to tyrosine kinase inhibitors (TKIs) remains an urgent problem. Aim. To study the correlation between the level of expression of regulatory proteins p53, c-myc, ki-67 and caspase-3 on the bone marrow cells and the concentration of certain pro- and anti-inflammatory cytokines in blood serum with the effectiveness of therapy in patients with CML. Materials and methods. Seventy-four CML patients with chronic phase of the disease receiving TKI therapy were examined. In all patients, the concentration of certain cytokines and growth factors (TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IL-18, IFN-α, and VEGF-A) was determined in blood serum by enzyme immunoassay and immunocytochemical study of bone marrow smears with monoclonal antibodies against antigens of regulatory molecules ki-67, p53, c-myc, and caspase-3. To determine the role of the biomarkers in predicting the therapy effectiveness, a comparative analysis of their values in groups of patients with a major molecular response (MMR) (n = 50) and without MMR (n = 24) was performed. Results. A comparative analysis of the expression of regulatory molecules on the bone marrow cells and the blood serum concentration of cytokines and growth factors of CML patients, depending on depth of the response to TKI therapy, showed that patients who did not achieve MMR had a significantly higher level of caspase-3 expression and concentration of pro-inflammatory cytokines IL-1β, IL-2, IL-6 and IL-17, as well as growth factor VEGF-A compared with those in patients with MMR. In turn, the achievement of MMR was characterized by a higher level of expression of regulatory molecules p53 and c-myc, as well as an increase in the IL-10 concentration and a decrease in the IL-1β, IL-2, IL-6 and IL-17 concentration. Analysis of the correlation between the level of expression of regulatory molecules and the single cytokine concentration showed a negative correlation between c-myc and p53 with IL-2, IL-1β, IL-17 and a positive (direct) correlation between c-myc and p53 with IL-10, a positive correlation between caspase-3 and IL-2, IL-1β, IL-6, IL-17 and a negative correlation between caspase-3 and IL-10. Thus, the achievement of MMR in patients with CML is more likely with a higher expression of regulatory molecules c-myc and p53 on the bone marrow cells, low expression of caspase-3, and low serum concentrations of IL-2, IL-1β, IL-17, IL-6 and a high concentration of IL-10, which indicates synergism of the biomarkers in the pathogenesis of CML and its tumor progression. The ROC analysis results showed the high quality of predictive models characterizing the achievement of MMR at the level of expression of c-myc > 6%, p53 > 4% in the bone marrow, which correlates with a low serum concentrations of IL-2, IL-1β, IL-17 and a high concentration of IL-10, and indicates the possibility of using these indicators as potential biomarkers of effectiveness of CML therapy and achievement of MMR. Conclusion. The results of the study showed that the concentration of cytokines in the blood serum of CML patients correlates with the intensity of expression of c-myc, p53 and caspase-3 proteins and is important in predicting the effectiveness of therapy.

Hexokinase inhibitor 2-deoxyglucose coordinates citrullination of vimentin and apoptosis of fibroblast-like synoviocytes by inhibiting HK2 /mTORC1-induced autophagy.

High hexokinase 2 (HK2) expression is associated with aberrant activation of fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA). However, the mechanism by which this occurs has not been fully elucidated. To investigate the role of HK2 and its underlying mechanism, adjuvant arthritis (AA) rats were treated with the HK2 inhibitor, 2-deoxyglucose (2-DG). In conjunction with HK2 knockdown experiments in FLSs, we evaluated the effect of HK2 on the citrullination of vimentin (cVIM), autophagy and apoptosis-associated protein expression, including that of cVIM, LC3, p62, Beclin1, Bax, Bcl2, and caspase 3. We further investigated the interaction of HK2 with downstream mTORC1 signaling effectors. Correlation analysis revealed that 2-DG treatment and HK2 knockdown upregulated the expression levels of caspase3, Bax, and p62 and downregulated the expression levels of LC3, Bcl2, and Beclin1, as well as decreasing vimentin citrullination. Furthermore, interactions between HK2 and mTOR decreased, coinciding with mTORC1 pathway activation. These findings suggest that the regulation of apoptosis and cVIM by HK2/mTORC1-dependent autophagy involves the inhibition of aberrant FLSs activation in the rat model of arthritis.

Lipopolysaccharide and lipoteichoic acid regulate the PI3K/AKT pathway through osteopontin/integrin β3 to promote malignant progression of non-small cell lung cancer.

Lung cancer (LC) is a malignancy with one of the highest mortality rates. Respiratory microbiota is considered to play a key role in the development of LC, but the molecular mechanisms are rarely studied. We used lipopolysaccharide (LPS) and lipoteichoic acid (LTA) to study human lung cancer cell lines PC9 and H1299. The gene expression of CXC chemokine ligand (CXCL)1/6, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The Cell-Counting Kit 8 (CCK-8) was used to analyze cell proliferation. Transwell assays were performed to analyze cell migration ability. Flow cytometry was used to observe cell apoptosis. Western blot and qRT-PCR were used to analyze the expression of secreted phosphoprotein 1 (SPP1), toll-like receptor (TLR)-2/4, and NLR family pyrin domain containing 3 (NLRP3) to determine the mechanism of LPS + LTA. We evaluated the effect of LPS + LTA on cisplatin sensibility by analyzing cell proliferation, apoptosis, and caspase-3/9 expression levels. We observed the proliferation activity, apoptosis, and migration ability of cells in which SPP1 had been transfected small interfering (si) negative control (NC) and integrin β3 siRNA. Then the mRNA expression level and protein expression of PI3K, AKT, and ERK were analyzed. Finally, the nude mouse tumor transplantation model was conducted to verify. We studied that in two cell lines, the expression level of inflammatory factors in LPS+LTA group was significantly higher than that in single treatment group (P<0.001). We explored LPS + LTA combined treatment group significantly increased the expression of NLRP3 and genes and proteins. LPS + LTA + Cisplatin group could significantly reduce the inhibitory effect of LPS on cell proliferation (P<0.001), reduce the apoptosis rate (P<0.001) and significantly reduce the expression levels of caspase-3/9 (P<0.001) compared with Cisplatin group. Finally, we verified that LPS and LTA could increase osteopontin (OPN)/integrin β3 expression and activate the PI3K/AKT pathway to promote malignant progression of LC in vitro studies. This study provides a theoretical basis for further exploration of the influence of lung microbiota on NSCLC and the optimization of LC treatment in the future.

Effects of Jatrorrhizine on Proliferation, Apoptosis and Invasion of Breast Cancer Cells by Regulating Wnt/Beta-Catenin Signaling Pathway

To explore the effects of jatrorrhizine on proliferation, apoptosis, migration and invasion of breast cancer cells via the regulation of Wnt/β-catenin signaling pathway. Breast cancer cell line MDA-MB-468 was treated with varying concentrations of jatrorrhizine and then the effects of varying concentrations of jatrorrhizine on apoptosis of the cell were analyzed by flow cytometry. Western blotting was performed to test the protein expression of Wnt/beta-catenin signaling pathway. The breast cancer cells treated with 0 μM jatrorrhizine, breast cancer cells treated with 5 μM showed significant down-regulation in proliferation (p>0.05) and the breast cancer cells treated with 20 μM jatrorrhizine obviously decreased in proliferation activity, indicating the presence of statistical difference (p<0.05). The breast cancer cells treated with 0 μM jatrorrhizine and those treated with 5 μM showed an increase in apoptosis rate, the breast cancer cells treated with 20 μM jatrorrhizine obviously increased in apoptosis rate, indicating the presence of statistical difference (p<0.05). 0 μM group, the experiment of cell invasion and migration showed that 5 μM group experienced remarkable decline in the number of invasive and migratory cells vs. 0 μM group (p<0.05); and in this respect, 20 μM group had a marked decrease vs. 5 μM group, showing significant difference (p<0.05). In contrast with 0 μM group, apoptosis rate and B-cell lymphoma 2 expression level were observed to be an obvious decline and caspase-3 expression level presented an apparent increase in 5 μM group (p<0.05). In contrast with 5 μM group, apoptosis rate and B-cell lymphoma 2 expression level were observed to be an obvious decline and caspase-3 expression level presented an apparent increase in 20 μM group (p<0.05). After 48 h of treatment with jatrorrhizine, the beta-catenin expression in breast cancer cells was down-regulated in a concentrationdependent manner and whereas those of glycogen synthase kinase-3 and E-cadherin were up-regulated in a concentration-dependent manner, but that of interstitial marker protein N-cadherin in 5 μM group and 20 μM group was remarkably lower vs. 0 μM group. The regulation of Wnt/beta-catenin signaling pathway following the treatment of MDA-MB-468 with jatrorrhizine can promote apoptosis, inhibit proliferation and metastasis of breast cancer cells in a dose-dependent manner.

Muscle fiber characteristics and apoptotic factor differences in beef Longissimus lumborum and Psoas major during early postmortem

This study was conducted to study muscle fiber characteristics and apoptotic factor differences within 24 h postmortem of bovine longissimus lumborum (LL) and psoas major (PM). Compared to LL, PM had a higher proportion of type I fibers and lower proportion of type II fibers. PM also had higher ROS levels. For bcl-2 family proteins, anti-apoptotic BCL-2 level was lower and pro-apoptotic BAX level was higher in PM. For caspases, at 1 h postmortem, gene and protein expression level of caspase-3 and caspase-9 was higher in PM than that of LL. The levels of DNA damage apoptotic factors ABL1, AIF and ENDOG was higher in PM than in LL. The results suggested that apoptotic gene and protein expression were different in muscles with different fiber type composition. These findings provided insights into muscle fiber and apoptotic factor differences during early postmortem in bovine PM and LL.

Acupotomy relieves pain and improves motor function by regulating autophagy and apoptosis of cartilage in rabbits with knee osteoarthritis apoptosis

To observe the effect of acupotomy on the expression of Beclin-1, Bcl-2 and Caspase-3 in the cartilage tissue in rabbits with knee osteoarthritis (KOA), so as to explore its mechanism underling improvement of KOA. Twenty-four healthy male New Zealand rabbits were randomly and equally divided into blank control, model and acupotomy groups, with 8 rabbits in each group. By using the modified Videman's methods, the KOA model was established by left hind limb immobilization with a plaster cast for 6 weeks. The severity of KOA (knee pain, swelling and motor function) was assessed using Lequesne score, and the rabbits with a score below 4 were excluded. The acupotomy was applied to "Hedingci" (the attachment of the quadriceps tendon to the patella at the upper edge), "Binneixia" (the medial patellar supporting band attachment of medial inferior patellar margin), "Binwaixia" (the lateral patellar supporting band attachment of the lower lateral patellar margin), "Chengfeijian" (the lateral collateral ligament of the knee passes over the lateral joint space), "Weiyangci" (the medial margin of biceps femoris at the lateral end of popliteus), "Yinlingci" (the medial tibial attachment of anserinus tendon) on the left hind limb once a week for 4 weeks. One week after the last intervention, the left knee joint dysfunction severity(pain, maximum walking distance, and some activities of daily living) was evaluated by using modified Lequesne score. Histopathological changes of the cartilage were observed under light microscope after H.E. staining. The apoptosis of chondrocytes was observed after terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) staining. The autophagolysosomes of chondrocytes were observed using transmission electron microscopy. The expression levels of Beclin-1, Bcl-2 and Caspase-3 (related factors of autophagy and apoptosis) were detected using Real-time PCR and Western blot separately. In comparison with the blank control group, the Lequesne score, apoptosis rate, expression levels of Caspase-3 mRNA and protein were significantly increased (P<0.001), and the number of autophagolysosomes, expression levels of Beclin-1 and Bcl-2 mRNAs and proteins considerably decreased (P<0.001) in the model group. Relevant to the model group, the acupotomy group had an obvious decrease in Lequesne score, rate of apoptosis, and expression levels of Caspase-3 mRNA and protein (P<0.001) and an apparent increase in the number of autophagolysosomes and expression levels of Beclin-1 and Bcl-2 mRNAs and proteins (P<0.001). Findings of H.E. staining showed severe damaged cartilage surface, with a large number of exfoliation defects, few chondrocytes on the surface and disordered arrangement of transitional cells in the model group, which was relatively milder in the acupotomy group. Acupotomy can mitigate knee-joint pain and improve functional activity in KOA rabbits, which may be associated with its functions in promoting autophagy and suppressing apoptosis by up-regulating expressions of Beclin-1 and Bcl-2 mRNAs and proteins and down-regulation of Caspase-3 mRNA and protein.

Silencing tumor-intrinsic HHLA2 potentiates the anti-tumoral effect of paclitaxel on MG63 cells: Another side of immune checkpoint

BackgroundOsteosarcoma is common type of bone cancer; however, the prognosis of patients with metastatic osteosarcoma is poor. As a new inhibitory immune checkpoint molecule, HHLA2 is upregulated in osteosarcoma. Herein, we studied the significance of tumor-intrinsic HHLA2 in MG-63 growth. Also, we examined the influence of combined therapy of HHLA2 knockdown with paclitaxel on the apoptosis, cell cycle, migration, and stemness of MG-63 cells. MethodsThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to study the half-maximal inhibitory concentration (IC50) of paclitaxel and the cytotoxicity of HHLA2-small interfering RNA (siRNA) on MG-63 cells. The apoptosis and cell cycle were analyzed using flow cytometry. The wound-healing and colony formation assays were conducted to investigate the effect of paclitaxel and HHLA2 knockdown on the migration and stemness of MG-63 cells, respectively. QRT-PCR was used to determine the Bax, caspase-3, and Bcl-2 mRNA expression levels. ResultsHHLA2 silencing has enhanced the chemosensitivity of MG-63 cells to paclitaxel. Besides, HHLA2 knockdown has increased the paclitaxel-induced cytotoxic effect on MG-63 cells. In terms of stimulating apoptosis, decreasing clonogenicity, halting the cell cycle at the sub G1 phase, and inhibiting migration, tumor-intrinsic HHLA2 silencing has increased these anti-tumor effects of paclitaxel on MG-63 cells. Besides, HHLA2 knockdown has potentiated paclitaxel-mediated Bcl-2 downregulation and paclitaxel-mediated caspase-3 and Bax upregulation in MG-63 cells. ConclusionTumor-intrinsic HHLA2 knockdown increases the anti-tumoral effect of paclitaxel on MG-63 cells and enhances the chemosensitivity of MG-63 cells to paclitaxel.

Apoptotic effects of Phlomis armeniaca mediated biosynthesized silver nanoparticles in monolayer (2D) and spheroid (3D) cultures of human breast cancer cell lines.

The purpose of current research was to assess the apoptotic effects of biofabrication silver nanoparticles (AgNPs) mediated by the aqueous extract of Phlomis armeniaca on human breast cancer cells (MCF-7 and MDA-MB-231) in monolayer (2D) and spheroid (3D) cultures. The biosynthesized AgNPs were characterized by UV-Vis spectrophotometer (the peaks of resonances at 432nm), scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDS). 1-20µM/mL AgNPs were applied to MCF-7 and MDA-MB-231 cell lines to determine IC50 values at 24, 48 and 72nd h and were found to be 10µM/mL for both cell lines. Immunohistochemical staining results of BrdU, TUNEL, caspase-3 and Endo G in both 2D and 3D cultures and gene expression levels of caspases (caspase-3, -8 and -9) and Endo G were evaluated. Moreover, the total oxidant/antioxidant status (TOS-TAS) due to AgNPs application in both cell culture mediums was evaluated. AgNPs treatment results in both cell lines in both 2D and 3D cultures showed a significant decrease in the BrdU labeling index, while large amounts of cells were labelled with TUNEL and Endo G. In 2D culture, Endo G expression increased in MCF-7 cells at 48 and 72nd hours, while it increased significantly in MDA-MB-231 cells at all hours. OSI results show that ROS production is increased in cell medium treated with AgNPs. In conclusion, AgNPs mediated by Phlomis armeniaca, synthesized by a green method, successfully induced damage to mitochondria, resulting in cell cycle arrest and consequent cell proliferation blockade and death in both MCF-7 and MDA-MB-231 cells.

Xinyang Tablet attenuates chronic hypoxia-induced right ventricular remodeling via inhibiting cardiomyocytes apoptosis

BackgroundHypoxia-induced pulmonary hypertension (HPH) is one of the fatal pathologies developed under hypobaric hypoxia and eventually leads to right ventricular (RV) remodeling and RV failure. Clinically, the mortality rate of RV failure caused by HPH is high and lacks effective drugs. Xinyang Tablet (XYT), a traditional Chinese medicine exhibits significant efficacy in the treatment of congestive heart failure and cardiac dysfunction. However, the effects of XYT on chronic hypoxia-induced RV failure are not clear.MethodsThe content of XYT was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS). Sprague–Dawley (SD) rats were housed in a hypobaric chamber (equal to the parameter in altitude 5500 m) for 21 days to obtain the RV remodeling model. Electrocardiogram (ECG) and hemodynamic parameters were measured by iWorx Acquisition & Analysis System. Pathological morphological changes in the RV and pulmonary vessels were observed by H&E staining and Masson’s trichrome staining. Myocardial apoptosis was tested by TUNEL assay. Protein expression levels of TNF-α, IL-6, Bax, Bcl-2, and caspase-3 in the RV and H9c2 cells were detected by western blot. Meanwhile, H9c2 cells were induced by CoCl2 to establish a hypoxia injury model to verify the protective effect and mechanisms of XYT. A CCK-8 assay was performed to determine the viability of H9c2 cells. CoCl2-induced apoptosis was detected by Annexin-FITC/PI flow cytometry and Hoechst 33,258 staining.ResultsXYT remarkably improved RV hemodynamic disorder and ECG parameters. XYT attenuated hypoxia-induced pathological injury in RV and pulmonary vessels. We also observed that XYT treatment decreased the expression levels of TNF-α, IL-6, Bax/Bcl-2 ratio, and the numbers of myocardial apoptosis in RV. In H9c2 myocardial hypoxia model, XYT protected H9c2 cells against Cobalt chloride (CoCl2)-induced apoptosis. We also found that XYT could antagonize CoCl2-induced apoptosis through upregulating Bcl-2, inhibiting Bax and caspase-3 expression.ConclusionsWe concluded that XYT improved hypoxia-induced RV remodeling and protected against cardiac injury by inhibiting apoptosis pathway in vivo and vitro models, which may be a promising therapeutic strategy for clinical management of hypoxia-induced cardiac injury.

Integrinβ1/FAK/ERK signalling pathway is essential for Chinese mitten crab Eriocheir sinensis hemocyte survival

Integrins are cellular adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Integrins can stimulate various signaling pathways by binding to different ligands, thereby exerting immunological functions. While integrins have been found to primarily play a role in bacterial agglutination, phagocytosis, and inhibition of apoptosis in invertebrates, the specific signaling pathway and mechanism of action remain unclear. In vertebrates, β1 integrin and extracellular matrix interactions can associate with focal adhesion kinase (FAK) to initiate MAPK/ERK signaling and regulate cell survival; however, in invertebrates (e.g., Chinese mitten crab), the mechanisms of integrins are poorly understood. The purpose of this study was to investigate whether integrinβ1/FAK activation of the MAPK/ERK pathway regulates hemocyte survival and the associated mechanism. Treatment with an integrinβ1 inhibitor RGD (a conserved tripeptide Arg-Gly-Asp), decreased the levels of FAK and ERK expression and phosphorylation, followed by an intensification of apoptosis. Similar results were obtained following siRNA knockdown of integrinβ1 expression. We further found that the attenuation of ERK phosphorylation enhanced the level of Caspase-3 expression. Together, these findings suggest that integrinβ1 activates the FAK/ERK signaling cascade and is involved in the survival of Chinese mitten crab hemocytes.

Design, Synthesis, Docking Studies and Biological Evaluation of Novel Benzochromenopyrimidines via Silica Sulfuric Acid Catalyzed Reaction on Apoptosis in Human Cancer Cells

Via one-pot reaction of 2-naphthol, thiobarbituric acid and aldehyde in the presence of silica sulfuric acid (SSA) as a catalyst to synthesis benzochromenopyrimidines derivatives. Then we studied the reaction of benzochromenopyrimidines derivatives with bromoacetic acid to obtain benzo[5,6]chromeno[2,3-d]thiazolo[3,2-a]pyrimidine-11,13(10H,14H)-dione derivatives. Meanwhile, dichloroethane was reacted with 12-(4-fluorophenyl)-9-thioxo-9,10-dihydro-8H-benzo[5,6]chromeno[2,3-d]pyrimidin-11(12H)-one to form 14-(4-fluorophenyl)-10,11-dihydrobenzo[5,6]chromeno[2,3-d]thiazolo[3,2-a]pyrimidin-13(14H)-one. All new products structure was elucidated based on analytical and spectroscopic analyses. Anti-cancer activity of newly synthesized structures was investigated. Cancer cells (MDA-MB-231 and HepG2) were used to evaluate the anti-cancer activity of tested compounds using neutral red uptake assay. Treatment of MDA-MB-231 and HepG2 cells with tested compound 6h revealed more inhibitory influence after 48 h. The expression levels of BCL2, BAX, Caspase3, Survivin and P53 genes were investigated using QRT-PCR. This study exhibited that compound 6h revealed significant pro-apoptotic effect via down regulation of BCL2 and Survivin genes and up-regulation of BAX, Caspase3 and P53 genes when treated with MDA-MB-231 and HepG2 cells as compared to control values. The biological studies of these compounds were proved through molecular docking studies with human cyclin-dependent kinase 2 (PDB code: 2A4L) and compound 6h showed low binding energy and shortage bond length with different amino acids. Noteworthy, the tested compound 6h exhibited the most pronounced effect in this respect.

A novel angiogenic effect of PCSK9- regulated genes

BackgroundSince the discovery of the Proprotein Convertase Subtilisin/Kexin Type 9(PCSK9) gene has been involved in regulating low-density lipoprotein metabolism and cardiovascular disease (CVD), many therapeutic strategies directly targeting PCSK9 have been introduced. PCSK9 gain of function (GoF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis. In contrast, PCSK9 loss of function (LOF) mutations have cardioprotective effects and can lead to familial hypo cholesterol in some instances. However, its potential impacts beyond the typical effects on lipid metabolism have not been elucidated. Therefore the study aimed to identify and verify PCSK9′s possible effects beyond its traditional role in lipid metabolism. MethodsThe S127R is a PCSK9 gain of function mutation. Firstly, We used the data of the gene expression Omnibus(GEO) database to identify the differentially expressed genes between S127R mutation carriers and ordinary people. Secondly, the identification and analysis of significant genes were performed with various bioinformatics programs. Thirdly, to verify the possible effect and the potential pathways of PCSK9 on angiogenesis, we constructed PCSK9 low and high expression models by transfecting PCSK9-siRNA (small interfering RNA) and PCSK9-plasmid complex into human umbilical vein endothelial cells (HUVECs), respectively. Furthermore, Wound-Healing Assay and Capillary tube formation assay were applied to measure the effect of PCSK9 on angiogenesis. Fourthly, the expression level of VEGFR2 and the significant genes between PCSK9 low and high expression models were verified by quantitative real-time PCR. All data were analysed by GraphPad Prism 8 software. Results88 DEGs were identified, including 45 up-regulated and 43 down-regulated DEGs. Furthermore, we identified the six genes (MMP9, CASP3, EGR1, NGFR, LEFTY1 and NODAL) as significantly different genes between PCSK9-S127R and Control hiPSC. Further, we found that these significant difference genes were mainly associated with angiogenesis after enrichment analysis. To verify the possible effect of PCSK9 on angiogenesis, we constructed low and high-expression PCSK9 models by transfecting siRNA and PCSK9-plasmid complex into human umbilical vein endothelial cells (HUVECs), respectively. The tubule formation test and Wound healing assays showed that overexpression of PCSK9 had an inhibitory effect on angiogenesis, which could be reversed by decreasing the expression of PCSK9. Moreover, bioinformatics analysis indicated that the six hub genes (MMP9, CASP3, EGR1, NGFR, LEFTY1 and NODAL) might play a vital role in the biological function of PCSK9 in angiogenesis. Real-time quantitative PCR was applied to clarify the expression profiles of these critical genes in overexpression/knockdown PCSK9. Finally, the expression levels of MMP9, Caspase3, LEFTY1, and NODAL were suppressed by overexpression of PCSK9 and could be alleviated by PCSK9 knockdown. Otherwise, EGR1 had the opposite expression trend, and there was no specific trend of NGFR after repeated experiments. ConclusionPCSK9 might play an essential role in angiogenesis, unlike its typical role in lipid metabolism, and MMP9, Caspase3, LEFTY1, NODAL, and EGR1 may be involved in the regulation of angiogenesis as critical genes.

Decreased thioredoxin reductase 3 expression promotes nickel-induced damage to cardiac tissue via activating oxidative stress-induced apoptosis and inflammation.

Thioredoxin reductase 3 (Txnrd3) plays a crucial role in antioxidant and anti-cancer activities, and sperm maturation. The damage of heavy metals, including Nickel (Ni), is the most prominent harm in social development, and hampering Txnrd3 might exacerbate Ni-induced cardiac damage. In this study, a total of 160 8-week-old C57BL/N male mice with 25-30 g weight of Txnrd3+/+ wild-type and Txnrd3-/- homozygote-type were randomly divided into eight groups. The mice in the control and Ni groups were gavaged with distilled water and a freshly prepared 10mg/kg NiCl2 solution. Melatonin (Mel) groups were administered at a concentration of 2mg/kg for 21 days at the mice's 0.1ml/10g body weight. Ni exposure up-regulated the messenger RNA (mRNA) levels of mitochondrial apoptosis (caspase-3, caspase-9, cytochrome c, p53, and BAX), autophagy (LC3, ATG 1, ATG 7, and Beclin-1), and inflammation (TNF-α, COX 2, IL-1β, IL-2, IL-6, and IL-7)-related markers, but down-regulated the mRNA levels of BCL-2, p62 and mTOR (p < .05). Ni exposure decreased the expression of BCL-2 and p62 protein but increased the expression levels of caspase-3, caspase-9, cytochrome c, p53, BAX, ATG 7, Beclin-1, TNF-α, COX 2, IL-1β and IL-2 protein (p < .05). Ni increased the contents of glutathione disulfide (GSSG) and malondialdehyde (MDA) and decreased the activities of catalase (CAT) and total superoxide dismutase (T-SOD) (p < .05). Decreased Txnrd3 expression significantly exacerbated changes compared to the Ni exposure (p < .05). Mel significantly attenuated these changes, but the effect decreased when Txnrd3 was inhibited (p < .05). In conclusion, decreased Txnrd3 expression promoted Ni-induced mitochondrial apoptosis and inflammation via oxidative stress and aggravated heart damage in mice. Decreased Txnrd3 expression significantly reduced the protective effect of Mel to Ni exposure.

Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells

To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism. The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 μmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells. Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G0/G1 (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01). Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.

Effect of intraperitoneal docetaxel on ovarian function in mice

Taxanes are important chemotherapeutic agents used to manage breast cancer and gynaecological malignancies. However, ovarian toxicity induced by the taxane docetaxel (DOC) is of great concern. We investigated DOC-induced toxicity in the ovaries of female CD1 strain mice. The mice were divided into control (saline), DOC-5 (5 mg/kg DOC), and DOC-10 (10 mg/kg DOC) groups and administered saline or DOC on the first day of the study and two weeks later. Two weeks after the second dose, the ovaries were removed for analysis after inducing superovulation. Ovary weight, the number of secondary follicles, and the total number of follicles were reduced after DOC administration. Additionally, the expression levels of caspase-3 and the pro-apoptotic protein Bcl-2 interacting mediator of cell death (BIM) increased. Our findings suggest that high-dose DOC induces damage to growing follicles; however, it may not affect primordial follicles. Impact statement What is already known on this subject? Docetaxel (DOC) is one of the most effective chemotherapeutic agents used to manage various cancers. Some in-vitro studies have examined paclitaxel-induced ovarian toxicity; however, limited research on DOC is available. What do the results of this study add? We investigated DOC-induced ovarian toxicity in female CD1 strain mice at 5 mg/kg and 10 mg/kg. We found that DOC reduced ovary weight, the number of secondary follicles, and the total number of follicles, with the higher dose having a higher effect. What are the implications of these findings for clinical practice and/or further research? We believe that our study makes a significant contribution to the knowledge about the effect of DOC on ovarian function.

Design, Synthesis, Molecular Modeling, and Anticancer Evaluation of New VEGFR-2 Inhibitors Based on the Indolin-2-One Scaffold.

A new series of indoline-2-one derivatives was designed and synthesized based on the essential pharmacophoric features of VEGFR-2 inhibitors. Anti-proliferative activities were assessed for all derivatives against breast (MCF-7) and liver (HepG2) cancer cell lines, using sunitinib as a reference agent. The most potent anti-proliferative derivatives were evaluated for their VEGFR-2 inhibition activity. The effects of the most potent inhibitor, 17a, on cell cycle, apoptosis, and expression of apoptotic markers (caspase-3&-9, BAX, and Bcl-2) were studied. Molecular modeling studies, such as docking simulations, physicochemical properties prediction, and pharmacokinetic profiling were performed. The results revealed that derivatives 5b, 10e, 10g, 15a, and 17a exhibited potent anticancer activities with IC50 values from 0.74-4.62 µM against MCF-7 cell line (sunitinib IC50 = 4.77 µM) and from 1.13-8.81 µM against HepG2 cell line (sunitinib IC50 = 2.23 µM). Furthermore, these compounds displayed potent VEGFR-2 inhibitory activities with IC50 values of 0.160, 0.358, 0.087, 0.180, and 0.078 µM, respectively (sunitinib IC50 = 0.139 µM). Cell cycle analysis demonstrated the ability of 17a to induce a cell cycle arrest of the HepG2 cells at the S phase and increase the total apoptosis by 3.5-fold. Moreover, 17a upregulated the expression levels of apoptotic markers caspase-3 and -9 by 6.9-fold and 3.7-fold, respectively. In addition, 17a increased the expression level of BAX by 2.7-fold while decreasing the expression level of Bcl-2 by 1.9-fold. The molecular docking simulations displayed enhanced binding interactions and similar placement as sunitinib inside the active pocket of VEGFR-2. The molecular modeling calculations showed that all the test compounds were in accordance with Lipinski and Veber rules for oral bioavailability and had promising drug-likeness behavior.

Expression of autophagy and apoptosis biomarkers in patients with acute ischemic stroke

Introduction and Aim: Apoptosis, autophagy, and necrosis are the main mechanisms of neuron death in acute ischemic stroke (AIS). This study aimed to evaluate the expression of apoptosis and autophagy biomarkers in peripheral blood of patients with AIS. Materials and Methods: Sixty-eight patients (32 men and 36 women) aged 30-60 years with AIS underwent a clinical and neurological examination on the 1st, 7th, and 14th days after the disease onset. The expression of apoptosis and autophagy biomarkers in peripheral blood was evaluated by flow cytometry and compared with the severity of neurological deficit and injury on the 1st, 7th, and 14th days, using correlation analysis. Results: There is a statistical significance compared with the control group and an increase in the expression of key biomarkers of apoptosis and autophagy was revealed. Increased expression levels of annexin A5 and caspase-3 positively correlate with the severity of neurological deficit and injury on the 1st and 7th days from the onset of the disease. Conclusion: A direct correlation was revealed between elevated levels of apoptosis and autophagy biomarkers in peripheral blood and severity of neurological deficit and injury on the 1st, 7th, and 14th days from the onset of AIS.

HIF-1α and Caspase-3 expression in aggressive papillary thyroid carcinoma

BackgroundTumor cells adapt to hypoxia by regulating transcription factors that involved in regulation of metabolism, angiogenesis, cell proliferation, and apoptosis. Under hypoxic conditions, hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1α and HIF-1β subunits, acts as a key transcription factor mediating the adaptive cellular responses. Caspase-3 is a key apoptosis-related protease that plays a role in tumor growth and development. Studies have shown that caspase-3 could be regulated by HIF-1α under pathological conditions. Therefore, HIF-1α and caspase-3 expression may be related to the poor prognosis of tumors. In this study, we analyzed the possible relationships between these two signaling factors in correlation with the clinical behavior of PTC.MethodsWe detected the expression levels of HIF-1α and caspase-3 in 70 samples of PTC and para-cancerous tissues (control group) by immunohistochemistry (IHC). Furthermore, various clinicopathological parameters were assessed to determine their correlations with HIF-1α and caspase-3 expressions.ResultsFirst, HIF-1α and caspase-3 expressions (60% and 37.1%, respectively) increased significantly in the PTC samples as compared to normal tissues (2.9% for both HIF-1α and caspase-3) (p < 0.05) as determined by IHC. Second, although there was no significant difference between the expression of HIF-1α and caspase-3 in regard to gender, age distribution, tumor size, lymph node metastasis, and BRAFV600E mutation (all p > 0.05), HIF-1α and caspase-3 expressions were associated with capsule invasion and cell subtypes of PTC (p < 0.05). The percent positivity of caspase-3 expression in tall-cell variant (TCV) was the highest (63.6%). Third, HIF-1α expression was positively correlated with that of caspase-3 (rs = 0.326; p < 0.05).ConclusionsOverexpression of HIF-1α and caspase-3 is associated with carcinogenesis. These factors might serve as promising predictors of aggressive PTC. These findings also suggest their potential as therapeutic targets.

Excessive dietary Cr6+ had no adverse effect on mud crab (Scylla paramamosain) but provoke consumers health risk

Hexavalent chromium (Cr6+) is a metallic contaminant that in organisms and poses a great threat to biological health and ecology due to its toxicity and biomagnification. However, the potential health risks to mud crabs and humans from dietary excessive Cr6+ were currently unknown. Therefore, a 9-week feeding trial was conducted to evaluate the toxic effects of dietary Cr6+ levels (1.4, 77.5 and 312.2 mg/kg) for mud crab (Scylla paramamosain). The results indicated that dietary Cr6+ levels had no significant effect on growth performance for mud crab. Cr concentrations in hepatopancreas and muscle significantly increased with dietary Cr6+ levels increasing from 1.4 to 312.2 mg/kg. The antioxidant capacity of mud crab was enhanced by increased the activities of superoxide dismutase (SOD) and scavenging capability for hydroxyl free radical (SCHR) to resist the Cr6+ toxicity. However, there was no statistical difference in the rate of apoptosis in hemocytes among all treatments. Expression levels of caspase 2 gene related to apoptosis in hemocytes significantly up-regulated with dietary Cr6+ level increasing from 1.4 to 312.2 mg/kg. For food safety, crabs fed diets with 77.5 and 312.2 mg/kg Cr6+ had target hazard quotient (THQ) values greater than 1 for the hepatopancreas, indicating a serious risk for human consumption. According to the target cancer risk (TCR) analysis, people who consumed muscle in mud crab fed diet with 312.2 mg/kg Cr6+ were at unacceptable cancer risk. Overall, the results of the present study indicated mud crab had a strong resistance to Cr6+ toxicity, and dietary excessive Cr6+ did not decline growth performance and survival, oxidative stress and apoptosis in mud crab. Excess Cr6+ were accumulated in tissues such as hepatopancreas and muscle, and consumption of crabs tissues fed diets with 77.5 and 312.2 mg/kg Cr6+ will provoke health and cancer risk in humans.