Modulation of apoptosis and Inflammasome activation in chondrocytes: co-regulatory role of Chlorogenic acid.

BACKGROUND: The B-cell lymphoma 2 (Bcl-2) protein regulates programmed cell death throughout the disease conditions by upholding apoptotic pathways. However, the mechanism by which it's expressed in chondrocytes still needs to be studied in chondrocyte-related disorders. Additionally, exploring the potential therapeutic role of Chlorogenic acid (CGA) in confluence with Bcl-2 modulation is of significant interest. METHODS: In vivo and in vitro studies were performed according to our previous methodologies. The chondrocytes were cultured in specific growth media under standard conditions after expression verification of different microRNAs through high-throughput sequencing and verification of Bcl-2 involvement in tibial growth plates. The effect of Bcl-2 expression was investigated by transfecting chondrocytes with miR-460a, siRNA, and their negative controls alone or in combination with CGA. The RNA was extracted and subjected to a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blot analysis and immunofluorescence assays were performed to visualize the intracellular localization of Bcl-2 and associated proteins related to apoptotic and inflammasome pathways. Moreover, apoptosis through flow cytometry was also performed to understand the modulation of concerning pathways. RESULTS: The suppression of Bcl-2 induced higher apoptosis and mitochondrial dysfunction, leading to IL-1ß maturation and affecting the inflammasome during chondrocyte proliferation. Conversely, overexpression attenuated the activation, as evidenced by reduced caspase activity and IL-1ß maturation. In parallel, CGA successfully reduced siRNA-induced apoptosis by decreasing Cytochrome C (Cyto C) release from the mitochondria to the cytoplasm, which in turn decreased Caspase-3 and Caspase-7 cleavage with Bcl-2-associated X protein (Bax). Furthermore, siBcl-2 transfection and CGA therapy increased chondrocyte proliferation and survival. The CGA also showed a promising approach to maintaining chondrocyte viability by inhibiting siRNA-induced apoptosis. CONCLUSIONS: Targeting Bcl-2-mediated regulation might be a possible treatment for chondrocyte-related conditions. Moreover, these results add knowledge of the complicated processes underlying chondrocyte function and the pathophysiology of related diseases, highlighting the significance of target specific therapies. Video Abstract.

In Silico Subtractive Proteomics and Molecular Docking Approaches for the Identification of Novel Inhibitors against Streptococcus pneumoniae Strain D39.

Streptococcus pneumoniae is a notorious Gram-positive pathogen present asymptomatically in the nasophayrnx of humans. According to the World Health Organization (W.H.O), pneumococcus causes approximately one million deaths yearly. Antibiotic resistance in S. pneumoniae is raising considerable concern around the world. There is an immediate need to address the major issues that have arisen as a result of persistent infections caused by S. pneumoniae. In the present study, subtractive proteomics was used in which the entire proteome of the pathogen consisting of 1947 proteins is effectively decreased to a finite number of possible targets. Various kinds of bioinformatics tools and software were applied for the discovery of novel inhibitors. The CD-HIT analysis revealed 1887 non-redundant sequences from the entire proteome. These non-redundant proteins were submitted to the BLASTp against the human proteome and 1423 proteins were screened as non-homologous. Further, databases of essential genes (DEGG) and J browser identified almost 171 essential proteins. Moreover, non-homologous, essential proteins were subjected in KEGG Pathway Database which shortlisted six unique proteins. In addition, the subcellular localization of these unique proteins was checked and cytoplasmic proteins were chosen for the druggability analysis, which resulted in three proteins, namely DNA binding response regulator (SPD_1085), UDP-N-acetylmuramate-L-alanine Ligase (SPD_1349) and RNA polymerase sigma factor (SPD_0958), which can act as a promising potent drug candidate to limit the toxicity caused by S. pneumoniae. The 3D structures of these proteins were predicted by Swiss Model, utilizing the homology modeling approach. Later, molecular docking by PyRx software 0.8 version was used to screen a library of phytochemicals retrieved from PubChem and ZINC databases and already approved drugs from DrugBank database against novel druggable targets to check their binding affinity with receptor proteins. The top two molecules from each receptor protein were selected based on the binding affinity, RMSD value, and the highest conformation. Finally, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses were carried out by utilizing the SWISS ADME and Protox tools. This research supported the discovery of cost-effective drugs against S. pneumoniae. However, more in vivo/in vitro research should be conducted on these targets to investigate their pharmacological efficacy and their function as efficient inhibitors.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

Enhanced anti-cancer effects of oestrogen and progesterone co-therapy against colorectal cancer in males.

Although ovarian sex steroids could have protective roles against colorectal cancer (CRC) in women, little is currently known about their potential anti-tumorigenic effects in men. Hence, this study measured the therapeutic effects of 17ß-oestradiol (E2) and/or progesterone (P4) against azoxymethane-induced CRC in male mice that were divided into (n = 10 mice/group): negative (NC) and positive (PC) controls, E2 (580 µg/Kg/day; five times/week) and P4 (2.9 mg/Kg/day; five times/week) monotherapies, and concurrent (EP) and sequential (E/P) co-therapy groups. Both hormones were injected intraperitoneally to the designated groups for four consecutive weeks. Similar treatment protocols with E2 (10 nM) and/or P4 (20 nM) were also used in the SW480 and SW620 human male CRC cell lines. The PC group showed abundant colonic tumours alongside increased colonic tissue testosterone levels and androgen (AR) and oestrogen (ERα) receptors, whereas E2 and P4 levels with ERß and progesterone receptor (PGR) decreased significantly compared with the NC group. E2 and P4 monotherapies equally increased ERß/PGR with p21/Cytochrome-C/Caspase-3, reduced testosterone levels, inhibited ERα/AR and CCND1/survivin and promoted apoptosis relative to the PC group. Both co-therapy protocols also revealed better anti-cancer effects with enhanced modulation of colonic sex steroid hormones and their receptors, with E/P the most prominent protocol. In vitro, E/P regimen showed the highest increases in the numbers of SW480 (2.1-fold) and SW620 (3.5-fold) cells in Sub-G1 phase of cell cycle. The E/P co-therapy also disclosed the lowest percentages of viable SW480 cells (2.8-fold), whilst both co-therapy protocols equally showed the greatest SW620 apoptotic cell numbers (5.2-fold) relative to untreated cells. Moreover, both co-therapy regimens revealed maximal inhibitions of cell cycle inducers, cell survival markers, and AR/ERα alongside the highest expression of cell cycle suppressors, pro-apoptotic molecules, and ERß/PGR in both cell lines. In conclusion, CRC was associated with abnormal levels of colonic sex steroid hormones alongside aberrant protein expression of their receptors. While the anti-cancer effects of E2 and P4 monotherapies were equal, their combination protocols showed boosted tumoricidal actions against CRC in males, possibly by promoting ERß and PGR-mediated androgen deprivation together with inhibition of ERα-regulated oncogenic pathways.

Enhanced renoprotective actions of Paricalcitol and omega-3 fatty acids co-therapy against diabetic nephropathy in rat.

Introduction: Although the synthetic vitamin D analogue, Paricalcitol, and omega-3 Fatty acids (ω-3) alleviated diabetic nephropathy (DN), their combination was not previously explored. Objectives: This study measured the potential ameliorative effects of single and dual therapies of Paricalcitol and/or ω-3 against DN. Methods: Forty rats were assigned as follow: negative (NC) and positive (PC) controls, Paricalcitol, ω-3 and Paricalcitol + ω-3 groups. Diabetes was generated by high-fat/high-fructose diet and a single streptozotocin injection (40 mg/kg). DN was confirmed by raised fasting blood glucose (FBG), polyuria, proteinuria, and decreased urine creatinine levels. Paricalcitol intraperitoneal injections (0.25 µg/Kg/day; 5 times/week) and oral ω-3 (415 mg/kg/day; 5 times/week) started at week-9 and for eight weeks. Results: The PC group showed hyperglycaemia, dyslipidaemia, abnormal renal biochemical parameters, elevated caspase-3 expression, and increased apoptosis by TUNEL technique. The mRNAs and proteins of the pathogenic molecules (TGF-ß1/iNOS) and markers of tissue damage (NGAL/KIM-1) augmented substantially in the PC renal tissues relative to the NC group. The oxidative stress (MDA/H2O2/protein carbonyl groups) and pro-inflammatory (IL1ß/IL6/TNF-α) markers increased, whereas the anti-inflammatory (IL10) and anti-oxidative (GSH/GPx1/GR/SOD1/CAT) declined, in the PC renal tissues. The monotherapy groups were associated with ameliorated FBG, lipid profile and renal functions, and diminished TGF-ß1/iNOS/NGAL/KIM-1/Caspase-3 alongside the apoptotic index than the PC group. The oxidative stress and pro-inflammatory markers decreased, whilst the anti-oxidative and anti-inflammatory molecules escalated, in the monotherapy groups than the PC group. Although the Paricalcitol renoprotective actions were better than ω-3, all the biomarkers were abnormal than the NC group. Alternatively, the Paricalcitol + ω-3 protocol exhibited the best improvements in metabolic control, renal functions, oxidative stress, inflammation, and apoptosis. However, FBG and tissue damage were persistently higher in the co-therapy group than controls. Conclusions: Both monotherapies showed modest efficacy against DN, whereas their combination displayed boosted renoprotection, possibly by enhancing renal anti-oxidant and anti-inflammatory pathways.

Profiling Activins and Follistatin in Colorectal Cancer According to Clinical Stage, Tumour Sidedness and Smad4 Status.

This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded (n = 90 patients) alongside fresh (n = 40 patients) specimen cohorts. Activin ß-subunits, follistatin and Smad4 mRNAs and proteins were measured by real-time PCR and immunohistochemistry (IHC). Mature activin-A, -B, -AB and follistatin proteins were measured by ELISA. Cancer tissues having ≤ the 20th percentile of the Smad4 IHC score were considered as low (L-S4) group. The Smad4-intact SW480 and Smad4-null HT29 colon cancer cell lines were treated with activins and follistatin, and cell cycle was analysed by flow cytometry. The cell cycle inducing (CCND1/CCND3) and inhibitory (p21/p27) proteins alongside the survival (survivin/BCL2) and pro-apoptosis (Casp-8/Casp-3) markers were measured by immunofluorescence. Thirty-nine patients had right-sided cancers (30%) and showed higher rates of L-S4 tumours (n = 17; 13.1%) alongside worse clinicopathological characteristics relative to left-sided cancers. The ßA-subunit and activin-A increased, whilst ßB-subunit and activin-AB decreased, in malignant sites and the late-stage cancers revealed the greatest abnormalities. Interestingly, follistatin declined markedly in early-stage malignant tissues, whilst increased significantly in the advanced stages. All activin molecules were comparable between the early stage right- and left-sided tumours, whereas the late-stage right-sided cancers and L-S4 tumours showed more profound deregulations. In vitro, activin-A increased the numbers of the SW480 cells in sub-G1 and G0/G1-phases, whereas reduced the HT29 cell numbers in the sub-G1 phase with simultaneous increases in the G0/G1 and S phases. The p21/p27/Casp-8/Casp-3 proteins escalated, whilst CCND1/CCND3/BCL2/survivin declined in the SW480 cells following activin-A, whereas activin-A only promoted p21 and p27 alongside reduced CCND3 in the HT29 cells. By contrast, activin-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases and promoted the anti-cancer and reduced the oncogenic proteins in both cell lines. In conclusion, activins and follistatin displayed stage-dependent dysregulations and were markedly altered during the advanced stages of right-sided and L-S4 cancers. Moreover, the activin-A actions in CRC could be Smad4-dependent, whereas activin-AB may act as a Smad4-independent tumour suppressor protein.

Chemopreventive effects of vitamin D3 and its analogue, paricalcitol, in combination with 5-fluorouracil against colorectal cancer: The role of calcium signalling molecules.

BACKGROUND: Although vitamin D (VD) is chemoprotective and enhances 5-fluorouracil (5-FU) cytotoxicity against colorectal cancer (CRC), little is known about its potential calcium (Ca2+)-mediated anti-tumorigenic actions. Therefore, this study compared between VD and its non-calcaemic analogue, Paricalcitol (Pcal), ± 5-FU in relation to chemoprevention and Ca2+-mediated apoptosis in vivo and in vitro. METHODS: Seventy male mice were distributed to: negative controls, positive controls (PC), VD, Pcal, 5-FU, VD + 5-FU and Pcal+5-FU groups. All groups, except negative, received two consecutive azoxymethane (AOM)-injections (10 mg/Kg/week) for CRC induction. VD3 (1000 IU/kg; three times/week) and Pcal (1.25 µg/kg; three times/week) injections started week-16 post-AOM and for 10 weeks. Three successive 5-FU cycles began at week-21 (50 mg/Kg/week). Similar protocols with VD3, Pcal and/or 5-FU were applied in the HT29 colon cancer cells. RESULTS: The PC group had abundant malignant tumours, markedly elevated proliferation markers (survivin/CCND1) and declines in cyclin-dependent kinase-inhibitor-1A, pro-apoptotic molecules (p53/BAX/cytochrome_C/caspase-3), tissue Ca2+ concentrations and Ca2+-dependent proteins (CaSR/CAM/CAMKIIA). All monotherapies equally reduced tumour numbers and proliferation markers whilst promoting the anti-tumorigenic molecules. VD and/or 5-FU, but not Pcal monotherapy, enhanced Ca2+ levels and Ca2+-related molecules (CaSR/CAM/CAMKIIA/BAX/cytochrome_C) in vivo and in vitro. However, VD + 5-FU co-therapy showed the lowest tumour numbers, the highest cell numbers in sub-G1 phase of cell cycle, alongside the most effective modulations of oncogenes, tumour suppressors and Ca2+-related molecules at the gene and protein levels in vivo and in vitro. CONCLUSIONS: VD3 was superior than Paricalcitol in potentiating 5-FU cytotoxicity, possibly by upregulating several Ca2+-related molecules involved in tumour suppression.

Augmentation of DTH reaction of mycobacterial antigenic cocktail using synthetic mycobacterial 19-kDa lipoprotein as a TLR-stimulant.

The current study proposed that previously characterized individual antigenic proteins could represent potential replacement for conventional purified protein derivative (PPD) in tuberculosis skin testing when used in cocktails triggered by suitable TLR-stimulants that would provide the missing pro-inflammatory stimulus. Three different cocktails of previously selected antigens, including C1 (ESAT-6/CPF-10/MPB-83); C2 (ESAT-6/MPB-64/MPB-83); and C3 (CPF-10/MPB-64/MPB-83), were evaluated in vitro using lymphocytic proliferation and IFN-γ production assays, as well as mRNA and protein expression levels of TNF-α, IL-12p40, and IL-2 as pro-inflammatory molecules. C1 showed the highest significant induction of pro-inflammatory molecules as compared to other cocktails, yet still significantly lower than that induced by conventional PPD. Interestingly, inclusion of the synthetic Mycobacterium tuberculosis 19-kDa lipoprotein (Pam3Cys-SSNKSTTGSGETTTA) as a TLR-stimulant resulted in obvious augmentation of C1-induced pro-inflammatory molecules to levels comparable to that of PPD. In addition, skin testing using sensitized guinea pig model revealed comparable significant reaction to that of conventional PPD. ESAT-6/CPF-10/MPB-83 cocktail is suggested as a potential alternative skin-testing reagent when used in combination with the M. tuberculosis 19-kDa lipoprotein as a TLR-stimulant.

Pharmacotherapy with Thymoquinone Improved Pancreatic ß-Cell Integrity and Functional Activity, Enhanced Islets Revascularization, and Alleviated Metabolic and Hepato-Renal Disturbances in Streptozotocin-Induced Diabetes in Rats.

AIMS: This study is aimed at evaluating the antidiabetic effects of thymoquinone (TQ) on streptozotocin (STZ)-induced diabetes in rats, and exploring the possible underlying mechanisms. METHODS: Diabetes was induced in adult male Wistar rats by intraperitoneal injection of freshly prepared STZ (65 mg/kg). After disease induction, 42 rats were equally assigned to: controls, STZ-diabetic group, and STZ-diabetic group treated with oral TQ (35 mg/kg/day) for 5 weeks. Fasting blood glucose levels were determined weekly, and the animals were euthanized at day 38 post-STZ injection. Blood samples were assessed for glucose-insulin homeostasis parameters (plasma glucose, glycated hemoglobin, serum insulin, homeostatic model assessment of insulin resistance, and insulin sensitivity index) and lipid profile. Resected pancreases were subjected to histological examination and immunohistochemical or enzyme-linked immunosorbent assay assessment to determine the pancreatic expression of insulin sensitizing ß-cells, anti-apoptotic protein "survivin," apoptosis-inducer "caspase-3," prototypic angiogenic factors (vascular endothelial growth factor [VEGF] and endothelial cluster of differentiation 31 [CD31]), pro- and anti-inflammatory cytokines (interleukin-1beta [IL-1ß] and interleukin-10 [IL-10], respectively), thiobarbituric acid reactive substances (TBARS), total glutathione (GSH), and superoxide dismutase (SOD). The hepato-renal statuses were assessed biochemically and histologically. RESULTS: Therapy with TQ markedly improved the integrity of pancreatic islets, glucose-insulin homeostasis-related parameters, lipid profile parameters, and hepato-renal functional and histomorphological statuses that collectively were severely deteriorated in untreated diabetic group. Mechanistically, TQ therapy efficiently increased insulin producing ß-cells, upregulated survivin, VEGF, CD31, IL-10, GSH and SOD, and downregulated caspase-3, IL-1ß, and TBARSs in the pancreatic tissues of STZ-diabetic rats. CONCLUSIONS: These findings prove the anti-diabetic potential of TQ and its efficacy in regenerating pancreatic ß-cells and ameliorating pancreatic inflammation and oxidative stress, and highlight its novelty in repressing apoptosis of ß-cells and enhancing islet revascularization in STZ-diabetic rats. Further studies are required to support these findings and realize their possible clinical significance.

The role of IREB2 and transforming growth factor beta-1 genetic variants in COPD: a replication case-control study.

BACKGROUND: Genetic factors are known to contribute to COPD susceptibility and these factors are not fully understood. Conflicting results have been reported for many genetic studies of candidate genes based on their role in the disease. Genome-wide association studies in combination with expression profiling have identified a number of new candidates including IREB2. A meta-analysis has implicated transforming growth factor beta-1 (TGFbeta1) as a contributor to disease susceptibility. METHODS: We have examined previously reported associations in both genes in a collection of 1017 white COPD patients and 912 non-diseased smoking controls. Genotype information was obtained for seven SNPs in the IREB2 gene, and for four SNPs in the TGFbeta1 gene. Allele and genotype frequencies were compared between COPD cases and controls, and odds ratios were calculated. The analysis was adjusted for age, sex, smoking and centre, including interactions of age, sex and smoking with centre. RESULTS: Our data replicate the association of IREB2 SNPs in association with COPD for SNP rs2568494, rs2656069 and rs12593229 with respective adjusted p-values of 0.0018, 0.0039 and 0.0053. No significant associations were identified for TGFbeta1. CONCLUSIONS: These studies have therefore confirmed that the IREB2 locus is a contributor to COPD susceptibility and suggests a new pathway in COPD pathogenesis invoking iron homeostasis.