The Difference of Milk-Derived Extracellular Vesicles from Cow Colostrum and Mature Milk on miRNAs Expression and Protecting Intestinal Epithelial Cells against Lipopolysaccharide Damage.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1ß). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.

Self-adjuvant Astragalus polysaccharide-based nanovaccines for enhanced tumor immunotherapy: a novel delivery system candidate for tumor vaccines.

The study of tumor nanovaccines (NVs) has gained interest because they specifically recognize and eliminate tumor cells. However, the poor recognition and internalization by dendritic cells (DCs) and insufficient immunogenicity restricted the vaccine efficacy. Herein, we extracted two molecular-weight Astragalus polysaccharides (APS, 12.19 kD; APSHMw, 135.67 kD) from Radix Astragali and made them self-assemble with OVA257-264 directly forming OVA/APS integrated nanocomplexes through the microfluidic method. The nanocomplexes were wrapped with a sheddable calcium phosphate layer to improve stability. APS in the formed nanocomplexes served as drug carriers and immune adjuvants for potent tumor immunotherapy. The optimal APS-NVs were approximately 160 nm with uniform size distribution and could remain stable in physiological saline solution. The FITC-OVA in APS-NVs could be effectively taken up by DCs, and APS-NVs could stimulate the maturation of DCs, improving the antigen cross-presentation efficiency in vitro. The possible mechanism was that APS can induce DC activation via multiple receptors such as dectin-1 and Toll-like receptors 2 and 4. Enhanced accumulation of APS-NVs both in draining and distal lymph nodes were observed following s.c. injection. Smaller APS-NVs could easily access the lymph nodes. Furthermore, APS-NVs could markedly promote antigen delivery efficiency to DCs and activate cytotoxic T cells. In addition, APS-NVs achieve a better antitumor effect in established B16-OVA melanoma tumors compared with the OVA+Alum treatment group. The antitumor mechanism correlated with the increase in cytotoxic T cells in the tumor region. Subsequently, the poor tumor inhibitory effect of APS-NVs on the nude mouse model of melanoma also confirmed the participation of antitumor adaptive immune response induced by NVs. Therefore, this study developed a promising APS-based tumor NV that is an efficient tumor immunotherapy without systemic side effects.

Advances in herbal polysaccharides-based nano-drug delivery systems for cancer immunotherapy.

The boom in cancer immunotherapy has provided many patients with a better chance of survival, but opportunities often come with challenges. Single immunotherapy is not good enough to eradicate tumours, and often fails to achieve the desired therapeutic effect because of the low targeting of immunotherapy drugs, and causes more side effects. As a solution to this problem, researchers have developed several nano Drug Delivery Systems (NDDS) to deliver immunotherapeutic agents to achieve good therapeutic outcomes. However, traditional drug delivery systems (DDS) have disadvantages such as poor bioavailability, high cytotoxicity, and difficulty in synthesis, etc. Herbal Polysaccharides (HPS), derived from natural Chinese herbs, inherently possess low toxicity. Furthermore, the biocompatibility, biodegradability, hydrophilicity, ease of modification, and immunomodulatory activities of HPS offer unique advantages in substituting traditional DDS. This review initially addresses the current developments and challenges in immunotherapy. Subsequently, it focuses on the immunomodulatory mechanisms of HPS and their design as nanomedicines for targeted drug delivery in tumour immunotherapy. Our findings reveal that HPS-based nanomedicines exhibit significant potential in enhancing the efficacy of cancer immunotherapy, providing crucial theoretical foundations and practical guidelines for future clinical applications.

Influence of Estrus on Dairy Cow Milk Exosomal miRNAs and Their Role in Hormone Secretion by Granulosa Cells.

Estrus is crucial for cow fertility in modern dairy farms, but almost 50% of cows do not show the behavioral signs of estrus due to silent estrus and lack of suitable and high-accuracy methods to detect estrus. MiRNA and exosomes play essential roles in reproductive function and may be developed as novel biomarkers in estrus detection. Thus, we analyzed the miRNA expression patterns in milk exosomes during estrus and the effect of milk exosomes on hormone secretion in cultured bovine granulosa cells in vitro. We found that the number of exosomes and the exosome protein concentration in estrous cow milk were significantly lower than in non-estrous cow milk. Moreover, 133 differentially expressed exosomal miRNAs were identified in estrous cow milk vs. non-estrous cow milk. Functional enrichment analyses indicated that exosomal miRNAs were involved in reproduction and hormone-synthesis-related pathways, such as cholesterol metabolism, FoxO signaling pathway, Hippo signaling pathway, mTOR signaling pathway, steroid hormone biosynthesis, Wnt signaling pathway and GnRH signaling pathway. Consistent with the enrichment signaling pathways, exosomes derived from estrous and non-estrous cow milk both could promote the secretion of estradiol and progesterone in cultured bovine granulosa cells. Furthermore, genes related to hormonal synthesis (CYP19A1, CYP11A1, HSD3B1 and RUNX2) were up-regulated after exosome treatment, while exosomes inhibited the expression of StAR. Moreover, estrous and non-estrous cow-milk-derived exosomes both could increase the expression of bcl2 and decrease the expression of p53, and did not influence the expression of caspase-3. To our knowledge, this is the first study to investigate exosomal miRNA expression patterns during dairy cow estrus and the role of exosomes in hormone secretion by bovine granulosa cells. Our findings provide a theoretical basis for further investigating milk-derived exosomes and exosomal miRNA effects on ovary function and reproduction. Moreover, bovine milk exosomes may have effects on the ovaries of human consumers of pasteurized cow milk. These differential miRNAs might provide candidate biomarkers for the diagnosis of dairy cow estrus and will assist in developing new therapeutic targets for cow infertility.

Isolation, Purification, and Structural Characterization of Polysaccharides from Codonopsis pilosula and Their Anti-Tumor Bioactivity by Immunomodulation.

The activity of polysaccharides is usually related to molecular weight. The molecular weight of polysaccharides is critical to their immunological effect in cancer therapy. Herein, the Codonopsis polysaccharides of different molecular weights were isolated using ultrafiltration membranes of 60- and 100-wDa molecular weight cut-off to determine the relationship between molecular weight and antitumor activities. First, three water-soluble polysaccharides CPPS-I (<60 wDa), CPPS-II (60-100 wDa), and CPPS-III (>100 wDa) from Codonopsis were isolated and purified using a combination of macroporous adsorption resin chromatography and ultrafiltration. Their structural characteristics were determined through chemical derivatization, GPC, HPLC, FT-IR, and NMR techniques. In vitro experiments indicated that all Codonopsis polysaccharides exhibited significant antitumor activities, with the tumor inhibition rate in the following order: CPPS-II > CPPS-I > CPPS-III. The treatment of CPPS-II exhibited the highest inhibition rate at a high concentration among all groups, which was almost as efficient as that of the DOX·HCL (10 µg/mL) group at 125 µg/mL concentration. Notably, CPPS-II demonstrated the ability to enhance NO secretion and the antitumor ability of macrophages relative to the other two groups of polysaccharides. Finally, in vivo experiments revealed that CPPS-II increased the M1/M2 ratio in immune system regulation and that the tumor inhibition effect of CPPS-II + DOX was superior to that of DOX monotherapy, implying that CPPS-II + DOX played a synergistic role in regulating the immune system function and the direct tumor-killing ability of DOX. Therefore, CPPS-II is expected to be applied as an effective cancer treatment or adjuvant therapy.