Citral alleviates peptidoglycan-induced inflammation and disruption of barrier functions in porcine intestinal epithelial cells.

Peptidoglycan (PGN) is a major polymer in bacterial cell walls and may constrain gut functionality and lower intestinal efficiencies in livestock. Citral has been reported to exhibit antibacterial and anti-inflammatory biological activities, improving the gastrointestinal function of swine. However, the protective effect of citral against PGN-elicited cellular responses and possible underlying mechanisms are unknown. In this study, the porcine jejunal epithelial cell line (IPEC-J2) was challenged with PGN from Staphylococcus aureus (S. aureus) or Bacillus subtilis (B. subtilis) to explore PGN-induced inflammatory responses. Our data showed that the inflammatory response stimulated by PGN from harmful bacteria (S. aureus) was more potent than that from commensal bacteria (B. subtilis) in IPEC-J2 cells. Based on the inflammatory model by PGN from S. aureus, it was demonstrated that PGN could significantly induce inflammatory cytokine production and influence nutrient absorption and barrier function in a dose-dependent manner. However, the PGN-mediated immune responses were remarkably suppressed by citral. In addition, citral significantly attenuated the effect of PGN on the intestine nutrient absorption and barrier function. The expression of TLR2 was strongly induced by PGN stimulation, which was suppressed by citral. All data nominated that citral downregulated PGN-induced inflammation via TLR2-mediated activation of the NF-κB signaling pathway in IPEC-J2 cells. Furthermore, the results also indicate that the PGN degradation through the inclusion of enzymes (e.g., muramidase) as well as the inclusion of citral for attenuating inflammation may improve pig gut health and functionality.

Monitoring collagen synthesis in fibroblasts using fluorescently labeled tRNA pairs.

There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET-based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti-fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells.

CCN2 expression by fibroblasts is not required for cutaneous tissue repair.

The CCN family of matricellular proteins, which includes CCN2 and CCN1, is believed to have a major in vivo role in controlling tissue morphogenesis and repair. In adult skin, the proadhesive matricellular protein connective tissue growth factor (CTGF/CCN2) is specifically up-regulated in fibrosis and wound healing. In mice, CCN2 is required for dermal fibrogenesis, but whether CCN2 is required for cutaneous tissue repair is unknown. To address this question, in this report we subjected adult mice bearing a fibroblast-specific deletion of CCN2 to the dermal punch model of cutaneous tissue repair. Loss of CCN2 did not appreciably affect the kinetics of tissue repair, collagen content, or the number of α-smooth muscle actin-positive cells. CCN1 (cyr61), which has in vitro effect similar to CCN2, is also induced in cutaneous tissue repair. Fibroblast-specific CCN1/CCN2 double knockout mice were also generated; loss of both CCN1 and CCN2 together did not appreciably affect cutaneous tissue repair. However, loss of CCN2 resulted in impaired recruitment of NG2-positive pericyte-like cells to the wound area. Collectively, these results indicate that neither CCN2 nor CCN1 is essential for cutaneous tissue repair; CCN2 appears to be required for recruitment of pericyte-like cells and may represent a specific antifibrotic target.

Fibrosis caused by loss of PTEN expression in mouse fibroblasts is crucially dependent on CCN2.

OBJECTIVE: Protein phosphatase and tensin homolog (PTEN) expression is reduced in dermal fibroblasts isolated from patients with diffuse cutaneous systemic sclerosis, a fibrotic autoimmune disease. In support of this finding, deletion of the PTEN gene in the dermal fibroblasts of mice has been shown to result in skin fibrosis and in vivo overexpression of connective tissue growth factor (CTGF; CCN2), a proadhesive matricellular protein; however, whether CCN2 is required for the fibrosis caused by loss of PTEN is unclear. This study was undertaken to investigate the role of CCN2 in fibrosis caused by reduced PTEN expression. METHODS: We generated conditional knockout mice in which PTEN was deleted in fibroblasts, either alone or in combination with CCN2. Skin samples were collected for histologic examination, immunohistochemical analysis, and collagen assay. RESULTS: Loss of CCN2 resulted in resistance to the increases in collagen production and myofibroblast recruitment that are caused by loss of PTEN. CCN2 deficiency did not impair Akt phosphorylation or the increases in the intensity of proliferating cell nuclear antigen staining that were caused by loss of PTEN. CONCLUSION: These data are consistent with the notion that CCN2 is required for particular aspects of the fibroproliferative response; therapeutic strategies blocking CCN2 may be of clinical benefit in combating fibrotic disease.

Impaired intervertebral disc development and premature disc degeneration in mice with notochord-specific deletion of CCN2.

OBJECTIVE: Currently, our ability to treat intervertebral disc (IVD) degeneration is hampered by an incomplete understanding of disc development and aging. The specific function of matricellular proteins, including CCN2, during these processes remains an enigma. The aim of this study was to determine the tissue-specific localization of CCN proteins and to characterize their role in IVD tissues during embryonic development and age-related degeneration by using a mouse model of notochord-specific CCN2 deletion. METHODS: Expression of CCN proteins was assessed in IVD tissues from wild-type mice beginning on embryonic day 15.5 to 17 months of age. Given the enrichment of CCN2 in notochord-derived tissues, we generated notochord-specific CCN2-null mice to assess the impact on the IVD structure and extracellular matrix composition. Using a combination of histologic evaluation and magnetic resonance imaging (MRI), IVD health was assessed. RESULTS: Loss of the CCN2 gene in notochord-derived cells disrupted the formation of IVDs in embryonic and newborn mice, resulting in decreased levels of aggrecan and type II collagen and concomitantly increased levels of type I collagen within the nucleus pulposus. CCN2-knockout mice also had altered expression of CCN1 (Cyr61) and CCN3 (Nov). Mirroring its role during early development, notochord-specific CCN2 deletion accelerated age-associated degeneration of IVDs. CONCLUSION: Using a notochord-specific gene targeting strategy, this study demonstrates that CCN2 expression by nucleus pulposus cells is essential to the regulation of IVD development and age-associated tissue maintenance. The ability of CCN2 to regulate the composition of the intervertebral disc suggests that it may represent an intriguing clinical target for the treatment of disc degeneration.

ß1 integrin-extracellular matrix interactions are essential for maintaining exocrine pancreas architecture and function.

Integrin receptors are responsible for integrating extracellular matrix signals inside the cell. The most prominent integrin receptor, ß1 integrin, has a role in cell function, survival and differentiation. Recently, we demonstrated a profound in vivo role of ß1 integrin expression in the pancreas on glucose homeostasis and islet function. Here, we extend these results by examining the role of ß1 integrin in exocrine pancreatic structure and function. Adult C57Bl/6 mice hemizygous for a collagen type Iα2 (Col1a2) promoter-controlled tamoxifen-inducible Cre recombinase gene and homozygous for loxP-ß1 integrin were injected with tamoxifen or corn oil to generate mice deleted or not for ß1 integrin. Pancreata derived from these male mice were analyzed by quantitative reverse transcriptase-polymerase chain reaction, western blot and immunofluorescence. Our results showed that ß1 integrin-deficient mice displayed a significant decrease in pancreas weight with a significant reduction of amylase, regenerating islet-derived protein II and carboxypeptidase-A expression (P<0.05-0.01). Compared with control pancreata, ß1 integrin-deficient pancreata showed reduced mRNA expression of extracellular matrix (collagen type Iα2, fibronectin and laminin) genes (P<0.05), detached acini clusters and lost focal adhesion structure. Moreover, ß1 integrin-deficient pancreatic acinar cells displayed decreased proliferation (P<0.05) and increased apoptosis (P<0.001). Apoptosis was reduced to that of controls when isolated exocrine clusters were cultured in media supplemented with extracellular matrix proteins. Taken together, these results implicate ß1 integrin as an essential component for maintaining exocrine pancreatic structure and function.

Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation.

OBJECTIVE: Enhanced adhesive signaling, including activation of focal adhesion kinase (FAK), is a hallmark of fibroblasts from lung fibrosis patients, and FAK has therefore been hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro. METHODS: FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor PF-562,271, or with small interfering RNA (siRNA)-mediated silencing of FAK were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and lungs were harvested for histologic and biochemical analysis. Using endothelin 1 (ET-1) as a stimulus, cell adhesion and contraction, as well as profibrotic gene expression, were studied in fibroblasts isolated from wild-type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild-type and ß1 integrin-deficient mouse fibroblasts. RESULTS: FAK expression and activity were up-regulated in fibroblast foci and remodeled vessels from lung fibrosis patients. Pharmacologic or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis in mice. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by JNK activation through ß1 integrin/FAK signaling. CONCLUSION: These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.

Expression of integrin beta1 by fibroblasts is required for tissue repair in vivo.

In tissue repair, fibroblasts migrate into the wound to produce and remodel extracellular matrix (ECM). Integrins are believed to be crucial for tissue repair, but their tissue-specific role in this process is poorly understood. Here, we show that mice containing a fibroblast-specific deletion of integrin ß1 exhibit delayed cutaneous wound closure and less granulation tissue formation, including reduced production of new ECM and reduced expression of α-smooth muscle actin (α-SMA). Integrin-ß1-deficient fibroblasts showed reduced expression of type I collagen and connective tissue growth factor, and failed to differentiate into myofibroblasts as a result of reduced α-SMA stress fiber formation. Loss of integrin ß1 in adult fibroblasts reduced their ability to adhere to, to spread on and to contract ECM. Within stressed collagen matrices, integrin-ß1-deficient fibroblasts showed reduced activation of latent TGFß. Addition of active TGFß alleviated the phenotype of integrin-ß1-deficient mice. Thus integrin ß1 is essential for normal wound healing, where it acts, at least in part, through a TGFß-dependent mechanism in vivo.

Focal adhesion kinase and reactive oxygen species contribute to the persistent fibrotic phenotype of lesional scleroderma fibroblasts.

OBJECTIVE: Fibrotic diseases such as SSc (systemic sclerosis, scleroderma) are characterized by the abnormal presence of the myofibroblast, a specialized type of fibroblast that overexpresses the highly contractile protein α-smooth muscle actin. Myofibroblasts display excessive adhesive properties and hence exert a potent mechanical force. We aim to identify the precise contribution of adhesive signalling, which requires integrin-mediated activation of focal adhesion kinase (FAK)/src, to fibrogenic gene expression in normal and fibrotic SSc fibroblasts. METHODS: We subject either FAK wild-type and knockout fibroblasts or normal and SSc fibroblasts treated with FAK/src inhibitors to real-time polymerase chain, western blot, cell migration and collagen gel contraction analyses. RESULTS: FAK operates downstream of both integrin ß1 and reactive oxygen species (ROS) to promote the expression of genes involved in matrix production and remodelling, including CCN2, α-smooth muscle actin and type I collagen. Blocking either FAK/src with PP2 or ROS with N-acetyl cysteine alleviates the elevated contractile and migratory capability of lesional SSc dermal fibroblasts. CONCLUSIONS: Excessive adhesive signalling is intimately involved with the fibrotic phenotype of lesional SSc fibroblasts; blocking adhesive signalling or ROS generation may be beneficial in controlling the fibrosis observed in SSc.

CCN2 is required for bleomycin-induced skin fibrosis in mice.

OBJECTIVE: No therapy for fibrotic disease is available. The proadhesive matricellular protein connective tissue growth factor CCN2 is a marker of fibrotic cells; however, the specific role of CCN2 in connective tissue biology in general and in fibrogenesis in particular is unclear. The aim of this study was to assess whether adult mice bearing a smooth muscle cell/fibroblast-specific deletion of CCN2 are resistant to bleomycin-induced skin scleroderma. METHODS: Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin. Untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast/smooth muscle cell-specific deletion of CCN2 were investigated for changes in dermal thickness, collagen content, and the number of α-smooth muscle actin (α-SMA)-positive cells. Dermal fibroblasts were isolated to assess whether the induction of collagen and α-SMA messenger RNA in response to transforming growth factor ß (TGFß) was impaired. RESULTS: The loss of CCN2 resulted in resistance to bleomycin-induced skin fibrosis. In response to bleomycin, wild-type mice possessed, but CCN2-deficient mice lacked, abundant α-SMA-expressing myofibroblasts within fibrotic lesions. Fibroblast responses to TGFß, a potent inducer of myofibroblast differentiation, were not affected. Collectively, these results indicate that CCN2 is essential for bleomycin-induced skin fibrosis, likely due to a defect in myofibroblast recruitment. CONCLUSION: These data indicate that therapeutic strategies that involve blocking CCN2 in vivo may be of benefit in combating fibrotic skin disease.

The Beneficial Effects of Hydrogen-Rich Saline Irrigation on Chronic Rhinitis: A Randomized, Double-Blind Clinical Trial.

Purpose: Chronic rhinitis (CR) is a common chronic inflammation of the nasal mucosa. Nasal saline irrigation has been demonstrated to be an effective treatment for CR. In this study, we investigated the beneficial effects of hydrogen-rich saline irrigation as an anti-inflammatory irrigation therapy for CR and compared its effectiveness over saline irrigation. Hydrogen-rich saline (HRS) was investigated due to its antioxidant and anti-inflammatory properties. Methods: A total of 120 patients with CR were randomly divided into two groups, patients irrigated with HR (HRS group) and the control group irrigated with saline (NS group). A randomized, double-blind control study was performed. The main observation index in this study was the total score of nasal symptoms (TNSS). In addition, eosinophilic protein (ECP) of the nasal secretions, nasal nitric oxide (nNO) levels, and levels of regulatory T cells (Treg) and regulatory B cells (Breg) were also compared between the two groups. Furthermore, patients with allergic rhinitis (AR) and non-allergic rhinitis (NAR) were also evaluated based on serum-specific IgE positivity. Results: After treatment, TNSS and nasal ECP in the two groups decreased significantly (P<0.05), with patients in the HRS group showing significantly lower levels compared to the NS group (P<0.05). There were no significant differences in Treg and Breg levels between the two groups. Subgroup analysis showed that TNSS in the AR-HRS group showed a more significant reduction compared to the AR-NS group (P<0.05); however, there were no significant differences for the other inflammatory biomarkers (P>0.05). ECP levels were reduced significantly in the NAR subgroup compared to NS irrigation (P<0.05). There were no obvious adverse events observed in patients during the entire treatment period. Conclusion: Compared to saline irrigation, HRS nasal irrigation was found to improve CR clinical symptoms, especially in patients with AR. HRS could effectively be used for the clinical treatment of patients with CR.

GSK-3beta in mouse fibroblasts controls wound healing and fibrosis through an endothelin-1-dependent mechanism.

Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by 2 genes, GSK3A and GSK3B. GSK-3 is thought to be involved in tissue repair and fibrogenesis, but its role in these processes is currently unknown. To investigate the function of GSK-3beta in fibroblasts, we generated mice harboring a fibroblast-specific deletion of Gsk3b and evaluated their wound-healing and fibrogenic responses. We have shown that Gsk3b-conditional-KO mice (Gsk3b-CKO mice) exhibited accelerated wound closure, increased fibrogenesis, and excessive scarring compared with control mice. In addition, Gsk3b-CKO mice showed elevated collagen production, decreased cell apoptosis, elevated levels of profibrotic alpha-SMA, and increased myofibroblast formation during wound healing. In cultured Gsk3b-CKO fibroblasts, adhesion, spreading, migration, and contraction were enhanced. Both Gsk3b-CKO mice and fibroblasts showed elevated expression and production of endothelin-1 (ET-1) compared with control mice and cells. Antagonizing ET-1 reversed the phenotype of Gsk3b-CKO fibroblasts and mice. Thus, GSK-3beta appears to control the progression of wound healing and fibrosis by modulating ET-1 levels. These results suggest that targeting the GSK-3beta pathway or ET-1 may be of benefit in controlling tissue repair and fibrogenic responses in vivo.

Capsaicin Attenuates Lipopolysaccharide-Induced Inflammation and Barrier Dysfunction in Intestinal Porcine Epithelial Cell Line-J2.

Capsaicin is a spicy, highly pungent, colorless, vanilloid compound found in chili peppers with anti-inflammatory, antioxidant, anti-cancer, and analgesic properties. However, the protective effects of capsaicin on the pig intestine during inflammation are yet to be explored. This study investigated the effects of capsaicin on the gut inflammatory response, intestinal epithelial integrity, and gene expression level of nutrient transporters in a model of lipopolysaccharide (LPS)-induced inflammation in non-differentiated intestinal porcine epithelial cell line-J2 (IPEC-J2). The results showed that the pre-treatment of cells with capsaicin (100 µM) significantly decreased the gene expression and secretion of proinflammatory cytokines induced by LPS through Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. In addition, pre-treatment of cells with capsaicin also increased both gene and protein abundance of tight junction proteins. Furthermore, pre-treatment cells with capsaicin significantly increased trans-epithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-dextran (FD4) from the apical side to the basolateral side compared with the control (P < 0.05). Additionally, pre-treatment of cells with capsaicin upregulated the mRNA abundance of nutrients transporters such as Na+/glucose cotransporter 1 (SGLT1). These results suggested that capsaicin could attenuate LPS-induced inflammation response through TLR4/NF-κB pathway and improve barrier integrity and glucose absorption.

Molecular distribution and localization of extracellular calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) at three different laying stages in laying hens (Gallus gallus domesticus).

The extracellular calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) play important roles in regulating calcium mobilization, calcium absorption, and calcium homeostasis, and they could be potential therapeutic targets to osteoporosis in laying hens. The present study investigated the molecular distribution of CaSR and VDR and the localization of CaSR in the kidney, proventriculus (true stomach), duodenum, jejunum, ileum, colon, cecum, shell gland, and tibia of laying hens at 3 different laying stages (19, 40, and 55 wk). The results showed that the relative mRNA abundance of CaSR in the kidney, ileum, proventriculus, duodenum, and colon was higher (P < 0.05) than the other tissues at 40 and 55 wk. The relative mRNA abundance of CaSR in the tibia was higher (P < 0.05) at 55 wk than at 40 wk. However, there were no significant differences in the relative protein abundance of CaSR among all tested tissues at peak production or in each tissue at the 3 different laying stages (P > 0.05). The relative mRNA abundance of VDR was higher (P < 0.05) in the small intestine (duodenum, jejunum, and ileum) when compared with other tissues at the 3 different laying stages. The relative protein abundance of VDR in the duodenum was higher (P < 0.05) than that in the proventriculus, colon, and cecum. There were no significant differences in the VDR expression among the tested tissues at the 3 different laying stages (P > 0.05). The immunohistochemical results showed that the positive staining was found widely in each tissue. Moreover, different laying stages did not affect the localization of CaSR except for the tibia tissue. In conclusion, similar to VDR, CaSR was widely expressed not only in the gut but also in the tibia and shell gland in laying hens. The expression level of CaSR and VDR in all tested tissues was unchanged at the different laying stages.

Efficiency of Deoxynivalenol Detoxification by Microencapsulated Sodium Metabisulfite Assessed via an In Vitro Bioassay Based on Intestinal Porcine Epithelial Cells.

Deoxynivalenol (DON) contamination occurs in feeds and causes a reduction in growth performance, damage to the intestinal epithelial cells, and increased susceptibility to enteric pathogen challenge. Sodium metabisulfite (SMBS) has shown promise in reducing DON; however, SMBS quickly degrades under aqueous acidic conditions such as the environment within a stomach. Thus, protection of SMBS is required for effective delivery to the small intestine to detoxify DON. This study was to encapsulate SMBS into hydrogenated palm oil-based microparticles for its delivery to the small intestine and to evaluate its efficacy on DON detoxification in simulated intestinal fluids using IPEC-J2 cells in vitro. The diameter of the SMBS containing microparticles was 511 ± 135 µm, and the loading capacity of SMBS in the microparticles was 45.50%; 1.41% of the encapsulated SMBS (ES) was released into the simulated gastric fluid, and 66.39% of ES was progressively released into the simulated intestinal fluid within 4 h at 37 °C. In IPEC-J2 cells, when DON was treated with the simulated gastric fluid containing 0.5% ES for 2 h, then mixed with the simulated intestinal fluid (1:1) and incubated for 2 h, cytotoxicity was not observed. DON treated with 0.5 ES decreased the gene expression of inflammatory cytokines in the cells compared with DON alone and maintained the cell integrity. To conclude, the SMBS containing microparticles were stable in the simulated gastric fluid and allowed a progressive release of SMBS in the simulated intestinal fluid. The released SMBS in the simulated intestinal fluid effectively detoxified DON.

Effects of encapsulated cinnamaldehyde on growth performance, intestinal digestive and absorptive functions, meat quality and gut microbiota in broiler chickens.

Essential oils are potential antimicrobial alternatives and their applications in animal feeds are limited due to their fast absorption in the upper gastrointestinal tract. This study investigated the effects of encapsulated cinnamaldehyde (CIN) at 50 mg/kg or 100 mg/kg on the growth performance, organ weights, meat quality, intestinal morphology, jejunal gene expression, nutrient digestibility, and ileal and cecal microbiota. A total of 320 male day-old broiler Cobb-500 chicks were randomly allocated to four treatments with eight pens per treatment (10 birds per pen): 1) basal diet (negative control, NC); 2) basal diet supplemented with 30 mg/kg avilamycin premix (positive control, PC); 3) basal diet with 50 mg/kg encapsulated CIN (EOL); 4) basal diet with 100 mg/kg encapsulated CIN (EOH). Despite birds fed EOH tended to increase (P = 0.05) meat pH at 24 h, all pH values were normal. Similar to PC group, meats from birds fed EOL and EOH showed a reduced (P < 0.05) Warner-Bratzler force shear (WBFS) compared to the NC group. The highest villus to crypt ratios (VH/CD; P < 0.05) were observed in broilers fed either EOL or EOH, with an average of 14.67% and 15.13% in the duodenum and 15.13% and 13.58% in the jejunum, respectively. For jejunal gene expressions, only six out of the 11 studied genes showed statistically significant differences among the dietary treatments. Gene expressions of cationic amino acid transporter 1 (CAT-1) and neutral amino acid transporter 1 (B0AT-1) were upregulated in EOH-fed birds compared to PC and NC-fed birds (P < 0.05), respectively; while the expression of proliferating cell nuclear antigen (PCNA) was downregulated in EOL-fed birds when compared to NC birds (P < 0.05). Nonetheless, the expressions of cadherin 1 (CDH-1), zonula occludens 1 (ZO-1), and maltase-glucoamylase (MG) were all upregulated (P < 0.05) in EOH-fed birds compared to PC-fed birds. The apparent ileal digestibility (AID) of dry matter, crude protein, crude fat and of all 18 tested amino acids increased in EOL-fed birds (P < 0.01). Additionally, relative abundances (%) of ileal Proteobacteria decreased, while ileal and cecal Lactobacillus increased in EOH-fed birds (P < 0.05). In conclusion, dietary encapsulated CIN improved meat quality and gut health by reducing meat WBFS, increasing VH/CD in intestines, jejunal gene expressions, AID of nutrients and beneficial ileal and cecal microbiota composition.

Rac1 expression by fibroblasts is required for tissue repair in vivo.

Tissue repair requires that fibroblasts migrate into the wound to produce and remodel extracellular matrix, a process that requires adhesion. Failure to suppress the tissue repair program results in fibrotic disorders that are characterized by excessive adhesive signaling. The role of specific components of adhesive signaling in fibrogenic responses is unclear, but may involve small GTPases such as Rac1. To address the functions of Rac1 in fibroblasts, we generated mice containing a fibroblast-specific deletion of Rac1. These mice show delayed cutaneous wound closure, including reduced collagen production and myofibroblast formation. In cultured Rac1-deficient fibroblasts, adhesion, spreading, and migration were significantly inhibited. Rac1-deficient fibroblasts possessed impaired myofibroblast formation and function as visualized by reduced alpha-smooth muscle actin expression as well as matrix contraction. Both in vivo and in vitro, Rac1- deficient fibroblasts showed a reduced generation of reactive oxygen species; in vitro, hydrogen peroxide alleviated the phenotype of Rac1-deficient fibroblasts. Thus, Rac1 is an essential signaling integrator that is required for normal wound healing and dermal homeostasis.

FAK is required for TGFbeta-induced JNK phosphorylation in fibroblasts: implications for acquisition of a matrix-remodeling phenotype.

Transforming growth factor beta (TGFbeta) plays a critical role in connective tissue remodeling by fibroblasts during development, tissue repair, and fibrosis. We investigated the molecular pathways in the transmission of TGFbeta signals that lead to features of connective tissue remodeling, namely formation of an alpha-smooth muscle actin (alpha-SMA) cytoskeleton, matrix contraction, and expression of profibrotic genes. TGFbeta causes the activation of focal adhesion kinase (FAK), leading to JNK phosphorylation. TGFbeta induces JNK-dependent actin stress fiber formation, matrix contraction, and expression of profibrotic genes in fak+/+, but not fak-/-, fibroblasts. Overexpression of MEKK1, a kinase acting upstream of JNK, rescues TGFbeta responsiveness of JNK-dependent transcripts and actin stress fiber formation in FAK-deficient fibroblasts. Thus we propose a FAK-MEKK1-JNK pathway in the transmission of TGFbeta signals leading to the control of alpha-SMA cytoskeleton reorganization, matrix contraction, and profibrotic gene expression and hence to the physiological and pathological effects of TGFbeta on connective tissue remodeling by fibroblasts.

Eugenol attenuates inflammatory response and enhances barrier function during lipopolysaccharide-induced inflammation in the porcine intestinal epithelial cells.

Eugenol (4-allyl-2-methoxyphenol) is an essential oil component, possessing antimicrobial, anti-inflammatory, and antioxidative properties; however, the effect of eugenol on porcine gut inflammation has not yet been investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model in porcine intestinal epithelial cells (IPEC-J2) has been set up. Cells were pretreated with 100 µM (16.42 mg/L) eugenol for 2 h followed by 10 µg/mL LPS stimulation for 6 h. Proinflammatory cytokine secretion; reactive oxygen species; gene expression of proinflammatory cytokines, tight junction proteins, and nutrient transporters; the expression and distribution of zonula occludens-1 (ZO-1); transepithelial electrical resistance (TEER); and cell permeability were measured to investigate the effect of eugenol on inflammatory responses and gut barrier function. The results showed that eugenol pretreatment significantly suppressed the LPS-stimulated interleukin-8 level and the mRNA abundance of tumor necrosis factor-α and restored the LPS-stimulated decrease of the mRNA abundance of tight junction proteins, such as ZO-1 and occludin, and the mRNA abundance of nutrient transporters, such as B0 1 system ASC sodium-dependent neutral amino acid exchanger 2, sodium-dependent glucose transporter 1, excitatory amino acid transporter 1, and peptide transporter 1. In addition, eugenol improved the expression and even redistribution of ZO-1 and tended to increase TEER value and maintained the barrier integrity. In conclusion, a low dose of eugenol attenuated inflammatory responses and enhanced selectively permeable barrier function during LPS-induced inflammation in the IPEC-J2 cell line.

Effects of antibiotic growth promoter and dietary protease on growth performance, apparent ileal digestibility, intestinal morphology, meat quality, and intestinal gene expression in broiler chickens: a comparison.

This study aimed to evaluate the effects of supplementing broiler diets with a dietary protease on growth performance, digestive function, intestinal morphology, and meat quality as compared with feeding diets with or without an antibiotic growth promoter (AGP). A total of 240 1-day-old male chicks (Cobb 500, 48.3 ± 3.3 g) were distributed to three treatments with eight replicates (10 birds per replicate). Three treatments were: 1) corn-soybean meal basal diets (CTRL), 2) basal diets with 0.003% avilamycin (AB), and 3) basal diets with 0.0125% protease (PRT). The diets were provided as mash form, and birds were fed ad libitum during the whole experimental period. On day 45, birds were euthanized, and tissue and digesta samples were collected. On day 46, the remaining birds were processed in a commercial slaughterhouse, and breast muscle samples were collected. Despite a trend for a decreased feed conversion ratio (FCR) in the AB group during the whole phase (P = 0.071), no significant differences in growth performance parameters and relative weights of organs were observed (P > 0.05) among the groups. The AB and PRT groups showed significantly greater apparent ileal digestibility of amino acids (AA) compared with the CTRL group (P < 0.05). The PRT group significantly improved the morphology of duodenum and jejunum (P < 0.05). No differences were detected for meat quality, white striping, and woody breast among the groups (P > 0.05). For the gene expressions, the AB group showed a greater level of B0-system neutral amino acid co-transporter 1 and excitatory amino acid transporter 1 mRNA abundance compared with PRT group, while a significantly lesser level of cationic amino acid transporter 1 mRNA abundance was observed in the AB group compared with CTRL group (P < 0.05). The PRT group had a lesser level of peptide transporter 1 mRNA abundance in the jejunum than the CTRL group (P < 0.05). The highest mRNA abundances of zonula occludens-1 and cadherin 1 were observed in the CTRL group (P < 0.05). In conclusion, supplementation of avilamycin tended to reduce FCR and significantly improved AA utilization, and supplementation of dietary protease significantly enhanced intestinal morphology and AA utilization in broilers. In that respect, exogenous protease use appears to be an interesting tool to be considered in AGP reduction strategies.