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.

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.

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.

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.

Key Role of Pro-inflammatory Cytokines in the Toxic Effect of Fluoride on Hepa1-6 Cells.

The role of pro-inflammatory cytokines in the toxicity of fluoride to tumor cells was investigated by culturing Hepa1-6 cells in medium containing gradient concentrations of fluoride (0, 0.5, 1, 1.5, 2, 3, 4, and 5 mmol/L). The viability of Hepa1-6 cells was detected via MTT assay. Interleukin (IL)-2, IL-6, tumor necrosis factor (TNF)-α, and IL-1ß levels in the supernatant were determined via an enzyme-linked immunosorbent assay, and the protein expression levels of these enzymes in Hepa1-6 cells were evaluated by immunofluorescence staining. Results showed that the viability of Hepa1-6 cells remarkably decreases after fluoride exposure, especially at concentration of 3, 4, and 5 mmol/L fluoride. Levels of IL-2, TNF-α, and IL-1ß in the supernatant markedly decreased when cells were exposed to fluoride at concentrations of 1 mmol/L or higher. However, levels of TNF-α and IL-1ß substantially increased and IL-2 showed no remarkable change when the fluoride concentration was 0.5 mmol/L. The content of IL-6 remarkably increased with increasing fluoride concentrations up to 2 mmol/L, and then markedly decreased at 3, 4, and 5 mmol/L fluoride; the decreasing trend of IL-6 content under high fluoride exposure is consistent with the decrease in Hepa1-6 cell viability observed at the same concentration. The protein expression levels of IL-2, IL-6, TNF-α, and IL-1ß were in accordance with their contents in the supernatant. In summary, our study demonstrated that fluoride inhibits Hepa1-6 cell growth and results in disorders in the expression and secretion pro-inflammatory cytokines.

Mitochondrial respiratory chain damage and mitochondrial fusion disorder are involved in liver dysfunction of fluoride-induced mice.

Our previous study showed that excessive fluoride (F) intake can induce liver dysfunction. The aim of this study was to investigate the mechanisms of F-induced mitochondrial damage resulting in liver dysfunction. Damaged mitochondrial ultrastructure and state of liver cells were estimated by TEM, TUNEL staining and BrdU measurement. The ROS level and ATP content in the liver tissue were measured by ELISA kit. Meanwhile, optic atrophy (OPA1), mitofusin-1 (Mfn1), NDUFV2, SDHA, CYC1, and COX Ⅳ expression levels were measured through real-time PCR and Western-blot. Results showed that the ROS level increased, thereby resulting in mitochondrial ultrastructure damage and abundant liver cells presented evident apoptotic characteristics after F treatment. Decreased ATP content and the abnormal expression of OPA1, Mfn1, NDUFV2, SDHA, CYC1, and COX Ⅳ of the liver tissue were observed. In conclusion, excessive F-induced mitochondrial respiratory chain damaged and mitochondrial fusion disorder resulted in liver dysfunction.

Eukaryotic elongation factor 2 is involved in the anticoccidial action of diclazuril in the second-generation merozoites of Eimeria tenella.

Eimeria tenella, an obligate intracellular parasite, can actively invade the cecal epithelial cells of chickens and cause severe enteric disease. Eukaryotic elongation factor 2 (eEF2) plays a major role in protein synthesis and cell survival. This study aims to explore the exact mechanisms underlying diclazuril inhibition in second-generation merozoites of E. tenella. The eEF2 cDNA of the second-generation merozoites of E. tenella (EtEF2) was cloned by reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Diclazuril-induced expression profiles of EtEF2 were also analyzed. The cloned full-length cDNA (2893 bp) of the EtEF2 nucleotide sequence encompassed a 2499 bp open reading frame (ORF) that encoded a polypeptide of 832 residues with an estimated molecular mass of 93.12 kDa and a theoretical isoelectric point of 5.99. The EtEF2 nucleotide sequence was submitted to the GenBank database with the accession number KF188423. The EtEF2 protein sequence shared 99 % homology with the eEF2 sequence of Toxoplasma gondii (GenBank XP_002367778.1). The GTPase activity domain and ADP-ribosylation domain were conserved signature sequences of the eEF2 gene family. The changes in the transcriptional and translational levels of EtEF2 were detected through quantitative real-time PCR and Western blot analyses. The mRNA expression level of EtEF2 was 2.706 fold increases and the protein level of EtEF2 was increased 67.31 % under diclazuril treatment. In addition, the localization of EtEF2 was investigated through immunofluorescence assay. Experimental results demonstrated that EtEF2 was distributed primarily in the cytoplasm of second-generation merozoites, and its fluorescence intensity was enhanced after diclazuril treatment. These findings indicated that EtEF2 may have an important role in understanding the signaling mechanism underlying the anticoccidial action of diclazuril and could be a promising target for novel drug exploration.

Mitochondrial respiratory chain dysfunction mediated by ROS is a primary point of fluoride-induced damage in Hepa1-6 cells.

To evaluate the mechanism of fluoride (F) mitochondrial toxicity, we cultured Hepa1-6 cells with different F concentrations (0, 1 and 2 mmoL/L) and determined cell pathological morphology, mitochondrial respiratory chain damage and cell cycle change. Results showed that the activities and mRNA expression levels of antioxidant enzymes considerably decreased, whereas the contents of reactive oxygen species (ROS), malondialdehyde (MDA) and nitric oxide (NO) markedly increased. Breakage of mitochondrial cristae and substantial vacuolated mitochondria were observed by transmission electron microscopy. These results indicate the F-induced oxidative damage in Hepa1-6 cells. The enzyme activities of mitochondrial complexes I, II, III and IV were disordered in Hepa1-6 cells treated by excessive F, thereby indicating a remarkable down-regulation. Further research showed that complex subunits also demonstrated the development of disorder, in which the protein expressions levels of NDUFV2 and SDHA were substantially down-regulated, whereas those of CYC1 and COX Ⅳ were markedly up-regulated. Reductions in ATP and mitochondrial membrane potential were detected with the dysfunction of the mitochondrial respiratory chain. The G2/M phase arrest of the cell cycle in Hepa1-6 cells was measured via flow cytometry, and the up-regulated protein expressions of Cyt c, caspase 9, caspase 3 and substantial apoptotic cells were determined. In summary, this study demonstrated that ROS-mediated mitochondrial respiratory chain dysfunction causes F-induced Hepa1-6 cell damage.

Effect of Fluoride on Small Intestine Morphology and Serum Cytokine Contents in Rats.

This study aimed to determine the effect of excessive fluoride (F) on the morphological characteristics of the small intestine and the contents of serum cytokines in rats. A total of 48 3-week-old healthy female Sprague-Dawley rats were randomly divided into four groups (n = 12). The control group was given deionized distilled water, while the F treatment groups were treated with water containing 25, 50, and 100 mg F-/L. After 70 days of treatment, the duodenum, the jejunum, and the ileum were collected to measure the developmental parameters and the distribution of intestinal glycoproteins, goblet cells, and mast cells through Pannoramic Viewer, Periodic Acid-Schiff (PAS) staining, Alcian blue and periodic acid-Schiff (AB-PAS) staining, and toluidine blue staining, respectively. The contents of cytokines, namely, interleukin (IL)-1ß, IL-2, IL-6, and tumor necrosis factor (TNF)-α, in serum were detected via enzyme-linked immunosorbent assay (ELISA). Results showed that the villus height, crypt depth, villus height to crypt depth ratio, goblet cells, glycoproteins, and mast cells of the small intestine significantly decreased (P < 0.05 or P < 0.01) in the F treatment group. The contents of IL-1ß, IL-2, IL-6, and TNF-α were significantly lower in the F treatment group than in the control group (P < 0.05 or P < 0.01). In summary, excessive F intake impaired intestinal development and immune function by decreasing the developmental parameters and the distribution of immune cells, glycoproteins, and cytokines.

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.

Mitochondrial respiratory chain complex abnormal expressions and fusion disorder are involved in fluoride-induced mitochondrial dysfunction in ovarian granulosa cells.

Excessive fluoride intake has a strong female reproductive toxicity, which can result in follicular developmental dysplasia and decrease oocytes developmental potential. The underlying mechanisms of fluoride-induced mitochondrial dysfunction in ovarian granulosa cells remain largely unknown. In this study, the ultrastructure changes of mitochondria and DNA damage in ovarian granulosa cells were observed under transmission electron microscope and TUNEL staining. Then, the ATP content and ROS level in granulosa cells were measured. The expression of mitochondrial fusion proteins and mitochondrial respiratory chain complexes, including OPA1 and Mfn1, and NDUFV2, SDHA and CYC1, in the ovarian tissues were measured by immunohistochemistry, Western blot and Quantitative real-time PCR analyses. The expression of ATP5j and ATP5h in the ovarian tissues was also measured. Results show that fluoride treatment considerably damages mitochondrial ultrastructure and enhances the apoptosis of granulosa cells. The ATP content greatly decreased, whereas the ROS level increased after fluoride treatment. The expression level of Mfn1 in the ovarian tissue was up-regulated, whereas OPA1 expression had no significant change. The expression levels of NDUFV2, SDHA and CYC1 were considerably up-regulated, and the expression of ATP5j and ATP5h were down-regulated after fluoride treatment. In summary, the damage in the mitochondrial ultrastructure, ATP content decrease, ROS level increase and the abnormal expression of OPA1, Mfn1, NDUFV2, SDHA, CYC1, ATP5j and ATP5h in ovary tissue are closely associated with fluoride-induced mitochondrial dysfunction, which might be responsible for the follicular developmental dysplasia and the potential decrease in oocyte development induced by fluoride in female mice.

JNK/STAT signalling pathway is involved in fluoride-induced follicular developmental dysplasia in female mice.

Excessive fluoride (F) intake decreases the development of potential oocytes by inducing oxidative stress and apoptosis in female mice in our previous study. This study aims to investigate the underlying mechanisms of F-induced follicular developmental dysplasia. Pathomorphological changes in the ovary tissues were observed under light and transmission electron microscopes. DNA damage and proliferation in granulosa cells were analysed by TUNEL staining and BrdU measurement. The protein expression of cell proliferation related regulatory factors including JNK, STAT3, STAT5, CDK2, CDK4, PCNA and Ki67 in the ovary tissues was measured by immunohistochemistry and Western blot analyses. Results indicated that the structure of granulosa cells in the ovary was seriously damaged by excessive F, evident by the swollen endoplasmic reticulum, mitochondria with vacuoles and nucleus shrinkage. F treatment also considerably enhanced the apoptosis and inhibited the proliferation of granulosa cells. The number of granulosa cells around the oocyte decreased after F treatment. The expression levels of STAT3, CDK2, CDK4 and Ki67 in the ovary tissues were up-regulated, and STAT5 and PCNA did not change significantly after F treatment, whereas JNK expression was down-regulated with increasing F dose. In summary, changes in the expression levels of JNK, STAT3, STAT5, CDK2, CDK4, PCNA and Ki67 in the JNK/STAT signalling pathway are involved in F-induced follicular dysplasia in the ovary.

PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells.

To investigate the mechanisms of fluoride-induced apoptosis, a fluoride-induced C2C12 skeletal muscle cell (C2C12 cell) model was established in this study, and the viability of the C2C12 cells was measured using an MTT assay. Cell morphological changes were observed via haematoxylin and eosin staining and transmission electron microscopy. Apoptosis was monitored through Hoechst staining. The mRNA and protein expression of PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and caspase-9 were detected through real-time PCR and western blotting, respectively. The results showed that the survival rates of C2C12 cells decreased gradually with an increasing fluoride doses. The C2C12 cell structure was seriously damaged by fluoride, presenting with pyknosis, mitochondrial ridge disruption and swollen endoplasmic reticulum. Furthermore, the expression of mRNA in PI3K, BAD, Bcl-2, Bax and caspase-9 were significantly increased in the fluoride group (P < 0.01), while the expression of PDK1 was markedly decreased (P < 0.01). The expression of protein in BAD, Bcl-2 and Bax were significantly increased in the fluoride group (P < 0.01), while the expression of PDK1 and P-AKT1 was markedly decreased (P < 0.01). In conclusion, fluoride-induced apoptosis in C2C12 cells is related to the PI3K/AKT signaling pathway.

ATP5J and ATP5H Proactive Expression Correlates with Cardiomyocyte Mitochondrial Dysfunction Induced by Fluoride.

To investigate the effect of excessive fluoride on the mitochondrial function of cardiomyocytes, 20 healthy male mice were randomly divided into 2 groups of 10, as follows: control group (animals were provided with distilled water) and fluoride group (animals were provided with 150 mg/L F- drinking water). Ultrastructure and pathological morphological changes of myocardial tissue were observed under the transmission electron and light microscopes, respectively. The content of hydrolysis ATP enzyme was observed by ATP enzyme staining. The expression levels of ATP5J and ATP5H were measured by Western blot and quantitative real-time PCR. The morphology and ultrastructure of cardiomyocytes mitochondrial were seriously damaged by fluoride, including the following: concentration of cardiomyocytes and inflammatory infiltration, vague myofilaments, and mitochondrial ridge. The damage of mitochondrial structure was accompanied by the significant decrease in the content of ATP enzyme for ATP hydrolysis in the fluoride group. ATP5J and ATP5H expressions were significantly increased in the fluoride group. Thus, fluoride induced the mitochondrial dysfunction in cardiomyocytes by damaging the structure of mitochondrial and interfering with the synthesis of ATP. The proactive ATP5J and ATP5H expression levels were a good response to the mitochondrial dysfunction in cardiomyocytes.

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.

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.

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.