Metabolic Alteration Bridging the Prediabetic State and Colorectal Cancer.

Prediabetes and colorectal cancer (CRC) represent compelling health burdens responsible for high mortality and morbidity rates, sharing several modifiable risk factors. It has been hypothesized that metabolic abnormalities linking prediabetes and CRC are hyperglycemia, hyperinsulinemia, and adipokines imbalance. The chronic stimulation related to these metabolic signatures can favor CRC onset and development, as well as negatively influence CRC prognosis. To date, the growing burden of prediabetes and CRC has generated a global interest in defining their epidemiological and molecular relationships. Therefore, a deeper knowledge of the metabolic impairment determinants is compelling to identify the pathological mechanisms promoting the onset of prediabetes and CRC. In this scenario, this review aims to provide a comprehensive overview on the metabolic alterations of prediabetes and CRC as well as an overview of recent preventive and therapeutic approaches for both diseases, focusing on the role of the metabolic state as a pivotal contributor to consider for the development of future preventive and therapeutic strategies.

Microplastics and Nanoplastics in Atheromas and Cardiovascular Events.

BACKGROUND: Microplastics and nanoplastics (MNPs) are emerging as a potential risk factor for cardiovascular disease in preclinical studies. Direct evidence that this risk extends to humans is lacking. METHODS: We conducted a prospective, multicenter, observational study involving patients who were undergoing carotid endarterectomy for asymptomatic carotid artery disease. The excised carotid plaque specimens were analyzed for the presence of MNPs with the use of pyrolysis-gas chromatography-mass spectrometry, stable isotope analysis, and electron microscopy. Inflammatory biomarkers were assessed with enzyme-linked immunosorbent assay and immunohistochemical assay. The primary end point was a composite of myocardial infarction, stroke, or death from any cause among patients who had evidence of MNPs in plaque as compared with patients with plaque that showed no evidence of MNPs. RESULTS: A total of 304 patients were enrolled in the study, and 257 completed a mean (±SD) follow-up of 33.7±6.9 months. Polyethylene was detected in carotid artery plaque of 150 patients (58.4%), with a mean level of 21.7±24.5 µg per milligram of plaque; 31 patients (12.1%) also had measurable amounts of polyvinyl chloride, with a mean level of 5.2±2.4 µg per milligram of plaque. Electron microscopy revealed visible, jagged-edged foreign particles among plaque macrophages and scattered in the external debris. Radiographic examination showed that some of these particles included chlorine. Patients in whom MNPs were detected within the atheroma were at higher risk for a primary end-point event than those in whom these substances were not detected (hazard ratio, 4.53; 95% confidence interval, 2.00 to 10.27; P<0.001). CONCLUSIONS: In this study, patients with carotid artery plaque in which MNPs were detected had a higher risk of a composite of myocardial infarction, stroke, or death from any cause at 34 months of follow-up than those in whom MNPs were not detected. (Funded by Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale and others; ClinicalTrials.gov number, NCT05900947.).

Dietary Epigenetic Modulators: Unravelling the Still-Controversial Benefits of miRNAs in Nutrition and Disease.

In the context of nutrient-driven epigenetic alterations, food-derived miRNAs can be absorbed into the circulatory system and organs of recipients, especially humans, and potentially contribute to modulating health and diseases. Evidence suggests that food uptake, by carrying exogenous miRNAs (xenomiRNAs), regulates the individual miRNA profile, modifying the redox homeostasis and inflammatory conditions underlying pathological processes, such as type 2 diabetes mellitus, insulin resistance, metabolic syndrome, and cancer. The capacity of diet to control miRNA levels and the comprehension of the unique characteristics of dietary miRNAs in terms of gene expression regulation show important perspectives as a strategy to control disease susceptibility via epigenetic modifications and refine the clinical outcomes. However, the absorption, stability, availability, and epigenetic roles of dietary miRNAs are intriguing and currently the subject of intense debate; additionally, there is restricted knowledge of their physiological and potential side effects. Within this framework, we provided up-to-date and comprehensive knowledge on dietary miRNAs' potential, discussing the latest advances and controversial issues related to the role of miRNAs in human health and disease as modulators of chronic syndromes.

MiR-148a-3p Promotes Colorectal Cancer Cell Ferroptosis by Targeting SLC7A11.

Ferroptosis, an iron-dependent form of cell death, and dysregulated microRNA (miRNA) expression correlate with colorectal cancer (CRC) development and progression. The tumor suppressor ability of miR-148a-3p has been reported for several cancers. Nevertheless, the role of miR-148a-3p in CRC remains largely undetermined. Here, we aim at investigating the molecular mechanisms and regulatory targets of miR-148a-3p in the CRC cell death mechanism(s). To this end, miR-148a-3p expression was evaluated in SW480 and SW620 cells and normal colon epithelial CCD 841 CoN cells with quantitative real-time polymerase chain reaction (qRT-PCR). Data reported a reduction of miR-148a-3p expression in SW480 and SW620 cells compared to non-tumor cells (p < 0.05). Overexpression of miR-148a selectively inhibited CRC cell viability (p < 0.001), while weakly affecting normal CCD 841 CoN cell survival (p < 0.05). At the cellular level, miR-148a-3p mimics promoted apoptotic cell death via caspase-3 activation (p < 0.001), accumulation of mitochondrial reactive oxygen species (ROS) (p < 0.001), and membrane depolarization (p < 0.001). Moreover, miR-148a-3p overexpression induced lipid peroxidation (p < 0.01), GPX4 downregulation (p < 0.01), and ferroptosis (p < 0.01), as revealed by intracellular and mitochondrial iron accumulation and ACSL4/TFRC/Ferritin modulation. In addition, levels of SLC7A11 mRNA and protein, the cellular targets of miR-148a-3p predicted by bioinformatic tools, were suppressed by miR-148a-3p's overexpression. On the contrary, the downregulation of miR-148a-3p boosted SLC7A11 gene expression and suppressed ferroptosis. Together, these in vitro findings reveal that miR-148a-3p can function as a tumor suppressor in CRC by targeting SLC7A11 and activating ferroptosis, opening new perspectives for the rationale of therapeutic strategies through targeting the miR-148a-3p/SLC7A11 pathway.

Chemopreventive effect of a milk whey by-product derived from Buffalo (Bubalus bubalis) in protecting from colorectal carcinogenesis.

BACKGROUND: Several studies show that natural foods are a source of compounds with anticancer properties that affect the gut microbiota and its metabolites. In the present study, we investigate the effect of a delactosed buffalo milk whey by-product (DMW) on colorectal carcinogenesis. METHODS: The effect of DMW on colorectal carcinoma (CRC) was investigated in the established mouse model of azoxymethane (AOM)-induced colon carcinoma, which closely resembles the human clinical condition of CRC. The effect of DMW on CRC immortalized cell lines was also evaluated to further identify the antineoplastic mechanism of action. RESULTS: Pretreatment of AOM-treated mice with DMW significantly (P < 0.05) reduced the percentage of mice bearing both aberrant crypt foci with more than four crypts (which are early precancerous lesions that progress to CRC) and tumors. In addition, DMW completely counteracted the effect of AOM on protein expression of caspase-9, cleaved caspase-3 and poly ADP-ribose polymerase in colonic tissue. Administration of DMW alone (i.e. without AOM) resulted in changes in the composition of the gut microbiota, leading to enrichment or depletion of genera associated with health and disease, respectively. DMW was also able to restore AOM-induced changes in specific genera of the gut microbiota. Specifically, DMW reduced the genera Atopobiaceae, Ruminococcus 1 and Lachnospiraceae XPB1014 and increased the genera Parabacteroides and Candidatus Saccharimonas, which were increased and reduced, respectively, by AOM. Blood levels of butyric acid and cancer diagnostic markers (5-methylcytidine and glycerophosphocholine), which were increased by AOM treatment, were reduced by DMW. Furthermore, DMW exerted cytotoxic effects on two human CRC cell lines (HCT116 and HT29) and these effects were associated with the induction of apoptotic signaling. CONCLUSIONS: Our results suggest that DMW exerts chemopreventive effects and restores the gut microbiota in AOM-induced CRC, and induces cytotoxic effect on CRC cells. DMW could be an important dietary supplement to support a healthy gut microbiota and reduce the prevalence of CRC in humans. Video Abstract.

MiR-15b-5p and PCSK9 inhibition reduces lipopolysaccharide-induced endothelial dysfunction by targeting SIRT4.

BACKGROUND: Endothelial dysfunction and deregulated microRNAs (miRNAs) participate in the development of sepsis and are associated with septic organ failure and death. Here, we explored the role of miR-15b-5p on inflammatory pathways in lipopolysaccharide (LPS)-treated human endothelial cells, HUVEC and TeloHAEC. METHODS: The miR-15b-5p levels were evaluated in LPS-stimulated HUVEC and TeloHAEC cells by quantitative real-time PCR (qRT-PCR). Functional experiments using cell counting kit-8 (CCK-8), transfection with antagomir, and enzyme-linked immunosorbent assays (ELISA) were conducted, along with investigation of pyroptosis, apoptosis, autophagy, and mitochondrial reactive oxygen species (ROS) by cytofluorometric analysis and verified by fluorescence microscopy. Sirtuin 4 (SIRT4) levels were detected by ELISA and immunoblotting, while proprotein convertase subtilisin-kexin type 9 (PCSK9) expression was determined by flow cytometry (FACS) and immunofluorescence analyses. Dual-luciferase reporter evaluation was performed to confirm the miR-15b-5p-SIRT4 interaction. RESULTS: The results showed a correlation among miR-15b-5p, PCSK9, and SIRT4 levels in septic HUVEC and TeloHAEC. Inhibition of miR-15b-5p upregulated SIRT4 content, alleviated sepsis-related inflammatory pathways, attenuated mitochondrial stress, and prevented apoptosis, pyroptosis, and autophagic mechanisms. Finally, a PCSK9 inhibitor (i-PCSK9) was used to analyze the involvement of PCSK9 in septic endothelial injury. i-PCSK9 treatment increased SIRT4 protein levels, opposed the septic inflammatory cascade leading to pyroptosis and autophagy, and strengthened the protective role of miR-15b-5p inhibition. Increased luciferase signal validated the miR-15b-5p-SIRT4 binding. CONCLUSIONS: Our in vitro findings suggested the miR-15b-5p-SIRT4 axis as a suitable target for LPS-induced inflammatory pathways occurring in sepsis, and provide additional knowledge on the beneficial effect of i-PCSK9 in preventing vascular damage by targeting SIRT4.

Evidence of an anti-inflammatory effect of PCSK9 inhibitors within the human atherosclerotic plaque.

BACKGROUND AND AIMS: Preclinical evidence suggests that proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors hold anti-inflammatory properties independently of their ability to lower LDL-cholesterol (C). However, whether PCSK9 inhibitors exert anti-inflammatory effects within the atherosclerotic plaque in humans is unknown. We explored the impact of PCSK9 inhibitors, used as monotherapy, compared with other lipid-lowering drugs (oLLD) on the expression of inflammatory markers within the plaque, assessing also the subsequent incidence of cardiovascular events. METHODS: In an observational study, we recruited 645 patients on stable therapy for at least six months and undergoing carotid endarterectomy, categorizing patients according to the use of PCSK9 inhibitors only (n = 159) or oLLD (n = 486). We evaluated the expression of NLRP3, caspase-1, IL-1ß, TNFα, NF-kB, PCSK9, SIRT3, CD68, MMP-9, and collagen within the plaques in the two groups through immunohistochemistry, ELISA, or immunoblot. A composite outcome including non-fatal myocardial infarction, non-fatal stroke, and all-cause mortality was assessed during a 678 ± 120 days follow-up after the procedure. RESULTS: Patients treated with PCSK9 inhibitors had a lower expression of pro-inflammatory proteins and a higher abundance of SIRT3 and collagen within the plaque, a result obtained despite comparable levels of circulating hs-CRP and observed also in LDL-C-matched subgroups with LDL-C levels <100 mg/dL. Patients treated with PCSK9 inhibitors showed a decreased risk of developing the outcome compared with patients on oLLD, also after adjustment for multiple variables including LDL-C (adjusted hazard ratio 0.262; 95% CI 0.131-0.524; p < 0.001). The expression of PCSK9 correlated positively with that of pro-inflammatory proteins, which burden was associated with a higher risk of developing the outcome, independently of the therapeutic regimen. CONCLUSIONS: The use of PCSK9 inhibitors is accompanied by a beneficial remodelling of the inflammatory burden within the human atheroma, an effect possibly or partly independent of their LDL-C lowering ability. This phenomenon might provide an additional cardiovascular benefit.

Whey Improves In Vitro Endothelial Mitochondrial Function and Metabolic Redox Status in Diabetic State.

Endothelial dysfunction plays a critical role in the progression of type 2 diabetes mellitus (T2DM), leading to cardiovascular complications. Current preventive antioxidant strategies to reduce oxidative stress and improve mitochondrial function in T2DM highlight dietary interventions as a promising approach, stimulating the deepening of knowledge of food sources rich in bioactive components. Whey (WH), a dairy by-product with a considerable content of bioactive compounds (betaines and acylcarnitines), modulates cancer cell metabolism by acting on mitochondrial energy metabolism. Here, we aimed at covering the lack of knowledge on the possible effect of WH on the mitochondrial function in T2DM. The results showed that WH improved human endothelial cell (TeloHAEC) function during the in vitro diabetic condition mimicked by treating cells with palmitic acid (PA) (0.1 mM) and high glucose (HG) (30 mM). Of note, WH protected endothelial cells from PA+HG-induced cytotoxicity (p < 0.01) and prevented cell cycle arrest, apoptotic cell death, redox imbalance, and metabolic alteration (p < 0.01). Moreover, WH counteracted mitochondrial injury and restored SIRT3 levels (p < 0.01). The SiRNA-mediated suppression of SIRT3 abolished the protective effects exerted by WH on the mitochondrial and metabolic impairment caused by PA+HG. These in vitro results reveal the efficacy of whey as a redox and metabolic modulator in the diabetic state and pave the way for future studies to consider whey as the source of dietary bioactive molecules with health benefits in preventive strategies against chronic diseases.

MicroRNA-nanoparticles against cancer: Opportunities and challenges for personalized medicine.

Micro-RNAs (miRNAs) control gene expression at the post-transcriptional level and are widely involved in carcinogenesis, playing a role as both oncogenes and tumor suppressors. MiRNAs act as potent therapeutic weapon in cancer, but their potential therapeutic use is limited by the off-target effect due to their nonspecific distribution in normal tissues. The encapsulation of miRNAs in nanostructured carriers allows targeted effects aimed to destroy cancer cells, without affecting healthy tissues. Due to their small size and the optimal surface/size ratio, nanoparticles (NPs) envelop, protect, and release miRNAs, representing a promising strategy in cancer treatment. In the present review, we discuss the latest advances in the field of miRNA-encapsulating NPs in cancer, focusing on colorectal cancer and its metastatic forms, one of the most common malignancies worldwide.

Use of former food products in dairy buffalo nutrition: In vitro and in vivo evaluation.

A feeding strategy that maintains high content of functional molecules in buffalo milk has been verified by giving Sorghum vulgare as green fodder, but it is not available all year round. The aim of this study was to evaluate the inclusion of former food products (FFPs) containing 87% biscuit meal (nonstructural carbohydrate: 60.1%; starch 14.7; crude protein 10.6), in the diet of buffaloes in terms of: (a) fermentation characteristics through gas production technique; (b) milk yield (MY) and quality; (c) content of some biomolecules and total antioxidant activity. The experiment was performed involving 50 buffaloes divided into two groups: Green group and FFPs group (animals fed Total Mixed Ration with either green forage or FFPs respectively). Daily MY was recorded and milk qualitative analyses were determined monthly for 90 days. Furthermore, fermentation characteristics of the diets were studied in vitro. No significant differences were recorded in feed intake, BCS and MY and quality. Similar in vitro fermentation data of two diets were found, with slight differences in terms of gas production and degradability. During the incubation, kinetic parameters showed a faster fermentation process with the diet of the FFPs group in relation to Green group (p < 0.05). Green group had higher levels (p < 0.01) of γ-butyrobetaine, glycine betaine, l-carnitine and propionyl l-carnitine in milk, whereas no differences were observed for δ-valerobetaine and acetyl l-carnitine. Total antioxidant capacity and iron reduction antioxidant assay were higher (p < 0.05) in the plasma and milk of the Green group. The administration of a diet high in simple sugars, obtained with FFPs, seems to favour the ruminal biosynthesis of some metabolites in milk, such as δ-valerobetaine and acetyl- l-carnitine, similar to green forage administration. Overall, the use of biscuit meal can be an alternative to green fodder when it is not available to ensure environmental sustainability and optimize costs without compromising milk quality.

MiR-27b attenuates mitochondrial oxidative stress and inflammation in endothelial cells.

MiR-27b is highly expressed in endothelial cells (EC) but its function in this context is poorly characterized. This study aims to investigate the effect of miR-27b on inflammatory pathways, cell cycle, apoptosis, and mitochondrial oxidative imbalances in immortalized human aortic endothelial cells (teloHAEC), human umbilical vein endothelial cells (HUVEC), and human coronary artery endothelial cells (HCAEC) exposed to TNF-α. Treatment with TNF-α downregulates the expression of miR-27b in all EC lines, promotes the activation of inflammatory pathways, induces mitochondrial alteration and reactive oxygen species accumulation, fostering the induction of intrinsic apoptosis. Moreover, miR-27b mimic counteracts the TNF-α-related cytotoxicity and inflammation, as well as cell cycle arrest and caspase-3-dependent apoptosis, restoring mitochondria redox state, function, and membrane polarization. Mechanistically, hsa-miR-27b-3p targets the 3'untranslated regions of FOXO1 mRNA to downregulate its expression, blunting the activation of the Akt/FOXO1 pathway. Here, we show that miR-27b is involved in the regulation of a broad range of functionally intertwined phenomena in EC, suggesting its key role in mitigating mithochondrial oxidative stress and inflammation, most likely through targeting of FOXO1. Overall, results reveal for the first time that miR-27b could represent a possible target for future therapies aimed at improving endothelial health.

Transcriptomic profiles of the ruminal wall in Italian Mediterranean dairy buffaloes fed green forage.

BACKGROUND: Green feed diet in ruminants exerts a beneficial effect on rumen metabolism and enhances the content of milk nutraceutical quality. At present, a comprehensive analysis focused on the identification of genes, and therefore, biological processes modulated by the green feed in buffalo rumen has never been reported. We performed RNA-sequencing in the rumen of buffaloes fed a total mixed ration (TMR) + the inclusion of 30% of ryegrass green feed (treated) or TMR (control), and identified differentially expressed genes (DEGs) using EdgeR and NOISeq tools. RESULTS: We found 155 DEGs using EdgeR (p-values < 0.05) and 61 DEGs using NOISeq (prob ≥0.8), 30 of which are shared. The rt-qPCR validation suggested a higher reliability of EdgeR results as compared with NOISeq data, in our biological context. Gene Ontology analysis of DEGs identified using EdgeR revealed that green feed modulates biological processes relevant for the rumen physiology and, then, health and well-being of buffaloes, such as lipid metabolism, response to the oxidative stress, immune response, and muscle structure and function. Accordingly, we found: (i) up-regulation of HSD17B13, LOC102410803 (or PSAT1) and HYKK, and down-regulation of CDO1, SELENBP1 and PEMT, encoding factors involved in energy, lipid and amino acid metabolism; (ii) enhanced expression of SIM2 and TRIM14, whose products are implicated in the immune response and defense against infections, and reduced expression of LOC112585166 (or SAAL1), ROR2, SMOC2, and S100A11, encoding pro-inflammatory factors; (iii) up-regulation of NUDT18, DNAJA4 and HSF4, whose products counteract stressful conditions, and down-regulation of LOC102396388 (or UGT1A9) and LOC102413340 (or MRP4/ABCC4), encoding detoxifying factors; (iv) increased expression of KCNK10, CACNG4, and ATP2B4, encoding proteins modulating Ca2+ homeostasis, and reduced expression of the cytoskeleton-related MYH11 and DES. CONCLUSION: Although statistically unpowered, this study suggests that green feed modulates the expression of genes involved in biological processes relevant for rumen functionality and physiology, and thus, for welfare and quality production in Italian Mediterranean dairy buffaloes. These findings, that need to be further confirmed through the validation of additional DEGs, allow to speculate a role of green feed in the production of nutraceutical molecules, whose levels might be enhanced also in milk.

SGLT-2 inhibitors and in-stent restenosis-related events after acute myocardial infarction: an observational study in patients with type 2 diabetes.

BACKGROUND: No study evaluated the incidence of intra-stent restenosis (ISR)-related events in patients with type 2 diabetes (T2DM) and acute myocardial infarction (AMI) treated or not with sodium/glucose cotransporter 2 inhibitors (SGLT2i). METHODS: We recruited 377 patients with T2DM and AMI undergoing percutaneous coronary intervention (PCI). Among them, 177 T2DM were treated with SGLT2 inhibitors before PCI. The primary outcome was major adverse cardiovascular events (MACE) defined as cardiac death, re-infarction, and heart failure related to ISR. In patients without ISR, minimal lumen area and minimal lumen diameter were assessed by coronary CT-angiography at 1-year follow-up. RESULTS: Glycemic control was similar in SGLT2i-treated patients and never SGLT2i-users. The incidence of ISR-related MACE was higher in never SGLT2i-users compared with SGLT2i-treated patients, an effect independent of glycemic status (HR = 0.418, 95% CI = 0.241-0.725, P = 0.002) and observed also in the subgroup of patients with HbA1c < 7% (HR = 0.393, 95% CI = 0.157-0.984, P = 0.027). In patients without the event, the stent patency was greater in SGLT2i-treated patients compared with never SGLT2i-users at 1-year follow-up. CONCLUSIONS: SGLT2i treatment in T2DM is associated with a reduced incidence of ISR-related events, independently of glycemic control.

SIRT3 mediates the effects of PCSK9 inhibitors on inflammation, autophagy, and oxidative stress in endothelial cells.

Background: Proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors (i) are a class of lipid-lowering drugs suggested to hold a plethora of beneficial effects independent of their LDL cholesterol-lowering properties. However, the mechanism underlying such observations is debated. Methods: Human aortic endothelial cells (TeloHAEC) were pre-treated with 100 µg/mL of the PCSK9i evolocumab and then exposed to 20 ng/mL of IL-6, a major driver of cardiovascular diseases (CVD), in both naïve state and after siRNA-mediated suppression of the NAD-dependent deacetylase sirtuin-3 (SIRT3). Inflammation, autophagy, and oxidative stress were assessed through Western Blots, ELISAs, and/or immunofluorescence coupled by flow cytometry. To explore the human relevance of the findings, we also evaluated the expression of IL-6, SIRT3, IL-1ß, the ratio LC3B II/I, and PCSK9 within the plaques of patients undergoing carotid endarterectomy (n=277), testing possible correlations between these proteins. Results: PCSK9i improved a range of phenotypes including the activation of inflammatory pathways, oxidative stress, and autophagy. Indeed, treatment with PCSK9i was able to counteract the IL-6 induced increase in inflammasome activation, the accrual of autophagic cells, and mitochondrial ROS accumulation. Of note, silencing of SIRT3 reverted the beneficial effects observed with PCSK9i treatment on all these phenomena. In atheroma specimens, the expression of PCSK9 was inversely related to that of SIRT3 while positively correlating with IL-6, IL-1ß, and the ratio LC3B II/I. Conclusions: Overall, these data suggest that PCSK9i bear intrinsic anti-inflammatory, anti-autophagic, and antioxidant properties in endothelial cells, and that these pleiotropic effects might be mediated, at least in part, by SIRT3. These results provide an additional mechanism supporting the emerging knowledge relative to the benefit of PCSK9i on CVD beyond LDL-lowering and uncover SIRT3 as a putative mediator of such pleiotropy.

Milk Exosomal miR-27b Worsen Endoplasmic Reticulum Stress Mediated Colorectal Cancer Cell Death.

The relationship between dietary constituents and the onset and prevention of colorectal cancer (CRC) is constantly growing. Recently, the antineoplastic profiles of milk and whey from Mediterranean buffalo (Bubalus bubalis) have been brought to attention. However, to date, compared to cow milk, the potential health benefits of buffalo milk exosome-miRNA are still little explored. In the present study, we profiled the exosomal miRNA from buffalo milk and investigated the possible anticancer effects in CRC cells, HCT116, and HT-29. Results indicated that buffalo milk exosomes contained higher levels of miR-27b, miR-15b, and miR-148a compared to cow milk. Mimic miR-27b transfection in CRC cells induced higher cytotoxic effects (p < 0.01) compared to miR-15b and miR-148a. Moreover, miR-27b overexpression in HCT116 and HT-29 cells (miR-27b+) induced apoptosis, mitochondrial reactive oxygen species (ROS), and lysosome accumulation. Exposure of miR-27b+ cells to the bioactive 3kDa milk extract aggravated the apoptosis rate (p < 0.01), mitochondrial stress (p < 0.01), and advanced endoplasmic reticulum (ER) stress (p < 0.01), via PERK/IRE1/XBP1 and CHOP protein modulation (p < 0.01). Moreover, GSK2606414, the ER-inhibitor (ER-i), decreased the apoptosis phenomenon and XBP1 and CHOP modulation in miR-27b+ cells treated with milk (p < 0.01 vs. miR-27b++Milk), suggesting the ER stress as a cell-death-aggravating mechanism. These results support the in vitro anticancer activity of 3kDa milk extract and unveil the contribution of miR-27b in the promising beneficial effect of buffalo milk in CRC prevention.

Breed and Feeding System Impact the Bioactive Anti-Inflammatory Properties of Bovine Milk.

In the present study, we aimed at assessing the influence of breed and feeding system on the bovine milk profile of betaines and carnitines and milk capacity in counteracting the inflammatory endothelial cell (EC) damage induced by interleukin (IL)-6. In the first experimental design, two breeds were chosen (Holstein vs. Modicana) to investigate the biomolecule content and antioxidant capacity in milk and dairy products. In the second experimental design, two feeding systems (pasture vs. total mixed ratio) were tested only in Holstein to evaluate the possible effect on the functional profile of milk and dairy products. Finally, the bulk milk from the two experimental designs was used to evaluate the efficacy of preventing IL-6-induced endothelial inflammatory damage. Results showed that Modicana milk and whey had higher biomolecule content and antioxidant activity compared to Holstein milk (p < 0.01). Milk from Holstein fed TMR showed higher concentration of γ-butyrobetaine, δ-valerobetaine (p < 0.01), and l-carnitine (p < 0.05). Similarly, whey from Holstein fed TMR also showed higher content of δ-valerobetaine, glycine betaine, l-carnitine, and acetyl-l-carnitine (p < 0.01) compared to the Holstein fed pasture. Conversely, the antioxidant activity of milk and dairy products was not affected by the feeding system. In ECs, all milk samples reduced the IL-6-induced cytokine release, as well as the accumulation of reactive oxygen species (ROS) and the induction of cell death, with the most robust effect elicited by Modicana milk (p < 0.01). Overall, Modicana milk showed a higher content of biomolecules and antioxidant activity compared to Holstein, suggesting that the breed, more than the feeding system, can positively affect the health-promoting profile of dairy cattle milk.

Sodium-glucose cotransporter-2 (SGLT2) expression in diabetic and non-diabetic failing human cardiomyocytes.

This study aimed at investigating the SGLT2 expression in human cardiomyocytes. Human studies evaluating cardiomyocyte SGLT2s expression are limited. To better clarify this issue, SGLT2 protein expression was assessed in human hearts of diabetic and non-diabetic patients, and in AC16 human cardiomyocyte cell line. A prospective study with a follow-up of patients who underwent their first heart transplant (HTX) was performed. Explanted heart, basal (1 week after HTX), and final (48 weeks after HTX) endomyocardial biopsies (EMBs) from patients were evaluated for SGLT2 occurrence in cardiomyocyte with immunohistochemistry, immunofluorescence and SGLT2 quantization with both real-time reverse transcription-polymerase chain reaction and Western blot analysis. The immunofluorescence co-localization of SGLT2 in cardiomyocyte evidenced that an increased expression in the explanted heart from diabetic patients compared to non-diabetic (p < 0.001). In all final EMBs from diabetic patients, the expression of SGLT2 in cardiomyocyte was increased compared to non-diabetic (p < 0.01). This evidence was confirmed by Western blot analysis of SGLT2 protein. In addition, PCR analysis revealed very low mRNA levels in basal EMBs from diabetic and non-diabetic patients (p = NS), whereas final EMBs from diabetic patients showed higher SGLT2 mRNA levels in diabetic compared to non-diabetic patients (p < 0.05). Cultured human cardiomyocytes exposed to high-glucose showed increased expression of SGLT2 protein compared to cells exposed to normal glucose (p < 0.05). The presence of SGLT2 in cardiomyocytes supports the hypothesis of SGLT2i-mediated impact on metabolic pathways within cardiomyocytes. Moreover, metabolic disorders linked to diabetes may lead promptly to upregulation of SGLT2 levels in human cardiomyocytes.

SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance.

Emerging evidence indicates that defects in sirtuin signaling contribute to impaired glucose and lipid metabolism, resulting in insulin resistance (IR) and endothelial dysfunction. Here, we examined the effects of palmitic acid (PA) treatment on mitochondrial sirtuins (SIRT2, SIRT3, SIRT4, and SIRT5) and oxidative homeostasis in human endothelial cells (TeloHAEC). Results showed that treatment for 48 h with PA (0.5 mM) impaired cell viability, induced loss of insulin signaling, imbalanced the oxidative status (p < 0.001), and caused negative modulation of sirtuin protein and mRNA expression, with a predominant effect on SIRT3 (p < 0.001). Restoration of SIRT3 levels by mimic transfection (SIRT3+) suppressed the PA-induced autophagy (mimic NC+PA) (p < 0.01), inflammation, and pyroptosis (p < 0.01) mediated by the NLRP3/caspase-1 axis. Moreover, the unbalanced endothelial redox state induced by PA was counteracted by the antioxidant δ-valerobetaine (δVB), which was able to upregulate protein and mRNA expression of sirtuins, reduce reactive oxygen species (ROS) accumulation, and decrease cell death. Overall, results support the central role of SIRT3 in maintaining the endothelial redox homeostasis under IR and unveil the potential of the antioxidant δVB in enhancing the defense against IR-related injuries.

Buffalo Milk Whey Activates Necroptosis and Apoptosis in a Xenograft Model of Colorectal Cancer.

Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo (Bubalus bubalis) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC.