Bacterial Community Influences the Effects of Lactobacillus acidophilus on Lipid Metabolism, Immune Response, and Antioxidant Capacity in Dogs.

Lactobacillus acidophilus (L. acidophilus), the most prevalent probiotic, has demonstrated the ability to improve the relative abundance of intestinal microorganisms and boost immunity. However, the underlying mechanisms of these effects remain unclear. This study evaluated body weight, nutrient apparent digestibility, serum indices, and bacterial communities in Chinese rural dogs from a L. acidophilus supplementation group (Lactobacillus acidophilus, n = 6) and a control group (CON, n = 6). The results indicated that L. acidophilus had no significant impact on the body weight and apparent nutrient digestibility of Chinese rural dogs. In comparison with the CON group, L. acidophilus significantly reduced the levels of cholesterol (CHO) and increased the levels of IgA, IFN-α, and T-AOC. Bacterial diversity indices were significantly reduced in the LAC group compared to the CON groups, and MetaStat analysis demonstrated notable distinctions in 14 bacterial genera between the groups. These bacterial genera exhibited correlations with physiological indices such as CHO, IgA, IFN-α, and T-AOC. In conclusion, L. acidophilus can modulate lipid metabolism, immunity, and antioxidant capacity by regulating the relative abundance of specific bacterial communities, which helps dogs to adapt to today's lifestyle.

Integrated Transcriptome and Microbiota Reveal the Regulatory Effect of 25-Hydroxyvitamin D Supplementation in Antler Growth of Sika Deer.

The level of plasma 25-hydroxyvitamin D (25(OH)D) is associated with the growth of the antler, a fast-growing bone organ of Cervidae. However, the benefits of 25(OH)D supplementation on antler growth and the underlying mechanisms remain unclear. Here, the antler growth profile and transcriptome, plasma parameters, rumen bacteria, and metabolites (volatile fatty acids and amino acids) were determined in sika deer in a 25(OH)D supplementation group (25(OH)D, n = 8) and a control group (Ctrl, n = 8). 25(OH)D supplementation significantly increased the antler weight and growth rate. The levels of IGF-1,25(OH)D and 1,25-dihydroxyvitamin D were significantly higher in the 25(OH)D group than in the Ctrl group, while the levels of LDL-C were lower. The levels of valerate and branched-chain amino acids in the rumen fluid were significantly different between the 25(OH)D and Ctrl groups. The bacterial diversity indices were not significantly different between the two groups. However, the relative abundances of the butyrate-producing bacteria (families Lachnospiraceae and Succinivibrionaceae) and the pyruvate metabolism pathway were higher in the 25(OH)D group. The transcriptomic profile of the antler was significantly different between the 25(OH)D and Ctrl groups, with 356 up- and 668 down-regulated differentially expressed genes (DEGs) in the 25(OH)D group. The up-regulated DEGs were enriched in the proteinaceous extracellular matrix and collagen, while the down-regulated DEGs were enriched in the immune system and lipid metabolism pathways. Overall, these results provide novel insights into the effects of 25(OH)D supplementation on the host metabolism, rumen microbiota, and antler transcriptome of sika deer.

Effect of Vitamin A Supplementation on Growth Performance, Serum Biochemical Parameters, Intestinal Immunity Response and Gut Microbiota in American Mink (Neovison vison).

This experiment investigated the effect of vitamin A supplementation on growth, serum biochemical parameters, jejunum morphology and the microbial community in male growing-furring mink. Thirty healthy male mink were randomly assigned to three treatment groups, with 10 mink per group. Each mink was housed in an individual cage. The mink in the three groups were fed diets supplemented with vitamin A acetate at dosages of 0 (CON), 20,000 (LVitA) and 1,280,000 IU/kg (HVitA) of basal diet. A 7-day pretest period preceded a formal test period of 45 days. The results show that 20,000 IU/kg vitamin A increased the ADG, serum T-AOC and GSH-Px activities, villus height and villus height/crypt depth ratio (p < 0.05). The mRNA expression levels of IL-22, Occludin and ZO-1 in the jejunum of mink were significantly higher in the LVitA group than those in the CON and HVitA groups (p < 0.05). Vitamin A supplementation increased the diversity of jejunum bacteria, decreased the ratio of Firmicutes to Bacteroidetes and increased the relative abundance of Akkermansia, uncultured bacterium f Muribaculaceae, Allobaculum, Lachnospiraceae NK4A136 group, Rummeliibacillus and Parasutterella. The comparison of potential functions also showed enrichment of glycan biosynthesis and metabolism, transport and catabolism pathways in the vitamin A supplementation groups compared with the CON group. In conclusion, these results indicate that dietary vitamin A supplementation could mediate host growth by improving intestinal development, immunity and the relative abundance of the intestinal microbiota.

Effects of Arginine Supplementation on Serum Metabolites and the Rumen Bacterial Community of Sika Deer (Cervus nippon).

Velvet antler is a regeneration organ of sika deer (Cervus nippon) and an important Chinese medicine, and nutrient metabolism affects its growth. Here, we investigated the effects of arginine supplementation on antler growth, serum biochemical indices, and the rumen bacterial community of sika deer during the antler growth period. Fifteen male sika deer (6 years old) were randomly assigned to three dietary groups, which were supplemented with 0 (n = 5, CON), 2.5 (n = 5, LArg), or 5.0 g/d (n = 5, HArg) L-arginine. The IGF-1, ALT and AST concentrations in the serum of LArg sika deer were significantly higher than those in the serum of CON (P < 0.05) and HArg deer (P < 0.05). The phyla Bacteroidetes, Firmicutes, and Proteobacteria were dominant in the rumen of sika deer among the three groups. Comparison of alpha diversities showed that the ACE and Chao1 indices significantly increased in the LArg and HArg groups compared with those in the CON group. PCoA and ANOSIM results showed that the bacterial community was significantly changed between the CON and LArg groups. Moreover, the relative abundances of Fibrobacter spp. and Prevotellaceae UCG-003 increased, but those of Clostridium sensu stricto 1 and Corynebacterium 1 decreased in the LArg and HArg groups compared with those in the CON group. Additionally, the relative abundances of 19 OTUs were significantly different between the LArg and HArg groups. These results revealed that arginine supplementation affected the sika deer rumen bacterial community and serum biochemical indices.

All-trans-retinoic acid inhibits mink hair follicle growth via inhibiting proliferation and inducing apoptosis of dermal papilla cells through TGF-ß2/Smad2/3 pathway.

Dermal papilla cells (DPCs), an important component of hair follicles, its proliferation and apoptosis directly regulate and maintain the growth of hair follicles. All-trans-retinoic acid (ATRA) plays a critical role in hair growth. In this study, the effects of ATRA on cultured mink hair follicle growth were studied by administration of different concentrations of ATRA for 12 days in vitro. In addition, the proliferation and apoptosis of DPCs were measured after treating with ATRA. The mRNA and protein levels of hair follicle growth associated factor transforming growth factor-ß2 (TGF-ß2) and the phosphorylation levels of Smad2/3 were determined. Moreover, TGF-ß type I and type II receptor inhibitor LY2109761 and specific inhibitor of Smad3 (SIS3) were administered to investigate the underlying molecular mechanism. The results showed that ATRA inhibited hair follicle growth, promoted TGF-ß2 expression and activated phosphorylation of Smad2/3. In addition, ATRA inhibited cell proliferation by arresting the cell cycle at G1 phase and induced apoptosis of DPCs by enhancing the ratio of Bax/Bcl-2 and promoted the cleavage of caspase-3. Furthermore, LY2109761 or SIS3 partially reversed the decreased cell viability, increased apoptosis that were induced by ATRA. In conclusion, ATRA could inhibit hair follicle growth via inhibiting proliferation and inducing apoptosis of DPCs partially through the TGF-ß2/Smad2/3 pathway.

Pantothenic acid promotes dermal papilla cell proliferation in hair follicles of American minks via inhibitor of DNA Binding 3/Notch signaling pathway.

AIMS: Pantothenic acid (PA) has been applied to treat alopecia, but the underlying mechanism is still unclear. Our study aims to explore the underlying mechanism of PA in regulating hair follicle (HF) growth. MAIN METHODS: Mink HFs and dermal papilla (DP) cells were isolated and cultured in vitro. HFs and DP cells were treated with 0, 10, 20, 40 µg/ml PA. The effect of PA on HF growth, DP cell proliferation, cell cycle distribution, cell migration, and insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) expressions in DP cells was measured. Moreover, the effect of PA on inhibitor of DNA binding 3 (ID3)/Notch signaling pathway was analyzed. Subsequently, ID3 was silenced to validate whether ID3/Notch signaling pathway was involved in regulating DP cell proliferation by PA. KEY FINDINGS: Both 20 µg/ml and 40 µg/ml PA promoted HF growth, G1/S transition of DP cells and IGF-1 and VEGF expressions in DP cells, while only 20 µg/ml PA promoted cell viability and the migration of DP cells. Thus 20 µg/ml PA was chosen for the following experiments. PA treatment was found to up-regulate ID3 expression but down-regulate Notch receptor 1 (Notch1) and Notch signaling targets expressions. Furthermore, ID3 knockdown reversed PA-induced cell proliferation and inhibition of Notch1 and Notch signaling targets expressions, indicating that PA-induced DP cell proliferation and inhibition of Notch signaling were mediated via up-regulation of ID3. SIGNIFICANCE: This study provides an underlying mechanism related to the effect of PA on stimulating DP cell proliferation.

Ginkgolide B and bilobalide promote the growth and increase ß-catenin expression in hair follicle dermal papilla cells of American minks.

The regeneration of proliferation potential of dermal papilla (DP) cells contributes to the treatment of hair loss disorders. Ginkgolide B (GKB) and bilobalide (BB) are two functional components isolated from Ginkgo biloba that can promote hair growth. In the current study, the effect of GKB or BB on DP cell viability and the related signaling was assessed. Hair follicles were isolated from minks, and the growth of hair follicles was measured under the administration of GKB or BB. DP cells isolated from minks were also subjected to GKB or BB. The administration of GKB or BB induced the growth of hair follicles. The viability of DP cells was also increased by GKB or BB as illustrated by methyl thiazolyl tetrazolium and flow cytometry detection. Moreover, the secretion of VEGF was enhanced by GKB or BB. At molecular level, the activities of Akt, ERK1/2, and ß-catenin were induced by GKB, whereas BB only increased the activities of Akt and ß-catenin. In conclusion, although the two components influenced the ß-catenin signaling activity in distinct mechanisms, they both increased the viability of DP cells and promoted the cycle of hair follicles.

Bacterial community and metabolome shifts in the cecum and colon of captive sika deer (Cervus nippon) from birth to post weaning.

Ruminant productivity is associated with the gastrointestinal tract bacterial community, which matures progressively with animal growth. However, knowledge of the bacteriome and metabolome dynamics in cecum and colon during the early lives of neonatal ruminants is limited. Thus, we examined the bacteriome and metabolomes of the cecum and colon in neonatal sika deer at days 1, 42 and 70. The bacterial diversity and richness in the cecum and colon increased with age, and the bacterial community significantly changed across three time points. For cecum and colon, the proportions of Bacteroides spp., Escherichia-Shigella, Clostridium sensu stricto 1, Lachnoclostridium spp. and Lactobacillus spp. were predominated at day 1 and decreased with age, while the proportions of Ruminococcaceae UCG 005, Ruminococcaceae UCG 010, Rikenellaceae RC9 and Prevotellaceae UCG 003 were predominated at days 42 and 70 and increased with age. The concentrations of creatine and serine were significantly decreased, whereas the concentrations of total short-chain volatile fatty acids, pelargonic acid and leucine were increased in both the cecum and colon during development. These data document the development of the bacterial community and the metabolites in the cecum and colon of sika deer, and suggest a possible importance of ecology niche on gut development.

Epigallocatechin-3-Gallate Promotes the Growth of Mink Hair Follicles Through Sonic Hedgehog and Protein Kinase B Signaling Pathways.

Background: Hair follicles play an essential role in the growth of hair. Epigallocatechin-3-gallate (EGCG), a catechin polyphenol in green tea, has various bioactivities. The present study aims to evaluate the effect of EGCG on the growth of mink hair follicles and investigate the possible molecular mechanisms. Methods: The length of hair follicles was recorded up to 6 days in presence of 0.1-5 µM EGCG. Primary dermal papilla cells (DPCs) and outer root sheath cells (ORSCs) were treated with 0.25-4 µM EGCG, and their growth was evaluated by MTT assay and cell cycle detection. The levels of key molecules in sonic hedgehog (Shh) and protein kinase B (AKT) signaling pathways were further assessed by quantitative real-time PCR, western blot and immunofluorescence. To determine the involvement of Shh and AKT pathways in EGCG-mediated growth-promotion of ORSCs and DPCs, Shh pathway inhibitors cyclopamine and GANT61 or AKT pathway inhibitor LY294002 were employed, and then cell proliferation and cell cycle were analyzed. Results: Data from ex vivo culture showed that, in presence of 0.5-2.5 µM EGCG, the growth of mink hair follicles was promoted. In vitro, the proliferation of DPCs and ORSCs was enhanced by 0.5-4 µM EGCG treatment. More cells entered S phase upon treatment of EGCG, accompanied with upregulation of cyclin D1 and cyclin E1. Furthermore, when exposed to EGCG, the Shh and AKT signaling pathways were activated in both hair follicles and primary DPCs and ORSCs. Inhibiting either of these two pathways partly reversed the effect of EGCG on proliferation and cell cycle of DPCs and ORSCs. Conclusion: EGCG promotes the growth of mink hair follicles at concentrations of 0.5-2.5 µM. This growth-promoting effect of EGCG may be associated with the increased proliferation of DPCs and ORSCs through activating Shh and AKT signaling pathways.

Balance between fibroblast growth factor 10 and secreted frizzled-relate protein-1 controls the development of hair follicle by competitively regulating ß-catenin signaling.

Growth of hairs depends on the regular development of hair follicles which are hypothesized to be regulated by fibroblast growth factor 10 (FGF10) and secreted frizzled-relate protein-1 (sFRP1). In the current study, the effect of FGF10 or sFRP1 on hair follicle cells was assessed and the possible mechanism mediating the interaction between FGF10 and sFRP1 in hair follicle cells was explored. Out root sheath (ORS) and dermal papilla (DP) cells were isolated from mink skin tissues and subjected to administrations of FGF10 (50 ng/ml) or sFRP1 (10 ng/ml). Then proliferation, cell cycle distribution, and migration potentials of both cell types were detected. Moreover, the nuclear translocation of ß-catenin was determined. The results showed that the administration of FGF10 increased cell proliferation and migration potential in both cell types, which was associated with the up-regulated nuclear level of ß-catenin. To the contrary, the administration of sFRP1 decreased cell proliferation and migration potentials while induced the G1 cell cycle arrest in both cell types by inhibiting nuclear translocation of ß-catenin. Compared with the sole administrations, the co-treatment of FGF10 and sFRP1 had a medium effect on cell proliferation, cell cycle distribution, cell migration, and nuclear ß-catenin level, representing an antagonistic interaction between the two factors, which was exerted by competitively regulating ß-catenin pathway. Conclusively, the cycle of hair follicles was promoted by FGF10 while blocked by sFRP1 and the interplay between the two factors controlled the development of hair follicles by competitively regulating ß-catenin signaling.

The Development of Microbiota and Metabolome in Small Intestine of Sika Deer (Cervus nippon) from Birth to Weaning.

The dense and diverse community of microorganisms inhabiting the gastrointestinal tract of ruminant animals plays critical roles in the metabolism and absorption of nutrients, and gut associated immune function. Understanding microbial colonization in the small intestine of new born ruminants is a vital first step toward manipulating gut function through interventions during early life to produce long-term positive effects on host productivity and health. Yet the knowledge of microbiota colonization and its induced metabolites of small intestine during early life is still limited. In the present study, we examined the microbiota and metabolome in the jejunum and ileum of neonatal sika deer (Cervus nippon) from birth to weaning at days 1, 42, and 70. The microbial data showed that diversity and richness were increased with age, but a highly individual variation was observed at day 1. Principal coordinate analysis revealed significant differences in microbial community composition across three time points in the jejunum and ileum. The abundance of Halomonas spp., Lactobacillus spp., Escherichia-Shigella, and Bacteroides spp. tended to be decreased, while the proportion of Intestinibacter spp., Cellulosilyticum spp., Turicibacter spp., Clostridium sensu stricto 1 and Romboutsia spp. was significantly increased with age. For metabolome, metabolites separated from each other across the three time points in both jejunum and ileum. Moreover, the amounts of methionine, threonine, and putrescine were increased, while the amounts of myristic acid and pentadecanoic acid were decreased with age, respectively. The present study demonstrated that microbiota colonization and the metabolome becomes more developed in the small intestine with age. This may shed new light on the microbiota-metabolome-immune interaction during development.

Knockdown of HIF-1α inhibits the proliferation and migration of outer root sheath cells exposed to hypoxia in vitro: An involvement of Shh pathway.

AIMS: Outer root sheath (ORS) is a highly proliferative component of a hair follicle. This study is performed to investigate whether hypoxia-induced elevation of hypoxia-inducible factor (HIF)-1α, a transcriptional activator, contributes to the outgrowth of ORS cells in vitro. MAIN METHODS: Hair follicles with intact ORS collected from 4-month old male American minks were cultured in normoxic or hypoxic condition (3% oxygen) for 7days. Primary ORS cells isolated from the mink hair follicles were exposed to hypoxia for 12, 24 or 48h, and their proliferation was analyzed with immunofluorescence assay using anti-proliferating cell nuclear antigen (PCNA) antibody. The migratory ability of ORS cells was detected via the transwell chamber. The endogenous HIF-1α was knocked down with its specific siRNA in ORS cells. KEY FINDINGS: Hypoxic exposure induced an elevation of HIF-1α in ex vivo cultured hair follicles. The mRNA and protein levels of sonic hedgehog (Shh), Shh receptor Patched 1, Smoothened and glioma-associated oncogene homologue 1 were upregulated. In vitro, hypoxia induced an increase in HIF-1α in ORS cells. Further, under hypoxic condition, the number of PCNA-positive cells was increased, and more cells migrated towards high serum media. Hypoxia-enhanced proliferation and migration of ORS cells were suppressed either by HIF-1α siRNA or by pharmacological inhibitors of Shh pathway, cyclopamine and GANT61. The activation of Shh pathway was attenuated in HIF-1α-silenced ORS cells under hypoxic condition. SIGNIFICANCE: Our work demonstrates a direct role of activated HIF-1/Shh biological axis in sustaining the development of ORS in vitro.

Epidermal Growth Factor Promotes Proliferation and Migration of Follicular Outer Root Sheath Cells via Wnt/ß-Catenin Signaling.

BACKGROUND/AIMS: To investigate the effect and molecular mechanism of EGF on the growth and migration of hair follicle outer root sheath (ORS) cells. METHODS: Intact anagen hair follicles were isolated from mink skin and cultured with EGF in vitro to measure ORS daily growth. Meanwhile, purified primary ORS cells were treated or transfected with EGF, and their proliferation and migration were assessed by MTT assay and transwell assay, respectively. The signaling pathway downstream of EGF was characterized by using the Wnt/ß-catenin signaling inhibitor, XAV-939. RESULTS: EGF of 2-20 ng/ml, not higher or lower, promoted the growth of follicular ORS in vitro. EGF treatment or overexpression promoted the proliferation and migration of ORS cells. Moreover, EGF stimulation induced nuclear translocation of ß-catenin, and upregulated the expression of Wnt10b, ß-catenin, EGF receptor and SOX9. Inhibition of Wnt/ß-catenin signaling by XAV-939 significantly reduced the basal and EGF-enhanced proliferation and migration of ORS cells. In addition, a number of follicle-regulatory genes, such as Survivin, Msx2 and SGK3, were upregulated by EGF in the ORS cells, which was also inhibited by XAV-939. CONCLUSION: EGF promotes the proliferation and migration of ORS cells and modulates the expression of several follicle-regulatory genes via Wnt/ß-catenin signaling.

Epidermal growth factor promotes proliferation of dermal papilla cells via Notch signaling pathway.

The effect of epidermal growth factor (EGF) on the development and growth of hair follicle is controversial. In the present study, 2-20 ng/ml EGF promoted the growth of mink hair follicles in vitro, whereas 200 ng/ml EGF inhibited follicle growth. Further, dermal papilla (DP) cells, a group of mesenchymal cells that govern hair follicle development and growth, were isolated and cultured in vitro. Treatment with or forced expression of EGF accelerated proliferation and induced G1/S transition in DP cells. Moreover, EGF upregulated the expression of DP mesenchymal genes, such as alkaline phosphatase (ALP) and insulin-like growth factor (IGF-1), as well as the Notch pathway molecules including Notch1, Jagged1, Hes1 and Hes5. In addition, inhibition of Notch signaling pathway by DAPT significantly reduced the basal and EGF-enhanced proliferation rate, and also suppressed cell cycle progression. We also show that the expression of several follicle-regulatory genes, such as Survivin and Msx2, were upregulated by EGF, and was inhibited by DAPT. In summary, our study demonstrates that the concentration of EGF is critical for the switch between hair follicle growth and inhibition, and EGF promotes DP cell proliferation via Notch signaling pathway.