The effect of selenium on the proliferation of bovine endometrial epithelial cells in a lipopolysaccharide-induced damage model.

BACKGROUND: Endometritis is a common bovine postpartum disease. Rapid endometrial repair is beneficial for forming natural defense barriers and lets cows enter the next breeding cycle as soon as possible. Selenium (Se) is an essential trace element closely related to growth and development in animals. This study aims to observe the effect of Se on the proliferation of bovine endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS) and to elucidate the possible underlying mechanism. RESULTS: In this study, we developed a BEECs damage model using LPS. Flow cytometry, cell scratch test and EdU proliferation assay were used to evaluate the cell cycle, migration and proliferation. The mRNA transcriptions of growth factors were detected by quantitative reverse transcription-polymerase chain reaction. The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/ß-catenin pathways were detected by Western blotting and immunofluorescence. The results showed that the cell viability and BCL-2/BAX protein ratio were significantly decreased, and the cell apoptosis rate was significantly increased in the LPS group. Compared with the LPS group, Se promoted cell cycle progression, increased cell migration and proliferation, and significantly increased the gene expressions of TGFB1, TGFB3 and VEGFA. Se decreased the BCL-2/BAX protein ratio, promoted ß-catenin translocation from the cytoplasm to the nucleus and activated the Wnt/ß-catenin and PI3K/AKT signaling pathways inhibited by LPS. CONCLUSIONS: In conclusion, Se can attenuate LPS-induced damage to BEECs and promote cell proliferation and migration in vitro by enhancing growth factors gene expression and activating the PI3K/AKT and Wnt/ß-catenin signaling pathways.

The effect and mechanism of selenium supplementation on the proliferation capacity of bovine endometrial epithelial cells exposed to lipopolysaccharide in vitro under high cortisol background.

Bovine endometritis severely inhibits uterine repair and causes considerable economic loss. Besides, parturition-induced high cortisol levels inhibit immune function, reduce cell proliferation, and further inhibit tissue repair. Selenium (Se) is an essential trace element for animals to maintain normal physiological function and has powerful antioxidant functions. This study investigated whether Se supplementation reduces endometrial damage and promotes tissue repair in cows with endometritis under stress and explored the underlying mechanism. Primary bovine endometrial epithelial cells were isolated and purified from healthy cows. The cells were treated with different combinations of lipopolysaccharide (LPS), cortisol, and various concentrations of Se. Data showed that LPS stimulation inhibited cell proliferation and increased cell apoptosis. High levels of cortisol further exacerbated these effects. Flow cytometry, scratch wound healing tests, and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays showed that Se supplementation promoted cell cycle progression, cell migration, and cell proliferation in the presence of LPS and cortisol. The quantitative PCR results showed that the expression of related growth factors was increased after Se supplementation. After administering various inhibitors, we further demonstrated that Se supplementation decreased the activity of glycogen synthetase kinase 3ß (GSK-3ß) through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway to reduce the degradation of ß-catenin except the Wnt signal to promote cell proliferation. In conclusion, Se supplementation attenuated the cell damage induced by LPS at high cortisol levels and increased cell proliferation to promote uterine repair by elevating the mRNA expression of TGFB3 and VEGFA and activating the PI3K/AKT/GSK-3ß/ß-catenin signaling pathway.

After parturition, endometritis is a common bovine disease, which hinders endometrial repair and reduces bovine economic value. Besides, parturition-induced high cortisol levels cause immunosuppression, aggravate infection, and further inhibit cell proliferation and tissue repair. As an essential trace element, adding selenium to feed helps to maintain the normal physiological function of animals. This study developed a cellular model using lipopolysaccharide (LPS) and cortisol to simulate cows with endometritis in stress conditions. The results showed that Se supplementation attenuated bovine endometrial epithelial cell damage and promoted their proliferation in the presence of LPS and high cortisol levels, which are positively correlated with the concentration of Se. Besides, this study proved another molecular mechanism for Se to regulate ß-catenin except for the Wnt signal by affecting the ß-catenin degradation pathway.

Selenomethionine Inhibits NF-κB-mediated Inflammatory Responses of Bovine Mammary Epithelial Cells Caused by Klebsiella pneumoniae by Increasing Autophagic Flux.

Klebsiella pneumoniae (K. pneumoniae) is one of the major pathogens causing bovine clinical mastitis. Autophagy maintains cellular homeostasis and resists excessive inflammation in eukaryotic organisms. Selenomethionine (Se-Met) is commonly used as a source of selenium supplementation for dairy cows. This study aimed to investigate the effects of Se-Met on inflammatory responses mediated by nuclear factor-kappa B (NF-κB) through autophagy. We infected bovine mammary epithelial cell line (MAC-T) with K. pneumoniae and examined the expression of autophagy-related proteins and changes in autophagic vesicles, LC3 puncta, and autophagic flux at various intervals. The results showed that K. pneumoniae activated the early-stage autophagy of MAC-T cells. The levels of LC3-II, Beclin1, and ATG5, as well as the number of LC3 puncta and autophagic vesicles, increased after 2 h post-treatment. However, the late-stage autophagic flux was blocked. Furthermore, the effect of autophagy on NF-κB-mediated inflammation was investigated with different autophagy levels. The findings showed that enhanced autophagy inhibited the K. pneumoniae-induced inflammatory responses of MAC-T cells. The opposite results were found with the inhibition of autophagy. Finally, we examined the effect of Se-Met on NF-κB-mediated inflammation based on autophagy. The results indicated that Se-Met alleviated K. pneumoniae-induced autophagic flux blockage, inhibited NF-κB-mediated inflammation, and decreased the adhesion of K. pneumoniae to MAC-T cells. The inhibitory effect of Se-Met on NF-κB-mediated inflammation could be partially blocked by the autophagy inhibitor chloroquine (CQ). Overall, Se-Met attenuated K. pneumoniae-induced NF-κB-mediated inflammatory responses by enhancing autophagic flux.

The Effect of Selenium on Endometrial Repair in Goats with Endometritis at High Cortisol Levels.

Endometritis is a common postpartum disease of female animals that causes significant losses to the goat industry. High levels of cortisol induced by various stresses after delivery severely inhibit innate immunity and tissue repair. The repair ability of the endometrium is closely related to the reproductive performance of goats. Selenium (Se) is an essential trace element in animals that has powerful antioxidant and immunity-enhancing functions. In this study, we established a goat model of endometritis at high cortisol (Hydrocortisone) levels to investigate the effect of Se (supplement additive) on endometrial repair. The results showed that the clinical symptoms, %PMN in uterine secretions, morphological endometrial damage, and the gene expression of BAX were reduced in the goats with Se supplementation compared with those in the model group. Se increased the gene expression of BCL2, VEGFA, TGFB1, and PCNA and activated the PI3K/AKT and Wnt/ß-catenin signaling pathways in goats with Se supplementation. In conclusion, Se reduced the inflammatory response, increased the proliferation, and decreased the apoptosis of endometrial cells to promote endometrial tissue repair in goats with endometritis at high cortisol levels. It probably achieved this effect of promoting repair by activating the Wnt/ß-catenin and PI3K/AKT pathways and affecting the gene expression of VEGFA, TGFB1, PCNA, BCL2, and BAX.

Selenium suppressed the LPS-induced inflammation of bovine endometrial epithelial cells through NF-κB and MAPK pathways under high cortisol background.

The bovine uterus is susceptible to infection, and the elevated cortisol level due to stress are common in cows after delivery. The essential trace element selenium plays a pivotal role in the antioxidant and anti-inflammatory defence system of body. This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high-level cortisol. The primary bovine endometrial epithelial cells were subjected to Escherichia coli lipopolysaccharide to establish cellular inflammation model. The gene expression of inflammatory mediators and proinflammatory cytokines was measured by quantitative PCR. The key proteins of NF-κB and MAPK signalling pathways were detected by Western blot and immunofluorescence. The result showed that pre-treatment of Na2 SeO3 (1, 2 and 4 µΜ) decreased the mRNA expression of proinflammatory genes, inhibited the activation of NF-κB and suppressed the phosphorylation of extracellular signal-regulated kinase, P38MAPK and c-Jun N-terminal kinase. This inhibition of inflammation was more apparent in the presence of high-level cortisol (30 ng/mL). These results indicated that selenium has an anti-inflammatory effect, which is mediated via NF-κB and MAPK signalling pathways and is augmented by cortisol in bovine endometrial epithelial cells.

Selenomethionine alleviates NF-κB-mediated inflammation in bovine mammary epithelial cells induced by Escherichia coli by enhancing autophagy.

Autophagy is crucial for the maintenance of homeostasis under stimuli related to infection. Selenium (Se) plays variable roles in defence against infection and Selenomethionine (Se-Met) is a common Se supplementation. This study aimed to understand whether Se-Met could regulate the nuclear factor-kappa B (NF-κB) signaling pathway through autophagy. Mammary alveolar cell-T (MAC-T) was challenged with Escherichia coli (E. coli). Western blotting and real-time quantitative PCR (RT-qPCR) were used to detect the protein expression and mRNA expression of cytokines. Immunofluorescence assays were performed to observe the expression of intracellular LC3. The results showed that E. coli inhibited autophagy by decreasing the LC3-Ⅱ protein levels, and the Atg5 and Beclin1 protein levels were increased after 4 h. Infection also decreased the number of LC3 puncta. E. coli increased the phosphorylation of p65 and IκBα protein. Concomitantly, the levels of interleukin (IL)-1ß, IL-6, IL-8 and tumour necrosis factor (TNF)-α mRNA increased at 3 and 4 h post-infection. We further explored the regulatory role of autophagy on NF-κB-mediated inflammation with autophagy modulators and shAtg5. The results indicated that the autophagy activator reduced the phosphorylation of p65 and IκBα and the mRNA expression of IL-1ß, IL-6, IL-8 and TNF-α. Additionally, activating autophagy weakened the adhesion to MAC-T of E. coli. Autophagy inhibitors exacerbated NF-κB-mediated inflammation and strengthened the adhesion of E. coli to cells. We then examined the effects of Se-Met on NF-κB-mediated inflammation through autophagy. The data suggested that Se-Met enhanced LC3-II expression, inhibited the E. coli-induced phosphorylation of p65 and IκBα, and suppressed the adhesion ability of E. coli to MAC-T and that the effects of Se-Met in attenuating NF-κB-mediated inflammation were partially blocked by an autophagy inhibitor. In summary, Se-Met alleviated NF-κB-mediated inflammation induced by E. coli by enhancing autophagy in bovine mammary epithelial cells.

Organic Selenium Ameliorates Staphylococcus aureus-Induced Mastitis in Rats by Inhibiting the Activation of NF-κB and MAPK Signaling Pathways.

Mastitis is an economically important disease in dairy cows, which is often caused by Staphylococcus aureus (S. aureus). Selenium is an indispensable element for physiological function and contributes to reduce injury of the mammary glands in mastitis. However, adequate sources of selenium have always been an important consideration for livestock. Therefore, the study aimed to explore the protective effect and mechanism of Selenohomolanthionine (SeHLan) on mastitis induced by S. aureus. The S. aureus-induced rat model was established and three doses (0.2, 2, 20 µg/kg body weight/day) of dietary OS were supplemented. The bacterial load, histopathology, and myeloperoxidase (MPO) of the mammary glands were performed and determined. Cytokines, including interleukin (IL)-1ß, TNF-α, and IL-6, were detected using qRT-PCR. The key proteins of NF-κB and MAPK signaling pathways were analyzed by Western blot. The results revealed that OS supplementation could reduce the recruitment of neutrophils and macrophages in mammary tissues, but did not decrease S. aureus load in the tissues. The overexpression levels of IL-1ß, TNF-α, and IL-6 induced by S. aureus were inhibited after OS treatment. Furthermore, the increased phosphorylation of NF-κB and MAPKs proteins were also suppressed. The results suggest that dietary supplementation with adequate OS during pregnancy contributes to protect the mammary glands from injury caused by S. aureus and alleviate the inflammatory response.

The effect of selenium on the autophagy of macrophage infected by Staphylococcus aureus.

Selenium can alleviate the inflammatory reaction infected by Staphylococcus aureus (S. aureus). However, the role of selenium on the autophagy in RAW264.7 macrophages infected by S. aureus has not been reported. The goal of this study was to clarify the effect of selenium on the autophagy and related inflammatory pathways (MAPK and NF-κB) in RAW264.7 macrophages infected by S. aureus. RAW264.7 macrophages were co-treated with Na2SeO3 and S. aureus. The expression of related inflammatory pathways (MAPK and NF-κB) and autophagy-related proteins were detected by Western blotting. The microtubule-binding protein light chain 3 (LC3) puncta were measured with immunofluorescence staining. The ultrastructure of RAW264.7 macrophages infected by S. aureus was detected by transmission electron microscope (TEM). And plate counting method was used to detect the proliferation of S. aureus in RAW264.7 macrophages. The results showed that the expression levels of LC3 II increased and the expression levels of p62 decreased after adding selenium, compared with S. aureus infection group. Compared with S. aureus infection group, the intracellular LC3 puncta and autophagic vesicles, autophagosomes, and autolysosomes increased with selenium supplementation. The number of S. aureus proliferation decreased with addition of selenium, compared with S. aureus infection group. Selenium could significantly inhibit the phosphorylation of MAPK and NF-κB signaling pathway key proteins, compared with S. aureus infection group. In summary, selenium could promote the autophagy in macrophages infected by S. aureus, alleviate the blockade of autophagic flow, depress the transcription of MAPK and NF-κB signaling pathways, and inhibit the proliferation of S. aureus in RAW264.7 macrophages.

Analgesic and physiological effect of electroacupuncture combined with epidural lidocaine in goats.

OBJECTIVE: To investigate physiological and antinociceptive effects of electroacupuncture (EA) with lidocaine epidural nerve block in goats. STUDY DESIGN: Prospective experimental trial. ANIMALS: Forty-eight hybrid male goats weighing 27 ± 2 kg. METHODS: The goats were randomly assigned to six groups: L2.2, epidural lidocaine (2.2 mg kg-1); L4.4, epidural lidocaine (4.4 mg kg-1); EA; EA-L1.1, EA with epidural lidocaine (1.1 mg kg-1); EA-L2.2, EA with epidural lidocaine (2.2 mg kg-1); and EA-L4.4, EA with epidural lidocaine (4.4 mg kg-1). EA was administered for 120 minutes. Epidural lidocaine was administered 25 minutes after EA started. Nociceptive thresholds of flank and thigh regions, abdominal muscle tone, mean arterial pressure (MAP), heart rate (HR), respiratory frequency (fR) and rectal temperature were recorded at 30, 60, 90, 120, 150 and 180 minutes. RESULTS: Lidocaine dose-dependently increased nociceptive thresholds. There were no differences in nociceptive thresholds between L4.4 and EA from 30 to 120 minutes. The threshold in EA-L2.2 was lower than in EA-L4.4 from 30 to 120 minutes, but higher than in EA-L1.1 from 30 to 150 minutes or in L4.4 from 30 to 180 minutes. The abdominal muscle tone in EA-L2.2 was higher at 30 minutes, but lower at 90 and 120 minutes than at 0 minutes. There were no differences in muscle tone between L4.4 and L2.2 or EA-L4.4, and between any two of the three EA-lidocaine groups from 0 to 180 minutes. The fR and HR decreased in L4.4 at 60 and 90 minutes compared with 0 minutes. No differences in fR, HR, MAP and temperature among the groups occurred from 30 to 180 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: EA combined with 2.2 mg kg-1 epidural lidocaine provides better antinociceptive effect than 4.4 mg kg-1 epidural lidocaine alone in goats. EA provided antinociception and allowed a decrease in epidural lidocaine dose.

MiRNAs are involved in chronic electroacupuncture tolerance in the rat hypothalamus.

Acupuncture tolerance is the gradual decrease in analgesic effect due to its prolonged application. However, its mechanism in terms of miRNA is still unknown. To explore the role of miRNAs in electroacupuncture (EA) tolerance of rats using deep sequencing, rats with more than a 50 % increase in tail flick latency (TFL) in response to EA were selected for this experiment. EA tolerance was induced by EA once daily for eight consecutive days. The hypothalami were harvested for deep sequencing. As a result, 49 differentially expressed miRNAs were identified and validated by real-time PCR. Of them, let-7b-5p, miR-148a-3p, miR-124-3p, miR-107-3p, and miR-370-3p were further confirmed to be related to EA tolerance by an intracerebroventricular injection of agomirs or antagomirs of these miRNAs. Potential targets of the 49 miRNAs were enriched in 9 pathways and 282 gene ontology (GO) terms. Five miRNAs were confirmed to participate in EA tolerance probably through the functional categories related to nerve impulse transmission, receptor signal pathways, and gene expression regulation, as well as pathways related to MAPK, neurotrophin, fatty acid metabolism, lysosome, and the degradation of valine, leucine, and isoleucine. Our findings reveal a characterized panel of the differentially expressed miRNAs in the hypothalamus in response to EA and thus provide a solid experimental framework for future analysis of the mechanisms underlying EA-induced tolerance.

Spinal Glutamate Transporters Are Involved in the Development of Electroacupuncture Tolerance.

BACKGROUND: Electroacupuncture (EA) tolerance is a gradual decline in EA antinociception because of its repeated or prolonged use. This study aims to explore the role of spinal glutamate transporters (GTs) in EA tolerance (EAT). METHODS: Rats were treated with EA once per day for eight consecutive days, and their L4-5 spinal cords were collected at days 0, 2, 4, 6 and 8. The levels of three spinal GTs and their mRNAs were detected with Western blot and pPCR, respectively. Then, riluzole, a positive GT regulator, was administered intrathecally in order to observe its effect on EA analgesia after repeated EA. RESULTS: The expression levels of the spinal GTs increased at days 2 and 4, and gradually decreased as the times of EA increased. At day 8, no difference was observed in the spinal GTs between the sham treatment and the EA treatment. Intrathecal administration of riluzole dose-dependently attenuated the decreased EA analgesia. CONCLUSION: These results indicated the participation of the spinal GTs in EAT.

Electroacupuncture relieves neuropathic pain via upregulation of glutamate transporters in the spinal cord of rats.

Glutamate transports (GTs), the only vehicle for removal of glutamate from the extracellular fluid, is reported to be related to chronic pain. To investigate whether the glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) participate in electroacupuncture (EA) analgesia, the EA effect was observed with paw withdraw threshold in a rat model of spared nerve injury. The expression levels of GLAST and GLT-1 were determined with Western Blot and RT-PCR. The results showed significantly upregulated GLAST and GLT-1, along with the relieved pain behaviors after EA treatment. In addition, intrathecal injection of GTs inhibitor, l-trans-pyrrolidine-2-4-dicarboxylate, attenuated the EA-induced analgesic effect. The experiment demonstrates that EA can increase the GTs of neuropathic pain rats, which might be one of the mechanisms underlying its effectiveness in the neuropathic pain.

[Progress of Researches on Central Mechanism of Electroacupuncture Tolerance].

Electroacupuncture (EA) has been demonstrated effective for pain relief. However, repeated application may decline analgesic effect of EA, which is termed EA tolerance, that reduces the clinical efficacy of EA. Therefore, it has attracted attention from researchers in recent years and the progresses include:(1) acute and chronic EA tolerance animal models have been established; (2) cross-tolerance between EA and morphine; (3) Anti-opioid substances, including cholecystokinin, orphanin FQ and angiotensin Ⅱ, have been reported to contribute to EA tolerance; (4) glutamate receptors and transporters, 5-hydroxytryptamine and norepinephrine have been revealed involvement in EA tolerance; (5) cyclic adenosine monophosphate, cyclic guanosine monophosphate and Ca2+ have been reported to be the second messengers cellularly in EA tolerance. The current EA tolerance effect lacks in-depth researches. Therefore, studies on its molecular mechanisms and signaling pathway are necessarily required.

The Expression Patterns of c-Fos and c-Jun Induced by Different Frequencies of Electroacupuncture in the Brain.

To investigate patterns of c-Fos and c-Jun expression induced by different frequencies of electroacupuncture (EA) in the brain, goats were stimulated by EA of 0, 2, 60, or 100 Hz at a set of "Baihui, Santai, Ergen, and Sanyangluo" points for 30 min. The pain threshold was measured using the potassium iontophoresis method. The levels of c-Fos and c-Jun were determined with Streptavidin-Biotin Complex immunohistochemistry. The results showed that the pain threshold induced by 60 Hz was 82.2% higher (P < 0.01) than that by 0, 2, or 100 Hz (6.5%, 35.2%, or 40.9%). EA induced increased c-Fos and c-Jun expression in most analgesia-related nuclei and areas in the brain. Sixty Hz EA increased more c-Fos or c-Jun expression than 2 Hz or 100 Hz EA in all the measured nuclei and areas except for the nucleus accumbens, the area septalis lateralis, the caudate nucleus, the nucleus amygdala basalis, and the locus coeruleus, in which c-Fos or c-Jun expressions induced by 60 Hz EA did not differ from those by 2 Hz or 100 Hz EA. It was suggested that 60 Hz EA activated more extensive neural circuits in goats, which may contribute to optimal analgesic effects.