ORIGINAL ARTICLE

All-Trans-Retinoic Acid Ameliorates the Inflammation by Inducing Transforming Growth Factor Beta 1 and Interleukin 10 in Mouse Epididymitis Wei Cao1*, Weigan Chen1*, Xiaoxuan Liang1, Jinlian Zhou2, Chao Wei1, Sheng Cui1, Jiali Liu1 1

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China; The 306th Hospital of PLA, Beijing, China

2

Keywords Epididymal epithelium, epididymitis, IL-10, retinoic acid, TGF-b1 Correspondence Jiali Liu, College of Biological Sciences, China Agricultural University, No. 2 Yuanmingyuanxi Road, Beijing 100193, China. E-mail: [email protected] *

Coauthors.

Submission October 18, 2013; accepted December 11, 2013. Citation Cao W, Chen W, Liang X, Zhou J, Wei C, Cui S, Liu J. All trans retinoic acid ameliorates the inflammation by inducing transforming growth factor beta 1 and interleukin 10 in mouse epididymitis. Am J Reprod Immunol 2014; 71: 312–321 doi:10.1111/aji.12197

Problem Epididymitis, one of the most common urological diseases, can lead to the destruction of the epididymal duct and cause transient or permanent sterility. The aim of this study was to investigate the functions and related mechanisms of all trans retinoic acid (atRA) in alleviating the acute inflammation of epididymitis. Method of study The mouse model of the epididymitis was induced by injecting Escherichia coli into the cauda epididymis. atRA was administrated for five consecutive days through intraperitoneal injection. The expression levels of inflammatory cytokines were measured by real-time PCR and Western blot. In addition, cultured primary mouse epididymal epithelial cells were treated with different concentrations of atRA and RAR antagonists to identify whether the effect of atRA was mediated through RAR. Results Our results demonstrate that atRA ameliorates the inflammation in mouse epididymitis by decreasing the expression of the pro-inflammatory cytokines and increasing the expression of anti-inflammatory factors including TGF-b1 and IL-10. Our results show that the upregulating effect of atRA on TGF-b1 was mediated by RARa, and the enhancing effect of atRA on IL-10 expression was mediated via RARb. Conclusion These new results suggest that atRA is involved in regulating the inflammatory response of epididymis.

Introduction Epididymitis is one of the most common urological diseases in adult males.1–3 The presumed mechanism for epididymitis is a bacterial infection ascending from the urinary tract into the male excurrent duct system.4,5 Epididymitis can lead to the destruction of the epididymal duct and to transient or permanent sterility, which is a more frequent outcome than infections and inflammation of the testis.6–8 Labora312

tory investigation has shown that severe infections are characterized by uncontrolled immune responses in the host, such as lymphocyte infiltration, leading to tissue injury.9–12 Therefore, downregulation of the inflammatory responses represents an approach for the treatment of acute epididymitis. All trans retinoic acid (atRA) is an active metabolite of vitamin A. Vitamin A is oxidized to retinal (all trans retinaldehyde) by cytosolic alcohol dehydrogenases (ADH) and microsomal retinol dehydrogenAmerican Journal of Reproductive Immunology 71 (2014) 312–321 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

atRA AMELIORATES INFLAMMATION IN EPIDIDYMITIS

ases (RDH). Further oxidization of retinal to atRA is catalyzed by aldehyde dehydrogenases (RALDH). The binding of atRA with its nuclear receptors RARa, RARb, or RARc activates or represses the transcription of the downstream target genes.13,14 It is well known that atRA elicits anti-inflammatory effects by modulating the humoral and cellular immune responses, maintaining immune homeostasis, and regulating immune cell differentiation.15,16 There is evidence that atRA is essential in various autoimmune diseases and inflammatory responses17,18 and plays an essential role in maintaining epithelial cells including the epithelia of the reproductive tract.19–22 All these studies suggest that atRA is closely associated with microbial infection. The aim of our study was to elucidate atRA’s role in inflammatory responses and investigate the related mechanisms. We will use the mouse model of acute epididymitis induced by E. coli. Our results demonstrate that atRA treatment ameliorates the development of epididymitis through enhancing the expressions of anti-inflammatory cytokines, IL-10, and TGF-b in the epididymal epithelial cells. In addition, atRA inhibits the expression of pro-inflammatory cytokines. These findings imply that atRA is a potential choice for treating human acute epididymitis. Materials and methods Animals All experimental protocols were approved by Chinese Association for Laboratory Animal Science. Adult male Kunming white mice were purchased from Beijing HFK Bioscience Co. Ltd (Beijing, China). The animals were maintained under controlled temperature (22–24°C), humidity (60–70%), and regular light (12L:12D) cycles with food and water ad libitum. Mouse Model of Epididymitis and atRA Treatment The mouse model of epididymitis was induced according to a report by K. Tanaka.23 Briefly, the mice were anesthetized and bilateral epididymides and testes were then exteriorized. A 50 lL E. coli suspension (106 cfu/mL) was carefully injected retrograde into the bilateral cauda epididymal stroma and ducts. Using the same method, 50 lL sterilized phosphate buffer solution (PBS) was administered in the control mice. After a 1-day recovery, 0 lg (control), 20 lg, American Journal of Reproductive Immunology 71 (2014) 312–321 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

and 100 lg atRA (Sigma–Aldrich, St. Louis, MO, USA) dissolved in 100 lL dimethyl sulfoxide (DMSO) were, respectively, admi-nistered for 5 days by intraperitoneal injections. On the 6th day, the mice were killed, and the cauda region was isolated for histopathological examination, RNA extraction, and cell lysate preparation. To determine the prophylactic effect of atRA on the epididymitis, atRA was daily injected, and 50 lL E. coli suspension (106 cfu/mL) was injected on day 4 as above. After 10 days, the mice were killed and the cauda region was isolated for histopathological examination. Epididymis Epithelial Cells Culture The culturing of mouse epididymal epithelium was originally described by Cuthbert and Wong.24,25 Briefly, the epididymis from adult mice was dissected, cut into small fragments, placed in a sterile tube containing Hank’s balanced salt solution (HBSS), and washed more than ten times until no sperm remained in the supernatant. The appropriate volume of HBSS containing 0.25% trypsin and 0.1% (w/v) collagenase type I was then added. After gentle shaking for 15 min at 34°C, the supernatant was discarded, and the pellet was resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.1% (w/v) collagenase type I and 5% fetal bovine serum and shaken for 45–60 min at 34°C. Cells were separated by centrifugation at 800 g for 5 min. The pellet was resuspended in DMEM containing non-essential amino acids (0.1 mM), sodium pyruvate (1 mM), glutamine (4 mM), 10% fetal bovine serum, penicillin (100 i.u./mL), and streptomycin (10 lg/mL). This suspension was allowed to stand for 6 hr, during which non-epithelial cells such as fibroblasts and smooth muscle cells attached rapidly to the plastic surface while epididymal epithelial cells remained unattached. The decanted cell suspension freed from non-epithelial cells was then seeded into the cell culture dish to form a monolayer of epididymal epithelial cells. Generally, the monolayer reached confluency after 24 hr in culture. The identity of the cultured epididymal epithelial cells was confirmed using a mouse anticytokeratin antibody (1:100 dilution; ZSGB-bio, Beijing, China) for immunofluorescence. After 4–6 days, all the cultures were confluent, atRA was added into the medium at different final concentration (0.1 nM, 1 nM, 10 nM, and 100 nM), and the equivalent 313

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amount of DMSO was added to the control group. The cells were harvested after 24 hr for RNA extraction and cell lysate preparation. Histopathology and Immunohistochemistry (IHC) Staining The caput and cauda epididymis were dissected, fixed in 4% paraformaldehyde solution, dehydrated in increasing concentrations of ethanol and xylene, and embedded in paraffin. Five-micrometer sections were cut and stained with hematoxylin and eosin for histopathological examinations. A rat monoclonal antibody against IL-10 (1:100 dilution, product no. ab33471; Abcam, Inc, Cambridge, UK) or a mouse monoclonal antibody against TGF-b1 (1:100 dilution, product no. sc-146; Santa Cruz, Dallas, TX, USA) was diluted in blocking solution and incubated with sections overnight at 4°C. Following three 5-min washes in blocking solution, sections were incubated with the appropriate secondary antibody conjugated to biotin (1:200 dilution; ZSGB-bio) diluted in blocking solution for 1 hr at room temperature. An Avidin-Biotin-HRP staining system (ZSGB-bio) was used to localize biotinylated antibody according to the manufacturer’s instructions.

A total of 2 lg RNA was used for reverse transcription in a 25-lL reaction system according to the operating instruction (Promega, Madison, WI, USA). Real-time PCR was performed in a total volume of 15 lL of reaction mixture containing 50 ng of cDNA samples, 3 pmol each of the forward and reverse primers, and 7.5 lL of 2 9 SYBR Green Real-time PCR Master Mix Kit (Takara) on 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). The primers are listed in Table I. Western Blot Tissues and cells were lysed in RIPA lysis solution (Beyotime, Nantong, China), and the protein concentration was measured using Coomassie Brilliant Blue G250 staining. The protein (80 lg) was subjected to electrophoresis on 15% sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred to PVDF membranes. The membranes were then immunoblotted with anti-IL-10 (dilution 1:500), anti-TGFb1 (dilution 1:250), and anti-GAPDH antibodies. The blots were incubated with goat anti-rat/anti-rabbit/ anti-mouse secondary antibody conjugated to horseradish peroxidase, and detection was performed using an enhanced chemiluminescence reagent kit (Thermo Pierce, Waltham, MA, USA).

RNA Extraction, Reverse Transcription PCR, and Real-Time PCR

Statistical Analysis

To determine the cytokine expression in the epididymis, total RNA was isolated from tissues and cells after treatment. One milliliter Trizol (Takara, Otus, Japan) was added into the samples, and total RNA was extracted according to the operating instruction.

Results were expressed as mean  S.E.M. of the indicated number of experiments. Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by a Newman–Keuls test. P < 0.05 was considered statistically significant.

Table I The sequences for the primers in fluorescent quantization PCR

Gapdh Il-1b Il-6 Il-10 Tgf-b1 Tnf-a Ifn-c Il-12 p35 Il-12 p40 Rara Rarb Rarc

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Forward primer

Reverse primer

GGTTGTCTCCTGCGACTTCA TGCTGGTGTGTGACGTTCCCA TGATGCTGGTGACAACCACGGC TGCTCCTAGAGCTGCGGACT GCTGGTGAAACGGAAGCGCA GGCCAACGGCATGGATCTCAA GGTCCAGCGCCAAGCATTCAA AGCTCCGCCTGAAAGCTCAAG GCTCGCAGCAAAGCAAGATGTG CCCACATCCCAGTCTTTGCTC CACCGAGGTTGTGGAGTAAA TCTGGGAGCAAACAGGAACAC

GGGTGGTCCAGGGTTTCTTA AGGGTGGGTGTGCCGTCTTTCA AGCCTCCGACTTGTGAAGTGGTA TTCTGGGCCATGCTTCTCTGCC AGATGGCGTTGTTGCGGTCCA TGTGGGTGAGGAGCACGTAGT ACCCCGAATCAGCAGCGACT TGAGAGAAGCGATGGAGGGCA CCTTTGTGGCAGGTGTACTGGC GCTTCACCCTTACCCTGATTATTG TCTGGGCAACATAAGGGA ACACCCGGCAGGAGGATTA

American Journal of Reproductive Immunology 71 (2014) 312–321 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Results Mouse Epididymitis Induced by E. coli Firstly, we induced mouse epididymitis by injecting 5 9 104 cfu of E. coli into the cauda region of the epididymis as reported by K. Tanaka.23 The epididymitis is characterized by interstitial edema and leukocytes infiltration in the cauda region (Fig. 1h,m) compared with the controls (Fig. 1g,l). However, the E. coli injection did not affect the caput region (Fig. 1c). In addition, we assessed the expressions of the pro-inflammatory cytokines Il-1b, Il-6, Tnfa, Ifnc, Il-12 p35, and Il-12 p40 by real-time RT-PCR, and the results demonstrated that E. coli significantly increased the expressions of these cytokines (Fig. 1p). These results confirm that the injection of E. coli into the cauda region of the epididymis results in acute epididymitis. Effect of atRA on Mouse Epididymitis To assess the effect of atRA on mouse epididymitis, 0 lg, 20 lg, and 100 lg atRA were, respectively, supplied to the mice by intraperitoneal injection after the E. coli infection for 5 days. A histological examination indicated that both the 20 lg and 100 lg atRA treatment ameliorated the inflammation characterized by a much weaker leukocyte infiltration (Fig. 1i,j,n,o), and drastic reduction in cytokine secretion including Il-1b, Il-6, Tnfa, Ifn-c, Il-12 p35, and Il-12 p40 expressing levels (Fig. 1p). These results suggest that atRA is a potential therapeutic candidate for treating epididymitis caused by E. coli infection. atRA Enhances IL-10 and TGF-b1 Expression Both IL-10 and TGF-b1 are important anti-inflammation cytokines.26 Immunohistochemistry results showed that IL-10 and TGF-b1 were located in the epididymal epithelial cells (Fig. 2d). We then assessed the effects of atRA on IL-10 and TGF-b1 expressions by real-time PCR and Western blot analysis. The results showed that the atRA treatment significantly increased IL-10 and TGF-b1 expressions, both at gene and protein levels, in the epididymis (Fig. 2a,b,c). Furthermore, we treated cultured epididymal epithelial cells with 0 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM atRA for 24 hr, and Il-10 and Tgf-b1 expressions were examined by real-time PCR. The results American Journal of Reproductive Immunology 71 (2014) 312–321 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

show that atRA significantly increased Il-10 expression but without an obvious dose dependence (Fig. 2e). 10 nM and 100 nM atRA treatment markedly increased Tgf-b1 mRNA levels. Western blot analysis indicated that atRA increased the protein expressions of IL-10 and TGFb1 at 10 nM atRA (Fig. 2f). These observations suggest that atRA plays an anti-inflammatory role by enhancing IL-10 and TGF-b1 expressions in the epididymal epithelial cells. atRA Enhances IL-10 and TGF-b1 Expressions Through Different RARs in Epididymal Epithelial Cells To analyze the molecular types of atRA receptors through which atRA exerts its functions in the epididymitis, we detected the mRNA expressions of Rara, Rarb, and Rarc in the epididymitis and cultured epididymal epithelial cells by real-time PCR (Fig. 3a,b). The results showed that Rara and Rarb were highly expressed in both the epididymis and cultured epididymal epithelial cells, and atRA significantly increased Rara and Rarb expression. However, Rarc mRNA expression was at a very low level and was not affected by the addition of atRA in the epididymal epithelial cells. We then used a specific RARa antagonist Ro415253 and a RARb antagonist LE135 to block the binding of atRA to RARa and RARb. The effects of atRA on Il-10 and Tgf-b1 expressions were analyzed by real-time PCR (Fig. 3c). The results showed that Ro41-5253 totally abolished the effect of atRA on Tgf-b1 mRNA expression in culture cells, while LE135 had no obvious inhibitory effect on atRAmediated induction in Tgf-b1 expression. Conversely, Ro41-5253 did not block the effect of atRA on Il-10 mRNA expression, but high concentration of LE135 significantly inhibited the effect of atRA on Il-10 mRNA expression. These indicate that the effect of atRA on Tgf-b1 expressions is through its binding to RARa, whereas the effect of atRA on Il-10 expressions is partially dependent on RARb. The Prophylactic Effect of atRA Pre-Treatment on the Mouse Epididymis To determine the protective effect of atRA on epididymitis, the mice were injected with atRA daily and challenged with E. coli on day 4. Histological examination was performed on day 10 after the first atRA injection (Fig. 4). A significant reduction in 315

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Fig. 1 atRA ameliorates the inflammation in the mouse epididymitis model. The morphology of the epididymis was examined with hematoxylin and eosin staining after atRA treatment for 5 days (a–o). a, f, and k represent the normal mouse epididymis. b, g, and l represent the mouse epididymis treated with DMSO after injection of sterilized PBS. c, h, and m represent the mouse epididymis treated with DMSO after injection of Escherichia coli. d, i, and n represent the mouse epididymis treated with atRA (20 lg) after injection of E. coli. e, j, and o represent the mouse epididymis treated with atRA (100 lg) after injection of E. coli (n = 5). The black arrows show representative leukocyte infiltration. The inflammatory cytokines expression in the epididymitis model tissues was measured by reverse transcription and real-time PCR (p). Control: no treatment group; PBS: treatment with DMSO after injection of sterilized PBS; E. coli: treatment with DMSO after injection of E. coli; RA 20 lg: treatment with atRA (20 lg) after injection of E. coli; RA 100 lg: treatment with atRA (100 lg) after injection of E. coli. Results are means  S.E.M. of three independent experiments, *P < 0.01.

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the inflammatory infiltration and epithelial lesions (Fig. 4i,j,n,o) was observed in the mice which received daily atRA treatment. This suggests that atRA enhances the resistance to E. coli-induced acute epididymitis. 316

Discussion The rat epididymitis model induced by E. coli has been used to evaluate the effects of antibiotic treatments27,28 and analyze the responses of American Journal of Reproductive Immunology 71 (2014) 312–321 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Fig. 2 All-trans-retinoic acid (atRA) increases the expression of IL-10 and TGF-b1 in the epididymis. The cytokine mRNA expression in the epididymal tissues was measured by reverse transcription and real-time PCR (a). The proteins were detected by Western blot (b), and the results were scanned and quantified for densitometric analysis (c). Control: no treatment group; PBS: treatment with DMSO after injection of sterilized PBS; Escherichia coli: treatment with DMSO after injection of E. coli; RA 20 lg: treatment with atRA (20 lg) after injection of E. coli; RA 100 lg: treatment with atRA (100 lg) after injection of E. coli. The immunolocalization of TGF-b1 and IL-10 in the sections of mouse epididymis was determined in the epithelial cells (d). The inflammation cytokines mRNA expression in the epididymis epithelial cells culture with the absence (control) and presence of atRA in different concentration was measured by reverse transcription and real-time PCR (e). The cytokine proteins were detected by Western blot after 10 nM atRA treatment (f). Results are means  S.E.M. of three independent experiments, **P < 0.01 versus control, ##P < 0.01 versus DMSO treatment group. American Journal of Reproductive Immunology 71 (2014) 312–321 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Fig. 3 RARs mediate the function of atRA in cultured epididymal epithelial cells. Three types of RAR mRNAs were measured by reverse transcription and real-time PCR in epididymis tissue (a) and cultured epididymal epithelial cells (b). Normal: control group; RA: atRA treatment group (100 lg in tissues and 10 nM in epithelial cells). We measured the mRNA expression of cytokines after blocking with RARa and RARb antagonists (c). Control: no treatment group; RA: treatment with atRA (10 nM); RA+Ro 1: treatment with atRA (10 nM) and Ro415253 (100 nM); RA+Ro 2: treatment with atRA (10 nM) and Ro41-5253 (200 nM); RA+LE 1: treatment with atRA (10 nM) and LE135 (500 nM); RA+LE2: treatment with atRA (10 nM) and LE135 (1000 nM); RA+Ro+LE: treatment with atRA (10 nM), Ro41-5253(200 nM) and LE135(1000 nM). Results are means  S.E.M. of three independent experiments, **P < 0.01 versus RA group.

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cytokines.23,29 In this study, we established the mouse epididymitis model by E. coli injection into the cauda epididymis, mainly based on the report by Tanaka.23 The E. coli-induced mouse model was evaluated by a histopathological examination and by a measurement of pro-inflammation cytokines, such as Il-1b, Il-6, Tnfa, Ifn-c, Il-12 p35, and Il-12 p40. These pathological changes were consistent with the signs of epididymitis that have been reported previously.23,27,29 These results suggest that the mouse model for experimental epididymitis induced by E. coli is suitable to evaluate the effect of atRA and other reagents on the epididymitis. It has been documented that vitamin A and its metabolites have beneficial effects on the development of various diseases, such as rheumatoid arthritis, type I diabetes, and experimental encephalomyelitis, ulcerative colitis30–33 through regulating the immune responses. The present study provides novel evidence that atRA functions to alleviate the inflammation by downregulating these pro-inflammation cytokines released in the mouse epididymitis. In addition, the pre-treatment with atRA prevents the occurrence of epididymitis induced by an E. coli infection. These findings are in accordance with previous studies that show the effects of atRA on the immune system in that atRA can regulate the differentiation of immune cells and the maintenance of immune homeostasis.17,33,34 The present study demonstrates that atRA plays its anti-inflammatory role in the mouse epididymis by enhancing TGF-b1 and IL-10 expressions. Firstly, it is reported that TGF-b1 and IL-10 act on important anti-inflammatory factors by suppressing cellular immune responses and delaying the progress of acute inflammatory responses.35,36 In support, our in vivo and in vitro results show that both TGF-b1 and IL-10 are specifically expressed in the epididymal epithelium, and atRA increases IL-10 and TGF-b1 levels in epididymal epithelial cells and alleviates the inflammation. These are in accordance with the recent reports that TGF-b and atRA have a synergistic effect on the differentiation of immune cells16,37 and that atRA signaling induces secretion of TGF-b and IL-10 in peripheral dendritic cells.15,16,34,38 It is known that the physiological functions of atRA are mediated primarily by the RARs, including RARa, RARb, and RARc. The present study shows RARa and RARb are the main isotypes in the epididymis and epididymal epitheliums, and exogenous American Journal of Reproductive Immunology 71 (2014) 312–321 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

atRA AMELIORATES INFLAMMATION IN EPIDIDYMITIS

Fig. 4 atRA pre-treatment protected the epididymis from Escherichia coli infection. The morphology of the epididymis was examined with hematoxylin and eosin staining after atRA treatment (DMSO as control) for 10 days (a–o). a, f, and k represent the normal epididymis of a mouse. b, g, and l represent the mouse epididymis injected with sterile PBS after being pretreated with DMSO. c, h, and m represent the mouse epididymis injected with E. coli after being pre-treated with DMSO. d, i, and n represent the mouse epididymis injected with E. coli after being pre-treated with atRA (20 lg). e, j, and o represent the mouse epididymis injected with E. coli after being pre-treated with atRA (100 lg) (n = 5). The black arrows show the representative leukocytes infiltration. The white arrow shows the destruction of the epididymal duct.

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mainly dependent on the RARb. These results suggest that atRA elicits a variety of downstream effects when binding to different receptors and activating different signaling pathways (Fig. 5). The underlying mechanisms of atRA and its potential therapeutic use in the treatment of epididymitis need to be further studied. Conclusions Fig. 5 atRA plays its anti-inflammatory role in the mouse epididymis by enhancing TGF-b1 and IL-10 expressions.

atRA significantly enhances the expression of RARa and RARb. It is of much interest that RARa and RARb play different roles after binding to atRA. RARa mediates the effects of atRA on TGF-b1, whereas the effect of atRA on IL-10 expression is American Journal of Reproductive Immunology 71 (2014) 312–321 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

In light of these novel results, atRA acts as an essential nutrition molecule and is involved in regulating the inflammatory response of epididymis by upregulating the expression of anti-inflammatory cytokines secreted from the epididymal epithelium. Its potential use in regulating the differentiation of immune cells and the maintenance of immune homeostasis needs to be investigated in further studies. 319

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All-trans-retinoic acid ameliorates the inflammation by inducing transforming growth factor beta 1 and interleukin 10 in mouse epididymitis.

Epididymitis, one of the most common urological diseases, can lead to the destruction of the epididymal duct and cause transient or permanent sterilit...
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