Structure elucidation and antiviral activity of a cold water-extracted mannogalactofucan Ts1-1A from Trametes sanguinea against human cytomegalovirus in vitro.

In this study, we purified a partially acetylated heteropolysaccharide (Ts1-1A) from the fruit bodies of Trametes sanguinea Lloyd through cold water extraction and serial chromatographic separation. The purified polysaccharide Ts1-1A (12.8 kDa) was characterized as a branched mannogalactofucan with a backbone of alternately connected 1,3-linked α-Fucp and 1,6-linked α-Galp, which was partially substituted by non-reducing end units of ß-Manp at O-2 and O-3 positions of 1,6-linked α-Galp. Ts1-1A showed pronounced anti-human cytomegalovirus activity at the concentration of 200 and 500 µg/mL in systematical assessments including morphological changes, western blotting, qPCR, indirect immunofluorescence and tissue culture infective dose assays. Moreover, Ts1-1A exerted its antiviral activity at two distinct stages of viral proliferation manifesting as significantly inhibiting viral protein (IE1/2 and p52) expression and reducing viral gene (UL123, UL44 and UL32) replication in the HCMV-infected WI-38 cells. At viral attachment stage, Ts1-1A interacted with HCMV and prevented HCMV from attaching to its host cells. While at early phase of viral replication stage, Ts1-1A suppressed HCMV replication by downregulating NQO1 and HO-1 proteins related to oxidative stress as an antioxidant. To sum up, Ts1-1A is a promising anti-HCMV agent which could be developed for HCMV infection prevention and therapy.

Renal macrophages monitor and remove particles from urine to prevent tubule obstruction.

When the filtrate of the glomerulus flows through the renal tubular system, various microscopic sediment particles, including mineral crystals, are generated. Dislodging these particles is critical to ensuring the free flow of filtrate, whereas failure to remove them will result in kidney stone formation and obstruction. However, the underlying mechanism for the clearance is unclear. Here, using high-resolution microscopy, we found that the juxtatubular macrophages in the renal medulla constitutively formed transepithelial protrusions and "sampled" urine contents. They efficiently sequestered and phagocytosed intraluminal sediment particles and occasionally transmigrated to the tubule lumen to escort the excretion of urine particles. Mice with decreased renal macrophage numbers were prone to developing various intratubular sediments, including kidney stones. Mechanistically, the transepithelial behaviors of medulla macrophages required integrin ß1-mediated ligation to the tubular epithelium. These findings indicate that medulla macrophages sample urine content and remove intratubular particles to keep the tubular system unobstructed.

4-Octyl itaconate inhibits poly(I:C)-induced interferon-ß secretion in mouse bone marrow-derived macrophages partially by activating Nrf2.

Viruses have become a major threat to human health. Interferon-ß (IFN-ß) has a key role in the antivirus process, as it can increase the expression of antivirus-associated genes. Itaconate and its derivatives can regulate the immune response, secretion of inflammatory factors, and pyroptosis of macrophages. The effect of itaconate on IFN-ß secretion of double-stranded RNA-induced macrophages are not well known. A derivative of itaconate, 4-octoyl itaconate (4-OI), was used to treat mouse bone marrow-derived macrophages (BMDM) induced with 100 µg/mL poly(I:C). The IFN-ß concentration was detected through ELISA, and IFN-ß mRNA expression was detected through quantitative PCR. High-throughput transcriptome sequencing was used to analyze changes in the BMDM transcriptome after 4-OI treatment. The Nrf2 expression was knocked down with siRNA.4-OI inhibited poly(I:C)-induced IFN-ß secretion and mRNA expression in BMDM. Results of transcriptome sequencing revealed that 4-OI downregulated 1047 genes and upregulated 822 genes. GO and KEGG enrichment of differently expressed genes revealed that many downregulated genes were related to the anti-virus process, whereas many upregulated genes were related to metabolism. The Nrf2 inhibitor ML385 and Nrf2 siRNA could partially reverse the inhibitory effect of 4-OI. In conclusion, 4-octyl itaconate could inhibit the poly(I:C)-induced interferon-ß secretion in BMDM partially by regulating Nrf2.

DNA damage response(DDR): a link between cellular senescence and human cytomegalovirus.

The DNA damage response (DDR) is a signaling cascade that is triggered by DNA damage, involving the halting of cell cycle progression and repair. It is a key event leading to senescence, which is characterized by irreversible cell cycle arrest and the senescence-associated secretory phenotype (SASP) that includes the expression of inflammatory cytokines. Human cytomegalovirus (HCMV) is a ubiquitous pathogen that plays an important role in the senescence process. It has been established that DDR is necessary for HCMV to replicate effectively. This paper reviews the relationship between DDR, cellular senescence, and HCMV, providing new sights for virus-induced senescence (VIS).

Impact of dietary plant flavonoids on 7,8-dihydroxyflavone transepithelial transport in human intestinal Caco-2 cells.

7,8-dihydroxyflavone (7,8-DHF) is a biologically active flavone with various physiological activities, including neuroprotection, anti-inflammation, and weight loss. Previous studies have found that the efflux protein P-glycoprotein (P-gp) significantly affects the transepithelial transport of 7,8-DHF in the intestine, resulting in its low oral bioavailability. Based on this, in this study, a Caco-2 monolayer cell model was used to investigate 14 dietary plant flavonoids as potential P-gp inhibitors, and their effects on the transepithelial transport and in vitro digestion of 7,8-DHF were explored. The results showed that among the 14 plant flavonoids, hesperetin, epigallocatechin gallate, fisetin, kaempferol, quercetin, and isoorientin increased and the apparent permeability coefficients (P app) of 7,8-DHF at AP → BL direction and lowered P app value at BL → AP direction to varying degrees, reducing the efflux ratio of 7,8-DHF less than 1.5. In particular, kaempferol and quercetin exhibited the best effect on promoting the transepithelial transport of 7,8-DHF, especially when used at molar concentration ratios of 1:1 and 1:2 with 7,8-DHF. This is beneficial for improving the oral bioavailability of 7,8-DHF. Meanwhile, 7,8-DHF was found to maintain structural stability in simulated saliva, gastric juice, and intestinal juice, and its stability was not affected by the coexistence of quercetin and kaempferol. Overall, this study provided a theoretical basis for seeking natural and safe P-gp inhibitors to improve the oral absorption of natural products.

Inhibition of mitochondrial calcium uptake by Ru360 enhances the effect of 1800 MHz radio-frequency electromagnetic fields on DNA damage.

Today, the existence of radio-frequency electromagnetic fields (RF-EMF) emitted from cell phones, wireless routers, base stations, and other sources are everywhere around our living environment, and the dose is increasing. RF-EMF have been reported to be cytotoxic and supposed to be a risk factor for various human diseases, thus, more attention is necessary. In recent years, interfere with mitochondrial calcium uptake by using mitochondrial calcium uniporter (MCU) inhibitor were suggested to be potential clinical treatment in mitochondrial calcium overload diseases, like neurodegeneration, ischemia/reperfusion injury, and cancer, but whether this approach increases the health risk of RF-EMF exposure are unknown. To address our concern, we did a preliminary study to determine whether inhibition of MCU will increase the genotoxicity of RF-EMF exposure in cells, and found that short-time (15 min) exposure to 1800 MHz RF-EMF induced significant DNA damage and cell apoptosis in mouse embryonic fibroblasts (MEFs) treated with Ruthenium 360 (Ru360), a specific inhibitor of MCU, but no significant effects on cell cycle, cell proliferation, or cell viability were observed. In conclusion, our results indicated that inhibiting MCU increases the genotoxicity of RF-EMF exposure, and more attention needs to be paid to the possible health impact of RF-EMF exposure under these treatments.

Protective effect of mussel polysaccharide on cyclophosphamide-induced intestinal oxidative stress injury via Nrf2-Keap1 signaling pathway.

The hard-shelled mussel (Mytilus coruscus) has been used as a traditional Chinese medicine and health food in China for centuries. Polysaccharides from mussel has been reported to have multiple biological functions, however, it remains unclear whether mussel polysaccharide (MP) exerts protective effects in intestinal functions, and the underlying mechanisms of action remain unclear. The aim of this study was to investigate the protective effects and mechanism of MP on intestinal oxidative injury in mice. In this study, 40 male BALB/C mice were used, with 30 utilized to produce an animal model of intestinal oxidative injury with intraperitoneal injection of cyclophosphamide (Cy) for four consecutive days. The protective effects of two different doses of MP (300 and 600 mg/kg) were assessed by investigating the change in body weight, visceral index, and observing colon histomorphology. Moreover, the underlying molecular mechanisms were investigated by measuring the antioxidant enzymes and related signaling molecules through ELISA, real-time PCR, and western blot methods. The results showed that MP pretreatment effectively protected the intestinal from Cy-induced injury: improved the colon tissue morphology and villus structure, increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, and reduced malondialdehyde (MDA) content in serum and colon tissues. Meanwhile, MP also significantly increased the expression levels of SOD, GSH-Px, heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) mRNA in colon tissues. Further, western blot results showed that the expression of Nrf2 protein was significantly upregulated while kelch-like ECH-associated protein 1 (Keap1) was significantly downregulated by MP in the colonic tissues. This study indicates that MP can ameliorate Cy-induced oxidative stress injury in mice, and Nrf2-Keap1 signaling pathway may mediate these protective effects.

Dietary flavonoids intake contributes to delay biological aging process: analysis from NHANES dataset.

BACKGROUND: Diet may influence biological aging and the discrepancy (∆age) between a subject's biological age (BA) and chronological age (CA). We aimed to investigate the correlation of dietary flavonoids with the ∆age of organs (heart, kidney, liver) and the whole body. METHOD: A total of 3193 United States adults were extracted from the National Health and Nutrition Examination Survey (NHANES) in 2007-2008 and 2017-2018. Dietary flavonoids intake was assessed using 24-h dietary recall method. Multiple linear regression analysis was performed to evaluate the association of dietary flavonoids intake with the ∆age of organs (heart, kidney, liver) and the whole body. BA was computed based on circulating biomarkers, and the resulting ∆age was tested as an outcome in linear regression analysis. RESULTS: The ∆age of the whole body, heart, and liver was inversely associated with higher flavonoids intake (the whole body ∆age ß = - 0.58, cardiovascular ∆age ß = - 0.96, liver ∆age ß = - 3.19) after adjustment for variables. However, higher flavonoids intake positively related to renal ∆age (ß = 0.40) in participants with chronic kidney disease (CKD). Associations were influenced by population characteristics, such as age, health behavior, or chronic diseases. Anthocyanidins, isoflavones and flavones had the strongest inverse associations between the whole body ∆age and cardiovascular ∆age among all the flavonoids subclasses. CONCLUSION: Flavonoids intake positively contributes to delaying the biological aging process, especially in the heart, and liver organ, which may be beneficial for reducing the long-term risk of cardiovascular or liver disease.

The association between dietary intake of flavonoids and its subclasses and the risk of metabolic syndrome.

Background: The healthiest way to prevent metabolic syndrome (MetS) is through behavioral and nutritional adjustments. We examined the relationship between total flavonoids intake, flavonoid subclasses, and clinically manifest MetS. Methods: A cross-sectional analysis was conducted among 28,719 individuals from the National Health and Nutrition Examination Survey (NHANES) and Food and Nutrient Database for Dietary Studies (FNDDS) 2007-2011 and 2017-2018. Two 24-h reviews were conducted to determine flavonoids intake and subclasses. The link between flavonoids intake and MetS was investigated using a multivariate logistic regression model. Results: Q2 and Q3 of total flavonoids intake were associated with 20 and 19% lower risk of incident MetS after adjusting age and sex. Anthocyanidins and flavanones intake in Q2 and Q3 substantially reduced the MetS risk compared to Q1. MetS risk decreased steadily as the total intake of flavonoids increased to 237.67 mg/d. Flavanones and anthocyanidins also displayed V-shaped relationship curves (34.37 and 23.13 mg/d). Conclusion: MetS was adversely linked with total flavonoids intake, flavanones, and anthocyanidins. Moreover, the most effective doses of total flavonoids, flavanones, and anthocyanidins were 237.67, 34.37, and 23.13 mg/d, respectively, potentially preventing MetS.

High FAAP24 expression reveals poor prognosis and an immunosuppressive microenvironment shaping in AML.

BACKGROUND: As a core member of the FA complex, in the Fanconi anemia pathway, FAAP24 plays an important role in DNA damage repair. However, the association between FAAP24 and patient prognosis in AML and immune infiltration remains unclear. The purpose of this study was to explore its expression characteristics, immune infiltration pattern, prognostic value and biological function using TCGA-AML and to verify it in the Beat AML cohort. METHODS: In this study, we examined the expression and prognostic value of FAAP24 across cancers using data from TCGA, TARGET, GTEx, and GEPIA2. To further investigate the prognosis in AML, development and validation of a nomogram containing FAAP24 were performed. GO/KEGG, ssGSEA, GSVA and xCell were utilized to explore the functional enrichment and immunological features of FAAP24 in AML. Drug sensitivity analysis used data from the CellMiner website, and the results were confirmed in vitro. RESULTS: Integrated analysis of the TCGA, TARGET and GTEx databases showed that FAAP24 is upregulated in AML; meanwhile, high FAAP24 expression was associated with poor prognosis according to GEPIA2. Gene set enrichment analysis revealed that FAAP24 is implicated in pathways involved in DNA damage repair, the cell cycle and cancer. Components of the immune microenvironment using xCell indicate that FAAP24 shapes an immunosuppressive tumor microenvironment (TME) in AML, which helps to promote AML progression. Drug sensitivity analysis showed a significant correlation between high FAAP24 expression and chelerythrine resistance. In conclusion, FAAP24 could serve as a novel prognostic biomarker and play an immunomodulatory role in AML. CONCLUSIONS: In summary, FAAP24 is a promising prognostic biomarker in AML that requires further exploration and confirmation.

Artificial intelligence predicts lung cancer radiotherapy response: A meta-analysis.

BACKGROUND: Artificial intelligence (AI) technology has clustered patients based on clinical features into sub-clusters to stratify high-risk and low-risk groups to predict outcomes in lung cancer after radiotherapy and has gained much more attention in recent years. Given that the conclusions vary considerably, this meta-analysis was conducted to investigate the combined predictive effect of AI models on lung cancer. METHODS: This study was performed according to PRISMA guidelines. PubMed, ISI Web of Science, and Embase databases were searched for relevant literature. Outcomes, including overall survival (OS), disease-free survival (DFS), progression-free survival (PFS), and local control (LC), were predicted using AI models in patients with lung cancer after radiotherapy, and were used to calculate the pooled effect. Quality, heterogeneity, and publication bias of the included studies were also evaluated. RESULTS: Eighteen articles with 4719 patients were eligible for this meta-analysis. The combined hazard ratios (HRs) of the included studies for OS, LC, PFS, and DFS of lung cancer patients were 2.55 (95 % confidence interval (CI) = 1.73-3.76), 2.45 (95 % CI = 0.78-7.64), 3.84 (95 % CI = 2.20-6.68), and 2.66 (95 % CI = 0.96-7.34), respectively. The combined area under the receiver operating characteristics curve (AUC) of the included articles on OS and LC in patients with lung cancer was 0.75 (95 % CI = 0.67-0.84), and 0.80 (95%CI = 0.0.68-0.95), respectively. CONCLUSION: The clinical feasibility of predicting outcomes using AI models after radiotherapy in patients with lung cancer was demonstrated. Large-scale, prospective, multicenter studies should be conducted to more accurately predict the outcomes in patients with lung cancer.

Efficacy and safety of nanopaclitaxel formulation for cancer treatment: evidence from randomized clinical trials.

Aim: We aimed to analyze efficacy and adverse events for nano-bound paclitaxel in cancer treatment, which remain controversial. Method: We obtained relevant previously published studies and extracted data on the efficacy and adverse events of nano-bound paclitaxel. Fifteen randomized clinical trials were included. Results: Nanoparticle albumin-bound (Nab-) paclitaxel was beneficial in terms of objective response rate (odds ratio [OR]: 1.08, 95% CI: 0.72-1.62) and partial response (OR: 1.28, 95% CI: 0.89-1.83), while polymeric micellar (PM-) paclitaxel was beneficial in terms of objective response rate (OR: 1.76) and partial disease (hazard ratio [HR]: 0.65). Both Nab-paclitaxel and PM-paclitaxel resulted in slightly longer overall survival (HR: 0.93 and 0.94) and progression-free survival (HR: 0.93 and 0.87) when compared with solvent-based paclitaxel. Peripheral sensory neuropathy (OR: 3.47), neutropenia (OR: 1.79) and anemia (OR: 1.79) were more frequent after Nab-paclitaxel treatment. Conclusion: Nanopaclitaxel formulations have a better efficacy in cancer treatment; however, they increase the risk of hematological adverse events and peripheral sensory neuropathy. The PM-paclitaxel treatment had a high safety effect.

This was a pooled analysis of the efficacy and adverse events of nano-bound paclitaxel (polymeric micellar [PM] or nanoparticle-bound formulation) in cancer treatment. Relevant studies published since 2016 were retrieved from the PubMed, ISI Web of Science and Embase databases. Fifteen randomized clinical trials (4925 patients) were included in this meta-analysis. Compared with solvent-based paclitaxel, nanoparticle albumin-bound paclitaxel (Nab-paclitaxel) had beneficial effects in terms of objective response rate and partial response, while PM-paclitaxel exhibited beneficial effects in terms of objective response rate and partial disease. Both Nab-paclitaxel and PM-paclitaxel were associated with a slightly longer overall survival and better progression-free survival when compared with solvent-based paclitaxel. Peripheral sensory neuropathy, neutropenia and anemia adverse events were more frequent after Nab-paclitaxel treatment. The nanopaclitaxel formulation had an improved efficacy in treatment of solid-organ tumors, but it increased the risk of hematological adverse events and peripheral sensory neuropathy. This study provided evidence on the efficacy and safety of the nanocarriers of paclitaxel.

RhoA promotes osteoclastogenesis and regulates bone remodeling through mTOR-NFATc1 signaling.

BACKGROUND: The cytoskeletal architecture of osteoclasts (OCs) and bone resorption activity must be appropriately controlled for proper bone remodeling, which is associated with osteoporosis. The RhoA protein of GTPase plays a regulatory role in cytoskeletal components and contributes to osteoclast adhesion, podosome positioning, and differentiation. Although osteoclast investigations have traditionally been performed by in vitro analysis, however, the results have been inconsistent, and the significance of RhoA in bone physiology and pathology is still unknown. METHODS: We generated RhoA knockout mice by specifically deleting RhoA in the osteoclast lineage to understand more about RhoA's involvement in bone remodeling. The function of RhoA in osteoclast differentiation and bone resorption and the mechanisms were assessed using bone marrow macrophages (BMMs) in vitro. The ovariectomized (OVX) mouse model was adopted to examine the pathological effect of RhoA in bone loss. RESULTS: Conditional deletion of RhoA in the osteoclast lineage causes a severe osteopetrosis phenotype, which is attributable to a bone resorption suppression. Further mechanistic studies suggest that RhoA deficiency suppresses Akt-mTOR-NFATc1 signaling during osteoclast differentiation. Additionally, RhoA activation is consistently related to the significant enhancement the osteoclast activity, which culminates in the development of an osteoporotic bone phenotype. Furthermore, in mice, the absence of RhoA in osteoclast precursors prevented occurring OVX-induced bone loss. CONCLUSION: RhoA promoted osteoclast development via the Akt-mTOR-NFATc1 signaling pathway, resulting a osteoporosis phenotype, and that manipulating RhoA activity might be a therapeutic strategy for osteoporotic bone loss.

Sulfated fuco-manno-glucuronogalactan alleviates pancreatic beta cell senescence via PI3K/AKT/FoxO1 pathway.

Appearance of senescent beta cells in the pancreas leads to the onset of type 2 diabetes (T2D). The structural analysis of a sulfated fuco-manno-glucuronogalactan (SFGG) indicated SFGG had the backbones of interspersing 1, 3-linked ß-D-GlcpA residues, 1, 4-linked α-D-Galp residues, and alternating 1, 2-linked α-D-Manp residues and 1, 4-linked ß-D-GlcpA residues, sulfated at C6 of Man residues, C2/C3/C4 of Fuc residues and C3/C6 of Gal residues, and branched at C3 of Man residues. SFGG effectively alleviated senescence-related phenotypes in vitro and in vivo, including cell cycle, senescence-associated ß-galactosidase, DNA damage and senescence-associated secretory phenotype (SASP) -associated cytokines and hall markers of senescence. SFGG also alleviated beta cell dysfunction in insulin synthesis and glucose-stimulated insulin secretion. Mechanistically, SFGG attenuated senescence and improved beta cell function via PI3K/AKT/FoxO1 signaling pathway. Therefore, SFGG could be used for beta cell senescence treatment and alleviation of the progression of T2D.

Comparison of anti-tumor activities and underlying mechanisms of glucuronomannan oligosaccharides and its sulfated derivatives on the hepatocarcinoma Huh7.5 cells.

Hepatocellular carcinoma (HCC) is an aggressive tumor triggered by various factors such as virus infection and alcohol abuse. Glucuronomannan polysaccharide (Gx) is a subtype of fucoidans that possesses many bioactivities, but its anti-tumor activities in HCC have not been reported. In this paper, the anti-tumor effects of glucuronomannan oligosaccharides (Gx) and its sulfated derivatives (GxSy) on hepatocarcinoma Huh7.5 cells were investigated. The anti-proliferation, anti-metastasis activities, and underlying mechanism of Gx and GxSy on Huh7.5 cells were analyzed and compared by MTT, wound healing, transwell, and western blotting assays, respectively. Results showed that the best anti-proliferation effects were G4S1 and G4S2 among 13 drugs, which were 38.67% and 30.14%, respectively. The cell migration rates were significantly inhibited by G2S1, G4S2, G6S2, and unsulfated Gn. In addition, cell invasion effects treated with G4S1, G4S2, and G6S1 decreased to 48.62%, 36.26%, and 42.86%, respectively. Furthermore, sulfated G4 regulated the expression of (p-) FAK and MAPK pathway, and sulfated G6 down-regulated the MAPK signaling pathway while activating the PI3K/AKT pathway. On the contrary, sulfated G2 and unsulfated Gx had no inhibited effects on the FAK-mTOR pathway. These results indicated that sulfated Gx derivatives have better anti-tumor activities than unsulfated Gx in cell proliferation and metastasis process in vitro, and those properties depend on the sulfation group levels. Moreover, degrees of polymerization of Gx also played a vital role in mechanisms and bioactivities. This finding shows the structure-activity relationship for developing and applying the marine oligosaccharide candidates.

Oligosaccharides from Sargassum thunbergii inhibit osteoclast differentiation via regulation of IRF-8 signaling.

Osteoporosis (OP) is a systemic bone degenerative disease characterized by low bone mass and deteriorated microarchitecture of bone tissue, causing high morbidity and mortality rates. Bone resorption by overactivated osteoclasts (OCs) is the main cause of osteoporosis. Glucuronomannan and its oligomers (Gs) and their sulfated derivatives (SGs) were previously prepared. The anti-osteoporosis activities of these glycans were evaluated. Firstly, we determined the viability of RAW264.7 by CCK-8 test. Nextly, we investigated the inhibitory effects of Gs and SGs on the differentiation of RAW264.7 cells into OCs using tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring staining, qualitative reverse-transcription polymerase chain reaction(qRT-PCR) and western blotting. TRAP staining revealed that Gs significantly blocked RANKL-induced OC generation while SGs did not exhibit this ability. F-actin staining assays demonstrated that Gs inhibits RANKL-induced actin ring formation. qRT-PCR analyses indicated that Gs dose-dependently inhibited the expression of OCs marker genes including Trap, NFATc1, c-Fos, DC-Stamp and ATP60 during the differentiation process, while SGs did not suppress. Regarding the mechanism of Gs, it was found that Gs suppressed osteoclastogenesis via inhibiting the degradation of IRF-8 and interfering with NF-κB pathway activation. Together, these results suggest that Gs have the ability to inhibit osteoclastogenesis by modulating IRF-8 signaling.

Suppressive effects of pterostilbene on human cytomegalovirus (HCMV) infection and HCMV-induced cellular senescence.

BACKGROUND: Human cytomegalovirus (HCMV), a member of the ß-herpesvirus family, causes the establishment of a latent infection that persists throughout the life of the host and can be reactivated when immunity is weakened. To date, there is no vaccine to prevent HCMV infection, and clinically approved drugs target the stage of viral replication and have obvious adverse reactions. Thus, development of novel therapeutics is urgently needed. METHODS: In the current study, we identified a naturally occurring pterostilbene that inhibits HCMV Towne strain replication in human diploid fibroblast WI-38 cells through Western blotting, qPCR, indirect immunofluorescence assay, tissue culture infective dose assays. The time-of-addition experiment was carried out to identify the stage at which pterostilbene acted. Finally, the changes of cellular senescence biomarkers and reactive oxygen species production brought by pterostilbene supplementation were used to partly elucidate the mechanism of anti-HCMV activity. RESULTS: Our findings revealed that pterostilbene prevented lytic cytopathic changes, inhibited the expression of viral proteins, suppressed the replication of HCMV DNA, and significantly reduced the viral titre in WI-38 cells. Furthermore, our data showed that pterostilbene predominantly acted after virus cell entry and membrane fusion. The half-maximal inhibitory concentration was determined to be 1.315 µM and the selectivity index of pterostilbene was calculated as 26.73. Moreover, cell senescence induced by HCMV infection was suppressed by pterostilbene supplementation, as shown by a decline in senescence-associated ß-galactosidase activity, decreased production of reactive oxygen species and reduced expression of p16, p21 and p53, which are considered biomarkers of cellular senescence. CONCLUSION: Together, our findings identify pterostilbene as a novel anti-HCMV agent that may prove useful in the treatment of HCMV replication.

Characterization of exosomes derived from IPEC-J2 treated with probiotic Bacillus amyloliquefaciens SC06 and its regulation of macrophage functions.

Probiotics can maintain or improve health by modulating the response of immune cells in the gastrointestinal tract. However, the mechanisms by which probiotics promote macrophage (Mφ) activity are poorly understood. Here, we evaluated exosomes derived from intestinal epithelial cells treated with Bacillus amyloliquefaciens SC06 (Ba) and investigated the regulation of Mφ phagocytosis, apoptosis, and polarization. We isolated two exosomes from intestinal porcine epithelial cell lines (IPEC-J2) with or without Ba-treatment, named Ba-Exo and Exo, respectively. They had typical sizes and a cup-shaped morphology, and their surfaces presented typical exosomes-associated proteins, including CD63, ALIX, and TSG101. Ba-Exo and Exo could entrer Mφ (3D4/21 cells) effectively. Moreover, an in vitro phagocytosis assay demonstrated that Ba-Exo can promote phagocytosis of Mφ. Similar to Exo, Ba-Exo had no effect on Mφ apoptosis. Furthermore, Ba-Exo significantly increased inducible nitric oxide synthase (iNOS), declined the expression of arginase 1 (Arg1) in Mφ, and stimulated Mφ polarization to M1. To explore the differences in the regulation of Mφ polarization between Ba-Exo and Exo, we performed reverse transcription quantitative polymerase chain reaction analysis of the small RNAs and found that miR-222 increased in the Ba-Exo group compared to that in the Exo group. These results provide a new perspective on the relationship between probiotics and intestinal immunity.