Are statins a risk factor in patients with atherosclerosis? / ¿Son las estatinas un factor de riesgo en pacientes con aterosclerosis?

SUMMARY: Statins inhibit cholesterol synthesis, but also have other pleiotropic effects. There are indications that they affect macrophage survival trough the regulation of apoptosis. We analyzed 50 samples of aortic wall, selected based on statins in patients' therapy (n=25, Th-S group) or statin-free therapy (n=25, Th-nonS group). Each group had 5 samples of healthy aortic tissue, 10 samples of mild and 10 samples of severe atherosclerotic changes in aortic wall. Tissue was stained with hematoxylin-eosin and immunohistochemical methods (anti-Bcl-2 antibody). Presence of Bcl2-positive macrophages (Bcl-2+ MP) was determined semiquantitatively, and data were processed in Microsoft Excell and IMB SPSS 23 Statistics. 60 % of patients in the Th-S group had a mild increase of Bcl-2+ MP The use of statins leads to a significantly more frequent increase in Bcl2+ macrophages in the intima of the healthy aortic tissue. Analysis of all aortic samples with pathohistological diagnosis showed that statin therapy was statistically significantly more often leading to a markedly increased presence of Bcl-2+ MP. In the media, all samples of the Th-S group have a mild increase of Bcl-2+ MP, and in adventitia 40 % of patients. The use of statins more often leads to a markedly increased presence of Bcl-2+ MP in aortic tissue with diagnosed mild and severe atherosclerosis. In samples of severe atherosclerosis, statins lead to a markedly increased presence of Bcl-2+ MP in the parts of the plaque towards the intima and towards the media. Statins lead to an increased presence of Bcl-2+ macrophages, prolong their life, both in healthy and atherosclerotic altered aortic tissue. This indicates potentiation of inflammation and damage to the aortic wall, and calls into question the positive effect of statins on the aortic wall with atherosclerosis.

RESUMEN: Las estatinas inhiben la síntesis de colesterol, pero también tienen otros efectos pleiotrópicos. Hay indicios de que afectan la supervivencia de los macrófagos a través de la regulación de la apoptosis.Se analizaron 50 muestras de pared aórtica, seleccionadas en base a estatinas en tratamiento de pacientes (n=25, grupo Th-S) o en tratamiento libre de estatinas (n=25, grupo Th- nonS). Cada grupo tenía 5 muestras de tejido aórtico sano, 10 muestras de cambios ateroscleróticos leves y 10 muestras de cambios ateroscleróticos severos en la pared aórtica. El tejido se tiñó con hematoxilina-eosina y métodos inmunohistoquímicos (anticuerpo anti-Bcl-2). La presencia de macrófagos positivos para Bcl2 (Bcl- 2+ MP) se determinó semicuantitativamente y los datos se procesaron en Microsoft Excell e IMB SPSS 23 Statistics. El 60 % de los pacientes del grupo Th-S tuvo un aumento leve de Bcl-2+ MP. El uso de estatinas conduce a un aumento significativamente más frecuente de macrófagos Bcl2+ en la íntima del tejido aórtico sano. El análisis de todas las muestras aórticas con diagnóstico anatomopatológico mostró que la terapia con estatinas fue significativamente más frecuente desde el punto de vista estadístico, lo que condujo a una presencia marcadamente mayor de Bcl-2+ MP. En los medios, todas las muestras del grupo Th-S tienen un leve aumento de Bcl-2+ MP, y en adventicia en el 40 % de los pacientes. El uso de estatinas con mayor frecuencia conduce a una presencia marcadamente mayor de MP Bcl-2+ en el tejido aórtico con aterosclerosis leve y grave diagnosticada. En muestras de aterosclerosis severa, las estatinas conducen a una presencia aumentada de Bcl-2+ MP en las partes de la placa hacia la íntima y hacia la media. Las estatinas conducen a una mayor presencia de macrófagos Bcl-2+, prolongan su vida, tanto en tejido aórtico sano como aterosclerótico alterado. Esto indica la potenciación de la inflamación y el daño a la pared aórtica y pone en duda el efecto positivo de las estatinas en la pared aórtica con aterosclerosis.

Hepato-protective effect of rutin via IL-6/STAT3 pathway in CCl4-induced hepatotoxicity in rats

BACKGROUND: Carbon tetrachloride (CCl4) induces hepatotoxicity in animal models, including the increased blood flow and cytokine accumulation that are characteristic of tissue inflammation. The present study investigates the hepato-protective effect of rutin on CCl4-induced hepatotoxicity in rats. RESULTS: Forty male Wistar rats were divided into four groups. Group I (control group) received 1 mL/kg of dimethyl sulfoxide intragastrically and 3 mL/kg olive oil intraperitoneally twice a week for 4 weeks. Group II received 70 mg/ kg rutin intragastrically. Groups III and IV received CCl4 (3 mL/kg, 30 % in olive oil) intraperitoneally twice a week for 4 weeks. Group IV received 70 mg/kg rutin intragastrically after 48 h of CCl4 treatment. Liver enzyme levels were determined in all studied groups. Expression of the following genes were monitored with real-time PCR: interleukin-6 (IL-6), dual-specificity protein kinase 5 (MEK5), Fas-associated death domain protein (FADD), epidermal growth factor (EGF), signal transducer and activator of transcription 3 (STAT3), Janus kinase (JAK), B-cell lymphoma 2 (Bcl2) and B-cell lymphoma-extra-large (Bcl-XL). The CCl4 groups showed significant increases in biochemical markers of hepatotoxicity and up-regulation of expression levels of IL-6, Bcl-XL, MEK5, FADD, EGF, STAT3 and JAK compared with the control group. However, CCl4 administration resulted in significant down-regulation of Bcl2 expression compared with the control group. Interestingly, rutin supplementation completely reversed the biochemical markers of hepatotoxicity and the gene expression alterations induced by CCl4. CONCLUSION: CCl4 administration causes alteration in expression of IL-6/STAT3 pathway genes, resulting in hepatotoxicity. Rutin protects against CCl4-induced hepatotoxicity by reversing these expression changes.

Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis

Background: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate the anti-apoptotic efficacy of Bcl-xL expressing dual promoter plasmid system as a candidate to be used for recombinant protein production and DNA vaccination approaches. For this purpose, Bcl-xL anti-apoptotic protein gene was inserted in a dual expressing vector system in frame with EGFP (enhanced green fluorescence protein) after IRES (internal ribosomal site). The plasmid has a multiple cloning site after CMV (cytomegalovirus promoter) left empty to be inserted a biopharmaceutical protein gene region or DNA vaccine antigens. Results: In order to determine the anti-apoptotic efficacy of Bcl-xL inserted dual expressing vector, BHK-21 cells were transfected both with this plasmid and empty vector as control. Apoptosis was stimulated by several apoptosis inducing agents and serum deprivation in the transfected cells for 48 hrs. Cells expressing Bcl-xL protein in frame with EGFP were determined by flow cytometry as an indicator of cell viability. Additionally, apoptosis were determined by intracellular cleaved Casp 3 staining in Bcl-xL expressing EGFP positive cells. The dual expression plasmid bearing Bcl-xL anti-apoptotic protein prolonged the cell survival rate and protected cells from apoptosis upon apoptosis induction by doxorubicin and camptothecin in which the anti-apoptotic efficacies are inhibited through over expressing of Bcl-xL. pIRES2EGFP/Bcl-xL transfected cell ratio was significantly higher compared to empty vector transfected cells (P < 0.001). In contrast, apoptotic cell ratio was significantly lower in pIRES2EGFP/Bcl-xL transfected cell population compared to empty vector transfected cells (P < 0.001). Conclusion: In conclusion...

Parthenolide-induced apoptosis of hepatic stellate cells and anti-fibrotic effects in an in vivo rat model

Parthenolide (PT), a sesquiterpene lactone derived from the plant feverfew, has pro-apoptotic activity in a number of cancer cell types. We assessed whether PT induces the apoptosis of hepatic stellate cells (HCSs) and examined its effects on hepatic fibrosis in an in vivo model. The effects of PT on rat HSCs were investigated in relation to cell growth inhibition, apoptosis, NF-kappaB binding activity, intracellular reactive oxygen species (ROS) generation, and glutathione (GSH) levels. In addition, the anti-fibrotic effects of PT were investigated in a thioacetamide-treated rat model. PT induced growth inhibition and apoptosis in HSCs, as evidenced by cell growth inhibition and apoptosis assays. PT increased the expression of Bax proteins during apoptosis, but decreased the expression of Bcl-2 and Bcl-XL proteins. PT also induced a reduction in mitochondrial membrane potential, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. PT inhibited TNF-alpha-stimulated NF-kappaB binding activity in HSCs. The pro-apoptotic activity of PT in HSCs was associated with increased intracellular oxidative stress as evidenced by increased intracellular ROS levels and depleted intracellular GSH levels. Furthermore, PT ameliorated hepatic fibrosis significantly in a thioacetamide-treated rat model. In conclusion, PT exhibited pro-apoptotic effects in rat HSCs and ameliorated hepatic fibrosis in a thioacetamide-induced rat model.

Calcium overload is essential for the acceleration of staurosporine-induced cell death following neuronal differentiation in PC12 cells

Differentiation of neuronal cells has been shown to accelerate stress-induced cell death, but the underlying mechanisms are not completely understood. Here, we find that early and sustained increase in cytosolic ([Ca2+]c) and mitochondrial Ca2+ levels ([Ca2+]m) is essential for the increased sensitivity to staurosporine-induced cell death following neuronal differentiation in PC12 cells. Consistently, pretreatment of differentiated PC12 cells with the intracellular Ca2+-chelator EGTA-AM diminished staurosporine-induced PARP cleavage and cell death. Furthermore, Ca2+ overload and enhanced vulnerability to staurosporine in differentiated cells were prevented by Bcl-XL overexpression. Our data reveal a new regulatory role for differentiation-dependent alteration of Ca2+ signaling in cell death in response to staurosporine.

Effects of betulinic acid on proliferation and apoptosis in Jurkat cells and its in vitro mechanism / 华中科技大学学报（医学）（英德文版）

The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 mumol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G(0)/G(1) phase and decrease in S phase. After treatment with 0, 20, 60, 100 mumol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00+/-1.25)% to (58.84+/-0.32)% in G(0)/G(1) phase, whereas it was decreased from (61.45+/-1.04)% to (35.82+/-1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G(0)/G(1) phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.

Up-regulation of Bax and endonuclease G, and down-modulation of Bcl-X(L) involved in cardiotoxin III-induced apoptosis in K562 cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced K562 cell apoptosis was confirmed by DNA fragmentation (DNA ladder, sub-G1 formation) and phosphatidylserine (PS) externalization with an IC50 value of 1.7 mug/ml at 48 h. A mechanistic analysis demonstrated that CTX III-induced apoptotic cell death was accompanied by up-regulation of both Bax and endonuclease G (Endo G), and downregulation of Bcl-X(L). CTX III had no effect on the levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. CTX III treatment caused loss of the mitochondrial membrane potential (delta psi m), release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and -3. CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. However, CTX III did not generate reactive oxygen species (ROS) and antioxidants, including N-acetylcysteine and catalase, did not block CTX III-induced apoptosis in K562 cells. Modulation of Bax, Bcl-X(L), and the Endo G proteins, release of mitochondrial cytochome c, and activation of caspase-3 and -9 all are involved in the CTX III-triggered apoptotic process in human leukemia K562 cells.

Immunohistochemical Expression of Bcl-2, Bcl-xL, Bax, p53 Proteins in Gastric Adenoma and Adenocarcinoma / 대한소화기학회지

BACKGROUND/AIMS: The aim of this study was to investigate the immunohistochemical expression of bcl-2, bcl-xL, bax, and p53 proteins according to the pathological parameters such as grade of dysplasia, histological type, depth of invasion, lymph node metastasis, and TNM stage in the gastric adenoma and gastric adenocarcinoma. METHODS: Immunohistochemical staining using monoclonal bcl-2, bcl-xL, bax, p53 antibodies were performed on paraffin embedded specimens from forty-one gastric adenomas and 100 gastric adenocarcinomas. RESULTS: The expression rate of bcl-2 was higher in adenomas (34.2%), especially in high grade dysplasia (52.4%), than adenocarcinomas (2.0%). The expression rate of bcl-xL was higher in adenocarcinomas (55.0%) than adenomas (22%). The expression rate of the bax was higher in adenocarcinomas (58.0%) than adenomas (14.6%). In the adenocarcinoma, the bax expression was significantly related with the depth of invasion, lymph node metastasis, and TNM stage. The expression rate of p53 was higher in adenocarcinomas (64.0%) than adenomas (14.6%). CONCLUSIONS: Bcl-2 protein would be related with the development of gastric adenoma, especially with high grade dysplasia. Bcl-xL and p53 proteins would be involved in the development of relatively early stage of gastric adenocarcinoma but not in tumor progression. Bax protein would be involved in the development of gastric adenocarcinoma and related with depth of invasion, lymph node metastasis, and TNM stage.