Estrogen deficiency aggravates fluoride-induced small intestinal mucosa damage and junctional complexes proteins expression disorder in rats.

To investigate the effect of estrogen deficiency on the small intestinal mucosal barrier induced by fluoride (F), F exposure models of ovariectomy (OVX) rats (surgically removed ovaries) and non-OVX rats (normal condition) were established by adding sodium fluoride (NaF) (0, 25, 50, and 100 mg/L, calculated by F ion) in drinking water for 90 days. The intestinal mucosal histomorphology, mucosal barrier function, and protein expression levels of tight junctions (TJs), adhesion junctions (AJs), and desmosomes were evaluated in the duodenum, jejunum, and ileum. Hematoxylin-eosin (HE) staining and 5-Bromo-2-deoxyUridine (BrdU) measurement showed that excessive F-induced damage to intestinal epithelial cells and inhibited the proliferation of intestinal epithelial cells, eventually decreasing the number of goblet cells and decreasing glycoprotein secretion, as indicated by Alcian blue and periodic acid-Schiff (AB-PAS) and periodic acid-Schiff (PAS) staining. Further immunofluorescence analysis demonstrated that excessive F decreased the protein expression levels of occludin, zonula occludens-1 (ZO-1), E-cadherin, and desmoplakin (P < 0.05, P < 0.01) and enhanced the expression of claudin-2 (P < 0.01), suggesting that cell-to-cell junctions were disrupted. Collectively, F exposure impaired the small intestinal mucosal barrier by inducing damage to intestinal epithelial cells and inhibiting intestinal epithelial cell proliferation. Disorders in the junctional complex protein expression blocked the synergy between intercellular communication and aggravated mucosal injury. In particular, estrogen deficiency exacerbated F-induced enterotoxicity, which provides new explanations for the development and severity of intestinal disease in postmenopausal women with high-F areas.

iTRAQ-based quantitative proteomic analysis of low molybdenum inducing thymus atrophy and participating in immune deficiency-related diseases.

Molybdenum is a trace element with extremely uneven distribution in the environment. It constitutes the active sites of molybdenum enzymes that can catalyze redox reactions in almost all organisms. In this study, a mouse model with a low molybdenum diet was established to investigate the differential protein expressions in the thymus and the mechanism of molybdenum regulating thymocyte development. Results showed that the thymus evidently atrophied, and the weight and organ index of the thymus substantially decreased under the condition of low molybdenum (P < 0.01). A total of 274 differentially expressed proteins (DEPs) were screened through isobaric tag for relative and absolute quantification; amongst them, ribosomal proteins (38) were the most abundant. Bioinformatics analysis revealed that DEPs were mainly involved in protein metabolism (18%), nucleus (15%) and nucleic acid binding activity (17%), corresponding to biological process, cellular component and molecular function, respectively. Moreover, DEPs induced by low molybdenum were enriched in 94 pathways, of which typical maps including ribosome, oxidative phosphorylation and systemic lupus erythematosus. Flow cytometry analysis indicated the prominent imbalances of CD4+ and CD8+ cell ratios (P < 0.05, P < 0.01), suggesting the disordered development of T cell subsets. Overall, low molybdenum resulted in thymus atrophy by interfering with ribosomal protein expression and protein metabolism. This study provides a data platform for revealing the linkage between molybdenum and thymus-dependent immunity.

Induction of pathological changes and impaired expression of cytokines in developing female rat spleen after chronic excess fluoride exposure.

This study was designed to investigate the effects of excessive fluoride on spleen toxicity. Twenty-four healthy female rats were randomly divided into two groups, each of 12 rats. Each group of female rats was given a control diet and either F- = 0 mg/L or an excessive F- = 150 mg/L in the drinking water for 120 days. The histomorphological and ultrastructural changes in their splenic tissues were observed under light and transmission electron microscopes. DNA damage and splenocyte apoptosis were examined using the micronucleus (MN) assay, single-cell gel electrophoresis (SCGE), and flow cytometry. The expression levels of cytokines, including interleukin (IL)-1ß, IL-2, IL-6, and tumor necrosis factor (TNF)-α, were determined through immunohistochemistry and Western-blot analysis. Results demonstrated that the histomorphological characteristics and ultrastructure of the splenic tissues were affected by excessive fluoride. Nuclear dying, nuclear membrane dissolution, mitochondrial vacuolation, and endoplasmic reticulum dilation were observed. SCGE and MN assays showed that the nuclear DNA of splenocytes was damaged by fluoride treatment, and splenocyte apoptosis was exacerbated in the fluoride group. With damage to the splenocyte structure and DNA, the protein expression levels of IL-1ß, IL-2, IL-6, and TNF-α were significantly downregulated by exposure to fluoride. Excessive fluoride ingestion caused splenic pathological damage and abnormal cytokine expression in female rats.

Effect of tributyltin on antioxidant ability and immune responses of zebrafish (Danio rerio).

Tributyltin (TBT) is a toxic compound released into aquatic ecosystems through antifouling paints. This study was designed to examine the effects of TBT on antioxidant ability and immune responses of zebrafish (Danio rerio). Three hundred sixty healthy zebrafish were randomly grouped into four groups and exposed to different doses of TBT (0, 1, 10 and 100ngL-1). At the end of 8 weeks, the fish were sampled, and antioxidant capability, immune parameters and immune-related genes were assessed. The results showed that with an increase in TBT dose, the concentration of malonaldehyde in the liver was significantly increased (p<0.05), whereas the activities of total superoxide dismutase, catalase and glutathione peroxidase were significantly decreased (p<0.05) compared to the control. The activity and expression of lysozyme and the content of immunoglobulin M were significantly decreased compared to those of the fish exposed to 0ngL-1 TBT (p<0.05). However, the expression of the HSP70, HSP90, tumor necrosis factor-α(TNF-α), interleukins (IL-1ß, IL-6), and nuclear factor-kappa B p65 (NF-κ B p65) genes were all enhanced with an increase in TBT dose. The results indicated that TBT induced oxidative stress and had immunotoxic effects on zebrafish.

Reproductive toxicity in male mice after exposure to high molybdenum and low copper concentrations.

To evaluate the effects of dietary high molybdenum (HMo) and low copper (LCu) concentrations on reproductive toxicity of male mice, 80 mice were divided into 4 groups of 20. These groups were fed with the following: (1) normal control (NC) diet (NC group); (2) NC and HMo diets (HMo group); (3) LCu diet (LCu group); and (4) HMo and LCu diets (HMoLCu group). On the 50th and 100th day, superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) were analyzed to determine oxidative stress states. Morphological changes in testicular tissue were evaluated with hematoxylin and eosin staining and ultrastructural changes were monitored by transmission electron microscopy. The results showed that administration of HMo, LCu, and HMoLCu not only decreased sperm density and motility but also increased the rate of teratosperm occurrence. A significant increase in MDA content and a decrease in SOD, GSH-Px, and T-AOC contents were observed in LCu, HMo, and HMoLCu groups. Testicular tissues and cells of mice were damaged by HMo and the damages were more serious in the case of Cu deficiency. Exposure to HMo adversely affected the reproductive system of male mice, and dietary LCu plays key roles in HMo-induced reproductive toxicity.

Effect of Diclazuril on the Bursa of Fabricius Morphology and SIgA Expression in Chickens Infected with Eimeria tenella.

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.

Effect of diclazuril on intestinal morphology and SIgA expression in chicken infected with Eimeria tenella.

Secretory immunoglobulin A (SIgA), as a vital actor involving in the mucosal immunity, plays a key role in defending a variety of pathogenic infections, such as bacteria, viruses and parasites. Eimeria tenella is an obligate intracellular apicomplexan parasite contacting with the digestive tract mucosa and specially parasitizes chicken caecum, causing a severe form of coccidiosis. Coccidiosis is currently mainly controlled using chemotherapeutic agents. Diclazuril, a classic coccidiostat, was used widely in the poultry industry. Because of the rising problem of drug resistance, it is therefore crucial to understand the pattern of the SIgA expression in the action of diclazuril against E. tenella. In this study, the intestinal morphology in the caecum was analyzed by haematoxylin-eosin (HE) staining, and the SIgA expression was examined by immunohistochemical technique. At the same time, the duodenum, jejunum and ileum tissues have also been evaluated. HE staining results showed that E. tenella infection caused severe damage characterized by structural disorder, haemorrhage, inflammatory cell infiltration, serous and fibrinous exudation in chicken caecum and invisible damage in the duodenum, jejunum and ileum. With the treatment of diclazuril, the damage in the caecum was alleviated obviously. Immunohistochemical analysis demonstrated that the SIgA level in the infected group was increased in the duodenum (p < 0.05), jejunum and ileum, respectively, but decreased (p < 0.01) in the caecum, compared with the control group. Interestingly, the SIgA level was decreased in the duodenum (p < 0.05), jejunum and ileum but increased (p < 0.05) in the caecum in the infected/diclazuril group in comparison to the infected group. The results showed that diclazuril effectively alleviated the damage in the caecum induced by E. tenella and provided a cure for coccidiosis by improving the immune function in chickens.

A novel serine/threonine protein phosphatase type 5 from second-generation merozoite of Eimeria tenella is associated with diclazuril-induced apoptosis.

Screening the anticoccidial drug targets is very important for developing novel drugs and revealing the molecular basis of drug resistance in coccidia. Due to high effectivity and safety, diclazuril was used widely in the poultry industry. To assess the roles of the serine/threonine protein phosphatase type 5 of second-generation merozoites in Eimeria tenella (EtPP5) in the anticoccidial activity of diclazuril against chicken coccidiosis, EtPP5 was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Ultrastructural changes in second-generation merozoites and mRNA expression level of EtPP5 were monitored by transmission electron microscopy (TEM) and quantitative real-time PCR, respectively. The results showed that the full length of the cloned EtPP5 cDNA (2,495 bp) encompassed a 1,647-bp open reading frame encoding a polypeptide of 548 residues with an estimated molecular mass of 60.82 kDa and a theoretical isoelectric point of 5.89. Molecular analysis of EtPP5 reveals the presence of a C-terminal phosphatase domain and an extended N-terminal tetratricopeptide repeat motif, a typical feature of protein phosphatases. The cDNA sequence has been submitted to the GenBank database with accession number JX987508. EtPP5 shared 89% homology with the published sequence of a PP5 ortholog of Toxoplasma gondii at the amino acid level (GenBank XP_002364442.1). TEM observed that diclazuril induced ultrastructural changes in second-generation merozoites. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtPP5 mRNA expression was significantly downregulated by 51.4% by diclazuril treatment. The high similarity of EtPP5 to previously described PP5 of other organisms, as well as its downregulated expression and connection with apoptosis in the second-generation merozoites induced by diclazuril, suggests that it could act an important role in understanding the signaling mechanism underlining the diclazuril-induced merozoites apoptosis.

Fluoride-induced osteoporosis via interfering with the RANKL/RANK/OPG pathway in ovariectomized rats: Oophorectomy shifted skeletal fluorosis from osteosclerosis to osteoporosis.

Osteosclerosis and osteoporosis are the two main clinical manifestations of skeletal fluorosis. However, the reasons for the different clinical manifestations are unclear. In this study, we established the fluoride (F) -exposed ovariectomized (OVX) and non-OVX rat models to assess the potential role of ovarian function loss in osteosclerosis and osteoporosis. Micro-CT scanning showed that excessive F significantly induced a high bone mass in non-OVX rats. In contrast, a low bone mass manifestation was presented in OVX F-exposed rats. Also, a prominent feature of increasing trabecular connectivity, collagen area, growth plate thickness, and reduced trabecular space was found by histopathological morphology in non-OVX F-exposed rats; an opposite result was observed in OVX F-exposed. These alterations indicated ovariectomy was a vital factor leading to osteosclerosis or osteoporosis in skeletal fluorosis. Furthermore, levels of bone alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase (TRAP) increased, combined with the increasing osteoclasts number, showing a sign of high bone turnover in both OVX and non-OVX F-exposed rats. Mechanistically, oophorectomy considerably activated the RANKL/RANK/OPG signaling pathway. Meanwhile, it was discovered that upregulated NF-κB positively facilitated the accumulation of nuclear factor of activated T-cells 1 (NFATC1), significantly promoting osteoclast differentiation. To sum up, this study greatly enriched the causes of clinical skeletal fluorosis and provided a new perspective for studying the pathogenesis of skeletal fluorosis.

Th17-Related Cytokines Involved in Fluoride-Induced Cecal and Rectal Barrier Damage of Ovariectomized Rats.

To investigate fluoride (F)-induced intestine barrier damage and the role of estrogen deficiency in this progress, a rat model of estrogen deficiency was established through bilateral surgical removal of ovaries. The F exposure model was then continued by adding sodium fluoride (0, 25, 50, and 100 mg/L, calculated on a fluorine ion basis) to drinking water for 90 days. Afterward, intestinal mucosal structure, barrier function, and inflammatory cytokines were evaluated. The results showed that excessive F decreased the developmental parameters (crypt depth) of the cecum and rectum and inhibited the proliferation capacity of the intestinal epithelia, which are more obvious in the state of estrogen deficiency. The distribution of goblet cells and glycoproteins in the intestinal mucosa decreased with the increase in F concentration, and estrogen deficiency led to a further decline, especially in the rectum. Using the immunofluorescence method, the study showed that excessive F caused interleukin-17A (IL-17A) significantly decrease in the cecum and increase in the rectum. Meanwhile, F treatment remarkably upregulated the expression of intestinal IL-1ß, IL-23, and IL-22, while the level of IL-6 was downregulated. In addition, estrogen deficiency increased IL-1ß, IL-6, IL-23, and IL-22, but decreased IL-17A expression in the cecum and rectum. Collectively, F exposure damaged intestinal morphological structure, inhibited epithelial cell proliferation and mucus barrier function, and resulted in the disturbance of T helper (Th) 17 cell-related cytokines expression. Estrogen deficiency may further aggravate F-induced damage to the cecum and rectum.

Molybdenum-Induced Apoptosis of Splenocytes and Thymocytes and Changes of Peripheral Blood in Sheep.

To investigate the effects of molybdenum (Mo) on apoptosis of lymphocytes and changes of peripheral blood in sheep, a total of 20 5-month-old healthy female sheep were randomly divided into five groups of 4 and orally administered with water containing Na2MoO4·2H2O (0, 5, 10, 20, and 50 mg/kg BW/day) for 28 days. Jugular vein blood was taken on the 0th, 7th, 14th, 21st, and 28th day of Mo treatment, respectively. On the 28th day, the spleen and thymus were removed for observing histopathology and apoptosis-related DNA damage by hematoxylin and eosin (HE) staining and TdT­mediated dUTP Nick-End Labeling (TUNEL) staining, respectively. The blood routine indexes were determined by an automatic blood analyzer. Further, the apoptosis of lymphocytes and changes in mitochondrial membrane potential (MMP) of peripheral blood were analyzed by flow cytometry. Results showed that excessive Mo induced apoptosis-related DNA damage in the splenocytes and thymocytes and significantly increased the apoptosis indexes of the splenocytes and thymocytes (P < 0.01). Furthermore, the treatment with excessive Mo significantly decreased the MMP (P < 0.01) and promoted apoptosis in peripheral blood lymphocytes (P < 0.01). And the number of WBC, Lymph, Gran, and RBC and the indexes of HGB and HCT were also significantly decreased (P < 0.05 or P < 0.01), while RDW was significantly increased by excessive Mo (P < 0.05 or P < 0.01). In conclusion, excessive Mo-induced DNA damage and apoptosis of the lymphocytes changed the RBC-related indexes of the peripheral blood in sheep.

Diclazuril-induced expression of CDK-related kinase 2 in the second-generation merozoites of Eimeria tenella.

Diclazuril is a classic anticoccidial drug. The key molecules of diclazuril in anticoccidial action allows target screening for the development of anticoccidial drugs. Cyclin-dependent kinases (CDK) are prominent target proteins in apicomplexan parasites. In this study, a diclazuril anticoccidiosis animal model was established, and the transcription and translation levels of the CDK-related kinase 2 of Eimeria tenella (EtCRK2) were detected. mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localised in the cytoplasm of the merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group. The anticoccidial drug diclazuril against E.tenella affects the expression pattern of EtCRK2 molecule, and EtCRK2 is a potential target for new drug development.

Receptor for activated C kinase ortholog of second-generation merozoite in Eimeria tenella: clone, characterization, and diclazuril-induced mRNA expression.

The receptor for activated C kinase (RACK) cDNA of second-generation merozoites of Eimeria tenella was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends, compared with other species, and then successfully expressed using the pET-28a vector in Escherichia coli BL21 (DE3) (EtRACK). Nucleotide sequence analysis revealed that the full length of the cloned cDNA (1,264 bp) encompassed a 957-bp open reading frame encoding a polypeptide of 318 residues with an estimated molecular mass of 34.94 kDa and a theoretical isoelectric point of 5.97. Molecular analysis of EtRACK reveals the presence of seven WD40 repeat motifs. EtRACK localizes to the cytoplasm and nucleus in second-generation merozoites of E. tenella. The cDNA sequence has been submitted to the GenBank Database with accession number JQ292804. EtRACK shared 98% homology with the published sequence of a RACK protein from Toxoplasma gondii at the amino acid level (GenBank XP_002370996.1). Recombinant protein expression was induced using 1 mM of isopropyl ß-D-1-thiogalactopyranoside in vitro at 30 °C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the 39.79-kDa fusion protein existed in unsolvable form. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtRACK mRNA expression in the treatment group was downregulated by 81.3% by diclazuril treatment. The high similarity of EtRACK to previously described RACKs of other organisms, as well as its downregulated expression in second-generation merozoites induced by diclazuril, suggests that it could play a key role in the signaling event that precedes protein secretion and parasite invasion. Moreover, the downregulation of EtRACK mRNA expression also enriches studies on the mechanism of action of diclazuril on E. tenella.

Estrogen deficiency aggravates fluorine ion-induced renal fibrosis via the TGF-ß1/Smad signaling pathway in rats.

To investigate the role and molecular mechanism of estrogen deficiency in fluorine ion (F-)-induced renal fibrosis, the models of F- exposure in ovary removed rats were established by drinking water with different doses of F- (0, 25, 50 and 100 mg/L) for 90 days. Results of H&E staining and BrdU labeled experiment showed that F- induced renal pathomorphological damage and inhibited cell proliferation. Further, Masson staining showed that F- induced renal glomerular and tubulointerstitial fibrosis. Meanwhile, renal fibrosis was confirmed by detecting the expression levels of collagen I, collagen III, collagen IV and fibronectin using immunofluorescence. In the state of estrogen deficiency, F--induced renal damage and fibrosis were aggravated. Moreover, the molecular mechanism of F--induced renal fibrosis was evaluated, and the results showed that F- induced TGF-ß1/Smad signaling pathway further dysregulation after ovariectomy, which manifested as the further up-regulated expression of TGF-ß1, Smad2, p-Smad2, Smad3 and p-Smad3, and further down-regulated of Smad7. Accompanied by renal damage and renal fibrosis, renal function was also disturbed, especially in ovariectomized rats. This study indicated that estrogen deficiency aggravated F--induced renal fibrosis via the TGF-ß1/Smad signaling pathway, leading to more serious renal dysfunction.

Fluoride exposure cause colon microbiota dysbiosis by destroyed microenvironment and disturbed antimicrobial peptides expression in colon.

Colon microenvironment and microbiota dysbiosis are closely related to various human metabolic diseases. In this study, a total of 72 healthy female mice were exposed to fluoride (F) (0, 25, 50 and 100 mg/L F-) in drinking water for 70 days. The effect of F on intestinal barrier and the diversity and composition in colon microbiota have been evaluated. Meanwhile, the relationship among F-induced colon microbiota alterations and antimicrobial peptides (AMPs) expression and short-chain fatty acids (SCFAs) level also been assessed. The results suggested that F decreased the goblet cells number and glycoprotein expression in colon. And further high-throughput 16S rRNA gene sequencing result demonstrated that F exposure induced the diversity and community composition of colonic microbiota significantly changes. Linear Discriminant Analysis Effect Size (LEfSe) analysis identified 11 predominantly characteristic taxa which may be the biomarker in response to F exposure. F-induced intestinal microbiota perturbations lead to the significantly decreased SCFAs levels in colon. Immunofluorescence results showed that F increased the protein expression of interleukin-17A (IL-17A) and IL-22 (P < 0.01) and disturbed the expression of interleukin-17 receptor A (IL-17RA) and IL-22R (P < 0.05 or P < 0.01). In addition, the increased expression of IL-17A and IL-22 cooperatively enhanced the mRNA expression of AMPs which response to F-induced microbiota perturbations. Collectively, destroyed microenvironment and disturbed AMPs are the primary reason of microbiota dysbiosis in colon after F exposure. Colonic homoeostasis imbalance would be helpful for finding the source of F-induced chronic systemic diseases.

Estrogen Deficiency Aggravates Fluoride-Induced Liver Damage and Lipid Metabolism Disorder in Rats.

Estrogen exerts essential role in liver metabolism, and its deficiency is frequently accompanied by a series of metabolic disorder diseases. To investigate the role of estrogen deficiency in fluorine ions (F-) induced liver injury, the ovariectomy (OVX) rat models were performed by surgically removing the ovaries, and the rats from OVX and non-OVX models were exposed to differential dose of F- (0, 25, 50 and 100 mg/L) in drinking water for 90 days. The liver morphological structure was evaluated by hematoxylin-eosin staining. Proliferation ability of hepatocytes was evaluated by 5-bromo-2-deoxyuridine (BrdU) assay. And distribution of lipid droplets in liver tissue was observed via oil red O staining. In addition, the liver function and lipid metabolism parameters in serum were detected by commercial kits. Results showed that F- induced hepatocytes morphological damage and inhibited the proliferation ability of hepatocytes; estrogen deficiency exacerbated these changes. The deposition of lipid droplets in the liver tissue was multiplicative with increased F- dose, especially after estrogen deficiency. In addition, F- exposure increased (P < 0.05 or P < 0.01) serum aminotransferase (ALT), aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyl transpeptidase (γ-GT) activities and total bilirubin (T-bil) level; meanwhile, serum triglyceride (TG) and cholesterol (TC) levels were also elevated (P < 0.05 or P < 0.01). F--induced liver function and lipid metabolism indexes were further increased (P < 0.05 or P < 0.01) in the state of estrogen deficiency. In conclusion, estrogen deficiency aggravated F--induced liver damage and lipid metabolism disorder.

Anticoccidial effect of toltrazuril and Radix Sophorae Flavescentis combination: Reduced inflammation and promoted mucosal immunity.

An anticoccidial model of chicken infected with Eimeria tenella was established to investigate the effect of toltrazuril (Tol) combined with the Radix Sophorae Flavescentis (RSF) on coccidiosis. The anticoccidial index (ACI) was evaluated, and the cecal developmental parameters (i.e., villus height, [VH], crypt depth, [CD], and VH/CD) were determined. The distributions of glycoproteins and goblet cells in the cecal tissue were determined through the Periodic Acid-Schiff (PAS) and Alcian blue PAS staining methods, respectively. The mRNA expression levels of interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-10, and IL-17 of the cecal tissue were determined through quantitative real-time PCR. The moderate ACI was obtained using the combination of Tol and RSF. Compared with the normal control (NC) group, the infected control (IC) group showed remarkably lower VH and VH/CD at five and seven days postinfection. Compared with the IC group, the IC + RSF and IC + TolRSF groups showed remarkably higher VH and VH/CD at five and seven days postinfection. Compared with the NC group, the IC group contained fewer glycoproteins and goblet cells, but the Tol and RSF treatment promoted more glycoproteins and goblet cells at five and seven days postinfection. The mRNA expression levels of IL-1ß, IL-2, IL-4, IL-6, IL-10, and IL-17 in the IC group were upregulated (P < 0.01) compared with those in the NC group. The IC + RSF and IC + TolRSF groups had downregulated mRNA expression levels of IL-1ß, IL-6, and IL-17 cytokines (P < 0.01), and upregulated mRNA expression levels of IL-2 and IL-4 cytokines (P < 0.01) compared with the IC group. Results showed that the combination of Tol and RSF exerts anticoccidial effect by reducing inflammation and promoting intestinal mucosal immunity.

Based on G-Series Mouse TH17 Array Study the Effect of Fluoride on C2C12 Cells Cytokines Expression.

C2C12 cells were cultured on medium containing fluoride (0, 1, and 2.5 mmol/L) for 48 h to investigate the effect of excessive fluoride on T helper 17 (Th17)-related cytokine expression profile in skeletal muscle cells, and the culture supernatant was collected and subjected for the detection of 18 cytokines via Th17 array. Results showed that compared with the control group, no differential expression proteins (DEPs) were found in the 1 mmol/L fluoride group; however, eight DEPs were upregulated in the 2.5 mmol/L fluoride group, including macrophage inflammatory protein-3α (MIP-3α), interleukin-21 (IL-21), IL-13, IL-17F, IL-28A, transforming growth factor type beta 1 (TGF-ß1), IL-23, and IL-17A. In addition, five DEPs (MIP-3α, IL-13, IL-21, TGF-ß1, and IL-17F) were upregulated in the 2.5 mmol/L fluoride group compared with the 1 mmol/L fluoride group. Gene ontology analysis revealed that the positive regulation of cytokine production, cytokine activity, receptor ligand activity, and cytokine receptor binding accounted for high percent of DEPs present. Kyoto Encyclopedia of Genes and Genomes analysis showed that these DEPs primarily involved 12 pathways enriched in the cytokine-cytokine receptor interaction and IL-17 signaling pathway after 2.5 mmol/L fluoride treatment. The results indicated that fluoride might induce cytotoxicity by disturbing Th17-related cytokine expression.

Eimeria tenella: effects of diclazuril treatment on microneme genes expression in second-generation merozoites and pathological changes of caeca in parasitized chickens.

The effects of diclazuril on mRNA expression levels of invasion-related microneme genes were examined in second-generation merozoites of Eimeria tenella (E. tenella) by quantitative real-time (QRT) PCR. Diclazruil treatment of infected chickens significantly decreased the number of second-generation merozoites by 65.13%, and resulted in downregulation of EtMIC genes: EtMIC1 by 65.63%, EtMIC2 by 64.12%, EtMIC3 by 56.82%, EtMIC4 by 73.48%, and EtMIC5 by 78.17%. SEM images of caecum tissue from uninfected chickens showed regular intestinal villus structure. In infected chickens, a distinct loss of the superficial epithelium, with a flattened mucosa and large-area necrosis and anabrosis, was evident. In diclazruil-treated chickens, a decrease in merozoite number and a visibly improved appearance of the caeca were noted. These improvements appeared to be mediated in part by downregulation of the expression of invasion-related EtMIC genes in response to diclazuril.

Actin-depolymerizing factor of second-generation merozoite in Eimeria tenella: clone, prokaryotic expression, and diclazuril-induced mRNA expression.

Actin depolymerizing factor (ADF) is an essential actin-binding protein that plays a key role in the control of actin dynamics and actin-based motility processes in intracellular parasites. To determine the effects of diclazuril on ADF gene of second-generation merozoites (mz-ADF) mRNA expression in Eimeria tenella, mz-ADF gene was cloned by RT-PCR from extracted RNA in second-generation merozoite of E. tenella and successfully expressed by pET-28a vector in Escherichia coli BL21(DE3). Results showed that the full length of the cloned cDNA sequence of the mz-ADF gene is 476 bp including an ORF of 375 bp. The sequence has 100% homology with a published sequence of sporozoite stage E. tenella ADF mRNA (GenBank EF195234.1). The recombinant protein was induced to be expressed by 1 mM isopropyl beta-D: -1-thiogalactopyranoside in vitro. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that 16.99 kDa fusion protein existed in solvable form. Compared with the infected/control group, mz-ADF mRNA expression level was downregulated by 63.86% in the infected/treatment group with the treatment of diclazuril. In conclusion, the data presented here indicate that mz-ADF gene participates in an important role in the invasion host of E. tenella. Downregulation of mz-ADF mRNA expression enrich the mechanism study of diclazuril on E. tenella.