MAPK Signaling Pathway Plays Different Regulatory Roles in the Effects of Boric Acid on Proliferation, Apoptosis, and Immune Function of Splenic Lymphocytes in Rats.

Boron is one of the essential trace elements in animals. Although boron supplementation can enhance immune function and promote cell proliferation, high-dose boron supplementation can negatively affect immune function and inhibit cell proliferation. Furthermore, its action pathway is unknown. In this study, ERK1/2, JNK, and p38MAPK signaling pathways were blocked using specific blockers to investigate the impact of low-dose and high-dose boron on proliferation, apoptosis, and immune function of lymphocytes, and the expression of genes related to cell proliferation and apoptosis in rats. The addition of 0.4 mmol/L boron did not affect the ratio of CD4+/CD8+ T cells (P>0.05), IgG and IFN-γ contents (P>0.05), the proliferation rate of lymphocytes (P>0.05), and mRNA and protein expressions of PCNA (P>0.05) in the spleen after ERK1/2 signal pathway was selectively inhibited. Moreover, the addition of 40 mmol/L boron did not affect the proportion of CD4+ T cells, contents of IgG and cytokines (IL-2 and IL-4), proliferation and apoptosis rates of lymphocytes, and expression of proliferation- and apoptosis-related genes in the spleen. Meanwhile, the addition of 0.4 mmol/l boron increased the ratio of CD4+/CD8+ T cells (P<0.05 or P<0.01), IFN-γ or IgG contents (P<0.05), and the proliferation rate of lymphocytes (P<0.05) in spleen after selective inhibition of JNK or p38MAPK signaling pathways, while the protein expression of Caspase-3 decreased (P<0.05 or P<0.01). Furthermore, 40 mmol/L boron decreased the proportion of lymphocyte subsets, cytokine contents, proliferation rate of lymphocytes, and mRNA and protein expressions of PCNA. In contrast, the mRNA and protein expressions of Caspase-3 and protein expression of Bax were increased. These results indicate that ERK1/2 signaling pathway mainly regulates the effects of low-dose and high-dose boron on proliferation, apoptosis, and immune function of splenic lymphocytes.

Proteomic Analysis of Rat Duodenum Reveals the Modulatory Effect of Boron Supplementation on Immune Activity.

The proper supplementation of boron, an essential trace element, can enhance animal immune function. We utilized the method of TMT peptide labeling in conjunction with LC-MS/MS quantitative proteomics for the purpose of examining the effects of boric acid on a rat model and analyzing proteins from the duodenum. In total, 5594 proteins were obtained from the 0, 10, and 320 mg/L boron treatment groups. Two hundred eighty-four proteins that exhibit differential expression were detected. Among the comparison, groups of 0 vs. 10 mg/L, 0 vs. 320 mg/L, and 10 vs. 320 mg/L of boron, 110, 32, and 179 proteins, respectively, demonstrated differential expression. The results revealed that these differential expression proteins (DEPs) mainly clustered into two profiles. GO annotations suggested that most of the DEPs played a role in the immune system process, in which 2'-5'-oligoadenylate synthetase-like, myxovirus resistance 1, myxovirus resistance 2, dynein cytoplasmic 1 intermediate chain 1, and coiled-coil domain containing 88B showed differential expression. The DEPs had demonstrated an augmentation in the signaling pathways, which primarily include phagosome, antigen processing, and presentation, as well as cell adhesion molecules (CAMs). Our study found that immune responses in the duodenum were enhanced by lower doses of boron and that this effect is likely mediated by changes in protein expression patterns in related signaling pathways. It offers an in-depth understanding of the underlying molecular mechanisms that lead to immune modulation in rats subjected to dietary boron treatment.

Transcriptome Profiling of Duodenum Reveals the Importance of Boron Supplementation in Modulating Immune Activities in Rats.

As an essential trace element, appropriate boron supplementation can promote immune function of animals. To illustrate the effects of boron in a rat model, RNA-Seq was conducted for the RNA from duodenum after treatment with different concentration of boron in which boron was given in the form of boric acid. More than 47 million reads were obtained in 0, 10, and 320 mg/L boron (0, 57.21, and 1830.66 mg/L boric acid) treatment groups that produced 58 965 402, 48 607 328, and 46 760 660 clean reads, respectively. More than 95% of the clean reads were successfully matched to the rat reference genome and assembled to generate 32 662 transcripts. A total of 624 and 391 differentially expressed candidate genes (DEGs) were found between 0 vs.10 and 0 vs. 320 mg/L boron comparison groups. We also identified transcription start site, transcription terminal site, and skipped exons as the main alternative splicing events. GO annotations revealed most of DEGs were involved in the regulation of immune activity. The DEGs were enriched in influenza A, herpes simplex infection, cytosolic DNA-sensing pathway, and antigen processing and presentation signaling pathways. The expression levels of genes enriched in these signaling pathways indicate that lower doses of boron could achieve better effects on promoting immune response in the duodenum. These effects on the immune system appear to be mediated via altering the expression patterns of genes involved in the related signaling pathways in a dose-dependent pattern. These data provide more insights into the molecular mechanisms of immune regulation in rats in response to dietary boron treatment.

GPR30 mediated effects of boron on rat spleen lymphocyte proliferation, apoptosis, and immune function.

Supplementing different quantities of boron can significantly affect immune function in rat spleen, but the mechanism of action behind this effect remains unclear. Our purpose was to study the involvement of the estrogen membrane receptor GPR30 in the effect of boron on the proliferation, apoptosis, and immune function of rat spleen lymphocytes. Results showed that the addition of 0.4 mmol/L boron had a beneficial effect on the immune function and proliferation of spleen lymphocytes, but the addition of 40 mmol/L boron had opposite effect. After using G-15 to selectively inhibit GPR30, the proportions of CD4+ and CD8+ T cells, the content of IL-2 and IFN-γ, and the expression of PCNA protein were significantly decreased, while lymphocyte apoptosis rate increased significantly (p < 0.05 or p < 0.01). After G-15 treatment, the addition of 0.4 mmol/L boron had no effects on T cell subsets, lymphocyte proliferation, PCNA protein expression, and IgG and cytokine content (P > 0.05), while the addition of 40 mmol/L boron did not change the effects on lymphocyte subsets, proliferation and apoptosis. The results suggested that GPR30 mediates the effects of 0.4 mmol/L boron boron on the proliferation, apoptosis and immune function of spleen lymphocytes.

Effects of Boron on Cytotoxicity, Apoptosis, and Cell Cycle of Cultured Rat Sertoli Cells In vitro.

The present study aimed to investigate the effects of the administration of boron on viability, apoptosis, and cell cycle of primary rat Sertoli cells (SCs) in vitro. SCs were aseptically isolated from 18-22-day-old male Sprague-Dawley (SD) rats. SCs were identified with immunofluorescence using anti-vimentin antibody. Further, to investigate the effects of boron on Sertoli cells, SCs of the boron treatment group were exposed to different concentrations (0.25, 0.5, 1, 5, 10, 40, and 80 mmol/L) of boric acid. Using MTT and Cell Counting Kit-8 assays, the impact of boron on SCs viability was analyzed. Cell apoptosis and cycle of SCs were analyzed using flow cytometry. A concentration of 0.5 mmol/L boric acid resulted in the highest viability and lowest necrosis and apoptosis. Above this concentration (even 1.0 mmol/L) showed lower viability and higher levels of necrosis and apoptosis. Administration of < 0.5 mmol/L boron significantly promoted the viability of Sertoli cells (P < 0.01); however, the exposure to high dose (> 10 mmol/L) of boron exhibited significant adverse effects on Sertoli cells (P < 0.01) and even toxic effects, inhibiting cell viability compared to the control group. Flow cytometry analysis showed that treatment with 0.5 mmol/L of boron significantly inhibited the apoptosis of Sertoli cells and the proportion of cells in S and G2/M phases was markedly increased; however, a higher concentration of 40 and 80 mmol/L of boron promoted Sertoli cell apoptosis and cells were completely arrested at G0/G1 phase. Boron at doses below 0.5 mmol/L could significantly improve the viable capacity of testicular Sertoli cells in vitro and inhibit their apoptosis. However, high dose of boron (at a concentration higher than 5.0 mmol/L) exhibited noticeable toxic effects, inhibiting cell viability, accelerating apoptosis of Sertoli cells, and arresting cell cycle at G0/G1 phase.

Isoleucine attenuates infection induced by E. coli challenge through the modulation of intestinal endogenous antimicrobial peptide expression and the inhibition of the increase in plasma endotoxin and IL-6 in weaned pigs.

Enteric infection is a major cause of morbidity and mortality in both humans and animals worldwide. Immunotherapy against intestinal infection is a well-known alternative to the antibiotic strategy. Herein, we demonstrated that isoleucine significantly suppressed the multiplication of E. coli in the presence of IPEC-J2 cells. Isoleucine supplementation enhanced the concentrations of total plasma protein and IgA in pigs compared to the alanine control diet, while inhibiting the increase in plasma endotoxin and IL-6 contents induced by E. coli challenge. A significant interaction between the E. coli challenge and the diet treatment was found in the red blood cell volume. Isoleucine improved the expression of porcine ß-defensin-1 (pBD-1), pBD-2, pBD-3, pBD-114 and pBD-129 in the jejunum and ileum of pigs with or without E. coli challenge. Conclusively, isoleucine attenuated the infection caused by the E. coli challenge possibly through increasing the intestinal ß-defensin expression and inhibiting the increase in plasma endotoxin and IL-6 in weaned pigs.

Effects of Selenium Yeast in Combination with Boron on Muscle Growth and Muscle Quality in Broilers.

The effect of selenium yeast in combination with boron on both growth and quality of the muscle in broilers was investigated. A total of 600 one-day-old Arbor Acres broilers were randomly divided into five groups with 120 broilers per group (6 replicates per group). The control group received a basal diet, and experimental groups I-IV received the same basal diet supplemented with 0.3 mg/kg selenium yeast and different doses of boron (0, 5, 10, and 15 mg/kg, respectively). The experiment was conducted for 42 days. Breast and thigh muscles were harvested and muscle quality were examined on day 21 and day 42 of the experiment. Compared to the control group, at 21 days of age, the thigh muscle weight and index were significantly increased in broilers of experimental group II (all P < 0.05); however, the drip loss and shear force of breast and thigh muscle were significantly decreased (P < 0.05). At 42 days of age, the breast muscle weight and index as well as the breast and thigh muscle water holding capability had significantly increased in broilers of experimental group II (all P < 0.05); the breast and thigh muscle drip loss, cooking loss and shear force, and thigh muscle fiber diameter were significantly reduced (all P < 0.05). Breast and thigh muscle fibers were tightly arranged with small cross-sectional areas in broilers of experimental group II. These results suggest that supplementation of 0.3 mg/kg selenium yeast in combination with 5 mg/kg boron in the basal diet can promote muscle growth and improved muscle quality in broilers.

Effect of Boron on Thymic Cytokine Expression, Hormone Secretion, Antioxidant Functions, Cell Proliferation, and Apoptosis Potential via the Extracellular Signal-Regulated Kinases 1 and 2 Signaling Pathway.

Boron is an essential trace element in animals. Appropriate boron supplementation can promote thymus development; however, a high dose of boron can lead to adverse effects and cause toxicity. The influencing mechanism of boron on the animal body remains unclear. In this study, we examined the effect of boron on cytokine expression, thymosin and thymopoietin secretion, antioxidant function, cell proliferation and apoptosis, and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in the thymus of rats. We found that supplementation with 10 and 20 mg/L boron to the drinking water significantly elevated levels of interleukin 2 (IL-2), interferon γ (IFN-γ), interleukin 4 (IL-4), and thymosin α1 in the thymus of rats (p < 0.05), increased the number of positive proliferating cell nuclear antigen (PCNA+) cells and concentrations of glutathione peroxidase (GSH-Px) and phosphorylated extracellular signal-regulated kinase (p-ERK) (p < 0.05), and promoted mRNA expression of PCNA and ERK1/2 in thymocytes (p < 0.05). However, the number of caspase-3+ cells and the expression level of caspase-3 mRNA were reduced (p < 0.05). Supplementation with 40, 80, and 160 mg/L boron had no apparent effect on many of the above indicators. In contrast, supplementation with 480 and 640 mg/L boron had the opposite effect on the above indicators in rats and elevated levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 1ß (IL-1ß), and tumor necrosis factor α (TNF-α) (p < 0.05). Our study showed that supplementation of various doses of boron to the drinking water had a U-shaped dose-effect relationship with thymic cytokine expression, hormone secretion, antioxidant function, cell proliferation, and apoptosis. Specifically, supplementation with 10 and 20 mg/L boron promoted thymocyte proliferation and enhanced thymic functions. However, supplementation with 480 and 640 mg/L boron inhibited thymic functions and increased the number of apoptotic thymocytes, suggesting that the effects of boron on thymic functions may be caused via the ERK1/2 signaling pathway.

Boron Affects Immune Function Through Modulation of Splenic T Lymphocyte Subsets, Cytokine Secretion, and Lymphocyte Proliferation and Apoptosis in Rats.

This study demonstrated the mechanisms of boron effects in a rat model and provided a scientific basis for the rational of boron use. These findings were achieved by investigating the effects of boron (10, 20, 40, 80, 160, 320, and 640 mg/L in drinking water or 1.5, 3, 6, 12, 24, 48, and 96 mg/kg BW) on rat serum immunoglobulins (IgGs), splenic cytokines, lymphocyte subsets, as well as on lymphocyte proliferation and apoptosis. Addition of 20 (3) and 40 (6) mg/L (mg/kg BW) of boron to drinking water significantly increased rat serum IgG concentrations, splenic IFN-γ and IL-4 expression as well as the number of splenic CD3+, CD4+ and proliferating cell nuclear antigen (PCNA)+ cells. Supplementation of drinking water with 40 mg/L (6 mg/kg BW) boron also markedly increased splenic IL-2 expression and the CD4+/CD8+ cell ratio and reduced splenic CD8+ cell number. Supplementation with 80 mg/L (12 mg/kg BW) boron significantly increased CD3+ and PCNA+ cell numbers (P < 0.05) and decreased the IL-10 expression in the spleen. Addition of 320 (48) and 640 (96) mg/L (mg/kg BW) boron markedly reduced the serum IgG concentrations; splenic IL-2 and IL-10 expression; the number of CD3+, CD4+ and PCNA+ cells; and increased the number of splenic CD8+ and caspase-3+ cells and promoted caspase-3 expression in CD3+ cells. In conclusion, these findings suggest that the supplementation of rat drinking water with 20(3) and 40(6) mg/L (mg/kg BW) boron can markedly enhance humoral and cellular immune functions, while boron concentrations above 320 mg/L (48 mg/kg BW) can have an inhibitory effect or even toxicity on immune functions. These results exhibit a U-shaped response characteristic of low and high doses of boron supplementation on immune function and imply that proper boron supplementation in food for humans and animals could be used as an immunity regulator.

[Effects of boron on serum biochemical indexes and microstructure of immune organs in male obese rats].

OBJECTIVE: Effects of boron( B) on the serum biochemical index and microstructure of immune organs in male obese rats were studied. METHODS: 40 male SD rats( 3-month old) were divided into five groups: normal control group, high-fat-diet control group and boron supplemental group of low, medium and high dose, randomly. The normal control group were fed with normal diet, the other 4 groups were fed with highfat diet to establish the model of obesity for 8 weeks. The boron supplemental group of low, medium and high dose were supplemented 20, 40 and 80 mg B / L in drinking water for 90 d, respectively. At the end, the rats were anesthetized and bled. The blood were collected from right atrium to detected the biochemical indexes related to liver function, and the thymus and spleen were obtained to weighted and fixed, then the samples were made into paraffin sections, stained with hematoxylin-eosin( HE) stain, observed and measured the histological parameters of immune organs. RESULTS: Compared with normal control group, the Lee's index and abdominal fat rate, the level of serum low density lipoprotein cholesterol( LDL-c) and the thymus weight were significantly increased( P <0. 05), but the level of serum total protein( TP) and high density lipoprotein cholesterol( HDL-c) were significantly decreased( P < 0. 05) in high-fat-diet control group, 40 mg /L and 80 mg / L supplement groups of boron. However, these detection indexes did not change significantly( P > 0. 05) in 20 mg / L supplement groups of boron. Compared with the high-fat-diet control group, the Lee's index and abdominal fat rate, the level of serum Apolipoprotein B( apo B) and LDL-c, the thymus weight and index were significantly decreased( P < 0. 05), but the level of serum TP and high density lipoprotein cholesterol( HDL-c) were significantly increased( P < 0. 05) in 20 mg /L supplement groups of boron. The level of serum LDL-c and thymus weight was significantly lower( P < 0. 05) in40 mg / L supplement groups of boron. But all the above detection indicators did not change significantly( P > 0. 05) in 80 mg / L supplement groups of boron. Under the microscope, compared with high-fat-diet control group, splenic nodule area was increased significantly( P < 0. 05), splenic periarterial lymphatic sheath, marginal zone and splenic cord were also thicker significantly( P < 0. 05), thymus medulla / cortex ratio decreased significantly( P < 0. 05), the cells arranged closely, vacuolar like structures were less in the thymus medulla of 20 mg / L and 40 mg / L supplement groups of boron. Microstructure of spleen and thymus did not change significantly in 80 mg / L supplement groups of boron. CONCLUSION: Supplementation of 20 mg B / L could decrease the level of serum apo B and LDL-c, and increase the level of serum HDL-c, and protect the liver function and immune organ of rat from damage caused obese by high fat diet.

Daidzein enhances immune function in late lactation cows under heat stress.

Heat stress decreases natural immunity making cows more vulnerable to diseases. A previous study reported that daidzein can enhance animal resistance to heat stress and regulate animal immunocompetence. However, it is unclear whether daidzein regulates the immune performance of late lactation cows under heat stress. In this study, late lactation cows in four groups were raised in hot weather and fed with basic diet, basic diet plus 200, 300, 400 mg/day daidzein, respectively, and the experimental period was 60 days. Blood was collected to examine the changes of serum total protein (TP), albumin (ALB), immunoglobulin G (IgG), interferon alpha (IFN-α), and interleukin-2 (IL-2). We found the levels of serum IgG and INF-α were significantly higher in late lactation cows after 300 and 400 mg/day daidzein treatment compared to those in the control group and 200 mg/day daidzein treatment (P < 0.05 or P < 0.01). Moreover, 300 and 400 mg/day daidzein treatment markedly increased serum IL-2 (P < 0.01), while the levels of serum TP and ALB were not changed by any concentration of daidzein treatment (P > 0.05). Daidzein can enhance the immunocompetence of late lactation cows and strengthen cow resistance to heat stress.

[Effect of drinking boron on microtructure of adrenal gland in rats].

OBJECTIVE: The effects of drinking boron exposure on the mass, organ indexes and structure of adrenal gland were studied in the paper. Methods 192 Sprague-Dawley rats (28 +/- 2 days) with no bacteria infecting were divided into six groups (n = 32, male = female) randomly. Treated rats drunk the distilled water which supplemented with boron of 0, 40, 80, 160, 320 and 640 mg/L, respectively, for 60 days. At the 30th and the 60th day of experiment, 16 rats (n = 8, male = female) of each group were selected and made into narcosis with 10% Chloral Hydrate. The adrenal glands were obtained, weighted and fixed after dissection, then the samples were made into paraffin sections, stained with HE stain and chromaffin, observed and photographed by Olympus CH-30 microphotograph system. RESULTS: Compared with control group, the average mass of adrenal gland of male rats in each experiment group decreased significantly or most significantly at the 30th day of experiment (P < 0.05 or P < 0.01), but the index of adrenal gland of male rats in the group of 640 mg/L boron at 60th day of experiment increased significantly (P < 0.05). Under the microscope, the microstructure of adrenal gland of rats in the group of 40 mg/L boron were better obviously than control group, and the numbers of chromaffin granules in chromaffin cell increased obviously. The histopathological changes of different degree could be observed in the group of 80 to 640 mg/L boron, and they became remarkable with the boron supplementation. By comparative observation, the damage of cells in adrenal medulla appeared ahead of them in adrenal cortex, and the pathological change of adrenal gland in male rats were obvious than female rats. CONCLUSION: Drinking supplemented with 40 mg/L boron could prompt the structure of adrenal gland in rats, but could cause different degree damage, or even obvious toxic effect when the concentration of boron supplementation in drinking from 80 to 640 mg/L.

[Effect of drinking boron on blood composition in rats].

OBJECTIVE: To observe the effects of boron supplemented in water on the blood composition in rats. METHODS: 192 Sprague-Dawley rats (28 +/- 2) d were randomly divided into six groups (n=32, 16 male rats and 16 female rats), and drank the distilled water supplemented with 0, 40, 80, 160, 320 and 640 mg/L boron for 60 days, respectively. At the 30th and the 60th day of experiment period, 16 rats (n=8, male=female) from each group were selected and made into narcosis with 10% Chloral Hydrate. Then the blood were obtained from the right atrium after dissection, the blood smear were made and stained with Wright's and Giemsa's stains at the same time. Then the red cells count, the white cells count, the reticulocyte counts, the hemoglobinometry and the differential leukocytes count were determined. RESULTS: In comparison with control group, the reticulocyte percentages of all experiment groups exhibited different degree increases, the RBC numbers and Hb contents in rats consuming 40 and 80 mg/L of boron more significantly raised (P < 0.05), but the changes of the leucocyte percentages were more no significant. The numbers of RBC and WBC and the Hb contents in rats supplemented with from 160 to 640 mg/L of boron all decreased significantly or most significantly (P < 0.05 or P < 0.01), and the percentages of neutrophil, eosinophil, basophil and monocytic showed similar changes on the 60th day of experiment time. CONCLUSION: The distilled water supplemented with 40 mg/L of boron could promote the hemopoiesis and Hb synthesis in rats, and the drinking water supplemented with 80 mg/L of boron would have some promotion on the erythropoiesis and Hb synthesis; but when the boron supplementation from 160 to 640 mg/L, the higher doses boron could inhibit the hemopoiesis and Hb synthesis obviously, or even exhibit apparent toxicity action.

Trophic effect of bee pollen on small intestine in broiler chickens.

In this study, the effects of bee pollen on the development of digestive organs were evaluated in broiler chickens. A total of 144 1-day-old AA broiler chickens were randomly and equally divided into two groups, assigned as the control group and the pollen group, respectively. The control group was fed with a basic diet, while the pollen group was fed with a basic diet supplemented with 1.5% bee pollen over a period of 6 weeks. At the end of each week, the digestive organs were obtained for comparison from 12 broilers randomly selected from each group. The results demonstrated that compared to the control group, the small intestine villi from the duodenum, jejunum, and ileum were longer and thicker in the pollen group. This difference was more significant during early development, especially through the first 2 weeks. Bee pollen increased the length of the villi by 37.1% and 29.4% in the duodenum, 28.1% and 33.7% in the jejunum, and 18.6% and 16.2% in the ileum in week 1 and 2, respectively. Furthermore, the small intestinal glands were developed at a higher density in the pollen group, and the depth of the glands was significantly increased by bee pollen in the first 2 weeks. These findings suggest that bee pollen could promote the early development of the digestive system and therefore is a potentially beneficial food supplement for certain conditions, such as short bowel syndrome.

[Effect of bee pollen on development of immune organ of animal].

OBJECTIVE: To study on the effect of been pollen on development of immune organ of animal. METHOD: A total of 144 one day-old broilers were randomly divided into 2 groups, in which each group included 72 chickens. The control group was fed on the basal diet for 42 days, and that of experiment group supplemented 1.5% bee pollen. Six chickens in each group were selected and slaughtered at 7, 14, 21, 28, 35, 42 days respectively, and the thymuses, cloacal bursa and spleens were obtained, weighted, fixed in Bouin liquid and made into paraffin section. RESULT: Compared with control group, the weight and the relative weight of thymuses, cloacal bursa and spleens of experiment group increased significantly (P < 0.05) or extremely significantly (P < 0.01). In experiment group, the cortex of thymic lobule, bursa nodule and Periarterial Lymphatic Sheaths thicken obviously; the volume of bursa nodule, splenic nodule and ellipsoid augmented, and the germinal center of splenic nodule were obvious; the thymic corpuscle increased; the plica of cloacal bursa developed well and the degenerating of it retarded. CONCLUSION: The diet supplemented bee pollen could boost the early development of thymus and cloacal bursa, retard the degenerating of cloacal bursa and promote the immune response of spleen.