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.

High and low dose of luzindole or 4-phenyl-2-propionamidotetralin (4-P-PDOT) reverse bovine granulosa cell response to melatonin.

Background: Communication between oocytes and granulosa cells ultimately dictate follicle development or atresia. Melatonin is also involved in follicle development. This study aimed to investigate the effects of melatonin and its receptor antagonists on hormone secretion, as well as gene expression related to hormone synthesis, TGF-ß superfamily, and follicle development in bovine granulosa cells, and assess the effects of melatonin in the presence of 4-P-PDOT and luzindole. Methods: Bovine ovaries were collected from a local abattoir and follicular fluid (follicle diameter 5-8 mm) was collected for granulosa cell isolation and culture. Granulosa cells and culture medium were collected 48 h after treatment with melatonin at high dose concentrations (10-5 M) and low dose concentrations (10-9 M) in the absence/presence of 4-P-PDOT and luzindole (10-5 M or 10-9 M). Furthermore, the expression level of genes related to hormonal synthesis (CYP11A1, CYP19A1, StAR, and RUNX2), TGF-ß superfamily (BMP6, INHA, INHBA, INHBB, and TGFBR3), and development (EGFR, DNMT1A, and FSHR) were detected in each experimental group by real-time quantitative PCR. In addition, the level of hormones in culture medium were detected using ELISA. Results: Both 10-5 M and 10-9 M melatonin doses promoted the secretion of inhibin A and progesterone without affecting the production of inhibin B and estradiol. In addition, both promoted the gene expression of INHA, StAR, RUNX2, TGFBR3, EGFR, and DNMT1A, and inhibited the expression of BMP6, INHBB, CYP11A1, CYP19A1, and FSHR. When combined with different doses of 4-P-PDOT and luzindole, they exhibited different effects on the secretion of inhibin B, estradiol, inhibin A, and progesterone, and the expression of CYP19A1, RUNX2, BMP6, INHBB, EGFR, and DNMT1A induced by melatonin. Conclusion: High and low dose melatonin receptor antagonists exhibited different effects in regulating hormone secretion and the expression of various genes in response to melatonin. Therefore, concentration effects must be considered when using luzindole or 4-P-PDOT.

Luzindole and 4P-PDOT block the effect of melatonin on bovine granulosa cell apoptosis and cell cycle depending on its concentration.

Granulosa cells play an essential physiological role in mediating the follicle development and survival or apoptosis of granulosa cells dictate the follicle development or atresia. The aim of this study was to investigate the role of high dose (10-5 M) and low dose (10-9 M) melatonin in bovine granulosa cells, and assess whether MT1 and MT2 inhibiter affect granulosa cells response to melatonin. We found that the high dose (10-5 M) and low dose (10-9 M) both could act as an essential role in modulating granulosa cells apoptosis, cell cycle and antioxidant. The beneficial effect could be related to that melatonin promoted the expression of Bcl2, Bcl-xl, SOD1 and GPX4, and inhibited Bax, caspase-3 and p53 expression. Moreover P21 expression was decreased in granulosa cells treated with the high dose (10-5 M) melatonin and increased in that treated with the low dose (10-9 M) melatonin. To further reveal the role of MT1 and MT2 in mediating the effect of melatonin on granulosa cells apoptosis, cell cycle and antioxidant, we found that the luzindole and 4P-PDOT did not affect the effect of high dose (10-5 M) melatonin on regulating Bcl2, Bax, caspase-3, SOD1, GPX4 and p53 expression, while blocked its effect on modulating Bcl-xl and P21expression. However, luzindole and 4P-PDOT disturbed the effect of low dose (10-9 M) melatonin on regulating Bcl2, Bax, caspase-3, Bcl-xl, SOD1, GPX4, and p53 expression. In conclusion, these results reveal that the effect of low dose (10-9 M) melatonin on granulosa cells apoptosis are mediated by MT1 and MT2, and the high dose (10-5 M) melatonin affect the granulosa cells apoptosis by other pathway, besides MT1 and MT2. Moreover MT1 and MT2 may work in concert to modulate bovine granulosa cells function by regulating cellular progression and apoptosis.

RNAi-mediated knockdown of MTNR1B without disrupting the effects of melatonin on apoptosis and cell cycle in bovine granulose cells.

Melatonin is well known as a powerful free radical scavenger and exhibits the ability to prevent cell apoptosis. In the present study, we investigated the role of melatonin and its receptor MTNR1B in regulating the function of bovine granulosa cells (GCs) and hypothesized the involvement of MTNR1B in mediating the effect of melatonin on GCs. Our results showed that MTNR1B knockdown significantly promoted GCs apoptosis but did not affect the cell cycle. These results were further verified by increasing the expression of pro-apoptosis genes (BAX and CASP3), decreasing expression of the anti-apoptosis genes (BCL2 and BCL-XL) and anti-oxidant genes (SOD1 and GPX4) without affecting cell cycle factors (CCND1, CCNE1 and CDKN1A) and TP53. In addition, MTNR1B knockdown did not disrupt the effects of melatonin in suppressing the GCs apoptosis or blocking the cell cycle. Moreover, MTNR1B knockdown did not affect the role of melatonin in increasing BCL2, BCL-XL, and CDKN1A expression, or decreasing BAX, CASP3, TP53, CCND1 and CCNE1 expression. The expression of MTNR1A was upregulated after MTNR1B knockdown, and melatonin promoted MTNR1A expression with or without MTNR1B knockdown. However, despite melatonin supplementation, the expression of SOD1 and GPX4 was still suppressed after MTNR1B knockdown. In conclusion, these findings indicate that melatonin and MTNR1B are involved in BCL2 family and CASP3-dependent apoptotic pathways in bovine GCs. MTNR1A and MTNR1B may coordinate the work of medicating the appropriate melatonin responses to GCs.

The effects of melatonin on bovine uniparental embryos development in vitro and the hormone secretion of COCs.

Melatonin is a unique multifunctional molecule that mediates reproductive functions in animals. In this study, we investigated the effects of melatonin on bovine parthenogenetic and androgenetic embryonic development, oocyte maturation, the reactive oxygen species (ROS) levels in parthenogenetic and androgenetic embryos and cumulus-oocyte complexes (COCs) hormone secretion with melatonin supplementation at four concentrations (0, 10, 20, and 30 pmol/mL), respectively. The results showed that melatonin significantly promoted the rates of bovine parthenogenetic and androgenetic embryonic cleavage and morula and blastocysts development (P < 0.05). The rate of cleavage was higher in the androgenetic embryo than that in the parthenogenetic embryo. Compared with the parthenogenetic embryos, the androgenetic embryos had a poor developmental competence from morula to blastocyst stage. Moreover, the levels of ROS were significantly lower in the parthenogenetic and androgenetic embryoes with melatonin-treated group than that of the control group (P < 0.05). Melatonin supplemented significantly increased the maturation rate of oocyte in vitro (P < 0.05). More importantly, melatonin significantly promoted the secretion of progesterone and estradiol by COCs (P < 0.05). To reveal the regulatory mechanism of melatonin on steroids synthesis, we found that steroidogenic genes (CYP11A1, CYP19A1 and StAR) were upregulated, suggesting that melatonin regulated estradiol and progesterone secretion through mediating the expression of steroidogenic genes (CYP11A1, CYP19A1 and StAR). In addition, MT1 and MT2 were identified in bovine early parthenogenetic and androgenetic embryos using western blot. It could be concluded that melatonin had beneficial effects on bovine oocyte in vitro maturation, COC hormone secretion, early development of subsequent parthenogenetic and androgenetic embryos. It is inferred that melatonin could be used to enhance the efficiency of in vitro developed embryos.

Arsenic speciation in Chinese Herbal Medicines and human health implication for inorganic arsenic.

Rice and drinking water are recognized as the dominant sources of arsenic (As) for human intake, while little is known about As accumulation and speciation in Chinese Herbal Medicines (CHMs), which have been available for many hundreds of years for the treatment of diseases in both eastern and western cultures. Inorganic arsenic was the predominant species in all of CHMs samples. The levels of inorganic arsenic in CHMs from fields and markets or pharmacies ranged from 63 to 550 ng/g with a mean of 208 ng/g and 94 to 8683 ng/g with a mean of 1092 ng/g, respectively. The highest concentration was found in the Chrysanthemum from pharmacies. It indicates that the risk of inorganic As in CHMs to human health is higher in medicines from markets or pharmacies than that collected directly from fields. Some CHMs may make a considerable contribution to the human intake of inorganic arsenic.

[Survey and assessment of heavy metals in soils and herbal medicines from Chinese herbal medicine cultivated regions].

Concentrations of As, Hg, Pb, Cd in soils and herbal medicine samples from cultivated regions of Anguo City in Hebei Province were analyzed and assessed, and the bioconcentration factors of different herbal medicines were studied and discussed as well. The results showed that the average contents of As, Hg, Pb, Cd in soils from herbal medicine cultivated regions were 12.9, 0.036, 15.6, 0.118 mg x kg(-1), respectively. Concentrations of heavy metals in soils were lower than class II of the soil environmental quality standard. When local soil background values of heavy metals were used as assessment standard, among the 16 cultivated regions the percentage of As, Hg, Ph, Cd belonging to lightly pollution class were 18.75%, 43.75%, 0%, 100%, respectively based on the single pollution index. And the Nemerow index results were between 1 and 2, which suggested the soils were at slight pollution level. However, when quality standard class II was used, both the single pollution index and Nemerow index did not exceed 0.7, which means that soils investigated were generally safe for cultivation of Chinese herbal medicines. The assessment of heavy metals in herbal medicines showed that the pollution indices of most herbal samples (< 95%) were lower then 1. Cd bioconcentration factors of Aster tataricus L. and Anemarrhena asphodeloides Bunge, Hg bioconcentration factors of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. and Glehnia littoralis F. Schmidt ex Miq. were above 1. Therefore, the accumulation characteristic of heavy metals in Chinese herbal medicines should be fully concerned when GAP base soil quality assessment was taken.

[Survey of arsenic concentrations in Chinese herbal medicines (CHMs) and preliminary risk assessment of As in CHMs on human health].

Characteristics and concentrations of As in Chinese Herbal Medicines (CHMs) from Hebei province (including samples collected from fields and from medicine markets) were investigated, and the ADI (allowable daily intake) standard value from FAO/WHO was used for preliminary assessment of As risk on human health. The results showed that the average of As concentrations in different roots which were used as medicines, ranged from 0.14 mg/kg to 0.54 mg/kg,while for shoots which were used as medicines, average value of As concentrations in Dendranthema morifolium was 1.42 mg/kg, and the range in other species was from 0.09 mg/kg to 0.27 mg/kg. Based on Green Standards of Medicinal Plants and Preparations for Foreign Trade and Economy (2.0 mg/kg), the percentage of samples with As concentrations exceeding standard was 3.27% (n = 153) in roots and 9.09% (n = 44) in shoots,and 4.57% (n = 197) in total CHMs samples. As concentrations in CHMs from fields ranged from 0.03 mg/kg to 0.73 mg/kg,which were all lower than 2.0 mg/kg. However, As concentrations in CHMs from markets ranged from 0.05 mg/kg to 7.05 mg/kg, and the concentrations of As in 6.36% of samples exceeded the limited standard. Arsenic concentrations in samples from markets were significantly higher than those from fields (p < 0.05). As concentrations in Compositae were distributed widely from 0.08 mg/kg to 7.05 mg/kg,and the average at 0.87 mg/kg,while the average of As concentrations in other six families were between 0.21 mg/kg and 0.41 mg/kg, and As levels in 75% of samples for each family were below 0.5 mg/kg. Compared to Compositae samples, the CHMs from other families have higher security. ADI regulated by FAO/WHO was used to assess As risk in CHMs on human health in this study as well. As daily intake through CHMs from markets varied from 0.90 microg/d to 19.7 microg/d, and percentages of the daily intake of As in CHMs to ADI were in the range of 0.70% to 15.4%, which indicated that As in CHMs from markets had a potential risk on human health after intake.