Macrophage-targeted Nanomedicine for Sepsis: Diagnosis and Therapy.

Sepsis is a syndrome involving complex pathophysiological and biochemical dysregulation. Nanotechnology can improve our understanding of the pathophysiology of sepsis and contribute to the development of novel diagnostic and therapeutic strategies to further reduce the risk of sepsis. Macrophages play a key role in the progression of sepsis, thus, macrophage-associated pathological processes are important targets for both diagnostic and treatment of sepsis. In this paper, we reviewed efforts in the past decade of nanotechnologybased solutions for manipulate macrophages in sepsis diagnosis and management according to the type of nanomaterial. We addressed the latest progress of nanoparticles targeting macrophages for early sepsis detection. Additionally, we summarized the unique advantages of macrophage-targeted nanoparticles in the treatment of sepsis. These nanoparticles can improve the dysregulation of inflammatory response in sepsis by inhibiting the release of inflammatory factors and regulating macrophage apoptosis, activity and polarization. Finally, we present future opportunities as well as challenges of novel diagnostic and therapeutic strategies with the aim of accelerating the clinical translation of nanomedicine for sepsis treatment.

Transcriptomic analysis of spleen B cell revealed the molecular basis of bursopentin on B cell differentiation.

The bursa of Fabricius, the acknowledged humoral immune organ unique to birds, plays a vital role in B cell development. Bursopentin (BP5) derived from the bursa is reported to induce the development and formation of B cells. However, the mechanism of BP5 on B cell differentiation is still unclear. In this paper, total B lymphocytes from mice immunized with H9N2 subtype AIV vaccine were stimulated with BP5. The results show that BP5 at the experimental dosages promoted B cell differentiation, including the total B cells, activated B cells, differentiated B cells, mature B cells and plasma cells. Then, the in vivo immune experiment proved that the percentages of activated and differentiated B cells from mice immunized with AIV vaccine and 0.25 mg/mL BP5 were increased. To investigate the molecular mechanism of BP5 on B cell differentiation, the gene expression profiles of B cells purified from the spleen cells of mice immunized with AIV vaccine and BP5 were detected following RNA sequencing technology. The results show that BP5 at 0.05 and 0.25 mg/mL induced the enrichment of various biological functions, and stimulated five common significant enrichment pathways in B cells from the immunized mice. Additionally, 120 and 59 differentially expressed genes (DEG) represented transcriptional factors in B cells following 0.05 and 0.25 mg/mL BP5 immunization, respectively. In summary, these results suggest that BP5 regulates various gene expression involved in regulation of B cell development, which provides the knowledge required for additional studies on B cell differentiation in response to bursal-derived peptides and also provides an important experimental basis for improving vaccine immunity.

Bursal-Derived BP7 Induces the miRNA Molecular Basis of Chicken Macrophages and Promotes the Differentiation of B Cells.

The bursa of Fabricius (BF) is a vital central humoral immune organ unique to birds. The bioactive peptide BP7 derived from bursa is reported to promote the vaccine immune response and antibody production. However, the regulatory effect on antigen presentation and B cell differentiation has been infrequently reported. In this paper, chicken macrophage HD11 cells were used for the cell model, and the cellular molecular expressions were determined by the fluorescent quantitative PCR (qPCR) after BP7 treatment. Then, the miRNA expression profile was analyzed by high-throughput sequencing. In addition, BALB/C mice were used as the animal model to detect B cell subtype with flow cytometry (FCM). The results showed that the expressions of four immune active molecules, IL-1ß, IL-6, iNOS, and IFN-α, in HD11 cells were significantly increased with 100 ng/mL BP7 treatment. Compared with the control group, there were 58 up-regulated and 61 down-regulated miRNAs in HD11 cells with BP7 treatment. The gene ontology (GO) function analysis found that BP7 mainly affected the various biological processes, molecular function, and MHC protein complex. Pathway analysis showed that 100 ng/mL BP7 stimulated various physiological metabolic pathways and signal transduction pathways, including the intestinal immune network producing IgA in HD11 cells. Furthermore, it was found that BP7 in vitro stimulated B cell populations, as well as plasma cells in spleen cells from the immunized mice. Additionally, B cell activation subpopulations were increased in mice immunized with the AIV vaccine and BP7. These results proved that BP7 stimulated various differentially expressed genes in chicken macrophage HD11 cells, and induced B cell differentiation in the immunized mice, which suggested that BP7 might participate in the antigen presentation process, thereby promoting the differentiation of B cells. These results provide an important basis for the mechanism of bursal-derived peptide on B cell development, and offer the experimental basis for the development of adjuvants.

The Immunomodulatory Functions of Various CpG Oligodeoxynucleotideson CEF Cells and H9N2 Subtype Avian Influenza Virus Vaccination.

CpG oligodeoxynucleotides (CpG ODN) present adjuvant activities for antigen proteins, which can induce humoral and cellular immune responses to antigens. However, the immunomodulatory functions of CpG ODNs with different sequences are very different. In this paper, six CpG ODNs with different sequences were designed based on CpG2007 as a template. Through the screening of CEF cells in vitro, the stimulating activity of CpG ODNs was determined. Then, two selected CpG ODN sequence backbones were modified by substituting the oxygen with sulfur (S-CpG) and verifying the immune activity. Next, to prove the feasibility of S-CpG as an immune potentiator, two immune models with or without white oil adjuvant were prepared in 20-day-old chicken vaccinations. The screening experiment in vitro showed that the inducing roles of CpG ODN 4 and 5 could strongly stimulate various immune-related molecular expressions. Additionally, CpG ODN 4 and 5 with sulfation modification significantly induced various cytokines' expressions. Furthermore, CpG ODN 4 and 5 induced the strongly humoral and cellular immune responses during vaccination, in which white oil, as an adjuvant, could significantly improve the immune effect of CpG ODN. These results provide an important experimental basis for exploring the structural characteristics and vaccine immunity of CpG ODN.

Tcf1 Sustains the Expression of Multiple Regulators in Promoting Early Natural Killer Cell Development.

T cell factor 1 (Tcf1) is known as a critical mediator for natural killer (NK) cell development and terminal maturation. However, its essential targets and precise mechanisms involved in early NK progenitors (NKP) are not well clarified. To investigate the role of Tcf1 in NK cells at distinct developmental phases, we employed three kinds of genetic mouse models, namely, Tcf7fl/flVavCre/+, Tcf7fl/flCD122Cre/+ and Tcf7fl/flNcr1Cre/+ mice, respectively. Similar to Tcf1 germline knockout mice, we found notably diminished cell number and defective development in BM NK cells from all strains. In contrast, Tcf7fl/flNcr1Cre/+ mice exhibited modest defects in splenic NK cells compared with those in the other two strains. By analyzing the published ATAC-seq and ChIP-seq data, we found that Tcf1 directly targeted 110 NK cell-related genes which displayed differential accessibility in the absence of Tcf1. Along with this clue, we further confirmed that a series of essential regulators were expressed aberrantly in distinct BM NK subsets with conditional ablating Tcf1 at NKP stage. Eomes, Ets1, Gata3, Ikzf1, Ikzf2, Nfil3, Runx3, Sh2d1a, Slamf6, Tbx21, Tox, and Zeb2 were downregulated, whereas Spi1 and Gzmb were upregulated in distinct NK subsets due to Tcf1 deficiency. The dysregulation of these genes jointly caused severe defects in NK cells lacking Tcf1. Thus, our study identified essential targets of Tcf1 in NK cells, providing new insights into Tcf1-dependent regulatory programs in step-wise governing NK cell development.

Addressless: A new internet server model to prevent network scanning.

Eliminating unnecessary exposure is a principle of server security. The huge IPv6 address space enhances security by making scanning infeasible, however, with recent advances of IPv6 scanning technologies, network scanning is again threatening server security. In this paper, we propose a new model named addressless server, which separates the server into an entrance module and a main service module, and assigns an IPv6 prefix instead of an IPv6 address to the main service module. The entrance module generates a legitimate IPv6 address under this prefix by encrypting the client address, so that the client can access the main server on a destination address that is different in each connection. In this way, the model provides isolation to the main server, prevents network scanning, and minimizes exposure. Moreover it provides a novel framework that supports flexible load balancing, high-availability, and other desirable features. The model is simple and does not require any modification to the client or the network. We implement a prototype and experiments show that our model can prevent the main server from being scanned at a slight performance cost.

Breast cancer exosomes contribute to pre-metastatic niche formation and promote bone metastasis of tumor cells.

Rationale: Breast cancer preferentially develops osteolytic bone metastasis, which makes patients suffer from pain, fractures and spinal cord compression. Accumulating evidences have shown that exosomes play an irreplaceable role in pre-metastatic niche formation as a communication messenger. However, the function of exosomes secreted by breast cancer cells remains incompletely understood in bone metastasis of breast cancer. Methods: Mouse xenograft models and intravenous injection of exosomes were applied for analyzing the role of breast cancer cell-derived exosomes in vivo. Effects of exosomes secreted by the mildly metastatic MDA231 and its subline SCP28 with highly metastatic ability on osteoclasts formation were confirmed by TRAP staining, ELISA, microcomputed tomography, histomorphometric analyses, and pit formation assay. The candidate exosomal miRNAs for promoting osteoclastogenesis were globally screened by RNA-seq. qRT-PCR, western blot, confocal microscopy, and RNA interfering were performed to validate the function of exosomal miRNA. Results: Implantation of SCP28 tumor cells in situ leads to increased osteoclast activity and reduced bone density, which contributes to the formation of pre-metastatic niche for tumor cells. We found SCP28 cells-secreted exosomes are critical factors in promoting osteoclast differentiation and activation, which consequently accelerates bone lesion to reconstruct microenvironment for bone metastasis. Mechanistically, exosomal miR-21 derived from SCP28 cells facilitates osteoclastogenesis through regulating PDCD4 protein levels. Moreover, miR-21 level in serum exosomes of breast cancer patients with bone metastasis is significantly higher than that in other subpopulations. Conclusion: Our results indicate that breast cancer cell-derived exosomes play an important role in promoting breast cancer bone metastasis, which is associated with the formation of pre-metastatic niche via transferring miR-21 to osteoclasts. The data from patient samples further reflect the significance of miR-21 as a potential target for clinical diagnosis and treatment of breast cancer bone metastasis.

Curdlan Prevents the Cognitive Deficits Induced by a High-Fat Diet in Mice via the Gut-Brain Axis.

A high-fat (HF) diet is a major predisposing factor of neuroinflammation and cognitive deficits. Recently, changes in the gut microbiota have been associated with neuroinflammation and cognitive impairment, through the gut-brain axis. Curdlan, a bacterial polysaccharide widely used as food additive, has the potential to alter the composition of the microbiota and improve the gut-brain axis. However, the effects of curdlan against HF diet-induced neuroinflammation and cognitive decline have not been investigated. We aimed to evaluate the neuroprotective effect and mechanism of dietary curdlan supplementation against the obesity-associated cognitive decline observed in mice fed a HF diet. C57Bl/6J male mice were fed with either a control, HF, or HF with curdlan supplementation diets for 7 days (acute) or 15 weeks (chronic). We found that acute curdlan supplementation prevented the gut microbial composition shift induced by HF diet. Chronic curdlan supplementation prevented cognitive declines induced by HF diet. In addition, curdlan protected against the HF diet-induced abnormities in colonic permeability, hyperendotoxemia, and colonic inflammation. Furthermore, in the prefrontal cortex (PFC) and hippocampus, curdlan mitigated microgliosis, neuroinflammation, and synaptic impairments induced by a HF diet. Thus, curdlan-as a food additive and prebiotic-can prevent cognitive deficits induced by HF diet via the colon-brain axis.

TLR-2-mediated metabolic reprogramming participates in polyene phosphatidylcholine-mediated inhibition of M1 macrophage polarization.

This study aimed to investigate whether the classic hepatoprotective drug polyene phosphatidylcholine (PPC) regulates macrophage polarization and explores the potential role of TLR-2 in this process. In RAW264.7 macrophages and murine bone marrow-derived macrophages (BMDMs) stimulated by lipopolysaccharide (LPS), PPC significantly inhibited the production of IL-6, TNF-α, and the mRNA expression of M1-type macrophage markers. Consistently, PPC reduced the mRNA expression of several key enzymes in the pathways of glycolysis and lipid synthesis while increasing the expression of key enzymes associated with lipid oxidation. Moreover, blocking the glycolytic pathway using 2-deoxy-D-glucose (2-DG) significantly enhanced the anti-inflammatory effect of PPC. However, inhibition of lipid oxidation using GW9662 (an inhibitor of PPAR-γ) and GW6471 (an inhibitor of PPAR-α) abolished the anti-inflammatory effect of PPC. Interestingly, TLR-2 expression in macrophages was significantly downregulated after exposure to PPC. Moreover, pre-activation of TLR-2 hampered the anti-inflammatory effect of PPC. In addition, PPC did not inhibit the secretion of IL-6 and TNF-α in TLR-2-/- BMDMs that were activated by LPS. This was consistent with the increased expression of M1 markers and glycolytic and lipid synthesis enzymes but decreased lipid oxidation-related enzymes. These results showed that PPC inhibits the differentiation of M1-type macrophages, which was most likely related to TLR-2-mediated metabolic reprogramming.

Supplement of microbiota-accessible carbohydrates prevents neuroinflammation and cognitive decline by improving the gut microbiota-brain axis in diet-induced obese mice.

BACKGROUND: Western pattern diets induce neuroinflammation and impair cognitive behavior in humans and animals. Neuroinflammation and cognitive impairment have been associated with microbiota dysbiosis, through the gut-brain axis. Furthermore, microbiota-accessible carbohydrates (MACs) found in dietary fiber are important in shaping the microbial ecosystem and have the potential to improve the gut-brain-axis. However, the effects of MACs on neuroinflammation and cognition in an obese condition have not yet been investigated. The present study aimed to evaluate the effect of MACs on the microbiota-gut-brain axis and cognitive function in obese mice induced by a high-fat and fiber deficient (HF-FD) diet. METHODS: C57Bl/6 J male mice were fed with either a control HF-FD or a HF-MAC diet for 15 weeks. Moreover, an additional group was fed with the HF-MAC diet in combination with an antibiotic cocktail (HF-MAC + AB). Following the 15-week treatment, cognitive behavior was investigated; blood, cecum content, colon, and brain samples were collected to determine metabolic parameters, endotoxin, gut microbiota, colon, and brain pathology. RESULTS: We report MACs supplementation prevented HF-FD-induced cognitive impairment in nesting building and temporal order memory tests. MACs prevented gut microbiota dysbiosis, including increasing richness, α-diversity and composition shift, especially in Bacteroidetes and its lower taxa. Furthermore, MACs increased colonic mucus thickness, tight junction protein expression, reduced endotoxemia, and decreased colonic and systemic inflammation. In the hippocampus, MACs suppressed HF-FD-induced neuroglia activation and inflammation, improved insulin IRS-pAKT-pGSK3ß-pTau synapse signaling, in addition to the synaptic ultrastructure and associated proteins. Furthermore, MACs' effects on improving colon-cognitive parameters were eliminated by wide spectrum antibiotic microbiota ablation. CONCLUSIONS: These results suggest that MACs improve cognitive impairments via the gut microbiota-brain axis induced by the consumption of an HF-FD. Supplemental MACs to combat obesity-related gut and brain dysfunction offer a promising approach to prevent neurodegenerative diseases associated with Westernized dietary patterns and obesity.

Oat-Derived ß-Glucans Induced Trained Immunity Through Metabolic Reprogramming.

Trained immunity has been recently identified in innate immune cells, which undergo long-term epigenetic and metabolic reprogramming after exposure to pathogens for protection from secondary infections. (1, 3)/(1, 6)-ß-glucan derived from fungi can induce potent trained immunity; however, the effect of (1, 3)/(1, 4)-ß-glucan (rich in dietary fiber oat) on trained immunity has not been reported. In the present study, two cell culture systems for trained immunity induction were validated in monocytes/macrophages from mouse bone myeloid and human THP-1 cells exposed to positive inducers of trained immunity, including ß-glucan from Trametes versicolor or human-oxidized low-density lipoprotein. Primed with oat ß-glucan, the mRNA expression and production of TNF-α and IL-6 significantly increased in response to re-stimulation of TLR-4/2 ligands. Moreover, the expression of several key enzymes in glycolytic pathway and tricarboxylic acid cycle was significantly upregulated. In addition, inhibiting these enzymes decreased the production of TNF-α and IL-6 boosted by oat ß-glucan. These results show that oat ß-glucan induces trained immunity through metabolic reprogramming. This provides important evidence that dietary fiber can maintain the long-term responsiveness of the innate immune system, which may benefit for prevention of infectious diseases or cancers.

Corrigendum: Genetic Removal of the CH1 Exon Enables the Production of Heavy Chain-Only IgG in Mice.

[This corrects the article DOI: 10.3389/fimmu.2018.02202.].

Hematopoietic stem cells and lineage cells undergo dynamic alterations under microgravity and recovery conditions.

Spaceflight leads to health risks including bone demineralization, skeletal muscle atrophy, cardiovascular dysfunction, and disorders of almost all physiologic systems. However, the impacts of microgravity on blood lineage cells and hematopoietic stem cells (HSCs) in vivo are largely unknown. In this study, we analyzed peripheral blood samples from 6 astronauts who had participated in spaceflight missions and found significant changes in several cell populations at different time points. These dynamic alterations of lineage cells and the role of HSCs were further studied in a mouse model, using hindlimb unloading (HU) to simulate microgravity. Large reductions in the frequency of NK cells, B cells, and erythrocyte precursors in the bone marrow of the HU mice were observed, together with an increased frequency of T cells, neutrophils, and HSCs. T cell levels recovered faster than those of B cells and erythrocyte precursors, whereas the recovery rates of NK cells and granulocytes were slow. In addition, competitive reconstitution experiments demonstrated the impaired function of HSCs, although these changes were reversible. Deep sequencing showed changes in the expression of regulatory molecules important for the differentiation of HSCs. This study provides the first determination of altered HSC function under simulated microgravity in vivo. The impairment of HSC function and differentiation provides an explanation for the immune disorders that occur under simulated microgravity. Thus, our findings demonstrated that spaceflight and simulated microgravity disrupt the homeostasis of immune system and cause dynamic alterations on both HSCs and lineage cells.-Cao, D., Song, J., Ling, S., Niu, S., Lu, L., Cui, Z., Li, Y., Hao, S., Zhong, G., Qi, Z., Sun, W., Yuan, X., Li, H., Zhao, D., Jin, X., Liu, C., Wu, X., Kan, G., Cao, H., Kang, Y., Yu, S., Li, Y. Hematopoietic stem cells and lineage cells undergo dynamic alterations under microgravity and recovery conditions.

The excretory-secretory products of Echinococcus granulosus protoscoleces stimulated IL-10 production in B cells via TLR-2 signaling.

BACKGROUND: Excretory-secretory products released by Echinococcus granulosus protoscoleces (EgPSC-ESPs) are well-known to regulate T cell responses. However, their direct influence on the differentiation of B cell subsets remains largely elusive. This study investigated the effects of EgPSC-ESPs on the differentiation of IL-10-producing B cells (B10), and explored the possible role of Toll-like receptor 2 (TLR-2) signaling in this process. RESULTS: In comparison to phosphate buffered saline (PBS), B cells exposed to the excretory-secretory products (ESPs) generated higher percentages of B10 cells, with higher expression of IL-10 mRNA, and larger amount of IL-10 production, which were in a dose dependent way. The mRNA and protein expression of TLR-2 in the ESPs-stimulated B cells were significantly higher than those in PBS, which was consistent to the results in B cells isolated from EgPSC infected mice. Moreover, TLR-2-/- B cells in response to ESPs stimulation expressed lower levels of IL-10 mRNA and produced undetectable IL-10 in comparison to those in normal B cells. In addition, Phosphatase and tensin homolog deleted on chromosome ten/AKT/Phosphatidylinositol-3 kinase (PTEN/AKT/PI3K) pathway was activated in ESPs-treated B cells, which was also dependent on TLR-2 signaling. Pam3CSK4, the agonist of TLR-2, could mock the effects of ESPs on the expression of PTEN, AKT and PI3K. CONCLUSION: Overall, this study revealed that TLR-2 signaling was required for B10 induction mediated by EgPSC-ESPs, which might be an immunomodulatory target against the parasite infection.

Genetic Removal of the CH1 Exon Enables the Production of Heavy Chain-Only IgG in Mice.

Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.

Redox-responsive self-assembly of ß-cyclodextrin and ferrocene double-headed amphiphilic molecules.

We present a redox-responsive self-assembly based on a unimolecular platform. Three double-headed amphiphilic molecules composed of ß-cyclodextrin (ß-CD) and ferrocene (Fc) each with an alkyl chain as a linker (ßCD-Cm-Fc, m = 2, 6, and 10) were synthesized, and their self-assembly behaviors were investigated. The molecules self-assembled into polydisperse micelles that transformed into vesicles upon oxidization of the Fc moieties to Fc+. 2D 1H NMR results suggest that although the three molecules formed aggregates with similar morphologies, their molecular configurations were different because of the different lengths of the alkyl chains. When the linker was a C2 chain, no host-guest complexes were formed, whereas host-guest recognition was detected for linker lengths of C6 and C10. For the oxidized state samples, there were no host-guest interactions for linker lengths of C2 and C6, whereas the alkyl chain was locked in the cavity of ß-CD by host-guest inclusion for the molecule with a C10 linker. Moreover, reversible redox-responsive self-assemblies based on the three ß-CD derivatives with a terminal Fc were successfully achieved. Our results enrich the field of ß-CD/Fc reversible self-assembly systems, and provide a possible unimolecular host-guest complexation model in host-guest chemistry.

Low-dose bortezomib increases the expression of NKG2D and DNAM-1 ligands and enhances induced NK and γδ T cell-mediated lysis in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy, although bortezomib has markedly improved its outcomes. Growing clinical evidence indicates that enhancing induced natural killer (NK) or γδ T cells for infusion is useful in the treatment of MM. However, whether combination treatment with bortezomib and induced NK and γδ T cells further improves outcomes in MM, and how the treatments should be combined, remain unclear. Herein, we found that low-dose bortezomib did not suppress the viability of induced NK and γδ T cells, but did induce MM cell apoptosis. Importantly, low-dose bortezomib increased the expression of NKG2D and DNAM-1 ligands on MM cells, which sensitized the multiple myeloma cells to lysis by induced NK and γδ T cells. Our results suggested that combination treatment with low-dose bortezomib and induced NK or γδ T cells had a synergistic cytotoxic effect on MM cells. This study provided a proof of principle for the design of future trials and investigation of this combination therapeutic strategy for MM treatment.

Effects of arsenic trioxide alone and in combination with bortezomib in multiple myeloma RPMI 8266 cells.

The aim of this study was to detect the efficiency of arsenic trioxide (ATO) alone or together with bortezomib to inhibit proliferation and induce apoptosis in a multiple myeloma (MM) RPMI 8266 cells. Mechanisms of action were also investigated. RPMI 8266 cells were treated with ATO alone and in combination with bortezomib for 24 hours, and cell viability was assessed by modified MTT. Annexin V-F1TC and PI staining was used to detect the apoptosis rate and cell cycling was investigated by flow cytometry, along with expression of cell surface death receptor-4(DR4) and death receptor-5 (DR5). Western blotting was applied to detect the expression of bcl-2, caspase-3, caspase-8, and caspase-9. As a result, the ATO combined with bortezomib group showed more inhibition of RPMI 8266 cell viability than the ATO group. Expression of DR4 and DR5 on the cell surfaces, and the apoptosis rate were increased after treatment by ATO alone or combined with bortezomib. The cells appeared to arrest in G2/M phase after treatment. Expression of bcl-2 was more significantly decreased in the combination group, and that of caspase-3, caspase-8 and caspase-9 was significantly increased as well. Therefore, bortezomib can enhance ATO actions to induce apoptosis in RPMI 8266 cells, with decrease in expression of bcl-2 and increase of caspase-3, caspase-8 and caspase-9 proteins.