Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in Pichia pastoris: Unveiling Antimicrobial and Anticancer Functionalities.

Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (PG1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number PG1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe3+) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications.

Benzyl isothiocyanate attenuates activation of the NLRP3 inflammasome in Kupffer cells and improves diet-induced steatohepatitis.

The NLRP3 inflammasome plays an important role in the pathogenesis of numerous inflammation-related diseases. Benzyl isothiocyanate (BITC) is rich in cruciferous vegetables and possesses potent antioxidant, anti-inflammatory, anti-cancer, and anti-obesogenic properties. In this study, we investigated the role of the NLRP3 inflammasome in the protection by BITC against steatohepatitis and insulin resistance. A mouse model of high-fat/cholesterol/cholic acid diet (HFCCD)-induced steatohepatitis, LPS/nigericin-stimulated primary Kupffer cells, and IL-1ß treated primary hepatocytes were used. BITC attenuated LPS/nigericin-induced activation of the NLRP3 inflammasome by enhancing protein kinase A-dependent NLRP3 ubiquitination, which increased the degradation of NLRP3 and reduced IL-1ß secretion in Kupffer cells. In hepatocytes, BITC pretreatment reversed the IL-1ß-induced decrease in the phosphorylation of IR, AKT, and GSK3ß in response to insulin. After 12 weeks of HFCCD feeding, increases in blood alanine aminotransferase (ALT) and glucose levels were ameliorated by BITC. Hepatic IL-1ß production, macrophage infiltration, and collagen expression induced by HFCCD were also mitigated by BITC. BITC suppresses activation of the NLRP3 inflammasome in Kupffer cells by enhancing the PKA-dependent ubiquitination of NLRP3, which leads to suppression of IL-1ß production and subsequently ameliorates hepatic inflammation and insulin resistance.

In Utero Cell Treatment of Hemophilia A Mice via Human Amniotic Fluid Mesenchymal Stromal Cell Engraftment.

Hemophilia is a genetic disorder linked to the sex chromosomes, resulting in impaired blood clotting due to insufficient intrinsic coagulation factors. There are approximately one million individuals worldwide with hemophilia, with hemophilia A being the most prevalent form. The current treatment for hemophilia A involves the administration of clotting factor VIII (FVIII) through regular and costly injections, which only provide temporary relief and pose inconveniences to patients. In utero transplantation (IUT) is an innovative method for addressing genetic disorders, taking advantage of the underdeveloped immune system of the fetus. This allows mesenchymal stromal cells to play a role in fetal development and potentially correct genetic abnormalities. The objective of this study was to assess the potential recovery of coagulation disorders in FVIII knockout hemophilia A mice through the administration of human amniotic fluid mesenchymal stromal cells (hAFMSCs) via IUT at the D14.5 fetal stage. The findings revealed that the transplanted human cells exhibited fusion with the recipient liver, with a ratio of approximately one human cell per 10,000 mouse cells and produced human FVIII protein in the livers of IUT-treated mice. Hemophilia A pups born to IUT recipients demonstrated substantial improvement in their coagulation issues from birth throughout the growth period of up to 12 weeks of age. Moreover, FVIII activity reached its peak at 6 weeks of age, while the levels of FVIII inhibitors remained relatively low during the 12-week testing period in mice with hemophilia. In conclusion, the results indicated that prenatal intrahepatic therapy using hAFMSCs has the potential to improve clotting issues in FVIII knockout mice, suggesting it as a potential clinical treatment for individuals with hemophilia A.

Lactoferrin Ameliorates Ovalbumin-Induced Asthma in Mice through Reducing Dendritic-Cell-Derived Th2 Cell Responses.

Asthma is a chronic respiratory disease with symptoms such as expiratory airflow narrowing and airway hyperresponsiveness (AHR). Millions of people suffer from asthma and are at risk of life-threatening conditions. Lactoferrin (LF) is a glycoprotein with multiple physiological functions, including antioxidant, anti-inflammatory, antimicrobial, and antitumoral activities. LF has been shown to function in immunoregulatory activities in ovalbumin (OVA)-induced delayed type hypersensitivity (DTH) in mice. Hence, the purpose of this study was to investigate the roles of LF in AHR and the functions of dendritic cells (DCs) and Th2-related responses in asthma. Twenty 8-week-old male BALB/c mice were divided into normal control (NC), ovalbumin (OVA)-sensitized, and OVA-sensitized with low dose of LF (100 mg/kg) or high dose of LF (300 mg/kg) treatment groups. The mice were challenged by intranasal instillation with 5% OVA on the 21st to 27th day after the start of the sensitization period. The AHR, cytokines in bronchoalveolar lavage fluid, and pulmonary histology of each mouse were measured. Serum OVA-specific IgE and IgG1 and OVA-specific splenocyte responses were further detected. The results showed that LF exhibited protective effects in ameliorating AHR, as well as lung inflammation and damage, in reducing the expression of Th2 cytokines and the secretion of allergen-specific antibodies, in influencing the functions of DCs, and in decreasing the level of Th2 immune responses in a BALB/c mouse model of OVA-induced allergic asthma. Importantly, we demonstrated that LF has practical application in reducing DC-induced Th2 cell responses in asthma. In conclusion, LF exhibits anti-inflammation and immunoregulation activities in OVA-induced allergic asthma. These results suggest that LF may act as a supplement to prevent asthma-induced lung injury and provide an additional agent for reducing asthma severity.

Kefir peptides exhibit antidepressant-like activity in mice through the BDNF/TrkB pathway.

Depression is a prevalent, stress-related mental disorder that can lead to serious psychiatric diseases with morbidity and high mortality. Although some functional fermented dairy drinks have promising anxiolytic and antidepressant effects, the mechanism is still not clear. To determine the antidepressant-like effect and the potential molecule mechanism of kefir peptides (KP), various behavioral tests, including the elevated plus maze test, open field test, forced swimming test, and tail suspension test, were used. Administration of 150 mg/kg KP in mice reduced the duration of immobility in the forced swimming test and tail suspension test, elevated the time spent in the open arm and center zone in the elevated plus maze test, and increased the total distance traveled, average speed, and time spent in the center zone in the open field test compared with the mock group. These results indicated that KP dramatically ameliorated the depression-like behaviors. Kefir peptides were further isolated and identified using high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry, from which 3 peptides were identified and designated KFP-1, KFP-3, and KFP-5. Among these peptides, administration of KFP-3 (15 AA residues) remarkably decreased immobility time in the forced swimming test and increased mobility time in the tail suspension test. Therefore, KFP-3 may be the major active peptide with antidepressant activity in KP. Overexpression of brain-derived neurotrophic factor, phosphorylated tropomyosin receptor kinase B, and phosphorylated ERK1/2 protein levels could be detected in the hippocampus under KP administration. Therefore, we suggest that KP improves depressive-like behaviors by activating the brain-derived neurotrophic factor-phosphorylated tropomyosin receptor kinase B signaling pathway. Kefir peptides may serve as a new type of antidepressant dairy product and may provide potent antidepressant effects for clinical use.

Effects of Mean Artery Pressure and Blood pH on Survival Rate of Patients with Acute Kidney Injury Combined with Acute Hypoxic Respiratory Failure: A Retrospective Study.

Background and Objectives: In the intensive care unit (ICU), renal failure and respiratory failure are two of the most common organ failures in patients with systemic inflammatory response syndrome (SIRS). These clinical symptoms usually result from sepsis, trauma, hypermetabolism or shock. If this syndrome is caused by septic shock, the Surviving Sepsis Campaign Bundle suggests that vasopressin be given to maintain mean arterial pressure (MAP) > 65 mmHg if the patient is hypotensive after fluid resuscitation. Nevertheless, it is important to note that some studies found an effect of various mean arterial pressures on organ function; for example, a MAP of less than 75 mmHg was associated with the risk of acute kidney injury (AKI). However, no published study has evaluated the risk factors of mortality in the subgroup of acute kidney injury with respiratory failure, and little is known of the impact of general risk factors that may increase the mortality rate. Materials and Methods: The objective of this study was to determine the risk factors that might directly affect survival in critically ill patients with multiple organ failure in this subgroup. We retrospectively constructed a cohort study of patients who were admitted to the ICUs, including medical, surgical, and neurological, over 24 months (2015.1 to 2016.12) at Chiayi Chang Gung Memorial Hospital. We only considered patients who met the criteria of acute renal injury according to the Acute Kidney Injury Network (AKIN) and were undergoing mechanical ventilator support due to acute respiratory failure at admission. Results: Data showed that the overall ICU and hospital mortality rate was 63.5%. The most common cause of ICU admission in this cohort study was cardiovascular disease (31.7%) followed by respiratory disease (28.6%). Most patients (73%) suffered sepsis during their ICU admission and the mean length of hospital stay was 24.32 ± 25.73 days. In general, the factors independently associated with in-hospital mortality were lactate > 51.8 mg/dL, MAP ≤ 77.16 mmHg, and pH ≤ 7.22. The risk of in-patient mortality was analyzed using a multivariable Cox regression survival model. Adjusting for other covariates, MAP ≤ 77.16 mmHg was associated with higher probability of in-hospital death [OR = 3.06 (1.374-6.853), p = 0.006]. The other independent outcome predictor of mortality was pH ≤ 7.22 [OR = 2.40 (1.122-5.147), p = 0.024]. Kaplan-Meier survival curves were calculated and the log rank statistic was highly significant. Conclusions: Acute kidney injury combined with respiratory failure is associated with high mortality. High mean arterial pressure and normal blood pH might improve these outcomes. Therefore, the acid-base status and MAP should be considered when attempting to predict outcome. Moreover, the blood pressure targets for acute kidney injury in critical care should not be similar to those recommended for the general population and might prevent mortality.

Benzyl isothiocyanate ameliorates high-fat/cholesterol/cholic acid diet-induced nonalcoholic steatohepatitis through inhibiting cholesterol crystal-activated NLRP3 inflammasome in Kupffer cells.

Incidence of nonalcoholic fatty liver disease is increasing worldwide. Activation of the NLRP3 inflammasome is central to the development of diet-induced nonalcoholic steatohepatitis (NASH). We investigated whether benzyl isothiocyanate (BITC) ameliorates diet-induced NASH and the mechanisms involved. C57BL/6 J mice fed a high-fat diet containing cholesterol and cholic acid (HFCCD) and Kupffer cells stimulated with LPS and cholesterol crystals (CC) were studied. LPS/CC increased the expression of the active form of caspase 1 (p20) and the secretion of IL-1ß by Kupffer cells, and these changes were reversed by MCC950, an NLRP3 inflammasome inhibitor. LPS/CC-induced NLRP3 inflammasome activation and IL-1ß production were dose-dependently attenuated by BITC. BITC decreased cathepsin B release from lysosomes and binding to NLRP3 induced by LPS/CC. Compared with a normal diet, the HFCCD increased serum levels of ALT, AST, total cholesterol, and IL-1ß and hepatic contents of triglycerides and total cholesterol. BITC administration (0.1% in diet) reversed the increase in AST and hepatic triglycerides in the HFCCD group. Moreover, BITC suppressed lipid accumulation, macrophage infiltration, fibrosis, crown-like structure formation, and p20 caspase 1 and p17 IL-1ß expression in liver in the HFCCD group. These results suggest that BITC ameliorates HFCCD-induced steatohepatitis by inhibiting the activation of NLRP3 inflammasome in Kupffer cells and may protect against diet-induced NASH.

14-Deoxy-11,12-didehydroandrographolide suppresses adipogenesis of 3 T3-L1 preadipocytes by inhibiting CCAAT/enhancer-binding protein ß activation and AMPK-mediated mitotic clonal expansion.

Obesity is highly correlated with several metabolic disorders. Adipocyte differentiation is a key process in determining obesogenesis. 14-Deoxy-11,12-didehydroandrographolide (deAND) is a diterpenoid rich in Andrographis paniculata (Burm.f.) Nees., a herbal medicine commonly used to treat colds, infections, and liver diseases. We investigated whether deAND inhibits the adipogenesis of 3T3-L1 cells and the underlying mechanisms. We found that deAND (0-15 µM) dose-dependently inhibits the mRNA and protein expression of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein 1c, fatty acid synthase, and stearoyl-CoA desaturase-1. Cellular lipid accumulation was decreased by deAND, and the early phase of adipocyte differentiation was critical for this inhibition. Immunoblotting revealed that deAND attenuated differentiation medium-induced protein kinase A (PKA) and cAMP response element-binding protein (CREB) activation, which leads to down-regulating C/EBPß transcription. Moreover, deAND inhibited ERK- and GSK3ß-mediated C/EBPß transcriptional activity. Flow cytometry analysis showed that deAND impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the G0/G1 phase, while the expression of cyclin D1, cyclin E, CDK6, and CDK2 was attenuated. deAND increased the phosphorylation of AMPK and raptor, an mTOR-interacting partner, which inhibited the mTOR-driven phosphorylation of P70S6K and eukaryotic translation initiation factor 4E binding protein. In the presence of compound C, deAND modulation of AMPK-mTOR signaling and inhibition of cell cycle regulator expression were reversed. Our results reveal that the anti-adipogenic effect of deAND is likely through inhibition of the PKA-CREB-C/EBPß and AMPK/mTOR pathways, which leads to down-regulating C/EBPß-driven lipogenic protein expression and halting MCE progression.

Aerosolized bovine lactoferrin reduces lung injury and fibrosis in mice exposed to hyperoxia.

This study investigated the ability of aerosolized bovine lactoferrin (bLF) to protect the lungs from injury induced by chronic hyperoxia. Female CD-1 mice were exposed to hyperoxia (FiO2 = 80 %) for 7 days to induce lung injury and fibrosis. The therapeutic effects of bLF, administered via an aerosol delivery system, on the chronic lung injury induced by this period of hyperoxia were measured by bronchoalveolar lavage, lung histology, cell apoptosis, and inflammatory cytokines in the lung tissues. After exposure to hyperoxia for 7 days, the survival of the mice was significantly decreased to 20 %. The protective effects of bLF against hyperoxia were further confirmed by significant reductions in lung edema, total cell numbers in bronchoalveolar lavage fluid, inflammatory cytokines (IL-1ß and IL-6), pulmonary fibrosis, and apoptotic DNA fragmentation. The aerosolized bLF protected the mice from oxygen toxicity and increased the survival fraction to 66.7 % in the hyperoxic model. The results support the use of an aerosol therapy with bLF in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure or chronic obstructive pulmonary disease.

Lactoferrin as a therapeutic agent for attenuating hepatic stellate cell activation in thioacetamide-induced liver fibrosis.

Liver fibrosis is a chronic liver disease caused by prolonged liver injuries. Excessive accumulation of extracellular matrix replaces the damaged hepatocytes, leading to fibrous scar formation and fibrosis induction. Lactoferrin (LF) is a glycoprotein with a conserved, monomeric signal polypeptide chain, exhibiting diverse physiological functions, including antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, and antitumoral activities. Previous study has shown LF's protective role against chemically-induced liver fibrosis in rats. However, the mechanisms of LF in liver fibrosis are still unclear. In this study, we investigated LF's mechanisms in thioacetamide (TAA)-induced liver fibrosis in rats and TGF-ß1-treated HSC-T6 cells. Using ultrasonic imaging, H&E, Masson's, and Sirius Red staining, we demonstrated LF's ability to improve liver tissue damage and fibrosis induced by TAA. LF reduced the levels of ALT, AST, and hydroxyproline in TAA-treated liver tissues, while increasing catalase levels. Additionally, LF treatment decreased mRNA expression of inflammatory factors such as Il-1ß and Icam-1, as well as fibrogenic factors including α-Sma, Collagen I, and Ctgf in TAA-treated liver tissues. Furthermore, LF reduced TAA-induced ROS production and cell death in FL83B cells, and decreased α-SMA, Collagen I, and p-Smad2/3 productions in TGF-ß1-treated HSC-T6 cells. Our study highlights LF's ability to ameliorate TAA-induced hepatocyte damage, oxidative stress, and liver fibrosis in rats, potentially through its inhibitory effect on HSC activation. These findings suggest LF's potential as a therapeutic agent for protecting against liver injuries and fibrosis.

Kefir peptides mitigate bleomycin-induced pulmonary fibrosis in mice through modulating oxidative stress, inflammation and gut microbiota.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and life-threatening lung disease with high mortality rates. The limited availability of effective drugs for IPF treatment, coupled with concerns regarding adverse effects and restricted responsiveness, underscores the need for alternative approaches. Kefir peptides (KPs) have demonstrated antioxidative, anti-inflammatory, and antifibrotic properties, along with the capability to modulate gut microbiota. This study aims to investigate the impact of KPs on bleomycin-induced pulmonary fibrosis. METHODS: Mice were treated with KPs for four days, followed by intratracheal injection of bleomycin for 21 days. Comprehensive assessments included pulmonary functional tests, micro-computed tomography (µ-CT), in vivo image analysis using MMPsense750, evaluation of inflammation- and fibrosis-related gene expression in lung tissue, and histopathological examinations. Furthermore, a detailed investigation of the gut microbiota community was performed using full-length 16 S rRNA sequencing in control mice, bleomycin-induced fibrotic mice, and KPs-pretreated fibrotic mice. RESULTS: In KPs-pretreated bleomycin-induced lung fibrotic mice, notable outcomes included the absence of significant bodyweight loss, enhanced pulmonary functions, restored lung tissue architecture, and diminished thickening of inter-alveolar septa, as elucidated by morphological and histopathological analyses. Concurrently, a reduction in the expression levels of oxidative biomarkers, inflammatory factors, and fibrotic indicators was observed. Moreover, 16 S rRNA sequencing demonstrated that KPs pretreatment induced alterations in the relative abundances of gut microbiota, notably affecting Barnesiella_intestinihominis, Kineothrix_alysoides, and Clostridium_viride. CONCLUSIONS: Kefir peptides exerted preventive effects, protecting mice against bleomycin-induced lung oxidative stress, inflammation, and fibrosis. These effects are likely linked to modifications in the gut microbiota community. The findings highlight the therapeutic potential of KPs in mitigating pulmonary fibrosis and advocate for additional exploration in clinical settings.

Bovine lactoferrin inhibits lung cancer growth through suppression of both inflammation and expression of vascular endothelial growth factor.

Lung cancers are among the most common cancers in the world, and the search for effective and safe drugs for the chemoprevention and therapy of pulmonary cancer has become important. In this study, bovine lactoferrin (bLF) was used in both in vitro and in vivo approaches to investigate its activity against lung cancer. A human lung cancer cell line, A549, which expresses a high level of vascular endothelial growth factor (VEGF) under hypoxia, was used as an in vitro system for bLF treatment. A strain of transgenic mice carrying the human VEGF-A165 (hVEGF-A165) gene, which induces pulmonary tumors, was used as an in vivo lung cancer therapy model. We found that bLF significantly decreased proliferation of A549 cells by decreasing the expression of VEGF protein in a dose-dependent manner. Furthermore, oral administration of bLF at 300 mg/kg of body weight 3 times a week for 1.5 mo to the transgenic mice overexpressing hVEGF-A165 significantly eliminated expression of hVEGF-A165 and suppressed the formation of tumors. Additionally, treatment with bLF significantly decreased the levels of proinflammatory cytokines, such as tumor necrosis factor-α, and antiinflammatory cytokines, such as IL-4 and IL-10. Levels of IL-6, which is both a proinflammatory and an antiinflammatory cytokine, were also reduced. Treatment with bLF decreased levels of tumor necrosis factor-α, IL-4, IL-6, and IL-10 cytokines, resulting in limited inflammation, which then restricted growth of the lung cancer. Our results revealed that bLF is an inhibitor of angiogenesis and blocks lung cell inflammation; as such, it has considerable potential for therapeutic use in the treatment of lung cancer.

Therapeutic effects of kefir peptides on adjuvant-induced arthritis in rats through anti-inflammation and downregulation of matrix metalloproteinases.

AIMS: Rheumatoid arthritis (RA) is a chronic autoimmune disease. Its pathological features are synovial inflammation, bone erosion, and joint structural damages. Our previous studies have shown that kefir peptides (KPs) can reduce cardiovascular disease, osteoporosis and renal inflammation. In this study, we further evaluate the efficacy of KPs on adjuvant-induced arthritis (AIA) in a rat model. MAIN METHODS: After the 14th day of adjuvant induction, rats were subsequently orally administered KPs (83 or 166 mg/day/kg) or tofacitinib (6.2 mg/day/kg) for 14 days. On the 28th day, the rats were anesthetized with isoflurane for ultrasonic, in vivo imaging system (IVIS), and radiographic imaging and then sacrificed for ankle tissues collection and analysis. In vitro, IL-1ß-treated human synovial cells (SW982) were subjected to anti-arthritis mechanism study in the presence of KPs. KEY FINDINGS: The results of ultrasonography, radiograph, histology, the expression of matrix metalloproteinases (MMPs), inflammatory cytokines and RANKL/OPG ratio demonstrated decreasing severity of synovitis and bone erosion in the ankle joints after KPs treatment. Activation of the NF-κB and MAPK pathways was significantly reduced in KPs treated AIA group. Furthermore, KPs attenuated IL-1ß-induced inflammatory cytokine production and the expression of MMPs in a human synovial cell line SW982. These results demonstrated that KPs alleviated adjuvant-induced arthritis in rats by inhibiting IL-1ß-related inflammation and MMPs production. SIGNIFICANCE: We concluded that KPs can exhibit anti-inflammatory effects by reducing the levels of macrophage-related inflammatory cytokines and MMPs, thus alleviating bone erosion in the ankle joint and constituting a potential therapeutic strategy for rheumatoid arthritis.

Andrographolide Inhibits Lipotoxicity-Induced Activation of the NLRP3 Inflammasome in Bone Marrow-Derived Macrophages.

Andrographolide is the major bioactive component of the herb Andrographis paniculata and is a potent anti-inflammatory agent. Obesity leads to an excess of free fatty acids, particularly palmitic acid (PA), in the circulation. Obesity also causes the deposition of ectopic fat in nonadipose tissues, which leads to lipotoxicity, a condition closely associated with inflammation. Here, we investigated whether andrographolide could inhibit PA-induced inflammation by activating autophagy, activating the antioxidant defense system, and blocking the activation of the NLRP3 inflammasome. Bone marrow-derived macrophages (BMDMs) were primed with lipopolysaccharide (LPS) and then activated with PA. LPS/PA treatment increased both the mRNA expression of NLRP3 and IL-1[Formula: see text] and the release of IL-1[Formula: see text] in BMDMs. Andrographolide inhibited the LPS/PA-induced protein expression of caspase-1 and the release of IL-1[Formula: see text]. Furthermore, andrographolide attenuated LPS/PA-induced mtROS generation by first promoting autophagic flux and catalase activity, and ultimately inhibiting activation of the NLRP3 inflammasome. Our results suggest that the mechanisms by which andrographolide downregulates LPS/PA-induced IL-1[Formula: see text] release in BMDMs involve promoting autophagic flux and catalase activity. Andrographolide may thus be a candidate to prevent obesity- and lipotoxicity-driven chronic inflammatory disease.

Kefir peptides attenuate atherosclerotic vascular calcification and osteoporosis in atherogenic diet-fed ApoE -/- knockout mice.

Aims: Vascular calcification (VC) and osteoporosis were previously considered two distinct diseases. However, current understanding indicates that they share common pathogenetic mechanisms. The available medicines for treating VC and osteoporosis are limited. We previously demonstrated that kefir peptides (KPs) alleviated atherosclerosis in high-fat diet (HFD)-induced apolipoprotein E knockout (ApoE -/- ) mice. The present study further addressed the preventive effects of KPs on VC and osteoporosis in ApoE -/- mice fed a high-cholesterol atherogenic diet (AD). Main methods: Seven-week-old ApoE -/- and wild-type C57BL/6 mice were randomly divided into five groups (n = 6). The development of VC and osteoporosis was evaluated after AD feeding for 13 weeks in KP-treated ApoE -/- mice and compared to C57BL/6 and ApoE -/- mice fed a standard chow diet (CD). Key findings: The results indicated that KP-treated ApoE -/- mice exhibited lower serum total cholesterol, oxidized low-density lipoprotein (ox-LDL), malondialdehyde (MDA) levels, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine kinase (CK) activities, which suggested that KPs prevented hyperlipidemia and possible damages to the liver and muscle in ApoE -/- mice. KPs reduced serum tumor necrosis factor-α (TNF-α) and the local expression of TNF-α, IL-1ß, and macrophage-specific CD68 markers in aortic tissues, which suggested that KPs inhibited inflammatory responses in AD-fed ApoE -/- mice. KPs reduced the deposition of lipid, collagen, and calcium minerals in the aortic roots of AD-fed ApoE -/- mice, which suggested that KPs inhibited the calcific progression of atherosclerotic plaques. KPs exerted osteoprotective effects in AD-fed ApoE -/- mice, which was evidenced by lower levels of the bone resorption marker CTX-1 and higher levels of the bone formation marker P1NP. KPs improved cortical bone mineral density and bone volume and reduced trabecular bone loss in femurs. Significance: The present data suggested that KPs attenuated VC and osteoporosis by reducing oxidative stress and inflammatory responses in AD-fed ApoE -/- mice. Our findings contribute to the application of KPs as preventive medicines for the treatment of hyperlipidemia-induced vascular and bone degeneration.

Novel Kefir Exopolysaccharides (KEPS) Mitigate Lipopolysaccharide (LPS)-Induced Systemic Inflammation in Luciferase Transgenic Mice through Inhibition of the NF-κB Pathway.

A novel kefir exopolysaccharides (KEPS) derived from kefir grain fermentation were found to have a small molecular weight (12 kDa) compared to the traditionally high molecular weight (12,000 kDa) of kefiran (KE). KE has been shown to possess antioxidant, blood pressure-lowering, and immune-modulating effects. In this study, we characterized KEPS and KE and evaluated their anti-inflammatory properties in vitro using RAW264.7 macrophages. The main monosaccharide components were identified as glucose (98.1 ± 0.06%) in KEPS and galactose (45.36 ± 0.16%) and glucose (47.13 ± 0.06%) in KE, respectively. Both KEPS and KE significantly reduced IL-6 secretion in lipopolysaccharide (LPS)-stimulated macrophages. We further investigated their effects in LPS-induced systemic injury in male and female NF-κB-luciferase+/+ transgenic mice. Mice received oral KEPS (100 mg/kg) or KE (100 mg/kg) for seven days, followed by LPS or saline injection. KEPS and KE inhibited NF-κB signaling, as indicated by reduced luciferase expression and phosphorylated NF-κB levels. LPS-induced systemic injury increased luciferase signals, especially in the kidney, spleen, pancreas, lung, and gut tissues of female mice compared to male mice. Additionally, it upregulated inflammatory mediators in these organs. However, KEPS and KE effectively suppressed the expression of inflammatory mediators, including p-MAPK and IL-6. These findings demonstrate that KEPS can alleviate LPS-induced systemic damage by inhibiting NF-κB/MAPK signaling, suggesting their potential as a treatment for inflammatory disorders.

SOD3 and IL-18 Predict the First Kidney Disease-Related Hospitalization or Death during the One-Year Follow-Up Period in Patients with End-Stage Renal Disease.

End-stage renal disease (ESRD) patients experience oxidative stress due to excess exogenous or endogenous oxidants and insufficient antioxidants. Hence, oxidative stress and inflammation cause endothelial damage, contributing to vascular dysfunction and atherosclerosis. Therefore, ESRD patients suffer more cardiovascular and hospitalization events than healthy people. This study aims to test the correlations between ROS, SOD3, IL-2, IL-6, and IL-18 and the first kidney disease-related hospitalization or death events in ESRD patients undergoing regular hemodialysis. A total of 212 participants was enrolled, including 45 normal healthy adults and 167 ESRD patients on regular dialysis. Blood samples from all participants were collected for ROS, SOD3, IL-2, IL-6, and IL-18 measurement at the beginning of the study, and every kidney disease-related admission or death was recorded for the next year. Multivariate analysis was conducted by fitting a linear regression model, logistic regression model, and Cox proportional hazards model to estimate the adjusted effects of risk factors, prognostic factors, or predictors on continuous, binary, and survival outcome data. The results showed that plasma SOD3 and serum IL-18 were two strong predictors of the first kidney disease-related hospitalization or death. In the Cox proportional hazards models (run in R), higher IL-18 concentration (>69.054 pg/mL) was associated with a hazard ratio of 3.376 for the first kidney disease-related hospitalization or death (95% CI: 1.2644 to 9.012), while log(SOD3) < 4.723 and dialysis clearance (Kt/V; 1.11 < value < 1.869) had a hazard ratio = 0.2730 (95% CI: 0.1133 to 0.6576) for reducing future kidney disease-related hospitalization or death. Other markers, including body mass index (BMI), transferrin saturation, total iron binding capacity, and sodium and alkaline phosphate, were also found to be significant in our study. These results reveal the new predictors SOD3 and IL-18 for the medical care of end-stage renal disease patients.

Human amniotic fluid mesenchymal stem cells attenuate pancreatic cancer cell proliferation and tumor growth in an orthotopic xenograft mouse model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a malignant cancer and chemotherapy ineffectively treats PDAC, leading to the requirement for alternative tumor-targeted treatment. Human amniotic fluid mesenchymal stem cells (hAFMSCs) have been revealed to suppress tumor growth in various cancers and they are a strong candidate for treating PDAC. METHODS: To evaluate the effects of hAFMSCs on human pancreatic carcinoma cells (PANC1, AsPC1 and BxPC3 cell lines) and the possible mechanism involved, an in vitro cell coculture system was used. A PANC1 orthotopic xenograft mouse model was established and hAFMSCs were injected intravenously at 4 weeks post-xenograft. RESULTS: An in vitro coculture assay showed that hAFMSCs inhibited PANC1 cell proliferation by inducing S phase cell cycle arrest and increased cell apoptosis in a time-dependent manner. In PANC1 cells, hAFMSCs caused the downregulation of Cyclin A and Cyclin B1 as well as the upregulation of p21 (CDKN1A) at 24 h post coculture. The upregulation of pro-apoptotic factors Caspase-3/-8 and Bax at 24 h post coculture reduced the migration and invasion ability of PANC1 cells through inhibiting the epithelial-mesenchymal transition (EMT) process. In a PANC1 orthotopic xenograft mouse model, a single injection of hAFMSCs showed significant tumor growth inhibition with evidence of the modulation of cell cycle and pro-apoptotic regulatory genes and various genes involved in matrix metallopeptidase 7 (MMP7) signaling-triggered EMT process. Histopathological staining showed lower Ki67 levels in tumors from hAFMSCs-treated mice. CONCLUSIONS: Our data demonstrated that hAFMSCs strongly inhibit PDAC cell proliferation, tumor growth and invasion, possibly by altering cell cycle arrest and MMP7 signaling-triggered EMT.

Therapeutic Effects of Kefir Peptides on Hemophilia-Induced Osteoporosis in Mice With Deficient Coagulation Factor VIII.

Osteoporosis is a clinically prevalent comorbidity in patients with hemophilia. A preventive effect of kefir peptides (KPs) on postmenopausal osteoporosis has been proved. The aim of this study was to evaluate the therapeutic effect of KPs for the treatment of osteoporosis in coagulation factor VIII (FVIII) gene knockout mice (F8KO), a model of hemophilia A. In this study, male F8KO mice at 20 weeks of age were orally administered different doses of KPs for 8 weeks. The therapeutic effects of KPs were shown in the femoral trabeculae and the 4th lumbar vertebrae, which increased the trabecular bone mineral density (BMD), bone volume (Tb.BV/TV), and trabecular number (Tb.N) and decreased the trabecular separation (Tb.Sp), and they were also observed in the femoral cortical bones, in which the mechanical properties were enhanced in a dose-dependent manner. Characterization of receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and interleukin 6 (IL-6) demonstrated that the serum RANKL/OPG ratio and IL-6 levels were significantly decreased in the F8KO mice after the KP treatment. Tartrate-resistant acid phosphatase (TRAP) staining of mature osteoclasts indicated that the therapeutic effect of KPs in F8KO mice was associated with the functions of KPs to inhibit RANKL-induced osteoclastogenesis by reducing serum RANKL/OPG ratio and IL-6 secretion. The present study is the first to address the potentials of KPs for the treatment of hemophilia-induced osteoporosis in mice and it also provides useful information for the application of KPs as a complementary therapy for the treatment of osteoporosis in hemophilic patients.

Kefir peptides ameliorate osteoporosis in AKR1A1 knockout mice with vitamin C deficiency by promoting osteoblastogenesis and inhibiting osteoclastogenesis.

The AKR1A1 protein is a member of the aldo-keto reductase superfamily that catalyzes the transformation of D-glucuronate to L-gulonate in the synthesis of L-ascorbic acid (vitamin C, Vit C). We previously demonstrated that AKR1A1 knockout mice (AKR1A1eGFP/eGFP) with Vit C deficiency exhibited aberrant bone formation and osteoporosis. In this study, we aimed to evaluate the osteoprotective effects of kefir peptides (KPs) in AKR1A1eGFP/eGFP mice and uncover the underlying mechanism of KPs in the modulation of bone remodeling. Six male CD-1 mice and 24 male AKR1A1eGFP/eGFP mice were used in this study, in which the AKR1A1eGFP/eGFP mice were randomly divided into four groups (n = 6). KPs treatment for 12 weeks exerted several effects in AKR1A1eGFP/eGFP mice including the reduction of serum proinflammatory cytokines (IL-1ß, IL-6, TNF-α), bone resorption markers (CTX-1, RANKL), and the increase of serum bone formation markers (P1NP, OPG, OC). µ-CT analysis indicated that KPs prevented the bone loss in the femurs of AKR1A1eGFP/eGFP mice by significantly increasing the trabecular parameters of bone mineral density, bone volume and bone number. Nanoindentation analysis demonstrated that KPs enhanced the elasticity and hardness of femoral cortical bones in AKR1A1eGFP/eGFP mice. KPs promoted bone marrow mesenchymal stem cells (BMMSCs)-derived osteoblast differentiation and mineralization by upregulating positive regulators of osteoblastogenesis (Runx2, ß-catenin, BMP-2, NFATc1). Conversely, KPs inhibited bone marrow macrophages (BMMs)-derived osteoclast differentiation and bone resorption, which was demonstrated by the facts that KPs suppressed RANKL-induced p38, NF-κB, Akt, PLCγ2 and CREB-1 phosphorylation, decreased the nuclear translocation of NFATc1 and c-Fos. Our findings demonstrate the efficacy of KPs in the prevention of osteoporosis in AKR1A1eGFP/eGFP mice and also unveil the dual effects of KPs in osteogenic promotion and osteoclastic inhibition. This study supports the use of KPs as nutritional supplements for the prevention of osteoporosis.