Effect Of bLF Research Articles

Bovine lactoferrin alleviates aflatoxin B1 induced hepatic and renal injury in broilers by mediating Nrf2 signaling pathway

Aflatoxin B1 (AFB1) a mycotoxin found in chicken feed that possess a global hazard to poultry health. However different potent compounds like bovine lactoferrin (bLF) may prove to be protective effects against AFB1. This study aims to explore the protective effect of bLF against AFB1-induced injury in the liver and kidney in broiler. For this purpose, 600 broilers chicks were randomly alienated into five groups (n=120 each): negative control; positive control (3mg/kg AFB1), and bLF high, medium, and low dosage groups (600 mg/kg, 300 mg/kg, and 150 mg/kg, respectively). The results highlight that AFB1 toxicity in birds exhibited low feed intake, reduction in weight gain, and a decrease in FCR while, bLF regulated these adverse effects. Meanwhile, AFB1 group showed higher levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) and lower levels of superoxide dismutase (SOD) and glutathione (GSHpx) in liver, while urea and creatinine were decline in kidney. Supplementation with bLF effectively controlled these biomarkers and control the negative effects of toxicity. Furthermore, hematoxylin and eosin (H&E) staining exhibited normal morphological structures within liver and kidney in the bLF treated groups, while degenerative changes were observed in AFB1 group. Similarly, bLF, decreased oxidative stress and thus prevented apoptosis in the liver and kidney cells of the birds. Whereas, mRNA level of mitochondrial apoptosis related gene including Bcl-2 (Bak and Bax), caspase-3 and caspase-9 was upregulated, while bcl2 gene were downregulated in AFB1 group. Dietary supplementation of bLF effectively normalizes the expression of these genes. AFB1 exposed birds shown to decrease gene expression level of the crucial component of Nrf2 pathway, responsible to regulate antioxidant defense. Interestingly, bLF reverse these detrimental effects of and restore the normal expression levels of Nrf2 pathway. Conclusively, our findings demonstrate that bLF mitigates the detrimental effects of AFB1, besides regulation of the apoptosis-related genes via mitochondrial pathways. These findings validate that the bLF (600 mg/kg) could be used as protective agent against AFB1-induced liver and kidney damage.

Bovine lactoferrin inhibits inflammatory response and apoptosis in lipopolysaccharide-induced acute lung injury by targeting the PPAR-γ pathway.

Lactoferrin (LF) is an iron-binding multifunctional cationic glycoprotein. Previous studies have demonstrated that LF may be a potential drug for treating acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In this study, we explored the anti-inflammatory effect and mechanism of bovine lactoferrin (bLF) in ALI using the RNA sequencing (RNA-seq) technology and transcriptome analysis. Based on the differentially expressed genes (DEGs) obtained from RNA-seq of the Lung from mouse model, the bioinformatics workflow was implemented using the BGISEQ-500 platform. The protein-protein interaction (PPI) network was obtained using STRING, and the hub gene was screened using Cytoscape. To verify the results of transcriptome analysis, the effects of bLF on Lipopolysaccharide (LPS)-induced BEAS-2B cells and its anti-reactive oxygen species (ROS), anti-inflammatory, and antiapoptotic effects were studied via Cell Counting Kit-8 (CCK-8) test, active oxygen detection test, ELISA, and western blot assay. Transcriptome analysis revealed that two hub gene modules of DEGs were screened via PPI analysis using the STRING and MCODE plug-ins of Cytoscape. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these core modules are enriched in the PPAR (peroxisome proliferator-activated receptor) and AMPK (AMP-activated protein kinase) signaling pathways. Through cell experiments, our study shows that bLF can inhibit ROS, inflammatory reaction, and LPS-induced BEAS-2B cell apoptosis, which are significantly antagonized by the PPAR-γ inhibitor GW9662. This study has suggested that the PPAR-γ pathway is the critical target of bLF in anti-inflammatory reactions and apoptosis of ALI, which provides a direction for further research.

Lactoferrin in the Prevention of Recurrent Respiratory Infections in Preschool Children: A Prospective Randomized Study.

Few studies have evaluated the effect of bovine lactoferrin (bLf) on reducing respiratory infections in preschool children. This randomized controlled trial evaluated the effect of bLf in preschool children with recurrent respiratory infections. Participants were randomly assigned bLf (n = 25) or control (n = 25). Outcomes included respiratory infection episodes (RIEs), symptom duration, school absence and medication. Fifty children aged 4.2 ± 0.1 years were included. During the active 4-month phase, median number of RIEs was reduced by 50% in the bLf group [1-episode, interquartile range (IQR): 0-2] vs. control (2, IQR: 1-3; p = 0.02). The proportion of participants with >3 RIEs was significantly lower in bLf (n = 1, 4%) vs. control (n = 7, 28%) with 80% lower odds of upper RIEs in the bLf arm (odds ratio: 0.20, 95% CI:0.06-0.74, p = 0.015). The duration of symptoms (3 vs. 6, p = 0.009) and days absent from school (3 vs. 6, p = 0.15) were lower in the active arm. Over the 2-month follow-up, no significant differences were observed between groups for infection episodes, symptom duration or school absence. However, bLf-treated children received significantly less corticosteroids over the entire 6-month study period (32% vs. 60%; p = 0.047). bLf supplementation significantly reduced the frequency and duration of RIEs in children with decreased corticosteroid use.

Regulation of HeLa cell proliferation and apoptosis by bovine lactoferrin.

Cervical cancer is one of the foremost common cancers in women. Lactoferrin (LF) has many biological functions, such as antitumor. This study aimed to explore the regulatory effect of bovine lactoferrin (bLF) on the proliferation and apoptosis of cervical cancer HeLa cells and to clarify the potential mechanism of action of bLF against HeLa cells. This study used CCK-8, Trypan blue staining, and colony formation assays to verify the effect of bLF on HeLa cell proliferation. Hoechst 33258 fluorescence staining, AO/EB staining, and western blotting were used to determine the effects of bLF on apoptosis and autophagy in HeLa cells. We discovered that bLF significantly reduced the proliferation of HeLa cells in a dose- and time-dependent manner compared to the control group. Furthermore, bLF primarily induced apoptosis in HeLa cells by increasing the expression of the proapoptotic proteins p53, Bax, and Cleaved-caspase-3 and downregulating the expression of the antiapoptotic protein Bcl-2. In addition, the present study also showed that bLF treatment significantly activated autophagy-related proteins LC3B-II and Beclin I and down regulated the autophagosome transporter protein p62, indicating that bLF treatment can induce autophagy in HeLa cells. After pretreatment with the autophagy inhibitor, 3-MA, which markedly found that autophagy inhibition by 3-MA reversed bLF-induced apoptosis, indicating that bLF can induce apoptosis by activating intracellular autophagy in HeLa cells. In the present study, our results support the theory of bLF significantly inhibited the proliferation of Hela cells by promoting apoptosis and reinforcing autophagy. The study will play an important role in therapying cervical cancer.

Bovine lactoferrin suppresses the cathepsin-dependent pathway of SARS-CoV-2 entry in vitro

Severe acute respiratory coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The frequent appearance of variants requires adjunct strategies to combat SARS-CoV-2 infections. Lactoferrin, a naturally occurring glycoprotein, exhibits promising antiviral properties. Here, we investigated the efficacy of bovine LF (bLF) against SARS-CoV-2 infection. SARS-CoV-2 pseudoviruses bearing the original Wuhan (wild-type), D614G mutant, or Omicron variant spike proteins were used to examine the effects of bLF on viral entry into host cells. We found that although bLF did not suppress entry of wild-type pseudoviruses into cells expressing both human angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), it did suppress entry of Omicron pseudoviruses into the cells. The results of this in vitro study highlight the spike protein-dependent effects of bLF on suppression of pseudovirus entry into host cells. Future clinical trials investigating the efficacy of bLF against SARS-CoV-2 infection should consider the differential impact of virus variants.

Bovine lactoferrin inhibits resistant Helicobacter pylori in vitro and protects gastric mucosal injury in vivo

Helicobacter pylori (H. pylori) is associated with gastric mucosal injury, gastric ulcer and other gastrointestinal diseases. Resistance of H. pylori to antibiotics has become an obstacle to clinical eradication therapy. Bovine lactoferrin (bLF) has a broad spectrum of antibacterial effects, high safety and no side effects, which may be conductive to improving the dilemma of antibiotic resistance. We aimed to explore the bactericidal effect of bLF on resistant H. pylori and the protective effect on gastric mucosal injury. In vitro experiments demonstrated that bLF has obvious antibacterial effect on resistant H. pylori and synergistic antibacterial effect when combined with clarithromycin. The MIC and MBC of bLF against resistant H. pylori ranged from 1.25 to 5 mg mL−1. In vivo experiments showed that bLF could improve the degree of gastric mucosa injury, decrease the area of gastric ulcer. Results indicate that bLF has a wide clinical application prospect in medical field.

Molecular Mechanisms of Inhibitory Effects of Bovine Lactoferrin on Invasion of Oral Squamous Cell Carcinoma

Lactoferrin (LF), an iron-binding glycoprotein, has been reported to have anticancer properties. However, the molecular mechanisms behind its anticancer effects on oral squamous cell carcinoma (OSCC) have not yet been elucidated. Therefore, we aimed to clarify the effects of LF on invasion of OSCC, and its underlying molecular mechanism. OSCC cell lines, HSC2 and HOC313, were treated with bovine LF (bLF). The effects of bLF on cell invasion were examined by a chamber migration assay, wound healing assay, and Boyden chamber method with a basement-membrane-analogue. Expression levels of MMP-1, MMP-3, and AP-1 were examined using RT-PCR, qRT-PCR, and western blotting. Roles of LRP1, a receptor of bLF, on cell invasion were analyzed using siLRP1 knockdown cells. Furthermore, to clarify the importance of LRP1 in invasion, the effects of bLF on tPA-induced invasion of OSCC cells were examined. The invasion assays showed that bLF suppressed invasion of the OSCC cells. Moreover, bLF down-regulated AP-1, and resulted in reductions of MMP-1 and MMP-3. With SiLRP1 knockdown, OSCC cells failed to induce their invasion, and bLF was not able to exert its effects on invasion. Furthermore, bLF remarkably inhibited tPA-induced cell invasion. These findings suggest the importance of LRP1 in bLF-suppressed invasion of OSCC cells via the reduction of AP-1 and MMP production.

Antitumor activity of bovine lactoferrin and its derived peptides against HepG2 liver cancer cells and Jurkat leukemia cells.

Liver cancer and leukemia are the fourth and first causes, respectively, of cancer death in children and adults worldwide. Moreover, cancer treatments, although beneficial, remain expensive, invasive, toxic, and affect the patient's quality of life. Therefore, new anticancer agents are needed to improve existing agents. Because bovine lactoferrin (bLF) and its derived peptides have antitumor properties, we investigated the anticancer effect of bLF and LF peptides (LFcin17-30, LFampin265-284 and LFchimera) on liver cancer HepG2 cells and leukemia Jurkat cells. HepG2 and Jurkat cells were incubated with bLF and LF peptides. Cell proliferation was quantified by an MTT assay, and cell morphology and damage were visualized by light microscopy or by phalloidin-TRITC/DAPI staining. The discrimination between apoptosis/necrosis was performed by staining with Annexin V-Alexa Fluor 488 and propidium iodide, and the expression of genes related to apoptosis was analyzed in Jurkat cells. Finally, the synergistic interaction of bLF and LF peptides with cisplatin or etoposide was assessed by an MTT assay and the combination index. The present study demonstrated that bLF and LF peptides inhibited the viability of HepG2 and Jurkat cells, inducing damage to the cell monolayer of HepG2 cells and morphological changes in both cell lines. bLF, LFcin17-30, and LFampin265-284 triggered apoptosis in both cell lines, whereas LFchimera induced necrosis. These results suggested that bLF and LF peptides activate apoptosis by increasing the expression of genes of the intrinsic pathway. Additionally, bLF and LF peptides synergistically interacted with cisplatin and etoposide. In conclusion, bLF and LF peptides display anticancer activity against liver cancer and leukemia cells, representing an alternative or improvement in cancer treatment.

Bovine Lactoferrin Suppresses Tumor Angiogenesis through NF-κB Pathway Inhibition by Binding to TRAF6.

Tumor angiogenesis is essential for tumor progression. The inhibition of tumor angiogenesis is a promising therapy for tumors. Bovine lactoferrin (bLF) has been reported as an anti-tumor agent. However, bLF effects on tumor angiogenesis are not well demonstrated. This study evaluated the inhibitory effects of bLF on tumor angiogenesis in vivo and in vitro. Herein, tumor endothelial cells (TECs) and normal endothelial cells (NECs) were used. Proliferation, migration, tube formation assays, RT-PCR, flow cytometry, Western blotting, siRNA experiments and immunoprecipitation were conducted to clarify the mechanisms of bLF-induced effects. CD-31 immunoexpression was examined in tumor tissues of oral squamous cell carcinoma mouse models with or without Liposomal bLF (LbLF)-administration. We confirmed that bLF inhibited proliferation/migration/tube formation and increased apoptosis in TECs but not NECs. TNF receptor-associated factor 6 (TRAF6), p-p65, hypoxia inducible factor-α (HIF-1α) and vascular endothelial growth factor (VEGF) were highly expressed in TECs. In TECs, bLF markedly downregulated VEGF-A, VEGF receptor (VEGFR) and HIF-1α via the inhibition of p-p65 through binding with TRAF6. Since NECs slightly expressed p-p65, bLF-TRAF-6 binding could not induce detectable changes. Moreover, orally administrated LbLF decreased CD31-positive microvascular density only in TECs. Hence, bLF specifically suppressed tumor angiogenesis through p-p65 inhibition by binding to TRAF6 and suppressing HIF-1α activation followed by VEGF/VEGFR down-regulation. Collectively, bLF can be an anti-angiogenic agent for tumors.

Bovine Lactoferrin and Current Antifungal Therapy Against Candida Albicans: A Systematic Review and Meta-Analysis.

Candida albicans is an important causative organism of opportunistic fungal infection, and it is a growing medical concern due to the increasing usage of broad-spectrum antibiotics, immunosuppressant agents, and other immunocompromising conditions. Currently, bLf and antifungal drugs have been known to have synergistic effects, increasing the drug's efficacy. This study aims to investigate the efficacy of the synergistic effect of bLf and antifungal drugs. This review addressed the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. We conducted literature searches to assess the association of lactoferrin and current antifungal therapy against Candida albicans in ProQuest, PubMed, MEDLINE, EBSCOhost, SAGE, JSTOR, GARUDA, and Open Gray with no date restriction (until March 5th, 2021). We used Jeffry's Amazing Statistical Program (JASP) to measure the overall size effect of MIC (minimum inhibitory concentration) between studies. A total of 7 studies retained were experimental in vitro studies. Based on the available data, 4 out of 7 studies were included in the quantitative analysis. This systematic review showed that bovine lactoferrin could help inhibit the development of azole-susceptible and azole-resistant C. albicans. Furthermore, there was synergistic activity between lactoferrin and various antifungals. Our meta-analysis showed that lactoferrin could significantly inhibit the C. albicans growth than the control group. Bovine lactoferrin and its peptide derivatives isolated from bovine milk can significantly inhibit the growth of C. albicans, both susceptible to azoles and those with azole resistance.

Impact of bovine lactoferrin fortification on pathogenic organisms to attenuate the risk of infection for infants

Cronobacter and Pseudomonas spp. are pathogenic organisms that are associated with neonatal morbidities such as bacteremia, sepsis, necrotizing enterocolitis (NEC), and infant meningitis. Lactoferrin (Lf) is a known bactericidal component of fresh human milk. Infants receiving formula or pasteurized donor milk however may not be receiving lactoferrin at same concentration as mother's own milk, so it is beneficial to understand an exogenous role for dairy bovine lactoferrin (bLf). Our aim was to investigate the potential for bLf to provide a novel way to inhibit the growth of these harmful organisms in infant feeding. Subinhibitory doses, Disc-diffusion assay, Minimum Inhibitory Concentrations (MIC), Minimum Bactericidal Concentrations (MBC), and challenge experiments in powder formula were used to investigate the effects of bLf on sepsis-causing bacteria such as C. muytjensii (ATCC-51329), C. sakazakii (ATCC-12868), C. sakazakii (ATCC-9027), P. aeruginosa (ATCC-19429), and P. aeruginosa (ATCC-9027). MIC and MBC of bLf in the range of 12.5–31.25 μM were sufficient to achieve a 3 log reduction of Cronobacter in nutrient broth (P < 0.0001). Similarly, minimum concentrations of 43.75–118.75 μM bLf against Pseudomonas (P < 0.0001) were demonstrated. After bactericidal concentrations were determined, higher resistant strains such as C. sakazakii (ATCC-12868) and P. aeruginosa (ATCC-19429) were chosen as ideal candidates for the bLf supplementation challenge studies. The bLf reduced the C. sakazakii (ATCC-12868) by 3.3 log at 35 °C, 2.8 log at 23 °C, and 1.9 log at 4 °C (P < 0.0001), whereas P. aeruginosa (ATCC-19429) showed 2.4 log reduction at 35 °C, 2.8 log at 23 °C (P < 0.0001), and 1.8 log at 4 °C (P < 0.01). Results indicated that bLf could effectively inactivate the sepsis-causing organisms in reconstituted infant formula and in bacterial growth media. Fortification of infant formula with bLf may address the problems of spoilage/contamination issues and provide a safety measure for feeding infants without mother's own milk.

Oral administration of bovine lactoferrin suppresses the progression of rheumatoid arthritis in an SKG mouse model.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory bone destruction in which tumor necrosis factor alpha (TNF-α) plays a key role. Bovine lactoferrin (bLF) is a multifunctional protein with anti-inflammatory and immunomodulatory properties. This study aimed to clarify the inhibitory effects of bLF on the pathological progression of RA. The mannan-induced arthritis model in SKG mice (genetic RA model) was used. Orally applied liposomal bLF (LbLF) markedly reduced ankle joint swelling and bone destruction. Histologically, pannus formation and osteoclastic bone destruction were prevented in the LbLF-treated animals. Moreover, orally administered LbLF improved the balance between Th17 cells and regulatory T cells isolated from the spleen of mannan-treated SKG mice. In an in vitro study, the anti-inflammatory effects of bLF on TNF-α-induced TNF-α production and downstream signaling pathways were analyzed in human synovial fibroblasts from RA patients (RASFs). bLF suppressed TNF-α production from RASFs by inhibiting the nuclear factor kappa B and mitogen-activated protein kinase pathways. The intracellular accumulation of bLF in RASFs increased in an applied bLF dose-dependent manner. Knockdown of the lipoprotein receptor-related protein-1 (LRP1) siRNA gene reduced bLF expression in RASFs, indicating that exogenously applied bLF was mainly internalized through LRP-1. Immunoprecipitated proteins with anti-TNF receptor-associated factor 2 (TRAF2; an adapter protein/ubiquitin ligase) included bLF, indicating that bLF binds directly to the TRAF2-TRADD-RIP complex. This indicates that LbLF may effectively prevent the pathological progression of RA by suppressing TNF-α production by binding to the TRAF2-TRADD-RIP complex from the RASFs in the pannus. Therefore, supplemental administration of LbLF may have a beneficial effect on preventive/therapeutic reagents for RA.

Temporal Dynamics of T Helper Populations in the Proximal Small Intestine after Oral Bovine Lactoferrin Administration in BALB/c Mice.

Bovine lactoferrin (bLf), a component of milk and a dietary supplement, modulates intestinal immunity at effector and inductor sites. Considering the regional difference in intestinal compartments and the dynamics of local cytokine-producing cells in the gut across time, the aim of this work was to characterize the effects of bLf on the proximal small intestine in a BALB/c murine model of oral administration. Male BALB/c mice were treated with oral bLf vs. saline control as mock by buccal deposition for 28 days. Intestinal secretions were obtained at different time points and cells were isolated from Peyer’s patches (PP) and lamina propria (LP) of the proximal small intestine as representative inductor and effector sites, respectively. Total and specific anti-bLF IgA and IgM were determined by enzyme-immuno assay; the percentages of IgA+ and IgM+ plasma cells (PC) and cytokine-producing CD4+ T cells of PP and LP were analyzed by flow cytometry. We found that total and bLf-specific IgA and IgM levels were increased in the intestinal secretions of the bLf group in comparison to mock group and day 0. LP IgA+ PC and IgM+ PC presented an initial elevation on day 7 and day 21, respectively, followed by a decrease on day 28 in comparison to mock. Higher percentages of CD4+ T cells in LP were found in the bLf group. Cytokines-producing CD4+ T cells populations presented a pattern of increases and decreases in the bLf group in both LP and PP. Transforming growth factor beta (TGF-β)+ CD4+ T cells showed higher percentages after bLf administration with a marked peak at day 21 in both LP and PP in comparison to mock-treated mice. Oral bLf exhibits complex immune properties in the proximal small intestine, where temporal monitoring of the inductor and effector compartments reveals patterns of rises and falls of different cell populations. Exceptionally, TGF-β+ CD4+ T cells show consistent higher numbers after bLf intervention across time. Our work suggests that isolated measurements do not show the complete picture of the modulatory effects of oral bLf in immunological sites as dynamic as the proximal small intestine.

Bovine lactoferrin and lactoferrin peptides affect endometrial and cervical cancer cell lines.

Cervical, uterine, and ovarian cancers are the most common malignancies of the female genital tract worldwide. Despite advances in prevention, early diagnosis, effective screening, and treatment programs, mortality remains high. Consequently, it is important to search for new treatments. The activity of bovine lactoferrin (bLF) and LF peptides against several types of cancer has been studied; however, only a few studies report the effect of bLF and LF peptides against cervical and endometrial cancers. In this study, we explored the effect of bLF as well as LFchimera and its constituent peptides LFcin17-30 and LFampin265-284 on the viability of cervical (HeLa, SiHa) and endometrial (KLE, HEC-1A) cancer cell lines. Cell proliferation was quantified with an MTT assay, cell morphological changes and damage were determined by Giemsa and phalloidin-TRITC and DAPI staining, and apoptotic and necrotic cells were identified by Alexa Fluor® 488 AnnexinV and propidium iodide staining. Additionally, the effect of combinations of bLF and LF peptides with cisplatin was assessed. bLF and LF peptides inhibited the proliferation of uterine cancer cells and caused cellular morphological changes and damage to cell monolayers. bLF induced apoptosis, LFcin17-30 and LFampin265-284 induced apoptosis and necrosis, and LF chimera induced necrosis. Additionally, bLF and LF chimera showed an additive interaction with cisplatin against uterine cancer cells.

Analysis of the intestinal IgA response in mice exposed to chronic stress and treated with bovine lactoferrin.

Immunoglobulin (Ig) A, an antibody with a pivotal role in gut homeostasis, can be modulated by stress and bovine lactoferrin (bLf). The aim of the present study was to analyze the impact of chronic stress on the IgA response in the small intestine during bLf treatment. Male BALB/c mice (n=6 mice/group) underwent 1h of chronic stress by immobilization for 7 consecutive days or were left unstressed, and were untreated or treated with bLf (50, 500 or 5,000<em>µ</em>g). Plasma corticosterone expression levels were determined by ELISA. The distal small intestine was dissected to analyze: i)total IgA, secretory IgA and IgG, as well as and specific IgA and IgG antibody levels in the intestinal liquid by ELISA; ii)α‑chain and polymeric immunoglobulin receptor (pIgR) protein expression in epithelial cell extracts analyzed by western blotting; iii)the mRNA expression levels of α‑/J‑chains, pIgR, IL‑2, IL‑4, IL‑5 and IL‑6 in whole mucosal samples by reverse transcription‑quantitative PCR. Data were analyzed by one‑way ANOVA, and the differences were analyzed by the Holm‑Šidák post hoc test and were considered significant if P&lt;0.05. Results from the present study revealed the upregulatory effects of chronic stress on the total antibody levels, protein (α‑chain; 78‑kDa pIgR) and mRNA (α‑ and J‑chains; pIgR; IL‑6) expression levels were restricted by bLf under stress. The effects of chronic stress on the downregulation of IL‑2 and IL‑4 mRNA expression were not changed by bLf under stress. The corticosterone response in unstressed mice treated with 5,000<em>µ</em>g bLf and the specific‑IgG levels in the unstressed and stressed groups treated with bLf at all doses were increased. The findings suggested an effect of bLf in maintaining homeostasis under stress.

Effect of bovine lactoferrin on prevention of late-onset sepsis in infants &lt;1500 g: a pooled analysis of individual patient data from two randomized controlled trials

We previously conducted two randomized controlled trials with bovine lactoferrin (bLF) for the prevention of late-onset sepsis (LOS) in infants with a birth weight <2500 g (Study 1) and <2000 g (Study 2). The aim of this study was to determine the preventative effects of bLF on culture-proven or probable LOS in infants with a birth weight <1500 g from both studies, and to determine the effect of bLF in relation to intake of human milk. Both trial designs had similar inclusion and exclusion criteria, the same dose of bLF [200 mg·(kg body mass)-1·day-1], and used the same control (maltodextrin). We fitted multivariate Cox regression models to estimate the effect of bLF on the risk of development of the composite outcome, adjusting for covariates. We included 335 neonates with a mean birth weight of 1162 ± 244 g; 27.5% were <1000 g. There were 33 first episodes of LOS in the bLF treatment group and 48 in the control group (19.5% vs. 28.9%). bLF had a protective effect on the risk of development of LOS [hazard ratio (HR) = 0.64; %95 CI = 0.41-0.99; p= 0.048]; particularly among infants weighing <1000 g [HR = 0.46; %95 CI = 0.22-0.96; p= 0.039] and infants with a low intake of human milk [HR = 0.40; %95 CI = 0.19-0.84; p= 0.015]. Therefore, bLF supplementation protects infants <1500 g from LOS, particularly those infants not receiving human milk.

Effects of oral bovine lactoferrin on a mouse model of inflammation associated colon cancer

Patients with ulcerative colitis or colonic Crohn's disease have a significantly increased risk of developing colorectal cancer. Bovine lactoferrin (bLF) reportedly inhibited the development of colon cancer in rats and mice, and in a placebo controlled trial, ingestion of bLF inhibited the growth of intestinal polyps. In addition, in a case study, a patient with Crohn's disease was reported to have remained in remission for over 7 years while ingesting 1g of bLF daily. Thus, bLF has an inhibitory effect on colon carcinogenesis, and it may also promote remission of Crohn's disease. The purpose of this study was to investigate the effects of bLF in a mouse model of colorectal cancer related to irritable bowel disease (IBD). The mice were divided into 4 groups: (i) no treatment; (ii) treated with bLF only; (iii) treated with azoxymethane plus dextran sulfate sodium (AOM + DSS); and (iv) treated with AOM + DSS + bLF. AOM was used to initiate intestinal cancer, and DSS was used to induce IBD-like inflammation in the intestine of the C57BL/6 mice. At the end of the study, the mice treated with AOM + DSS + bLF had a better fecal score, fewer lesions in the colon, and less weight loss than the mice treated with AOM + DSS without bLF. However, there were no statistically significant differences between the two groups with respect to tumor burden.

Bovine lactoferrin enhances osteogenesis through Smad2/3 and p38 MAPK activation

ObjectivesLactoferrin (LF) possesses diverse biological functions. We previously reported that bovine LF (bLF) attenuates lipopolysaccharide-induced bone resorption in osteoblasts. In addition to its ability to inhibit osteoclastogenesis, bLF has been implicated in stimulating bone formation. However, the molecular mechanisms of bLF in bone cell anabolism remain unclear. Here, we tried to analyse the molecular mechanisms involved in osteogenesis in the presence of bLF. MethodsAlkaline phosphatase activity, Runx2 activity, gene expression, and Alizarin red staining were analyzed to evaluate the osteogenic differentiation status. The expression of the Smads and mitogen-activated protein kinase (MAPK) signaling molecules was analyzed via western blotting. Ex vivo organ cultures of mouse calvariae were performed to evaluate the effect of bLF on bone regeneration. ResultsbLF enhanced the osteoblastic differentiation of mesenchymal stem cells through activation of Smad2/3 and p38 MAPK, which increased the transcriptional activity of Runx2. bLF treatment also enhanced osteoblastic differentiation and mineralized nodule formation of osteoblast-lineage cells, and repaired bone defects ex vivo. Moreover, inhibition of Smad2/3 or p38 MAPK signaling reduced the anabolic effects of bLF. Together, these results suggested that bLF is a potent osteogenic factor, which mediates its function via activation of the Smad2/3 and p38 MAPK signaling pathways. ConclusionsHere, we described a novel function of bLF and its signal transduction mechanisms in osseous tissue. Along with inhibiting osteoclastogenesis, bLF may limit further osteoclast formation and contribute to bone mass enlargement. Thus, bLF represents a potentially valuable therapeutic agent for bone regeneration and destructive bone diseases.

Comparative examination of levodopa pharmacokinetics during simultaneous administration with lactoferrin in healthy subjects and the relationship between lipids and COMT inhibitory activity in vitro

ABSTRACT Background: Lactoferrin (bLF) is an iron-binding multifunctional protein that is abundant in milk. In mice, it inhibits catechol-O-methyltransferase (COMT) activity and increases blood levodopa levels. However, the clinical effects are unknown. Objective: The objective of this study was to determine the effect of bLF on the kinetics of levodopa in blood. Design: The effects of the concomitant administration of a combined formulation of levodopa and an aromatic amino acid decarboxylase inhibitor and bLF on the concentration of levodopa in blood and its metabolism were assessed in eight healthy subjects. In addition, we analyzed the association with clinical factors and evaluated whether clinical factors affected the COMT inhibitory activity of bLF in vitro. Results: Although not statistically significant, the peak plasma concentration (C max) of levodopa increased by 18.5%. From the results of the stratified analysis of total cholesterol, a relationship with ΔC max was predicted. Therefore, bLF was reacted with cholesterol in the presence of lecithin and sodium deoxycholate in vitro to evaluate COMT inhibitory activity, and an increase in inhibitory activity was observed. By contrast, the ester compound cholesteryl oleate had no effect. The inhibitory activity of free fatty acids, which are known to interact with bLF, was also enhanced. Conclusion: The COMT inhibitory activity of bLF is not effective in elevating blood levodopa levels. However, in humans with high lipid levels, such as cholesterol, interactions may enhance the inhibitory effect, resulting in the enhanced absorption of levodopa. Trial registration: ID, UMIN000026787, registered 30 March 2017; URL, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000030749 Trial registration: UMIN Japan identifier: UMIN000026787

Effects of Bovine Lactoferrin on Rat Intestinal Epithelial Cells.

To determine the effects of bovine lactoferrin (bLF) on cell viability, proliferation, and the protective roles in intestinal epithelial cells-6 (IEC-6) treated by lipopolysaccharide (LPS). Cell viability and proliferation of IEC-6 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Brdu assay separately. Cell cycle distribution was analyzed by flow cytometry. Inflammatory cytokines were analysed by real-time PCR and ELISA. Western blot was utilized to measure the level of MAPK and NF-κβ nuclear translocation. Dose-dependent effects of bLF on cell viability and proliferation were observed in IEC-6 cells (both P < 0.05), especially at a dose of 100 μg/ml. The percentage of cells in the G2 and S phase was significantly higher than those of the control group (8.17 ± 0.49% vs 4.72 ± 0.55%, P < 0.01 and 12.75 ± 0.33% vs 9.48 ± 0.33%, P < 0.01, respectively). The mRNA level of IL-1β, IL-6 and TNF-α was decreased by co-stimulation of bLF and LPS compared with the LPS treatments alone in IEC-6 cells (all P < 0.001). The secretion of IL-6 and TNF-α were also decreased by co-stimulation of bLF and LPS (both P < 0.01). Bovine lactoferrin treatment at dose of 100 μg/ml could inhibit the activation of MAPK/NF-κβ signal pathway induced by LPS (both P < 0.001). Bovine lactoferrin could promote the cell viability and proliferation, and have anti-inflammatory effects via inhibition of the activation of MAPK and NF-κβ nuclear translocation. Supplementation of formula with bLF may be beneficial in preventing NEC in preterm infants.