Epidermal Growth Factor mRNA Expression Research Articles

Comparison of epidermal growth factor expression and secretion in human salivary glands

ObjectiveTo investigate the expression and secretion of epidermal growth factor (EGF) in major and minor salivary gland tissues of human subjects and to examine the potential influence of sex and age on EGF expression and secretion. DesignSaliva samples from the oral cavity at rest and after citric acid stimulation, as well as serum samples, were collected from 150 healthy subjects, and the concentrations of EGF were measured with enzyme-linked immunosorbent assay (ELISA) and compared. The expression of EGF mRNA and protein in normal salivary gland tissues was measured by real-time polymerase chain reaction (RT-PCR), Western blot (WB), and immunohistochemistry (IHC). ResultsThe EGF concentration in acid-stimulated saliva was significantly higher than that in resting saliva (P < 0.001), and significantly higher than that in serum (P < 0.001). No sex difference was observed in EGF levels of whole saliva and serum, whereas the EGF levels in saliva and serum were decreased with age (P < 0.001 and P < 0.001, respectively). The EGF concentration and compound secretion rate (CSR) in resting submandibular glands saliva were significantly higher than those in resting parotid glands saliva (P = 0.002 and P < 0.001, respectively). The EGF was expressed in all major and minor salivary glands and ranked in order of submandibular, parotid, sublingual, and labial glands. ConclusionAll salivary glands have the function of secreting EGF, and the submandibular gland is the main source of salivary EGF. Aging is a factor influencing the expression and secretion of EGF.

IL-33 Induces Paneth Cell Production of EGF and Soluble ST2, Regulating Epithelial Regeneration after Intestinal Injury

Crypt base intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but their frequencies are reduced in experimental models of graft vs. host disease (GVHD). Paneth cells provide an epithelial contribution to the stem cell niche, but their frequencies are also reduced in GVHD mouse models and in patients with GVHD. Mechanisms regulating ISCs and their niche after damage remain poorly understood. Interleukin-33 (IL-33) is an immunomodulatory alarmin typically thought to target lymphocytes such as T cells and innate lymphoid cells. In experimental models of allogeneic bone marrow transplantation, IL-33 can promote or attenuate GVHD, depending on the cellular target and the timing of the exposure. A secreted isoform of the IL-33 receptor, termed soluble ST2 (sST2), acts as a negative regulator by blocking IL-33 from binding to the membrane-bound isoform that mediates intracellular signaling. sST2 also has diagnostic and prognostic utility, serving as a biomarker for GVHD severity. While IL-33 has well-described effects on lymphocytes populations, there is limited understanding of the role of IL-33 signaling within the intestinal mucosa after damage. Utilizing experimental radiation injury to model conditioning-related intestinal pathology, we investigated the role of the IL-33/ST2 axis in epithelial damage and regeneration. Wild-type (WT) mice increased IL-33 mRNA and protein expression in the small intestine within days after 10 Gy total body irradiation (TBI). Intestinal sST2 expression also increased quickly in WT mice after TBI, but not in IL-33 -/- mice, suggesting that IL-33 itself was a regulator of sST2. While the ileal mucosa appeared similar at baseline in wild-type and IL-33 -/- mice, the IL-33 -/- mice demonstrated more severe radiation injury 5 days after 10 Gy TBI, with more crypt loss, loss of ISCs and Paneth cells, and an attenuated regenerative response with smaller crypts post-TBI containing fewer Ki67 + cells. 3-D whole-mount confocal microscopy of ileal tissue from IL-33-GFP reporter mice indicated that the crypt epithelium did not express IL-33 at baseline, but Olfm4 + ISCs adjacent to Paneth cells began to express IL-33 following radiation injury (Fig 1). Anti-Thy1-mediated lymphocyte depletion prior to irradiation of WT and IL-33 -/- mice did not abrogate the increased radiation sensitivity of IL-33 -/- mice, indicating a role for IL-33 beyond immunomodulation and suggesting that IL-33 could have direct effects within the epithelium. Intestinal organoid cultures confirmed epithelial expression of IL-33, confirmed increased radiation sensitivity of IL-33 -/- epithelium, and identified impaired epidermal growth factor (EGF) expression in the absence of IL-33. Furthermore, treatment of intestinal organoids with IL-33 induced EGF mRNA and protein expression, both of which were abrogated by the p38 inhibitor SB202190. Treatment of purified Lysozyme-DsRed + Paneth cells indicated that IL-33 directly promoted their production of EGF. In vivo, TBI-induced EGF upregulation was absent in IL-33 -/- mice. To further investigate this newly identified epithelial IL-33/ST2/EGF pathway in the ISC compartment, RiboTag mice, which facilitate analysis of lineage-specific proteome-relevant gene expression with minimal tissue processing and artifact introduction, were crossed to Olfm4-Cre and Lysozyme-Cre transgenics. Lineage-specific ribosome targeting was confirmed by immunostains and qPCR, and this approach also confirmed ISC upregulation of IL-33 following radiation injury and Paneth cell upregulation of EGF and sST2. Finally, to investigate the importance of this radiation-induced EGF upregulation, WT mice were treated with the EGFR inhibitor gefitinib following irradiation, which resulted in more severe radiation-induced ISC loss, similar to IL-33 deficiency. Furthermore, epithelial regeneration and ISC recovery were augmented in IL-33 -/- mice after TBI by treatment with exogenous EGF (Fig 2). These findings reveal an unknown pathway of stem cell niche regulation and crypt regeneration whereby the Paneth cell niche responds dynamically upon injury by upregulating EGF and the stem cells orchestrate regeneration by regulating their niche with IL-33. This regenerative circuit also highlights the breadth of IL-33 activity beyond immunomodulation and the therapeutic potential of EGF administration for treatment of intestinal injury.

The histologic comparison of submandibular gland between yak and yellow cattle.

The purpose of this study was to compare between the histochemical characteristics and the expression of epidermal growth factor (EGF) and it's receptor (EGFR) in the submandibular gland (SMG) of adult yaks and yellow cattle. The SMG tissues of yaks and yellow cattles were collected for histochemical, immunohistochemical (IHC), immunofluorescence (F-IHC)，real-time quantitative polymerasechain reaction (RT-qPCR) and Western blotting methods. The results showed that the striated ducts of SMG were highly developed and connected to the intercalated ducts, which were shorter and directly connected to the acini. Compared with yellow cattle, yak SMG contains more mucous acini. Immunofluorescence showed significant expression of EGF and its receptor in both striated and intercalated ducts of these two species of cattle. Statistical analysis divulged that the distribution density of EGF and EGFR in the SMG of the yak was both significantly higher than that in yellow cattle (p < 0.05). Furthermore, the mRNA expression of EGF and EGFR in yak SMG was also higher than that in yellow cattle. The above results indicated that the intercalated ducts and striated ducts are the main expression sites of EGF and EGFR, the acidic mucin and EGF secreted from SMG of yak were more than that from yellow cattle. The results of this study provide powerful data for the study of physiological functions of submaxillary gland in ruminants and also provide important clues for the study of adaptive physiological mechanisms in plateau organisms.

Lack of Association between Epidermal Growth Factor or Its Receptor and Reflux Esophagitis, Barrett's Esophagus, and Esophageal Adenocarcinoma: A Case-Control Study.

The epidermal growth factor (EGF) and its receptor (EGFR) gene-gene interactions were shown to increase the susceptibility to esophageal cancer. However, the role of the EGF/EGFR pathway in the development of gastroesophageal reflux disease (GERD) and its complications (reflux esophagitis (RE), Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC)) remains unclear. This association study is aimed at investigating functional EGF and EGFR gene polymorphisms, their mRNA expression in esophageal tissues, and EGF plasma levels in relation to RE, BE, and EAC development in the Central European population. 301 patients with RE/BE/EAC (cases) as well as 98 patients with nonerosive reflux disease (NERD) and 8 healthy individuals (controls) were genotyped for +61 A>G EGF (rs4444903) and +142285 G>A EGFR (rs2227983) polymorphisms using the TaqMan quantitative polymerase chain reaction (qPCR). In random subgroups, the EGF and EGFR mRNA expressions were analyzed by reverse transcription qPCR in esophageal tissue with and without endoscopically visible pathological changes; and the EGF plasma levels were determined by enzyme-linked immunosorbent assay. None of the genotyped SNPs nor EGF-EGFR genotype interactions were associated with RE, BE, or EAC development (p > 0.05). Moreover, mRNA expression of neither EGF nor EGFR differed between samples of the esophageal tissue with and without endoscopically visible pathology (p > 0.05) nor between samples from patients with different diagnoses, i.e., RE, BE, or EAC (p > 0.05). Nevertheless, the lower EGF mRNA expression in carriers of combined genotypes AA +61 EGF (rs4444903) and GG +142285 EGFR (rs2227983; p < 0.05) suggests a possible direct/indirect effect of EGF-EGFR gene interactions on EGF gene expression. In conclusion, EGF and EGFR gene variants and their mRNA/protein expression were not associated with RE, BE or EAC development in the Central European population.

Celastrol attenuates amphiregulin expression by inhibiting MAPK signaling pathway in triple-negative breast cancer cells

BackgroundAmphiregulin (AREG) plays an essential role in the metastasis and angiogenesis of various cancers. PurposeThe objective of this study was to investigate the pharmacological effects of celastrol on AREG expression in triple-negative breast cancer (TNBC) cells. MethodsWe analyzed the prognostic value of AREG, EGF, TGFA, and HB-EGF mRNA expression in TNBC patients. Cell proliferation was analyzed by MTT assay and colony-forming assay. Levels of protein and gene expression were analyzed by ELISA, Western blotting, confocal microscopy, and real-time PCR, respectively. The expression of angiogenic proteins was analyzed by using the Human angiogenesis array kit. Cell invasiveness was analyzed by Boyden chamber assay. ResultsOur results showed that abnormal AREG induction remarkably decreased the relapse-free survival (RFS) rate of TNBC patients, whereas epidermal growth factor (EGF), transforming growth factor-alpha (TGFA), or heparin-binding EGF-like growth factor (HBEGF) demonstrated no effect on RFS rate. In addition, the growth of TNBC cells was prevented by drugs targeting EGFR such as neratinib or lapatinib. On the contrary, cell growth and invasion were enhanced by AREG treatment. Interestingly, celastrol reduced AREG expression and the growth of TNBC cells. Furthermore, we found that levels of AREG expression were reduced by a potent inhibitor of MEK1/2, binimetinib (BIN). Our results showed that celastrol significantly decreased the levels of ERK phosphorylation in TNBC cells. Finally, we observed that celastrol also suppressed the AREG-induced EGFR signaling pathway. Taken together, these results suggest that celastrol can downregulate AREG expression by inhibiting the MEK/ERK signaling pathway. In addition, the AREG-induced EGFR signaling pathway was inhibited by celastrol. ConclusionCelastrol can be an effective drug for treating TNBC by inhibiting AREG/EGFR signaling pathways.

Blockade of epidermal growth factor and its receptor and axial elongation in experimental myopia.

To examine the influence of epidermal growth factor (EGF) and its receptor (EGFR) on axial ocular elongation, we intraocularly injected an EGF antibody and an EGFR antibody into young guinea pigs with lens-induced axial elongation (myopization). Mean axial elongation was reduced in the eyes injected with the EGF/EGFR-antibody compared with the contralateral control eyes injected with PBS (phosphate-buffered solution) (0.43±0.13mm vs 0.53±0.13mm; P<.001). The intereye difference in axial length increased (P=.005) as the doses of the EGF antibody and EGFR antibody increased. As a corollary, the thickness of the retina at the posterior pole was dose-dependently increased in the injected eyes compared to the contralateral control eyes. Immunohistochemical staining for EGF and the relative mRNA expression of EGF and EGFR were the highest in eyes not injected with the EGF antibody or EGFR antibody and decreased (P<.05) as the dose of EGF antibody or EGFR antibody increased. In an in vitro study, EGF had a stimulating effect and the EGF antibody had an inhibitory effect on the proliferation and migration of RPE cells. The findings showed that the intravitreal application of an EGF antibody and EGFR antibody is associated with a dose-dependent reduction in lens-induced axial elongation in young guinea pigs. The EGFR family may play a role in axial elongation of the eye and in the development of myopia.

The localization and expression of epidermal growth factor and epidermal growth factor receptor in bovine ovary during oestrous cycle.

Epidermal growth factor (EGF) is one of the important regulatory factors of EGF family. EGF has been indicated to effectively inhibit the apoptosis of follicular cells, to promote the proliferation of granulosa cells and the maturation of oocytes, and to induce ovulation process via binding to epidermal growth factor receptor (EGFR). However, little is known about the distribution and expression of EGF and EGFR in cattle ovary especially during oestrous cycle. In this study, the localization and expression rule of EGF and EGFR in cattle ovaries of follicular phase and luteal phase at different time points in oestrous cycle were investigated by using IHC and real-time qPCR. The results showed that EGF and EGFR in cattle ovary were mainly expressed in granulosa cells, cumulus cells, oocytes, zona pellucida, follicular fluid and theca folliculi externa of follicles. The protein and mRNA expression of EGF/EGFR in follicles changed regularly with the follicular growth wave both in follicular and in luteal phase ovaries. In follicular phase ovaries, the protein expression of EGF and EGFR was higher in antral follicles than that of those in other follicles during follicular growth stage, and the mRNA expression of EGFR was also increased in stage of dominant follicle selection. However, in luteal phase ovaries, the growth of follicles was impeded during corpus luteum development under the action of progesterone secreted by granular lutein cell. The mRNA and protein expressions of EGF and EGFR in ovarian follicles during oestrous cycle indicate that they play a role in promoting follicular development in follicular growth waves and mediating the selection process of dominant follicles.

The influence of retinol concentration in liquid crystal formula on epidermal growth factor, interleukin-6 and transglutaminase-1 mRNA expression in the epidermis.

Topical retinol application effectively reduces the effects of photoaging and improves skin condition, e.g. influences the process of keratinization of the epidermis, which improves stratum corneum structure and reduces transepidermal water loss. However, cosmetics use lower concentrations of retinol, which has been associated with emerging hypersensitivity reactions as well as redness and irritation of the skin. The question arises whether the vehicle used in the cosmetic may be important in stimulating the inflammatory reaction in the skin and if the concentration of retinol used could significantly affects the growth of epidermal cells. The aim of this study was to evaluate the effects of different concentrations of liquid crystal retinol (0.15%, 0.3% and 0.5%) on the clinical and histological characteristics of a reconstructed epidermis skin model. It also compares the effectiveness of 0.3% retinol formula in liquid crystal to that in lipid. The study used reconstructed human epidermis tissue containing normal human keratinocytes. Four original formulas containing retinol were tested: 0.15%, 0.3% and 0.5% with a liquid crystal base, and 0.3% with a lipid base. Interleukin 6 (IL-6), transglutaminase-1 (TGM1), and epidermal growth factor (EGF) mRNA expression was measured expression of the skin-equivalent tissue for 10 days of exposure. Histopathological analysis and mRNA quantification were performed. Gene expression was analyzed by total mRNA extraction. All liquid crystal formulas induced higher EGF mRNA expression than lipid base formula. IL-6 expression did not differ significantly from the DPBS reference values. Interestingly, TGM1 expression was found to increase together with increasing retinol concentration (0.15%, 0.3%, 0.5%). Histological examination revealed changes in epidermal structure, mainly hyperkeratinization of the stratum corneum. Our results support the hypothesis liquid crystal formula might be regarded as more beneficial since it inducess less pro-inflammatory action manifested by lowered expression IL-6. In addition, EGF expression was found to correlate significantly with the retinol concentration of the liquid crystal formula: 0.5% > 0.3% > 0.15% (P < 0.05). Lower concentrations may increase TGM1 expression, thus enhancing the formation of a protective layer of cornified envelope.

Effects of a prostaglandin F2alpha derivative glaucoma drug on EGF expression and E-cadherin expression in a corneal epithelial cell line

Purpose: We examined the effects of travoprost on cell proliferation-related signals and E-cadherin expression in vitro and in situ in order to obtain evidence to support the hypothesis that topical travoprost impairs the integrity of the corneal epithelium.Methods: A human corneal epithelial cell culture was treated with travoprost (0.4 mg/ml) and/or PD168393 (an EGF receptor inhibitor, 10 μM). The culture was then processed for cell proliferation, an mRNA expression analysis of epidermal growth factor (EGF) and E-cadherin, and protein expression analysis of E-cadherin by immunocytochemistry and Western blotting. The eyes of C57/BL6 mice were incubated in serum-free medium plus travoprost (0.4 mg/ml) and/or PD168393 (10 μM). After being cultured for 24 h, the expression patterns of phospho-EGFR, phospho-ERK, E-cadherin, and Ki67 were immunohistochemically examined in paraffin sections.Results: The addition of travoprost up-regulated EGF mRNA expression and cell proliferation in the corneal epithelial cell culture, and this was cancelled by the addition of PD168393. This FP agonist also decreased E-cadherin expression levels in the cell–cell contact zone, and this was cancelled by the addition of PD168393. In the organ culture, the addition of travoprost to the medium up-regulated the expression of phospho-EGFR and phospho-ERK as well as cell proliferation, and down-regulated the expression of E-cadherin in the corneal epithelium, particularly in basal cells, whereas PD168393 reversed these effects.Conclusions: Travoprost activates epithelial cell proliferation by up-regulating an EGF-related signal in association with the suppression of E-cadherin localization in the cell–cell contact zone. Modulation of the EGF signal may be a strategy to minimize the negative impact of this mitogen on reformation of corneal barrier function during epithelial renewal.

Interaction of epidermal growth factor with COX-2 products and peroxisome proliferator-activated receptor-γ system in experimental rat Barrett's esophagus.

Mixed acidic-alkaline refluxate is a major pathogenic factor in chronic esophagitis progressing to Barrett's esophagus (BE). We hypothesized that epidermal growth factor (EGF) can interact with COX-2 and peroxisome proliferator-activated receptor-γ (PPARγ) in rats surgically prepared with esophagogastroduodenal anastomosis (EGDA) with healthy or removed salivary glands to deplete salivary EGF. EGDA rats were treated with 1) vehicle, 2) EGF or PPARγ agonist pioglitazone with or without EGFR kinase inhibitor tyrphostin A46, EGF or PPARγ antagonist GW9662 respectively, 3) ranitidine or pantoprazole, and 4) the selective COX-2 inhibitor celecoxib combined with pioglitazone. At 3 mo, the esophageal damage and the esophageal blood flow (EBF) were determined, the mucosal expression of EGF, EGFR, COX-2, TNFα, and PPARγ mRNA and phospho-EGFR/EGFR protein was analyzed. All EGDA rats developed chronic esophagitis, esophageal ulcerations, and intestinal metaplasia followed by a fall in the EBF, an increase in the plasma of IL-1β, TNFα, and mucosal PGE2 content, the overexpression of COX-2-, and EGF-EGFR mRNAs, and proteins, and these effects were aggravated by EGF and attenuated by pioglitazone. The rise in EGF and COX-2 mRNA was inhibited by pioglitazone but reversed by pioglitazone cotreated with GW9662. We conclude that 1) EGF can interact with PG/COX-2 and the PPARγ system in the mechanism of chronic esophagitis; 2) the deleterious effect of EGF involves an impairment of EBF and the overexpression of COX-2 and EGFR, and 3) agonists of PPARγ and inhibitors of EGFR may be useful in the treatment of chronic esophagitis progressing to BE.NEW & NOTEWORTHY Rats with EGDA exhibited chronic esophagitis accompanied by a fall in EBF and an increase in mucosal expression of mRNAs for EGF, COX-2, and TNFα, and these effects were exacerbated by exogenous EGF and reduced by removal of a major source of endogenous EGF with salivectomy or concurrent treatment with tyrphostin A46 or pioglitazone combined with EGF. Beneficial effects of salivectomy in an experimental model of BE were counteracted by PPARγ antagonist, whereas selective COX-2 inhibitor celecoxib synergistically with pioglitazone reduced severity of esophageal damage and protected esophageal mucosa from reflux. We propose the cross talk among EGF/EGFR, PG/COX-2, and proinflammatory cytokines with PPARγ pathway in the mechanism of pathogenesis of chronic esophagitis progressing to BE and EAC.

Nucleolin promotes Ang II-induced phenotypic transformation of vascular smooth muscle cells by regulating EGF and PDGF-BB.

RNA‐binding properties of nucleolin play a fundamental role in regulating cell growth and proliferation. We have previously shown that nucleolin plays an important regulatory role in the phenotypic transformation of vascular smooth muscle cells (VSMCs) induced by angiotensin II (Ang II). In the present study, we aimed to investigate the molecular mechanism of nucleolin‐mediated phenotypic transformation of VSMCs induced by Ang II. Epidermal growth factor (EGF) and platelet‐derived growth factor (PDGF) inhibitors were used to observe the effect of Ang II on phenotypic transformation of VSMCs. The regulatory role of nucleolin in the phenotypic transformation of VSMCs was identified by nucleolin gene mutation, gene overexpression and RNA interference technology. Moreover, we elucidated the molecular mechanism underlying the regulatory effect of nucleolin on phenotypic transformation of VSMCs. EGF and PDGF‐BB played an important role in the phenotypic transformation of VSMCs induced by Ang II. Nucleolin exerted a positive regulatory effect on the expression and secretion of EGF and PDGF‐BB. In addition, nucleolin could bind to the 5′ untranslated region (UTR) of EGF and PDGF‐BB mRNA, and such binding up‐regulated the stability and expression of EGF and PDGF‐BB mRNA, promoting Ang II‐induced phenotypic transformation of VSMCs.

Urinary Epidermal Growth Factor as a Marker of Disease Progression in Children With Nephrotic Syndrome

IntroductionChildhood-onset nephrotic syndrome has a variable clinical course. Improved predictive markers of long-term outcomes in children with nephrotic syndrome are needed. This study tests the association between baseline urinary epidermal growth factor (uEGF) excretion and longitudinal kidney function in children with nephrotic syndrome.MethodsThe study evaluated 191 participants younger than 18 years enrolled in the Nephrotic Syndrome Study Network, including 118 with their first clinically indicated kidney biopsy (68 minimal change disease; 50 focal segmental glomerulosclerosis) and 73 with incident nephrotic syndrome without a biopsy. uEGF was measured at baseline for all participants and normalized by the urine creatinine (Cr) concentration. Renal epidermal growth factor (EGF) mRNA was measured in the tubular compartment microdissected from kidney biopsy cores from a subset of patients. Linear mixed models were used to test if baseline uEGF/Cr and EGF mRNA expression were associated with change in estimated glomerular filtration rate (eGFR) over time.ResultsHigher uEGF/Cr at baseline was associated with slower eGFR decline during follow-up (median follow-up = 30 months). Halving of uEGF/Cr was associated with a decrease in eGFR slope of 2.0 ml/min per 1.73 m2 per year (P < 0.001) adjusted for age, race, diagnosis, baseline eGFR and proteinuria, and APOL1 genotype. In the biopsied subgroup, uEGF/Cr was correlated with EGF mRNA expression (r = 0.74; P < 0.001), but uEGF/Cr was retained over mRNA expression as the stronger predictor of eGFR slope after multivariable adjustment (decrease in eGFR slope of 1.7 ml/min per 1.73 m2 per year per log2 decrease in uEGF/Cr; P < 0.001).ConclusionuEGF/Cr may be a useful noninvasive biomarker that can assist in predicting the long-term course of kidney function in children with incident nephrotic syndrome.

Study on effect of extract from Tibetan medicine Urtica hyperborean on anti-prostatic hyperplasia

In this study,mouse models of benign prostatic hyperplasia induced by subcutaneous injection of testosterone propionate was used to investigate the therapeutic effect and mechanism of Urtica hyperborean( UW) extracts on prostate hyperplasia in mice. The effects of UW extracts on prostate index,serum epidermal growth factor( EGF) and dihydrotestosterone( DHT) in model mice were observed,and the EGF and anti-apoptotic factor( Bcl-2) mRNA expression levels were detected as well as pathological changes in prostate tissue. The results showed that the ethyl acetate extraction and alcohol soluble fraction of the UW could significantly reduce the prostate index,reduce the serum DHT and EGF levels( P<0. 01),and significantly decrease the EGF and Bcl-2 mRNA expression( P<0. 01),significantly improved the morphological structure of prostate tissue. The above results confirmed that ethyl acetate extract and alcohol-soluble parts of UW have a good preventive effect on mice prostatic hyperplasia model,and its mechanism may be to reduce androgen levels by regulating polypeptide growth factors and/or inhibiting cell hyperproliferation and promoting apoptosis. This study laid the foundation for the further research on UW.

Effects of Bacillus subtilis DSM32315 supplementation and dietary crude protein level on performance, gut barrier function and microbiota profile in weaned piglets1

Seventy-two piglets aged at 25 d were chosen to investigate the effects of Bacillus subtilis DSM32315 supplementation in diets with different protein levels on growth performance, intestinal barrier function, and gut microbiota profile in a 42-d trial. The animals were allotted to four treatment groups in a randomized complete block design involving a 2 (protein levels) × 2 (probiotic levels) factorial arrangement of treatments. Two protein levels included the high CP (HP) diets (0 to 14 d, 20.5%; 15 to 42 d, 19.5%) and the low CP (LP) diets (0 to 14 d, 18%; 15 to 42 d, 17%), and added probiotic (PRO) levels included at 0 and 500 mg/kg diet. Two interactions between CP and PRO for ADG (P < 0.01) and F/G (P < 0.05) were observed in phase 1. Within the piglets given the LP diet, probiotic supplementation increased ADG and decreased F/G ratio. Likewise, there were interactions between CP and PRO on the digestibility of CP (P < 0.01) and EE (P < 0.05), and probiotic supplementation increased the digestibility of CP and ether extract (EE) of piglets fed with LP diet, but that was not the case for piglets fed with HP diet. Furthermore, there were interactions between CP and PRO on villus height (P < 0.01) and villus height:crypt depth ratio (P < 0.05) in ileum. Piglets fed with LP diet containing probiotic had the greatest villus height and villus height:crypt depth ratio in ileum among treatments. There were also main effects of PRO on the propionic acid (P < 0.05) and butyric acid (P < 0.05), and the concentrations of propionic acid and butyric acid in colonic digesta were increased with the inclusion of probiotic in diet. Piglets fed with LP diet containing probiotic had the greatest population of Bacillus and Bifidobacterium (P < 0.05) in colon. In addition, there were interactions between CP and PRO on the mRNA expressions of occludin-1 (P < 0.05), epidermal growth factor (EGF) (P < 0.05), and insulin-like growth factor 1 receptor (IGF-1R) (P < 0.05). The LP fed piglets plus probiotic exhibited the greatest mRNA expressions of occludin-1, EGF, and IGF-1R in ileum compared with other treatments. In conclusion, moderate dietary protein restriction combining with the addition of B. subtilis DSM32315 synergistically increased growth performance, altered hindgut bacterial composition and metabolites, maintained intestinal barrier function in ileum of piglets.

Abstract WP549: GRK5-Mediated Inhibitory Phosphorylation of the Mineralocorticoid Receptor Protects Against Aldosterone`s Stroke-Promoting Vascular Effects

Introduction: Stroke is one of the leading causes of death in the Western world. Mineralocorticoid receptor (MR) antagonism is a potential therapy for ischemic and hemorrhagic stroke patients, since aldosterone exerts deleterious effects in the cerebral vasculature via the MR. Prominent among these is activation of epidermal growth factor (EGF)-dependent vessel wall remodeling. GRK2 and GRK5 are the most abundant G protein-coupled receptor (GPCR)-kinases (GRKs) in the heart and vessels and both phosphorylate GPCRs but also non-GPCR substrates at Ser/Thr residues. The human MR gets phosphorylated at various Ser residues, including Ser-843 in the ligand binding domain (LBD) and Ser-601 in the N-terminal domain (NTD). These Ser phosphorylations occur in the cytoplasm and result in MR inhibition via direct transcriptional activity repression or cytosolic retention, respectively. Hypothesis: GRK5 phosphorylates and inhibits the MR in the heart and vessels. Methods: We used rat cardiomyocytes (H9c2) and aortic smooth muscle cells (ASMCs). We checked for GRK interactions with the MR via co-immunoprecipitation. We measured MR phosphorylation via phospho-Ser immunoblotting and MR transcriptional activity via the luciferase reporter assay. We also measured EGF and EGF receptor (EGFR) expression via real time-PCR. Results: GRK5, but not GRK2, phosphorylates the MR in H9c2 cardiomyocytes. Beta2-adrenoceptor activation stimulates this non-canonical effect of GRK5. The GRK5-phosphorylated MR is incapable of activating gene transcription, since aldosterone-induced MR transcriptional activity is markedly suppressed upon GRK5 overexpression in both cardiomyocytes and ASMCs. Conversely, CRISPR-mediated GRK5 gene deletion augments cardiac and ASMC aldosterone-dependent MR transcriptional activity. Finally, beta2-adrenoceptor-stimulated GRK5 phosphorylates and inhibits the MR in ASMCs, thereby suppressing aldosterone-induced EGF and EGFR mRNA expression. Conclusions: GRK5 blocks the toxic MR-dependent effects of aldosterone in the heart and vascular smooth muscles. Thus, GRK5 stimulation (e.g. via beta2-adrenoceptor activation) may protect against the stroke-promoting effects of aldosterone in the cerebral vasculature.

Effects of vascular endothelial growth factor and epidermal growth factor on biological properties of gastric cancer cells.

IntroductionThe exfoliation of exfoliative cells from gastric serosa into the peritoneum is the main cause of peritoneal metastasis, which is the most common form of postoperative recurrence in gastric cancer. This study investigates the effects of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) on the biological properties of gastric cancer cells.Material and methodsmRNA expression of VEGF and EGF in gastric cancer tissues from 80 patients suffering from serosa-infiltrated gastric cancer (T3) was examined. The differences of proliferation, movement, adhesion and invasion among 4 gastric cancer cell lines were analysed. The mRNA expression of EGF, EGFR, VEGF and VEGFR in the gastric cancer cell lines was examined before and after adding endostatin (Endostar) or cetuximab (Erbitux) to observe changes of gastric cancer cells.ResultsmRNA levels of EGF and VEGF in positive exfoliative cytology cases were significantly higher than negative cases (p < 0.05). The biological properties were reduced sequentially in MGC803, HGC27, BGC823 and SGC7901 (p < 0.05). The mRNA expression of EGF, EGFR, VEGF and VEGFR was the strongest in MGC803, but was attenuated significantly after treatment (p < 0.05).ConclusionsLower survival was related to positive exfoliative cytology, lymphatic node metastasis, serosa-infiltrated and poorly differentiated gastric cancer. The expression of VEGF and EGF was correlated with the properties of gastric cancer cells. Specific inhibition of VEGF and EGF may impair the biological properties of gastric cancer cells in vitro.

Therapeutic effect of the epidermal growth factor on diabetic foot ulcer and the underlying mechanisms.

The present study aimed to explore the therapeutic effect and underlying mechanism of epidermal growth factor (EGF) on the wound healing of diabetic foot ulcers (DFU). A total of 48 rabbits with DFU were randomly divided into 2 groups, comprising the treatment and control groups. Full-thickness skin (10×10 mm) was excised from the thigh of each rabbit. The wounds in the treatment group were treated with 100 mg/l EGF once a day for 1 month. The control group received no treatment. At 20 days following treatment, new granulation tissues that formed beyond the edge of the wound were collected for subsequent analysis. Tissues from rabbits in the treatment group produced a greater number of fibroblasts, which exhibited a fibroblastic morphology when compared with those in the control group. In the treatment group, a larger number of these fibroblasts were observed as clusters, and there were numerous blood vessels when compared with the control group. The fibroblasts in the control group exhibited an irregular morphology, contained fewer organelles and the surrounding collagenous fibers were sparse. These fibroblasts also demonstrated a disordered arrangement and it was revealed that the wound healed at a slower rate compared with the treatment group. Endogenous EGF mRNA detection revealed that there was a significant difference (P<0.05) in the relative gray value of EGF mRNA between the treatment (103.27±4.27) and control (63.88±4.36) groups. In conclusion, EGF may accelerate the healing of DFU, and exogenous EGF treatment may upregulate the expression of EGF mRNA in newly generated tissues.

Study on the Effect of Cerium Oxide Nanocubes on Full-Thickness Cutaneous Wound Healing of Type 2 Diabetic Rats

Objective: To explore the effect and mechanism of cerium oxide nanocubes on the cutaneous wound healing of Goto-Kakizaki (GK) rats. Methods: A total of 90 GK rats with full-thickness wounds were equally divided into three groups according to different treatments: normal saline (NS), recombinant human epidermal growth factor (rhEGF), and cerium oxide nanocubes solution (CONS). Two round cuts were made on the rats' dorsum from epidermis to the muscle layer. On days 3, 7, 10, 13, 16 and 21 post operation, five rats in each group were killed and made into specimens. Histological evaluation were conducted and the protein and mRNA levels of epidermal growth factor (EGF) and tumor necrosis factor (TNF-α) were analyzed with enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) technology. In addition, the levels of superoxide dismutase (SOD) and catalase (CAT) were detected by ELISA. Results: Compared with the other two groups, the CONS-treated group had less inflammatory cell infiltration, more fibroblasts and smaller granulation tissue area in HE stained tissue micrographs. Meanwhile, Masson stained tissue micrographs showed thicker, more compact and more regular collagenous fiber. In CONS-treated rats, the wound healing rate on days 7 and 10 after surgery was significantly higher compared with other groups (p&amp;lt;0.05). On days 7, 10 and 13, the levels of TNF-α protein as well as TNF-α mRNA in the CONS-treated group were lower than in the NS-treated group (p&amp;lt;0.05). On days 7 and 10, SOD, CAT and EGF protein levels in the CONS-treated group were higher when compared to the NS-treated group (p&amp;lt;0.05), while the expression of EGF mRNA in the CONS-treated obviously increased from day 3 till day 13. Conclusion: The mechanism of cerium oxide nanocubes promoting experimental diabetic wound healing may be related to reducing inflammatory reaction and promoting the synthesis of antioxidants and growth factors. Disclosure Y. Han: None. L. Li: None. Y. Liu: None. Y. Wang: None. C. Yan: None. L. Sun: None.

Analysis of FK506-mediated functional recovery and neuroprotection in a rat model of spinal cord injury indicates that EGF is modulated in astrocytes.

The present study aimed to evaluate whether the application of tacrolimus (FK506) could improve functional recovery in spinal cord injury (SCI) rat models by activating astrocytes, and to further investigate the underlying mechanisms of this action. Male Sprague-Dawley rats (n=56) were used to establish moderate SCI models, which were induced at the T10 spinal segment by dropping a 10-g weight from a height of 25 mm using a New York University Impactor device. The rats were randomly separated into the FK506 or control group (n=28 per group). Rats were treated with FK506 (0.5 mg/kg) or saline intravenously 30 min after sustaining the injury. Functional recovery was evaluated over 42 days following the injury, and epidermal growth factor (EGF) levels were detected. The astrocytes were treated with FK506 in vitro, and the EGF mRNA and protein expression levels were analyzed using reverse transcription-quantitative polymerase chain reaction and ELISA, respectively. DNA microarray analysis was also performed to evaluate the genes in astrocytes. Rats in the FK506 group had improved locomotor functional recovery compared with those of control group. Furthermore, FK506 upregulated EGF expression of astrocytes both in vivo and in vitro. Subsequent to treatment with FK506-conditioned medium (CM), the length of neuronal cells increased 61.06% on the first day, and increased 56.4% on the third day compared with those of C-CM group. Furthermore, addition of anti-EGF neutralizing antibodies could interrupt the promotion of neurite outgrowth by FK506-CM. The present study indicates that astrocytes have an important role as mediators of FK506-improved spinal cord function recovery, and this partially clarifies the role of cell-cell interaction through modulating EGF in this process.

Identification of breast cancer mechanism based on weighted gene coexpression network analysis.

Our gene expression-profiling analysis aimed to explain the mechanism of breast cancer development by identifying key pathways and constructing networks of related transcription factors (TFs) and microRNAs (miRNAs) in breast cancer tissues. Gene expression profiles of normal and breast cancer tissues were downloaded to identify differentially expressed genes (DEGs). Coexpression modules were explored using weighted gene coexpression network analysis (WGCNA). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to discover the enriched functionally associated gene groups and define pathways in breast cancer, respectively. miRNAs-DEGs and TF-DEG regulatory networks were constructed using Cytoscape. CDK6(cyclin-dependent kinase), miR-124, EGF(epidermal growth factor) and NF-κB(nuclear factor of kappa light polypeptide gene enhancer in B-cells 1) expression was also analyzed using real-time quantitative PCR. Totally, 7713 DEGs were identified for WGCNA. The results revealed that 1388 upregulated DEGs were associated with protein transport, protein localization and organic substance transport, whereas 1819 downregulated DEGs were associated with cancer and Wnt signaling pathways. Five miRNAs (miR-760, miR-1276, miR-124, miR-124-3p and miR-506-3p) with a degree of ⩾15 and one important TF (NF-κB) were identified in miRNA and TF regulatory networks. CDK6 mRNA and miR-124 expression was significantly reduced and EGF mRNA expression was clearly enhanced in cancer tissues compared with those in normal breast tissues. The CDK6 gene could be regulated by miR-124, which is involved in Wnt signaling and cancer pathways. NF-κB might initiate the breast cancer pathway by targeting EGF in human breast cancer tissues. This putative information on regulatory networks in breast cancer will be beneficial for future researches on mechanisms underlying its development.