Homologous-targeting biomimetic nanoparticles co-loaded with melittin and a photosensitizer for the combination therapy of triple negative breast cancer.

Melittin (Mel) is considered a promising candidate drug for the treatment of triple negative breast cancer (TNBC) due to its various antitumor effects. However, its clinical application is hampered by notable limitations, including hemolytic activity, rapid clearance, and a lack of tumor selectivity. Here, we designed novel biomimetic nanoparticles based on homologous tumor cell membranes and poly(lactic-co-glycolic acid) (PLGA)/poly(beta-aminoester) (PBAE), denoted MDM@TPP, which efficiently coloaded the cytolytic peptide Mel and the photosensitizer mTHPC. Both in vitro and in vivo, the MDM@TPP nanoparticles effectively mitigated the acute toxicity of melittin and exhibited strong TNBC targeting ability due to the homologous targeting effect of the tumor cell membrane. Under laser irradiation, the MDM@TPP nanoparticles showed excellent photodynamic performance and thus accelerated the release of Mel by disrupting cell membrane integrity. Moreover, Mel combined with photodynamic therapy (PDT) can synergistically kill tumor cells and induce significant immunogenic cell death, thereby stimulating the maturation of dendritic cells (DCs). In 4T1 tumor-bearing mice, MDM@TPP nanoparticles effectively inhibited the growth and metastasis of primary tumors and finally prevented tumor recurrence by improving the immune response.

Combination Nano-Delivery Systems Remodel the Immunosuppressive Tumor Microenvironment for Metastatic Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer for which effective therapies are lacking. Targeted remodeling of the immunosuppressive tumor microenvironment (TME) and activation of the body's immune system to fight tumors with well-designed nanoparticles have emerged as pivotal breakthroughs in tumor treatment. To simultaneously remodel the immunosuppressive TME and trigger immune responses, we designed two potential therapeutic nanodelivery systems to inhibit TNBC. First, the bromodomain-containing protein 4 (BRD4) inhibitor JQ1 and the cyclooxygenase-2 (COX-2) inhibitor celecoxib (CXB) were coloaded into chondroitin sulfate (CS) to obtain CS@JQ1/CXB nanoparticles (NPs). Then, the biomimetic nanosystem MM@P3 was prepared by coating branched polymer poly(ß-amino ester) self-assembled NPs with melittin embedded macrophage membranes (MM). Both in vitro and in vivo, the CS@JQ1/CXB and MM@P3 NPs showed excellent immune activation efficiencies. Combination treatment exhibited synergistic cytotoxicity, antimigration ability, and apoptosis-inducing and immune activation effects on TNBC cells and effectively suppressed tumor growth and metastasis in TNBC tumor-bearing mice by activating the tumor immune response and inhibiting angiogenesis. In summary, this study offers a novel combinatorial immunotherapeutic strategy for the clinical TNBC treatment.

Delivery of Interferon ß-Encoding Plasmid via Lipid Nanoparticle Restores Interferon ß Expression to Enhance Antitumor Immunity in Colon Cancer.

Type I interferon (IFN-I) plays a critical role in host cancer immunosurveillance, but its expression is often impaired in the tumor microenvironment. We aimed at testing the hypothesis that cationic lipid nanoparticle delivery of interferon ß (IFNß)-encoding plasmid to tumors is effective in restoring IFNß expression to suppress tumor immune evasion. We determined that IFN-I function in tumor suppression depends on the host immune cells. IFN-I activates the expression of Cxcl9 and Cxcl10 to enhance T cell tumor infiltration. RNA-Seq detected a low level of IFNα13 and IFNß in colon tumor tissue. scRNA-Seq revealed that IFNß is expressed in immune cell subsets in non-neoplastic human tissues and to a lesser degree in human colon tumor tissues. Forced expression of IFNα13 and IFNß in colon tumor cells up-regulates major histocompatibility complex I (MHC I) expression and suppresses colon tumor growth in vivo. In human cancer patients, IFNß expression is positively correlated with human leukocyte antigen (HLA) expression, and IFN-I signaling activation correlates with the patient response to PD-1 blockade immunotherapy. To translate this finding to colon cancer immunotherapy, we formulated a 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-cholesterol-encapsulated IFNß-encoding plasmid (IFNBCOL01). IFNBCOL01 transfects colon tumor cells to express IFNß to increase the level of MHC I expression. IFNBCOL01 therapy transfects tumor cells and tumor-infiltrating immune cells to produce IFNß to activate MHC I and granzyme B expression and inhibits colon tumor growth in mice. Our data determine that lipid nanoparticle delivery of IFNß-encoding plasmid DNA enhances tumor immunogenicity and T cell effector function to suppress colon tumor growth in vivo.

Hair Follicle Development of Rex Rabbits Is Regulated Seasonally by Wnt10b/ß-Catenin, TGFß-BMP, IGF1, and EGF Signaling Pathways.

This experiment was conducted to study the effects of different skinning seasons on the fur quality and hair follicle development of Rex rabbits. A total of 80,150-day-old Rex rabbits were slaughtered on 15 July 2022 (summer), 15 October 2022 (autumn), 15 January 2023 (winter), and 15 April 2023 (spring) in Shandong Province (10 males and 10 females in each season). The results show that the skin weight, skin area, skin thickness, and hair follicle density of the Rex rabbits (at 150 days of age) were lower in summer than in winter (p < 0.05). Moreover, the coat length was shorter in summer than in spring, autumn, and winter (p < 0.05). The shoulder fat weight, perirenal fat weight, and perigastric fat weight of the Rex rabbits in winter were higher than those in summer (p < 0.05). Furthermore, the leptin levels in serum were higher in winter than in summer in the Rex rabbits (p < 0.05). In terms of serum biochemistry, the glucose levels were higher in autumn and winter than in spring and summer (p < 0.05). The cholesterol, high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) in summer had higher values than in winter in the Rex rabbits (p < 0.05). In winter, the expression of the Wnt10b, catenin beta 1 (CTNNB1), glycogen synthase kinase 3 beta (GSK3ß), insulin like growth factor I (IGF-I), Type I insulin-like growth factor receptor (IGF-IR), and epidermal growth factor (EGF) genes was higher (p < 0.05), and the expression of the dickkopf-1 (DDK1), transforming growth factor beta 1 (TGFß-1), bone morphogenetic protein 2 (BMP2), and bone morphogenetic protein 4 (BMP4) genes was lower than in summer (p < 0.05). In summer, the heat shock 70 kDa protein (HSP70) expression and CTNNB1 protein phosphorylation levels in skin tissue were higher than in spring, autumn, and winter (p < 0.05). In winter, Wnt10b protein expression was higher (p < 0.05), and GSK-3ß protein phosphorylation levels were lower than in spring, autumn, and winter (p < 0.05). These results show that the skinning season can affect the production performance and hair follicle development of Rex rabbits. Compared with other seasons, the quality of skin from rabbits slaughtered in winter is better. Seasons may regulate hair follicle development via the Wnt10b/ß-catenin, TGFß-BMP, IGF1, and EGF signaling pathways in Rex rabbits.

Impact of different dietary fibre sources on production performance, bacterial composition and metabolites in the caecal contents of rabbits.

This study was conducted to investigate the effects of different dietary fibre sources on growth performance, gastrointestinal tract development, caecal fermentation and bacterial composition in the caecal contents of rabbits. A total of 120 35-day-old weaned Minxinan black rabbits were divided into three groups and fed a diet composed of peanut straw powder (Group A), alfalfa powder (Group B) and soybean straw powder (Group C) as the main fibre source. The final body weight and average daily gain in Group B were higher than those in Group C, and the average daily feed intake and feed conversion ratio in Group A were lower than those in Group C (p < 0.05). The relative weights of the stomach, small intestine and caecum of rabbits in Group C were higher than those in Groups of B and A, and the relative weights of the caecal contents in Group C were lower than those in Groups A or B (p < 0.05). The pH value and propionic acid, butyric acid and valeric acid concentrations in the caecum of Group C were lower than those in the caecum of Groups A or B, and the concentration of acetic acid in the caecum was lower (p < 0.05). The dominant microbes in the caecal contents of Minxinan black rabbits were Firmicutes, Bacteroidetes and Proteobacteria at the phylum level, and the number of species, Chao1 index and ACE index measured was different between the B-C and A-C groups (p < 0.05). Different dietary fibre sources could affect the growth performance, gastrointestinal tract development and intestinal microbiota of rabbits, and the nutritional value of alfalfa powder was better than that of peanut straw and soybean straw.

Oxyhemoglobin-Based Nanophotosensitizer for Specific and Synergistic Photothermal and Photodynamic Therapies against Porphyromonas gingivalis Oral Infection.

Photothermal therapy (PTT) and photodynamic therapy (PDT) are emerging alternative antibacterial approaches. However, due to the lack of selectivity of photosensitizers for pathogenic bacteria, these methods often show more or less different degrees of in vivo toxicity. Moreover, it is difficult for PDT to exert effective antibacterial effects against anaerobic infections due to the oxygen deficiency. As one of the major anaerobic pathogens in oral infections, Porphyromonas gingivalis (P. gingivalis) acquires iron and porphyrin mainly from hemoglobin in the host. Hence, we developed a nanophotosensitizer named as oxyHb@IR820 through stable complexation between oxyhemoglobin and IR820, which is a photosensitizer possessing both PTT and PDT performance, for fighting P. gingivalis oral infection specifically and efficiently. Owing to hydrophobic interaction, oxyHb@IR820 had much stronger photoabsorption at 808 nm than free IR820, and thus exhibited significantly enhanced photothermal conversion efficiency. As an oxygen donor, oxyHb played an important role in enhancing the photodynamic efficiency of oxyHb@IR820. More importantly, oxyHb@IR820 showed efficient and specific uptake in P. gingivalis and exerted synergistic PTT/PDT performance against P. gingivalis and oral infection in golden hamsters. In summary, this study provides an efficient strategy for delivering photosensitizers specifically to P. gingivalis and augmenting antibacterial PDT against anaerobic infections.

Versatile Red Blood Cells for Triple-Negative Breast Cancer Treatment via Stepwise Photoactivations.

Phototherapies have many advantages for triple-negative breast cancer (TNBC) treatment. However, their effects are often limited by short blood circulation time, poor tumor selectivity and weak penetration of phototherapeutic agents, and tumor hypoxia. For overcoming these limitations, a versatile biomimetic system is developed based on red blood cells (RBCs). Photothermal agent new indocyanine green (IR820) is conjugated with the cell/tissue-penetrating TAT peptide and further efficiently encapsulated into the intact RBCs by crossing cell membranes to realize the long blood circulation. Meanwhile, cyclic RGD peptide (cRGD) is linked to the surfaces of RBCs through phospholipid insertion to obtain tumor vessel-targeting ability. Photosensitizer temoporfin (mTHPC) is next loaded into the membranes of RBCs by spontaneous transferring. The acquired biomimetic system (cRGD-RBC@mTHPC/TAT-IR820) exhibits potent photodynamic performance upon 652 nm laser irradiation with the facilitation of oxyhemoglobin, which could not only trigger TAT-IR820 release but also destroy tumor vessels. TAT-IR820 penetrates deeply into tumor tissue via the mediation of TAT peptide, exerting greatly promoted photothermal ablation against TNBC upon 808 nm laser irradiation. In situ generated tumor antigens further induce robust immune responses to suppress TNBC recurrence and metastasis. In summary, this study provides a versatile biomimetic system for comprehensive TNBC treatment via stepwise photodynamic and photothermal activations.

Albiflorin ameliorates inflammation and oxidative stress by regulating the NF-κB/NLRP3 pathway in Methotrexate-induced enteritis.

Methotrexate (MTX) treats various diseases but also damages intestinal barrier and leads to enteritis. Albiflorin (ALB) has a variety of pharmacological effects, including antioxidant, anti-inflammation and anti-apoptosis. In the present study, we evaluated the therapeutic effect of ALB on MTX-induced enteritis and investigated the possible mechanisms involved. Male SD rats were intraperitoneally injected with 7 mg/kg MTX for three consecutive days to establish the enteritis model. ALB (20 or 40 mg/kg/day) was intragastrically administrated since two days prior MTX treatment and lasted for six days. We found that ALB treatment increased body weight and intestinal weight of rats with MTX injection. The disease activity index (DAI) score was also decreased after ALB administration. In histological examination, ALB treatment attenuated inflammatory cells infiltration and promoted survival of goblet cells. In detection of inflammatory-associated factors, ALB treatment decreased CD68+ cells infiltration, inhibited myeloperoxidase activity, and suppressed intercellular cell adhesion molecule-1 and cyclooxygenase-2 expression. Additionally, ALB reduced malondialdehyde, glutathione levels, inhibited superoxide dismutase activity and suppressed reactive oxygen species production. Moreover, ALB treatment effectively inhibited NLRP3, as well as caspase 1 p20 and interleukin (IL)-1ß and 18 expression. Finally, nuclear factor-κB (NF-κB) p65 phosphorylation and nuclear translocation were also demonstrated to be blocked upon ALB treatment. In conclusion, our findings indicated that ALB alleviated MTX-induced enteritis via inhibiting the NF-κB/NLRP3 pathway.

iRGD Tumor-Penetrating Peptide-Modified Nano-Delivery System Based on a Marine Sulfated Polysaccharide for Enhanced Anti-Tumor Efficiency Against Breast Cancer.

BACKGROUND: Breast cancer is a common malignancy in women. Conventional clinical therapies for breast cancer all display moderate clinical efficacies and limitations. It is urgent to explore the novel and combined therapeutic strategies for breast cancer to meet clinical demand. METHODS: An iRGD tumor-penetrating peptide-modified nano-delivery system (denoted as iRGD-PSS@PBAE@JQ1/ORI nanoparticles) based on a marine sulfated polysaccharide was developed by codelivery of JQ1 (BET inhibitor) and oridonin (ORI, bioactive diterpenoid derived from traditional Chinese medicine herb). The iRGD-PSS@PBAE@JQ1/ORI NPs, surface modified with iRGD peptide conjugated propylene glycol alginate sodium sulfate (iRGD-PSS). The antitumor efficacy was evaluated both in vitro and in vivo. RESULTS: The prepared iRGD-PSS@PBAE@JQ1/ORI NPs effectively enhanced the tumor targeting and cellular internalization of JQ1 and ORI. Thus, JQ1 exerted the reversal effect on immune tolerance by decreasing the expression of PD-L1, while ORI displayed multiple antitumor effects, such as antiproliferation, inhibition of intracellular ROS production and inhibition of lactic acid secretion. CONCLUSION: Our data revealed that iRGD peptide could significantly improve the cellular internalization and tumor penetration of the nano-delivery system. The combination of JQ1 and ORI could exert synergistic antitumor activities. Taken together, this study provides a multifunctional nanotherapeutic system to enhance the anti-tumor efficiency against breast cancer.

Self-assembled nanoparticles containing photosensitizer and polycationic brush for synergistic photothermal and photodynamic therapy against periodontitis.

BACKGROUND: Periodontitis is a chronic inflammatory disease in oral cavity owing to bacterial infection. Photothermal therapy (PTT) and photodynamic therapy (PDT) have many advantages for antibacterial treatment. As an excellent photosensitizer, indocyanine green (ICG) shows prominent photothermal and photodynamic performances. However, it is difficult to pass through the negatively charged bacterial cell membrane, thus limiting its antibacterial application for periodontitis treatment. RESULTS: In this work, self-assembled nanoparticles containing ICG and polycationic brush were prepared for synergistic PTT and PDT against periodontitis. First, a star-shaped polycationic brush poly(2-(dimethylamino)ethyl methacrylate) (sPDMA) was synthesized via atom transfer radical polymerization (ATRP) of DMA monomer from bromo-substituted ß-cyclodextrin initiator (CD-Br). Next, ICG was assembled with sPDMA to prepare ICG-loaded sPDMA (sPDMA@ICG) nanoparticles (NPs) and the physicochemical properties of these NPs were characterized systematically. In vitro antibacterial effects of sPDMA@ICG NPs were investigated in porphyromonas gingivalis (Pg), one of the recognized periodontitis pathogens. A ligature-induced periodontitis model was established in Sprague-Dawley rats for in vivo evaluation of anti-periodontitis effects of sPDMA@ICG NPs. Benefiting from the unique brush-shaped architecture of sPDMA polycation, sPDMA@ICG NPs significantly promoted the adsorption and penetration of ICG into the bacterial cells and showed excellent PTT and PDT performances. Both in vitro and in vivo, sPDMA@ICG NPs exerted antibacterial and anti-periodontitis actions via synergistic PTT and PDT. CONCLUSIONS: A self-assembled nanosystem containing ICG and polycationic brush has shown promising clinical application for synergistic PTT and PDT against periodontitis.

Pyridoxine regulates hair follicle development via the PI3K/Akt, Wnt and Notch signalling pathways in rex rabbits.

This study was conducted to evaluate the effect of pyridoxine on the development of hair follicles in Rex rabbits and the underlying molecular mechanism. Two hundred 3-month-old Rex rabbits were randomly divided into 5 groups and fed diets supplemented with 0, 5, 10, 20, or 40 mg/kg pyridoxine. The hair follicle density on the dorsal skin and the gene and protein expression levels of components of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB or Akt), Wnt, Notch and bone morphogenetic protein (BMP) signalling pathways were measured. In addition, free hair follicles were isolated from Rex rabbits and cultured with pyridoxine in vitro to measure hair shaft growth. Furthermore, dermal papilla cells (DPC) were isolated from the skin of Rex rabbits and cultured with pyridoxine in vitro to measure the gene and protein expression levels of components of the PI3K/Akt, Wnt, Notch and BMP signalling pathways. The results showed that the addition of dietary pyridoxine significantly increased the total follicle density, secondary follicle density, and secondary-to-primary ratio (S/P, P < 0.05), that the growth ratio of hair stems was promoted by pyridoxine in basic culture medium, and that the growth length of tentacle hair follicles cultured in the pyridoxine group was longer than that in the control group (P < 0.05). In addition, pyridoxine changed the DPC cycle progression and promoted cell proliferation, and appropriate concentrations of pyridoxine (10 and 20 µmol/L) significantly inhibited cell apoptosis (P < 0.05). Pyridoxine significantly affected the gene expression of components of the PI3K/Akt, Wnt and Notch signalling pathways in the skin and DPC of Rex rabbits (P < 0.05), increased the levels of phosphorylated catenin beta 1 (CTNNB1) and Akt, and decreased the level of phosphorylated glycogen synthase kinase 3 beta (GSK-3ß) (P < 0.05). Therefore, the molecular mechanism by which pyridoxine promotes hair follicle density in Rex rabbits probably occurs through activation of the PI3K/Akt, Wnt and Notch signalling pathways, prolonging hair follicle growth and delaying the onset of telogen.

A Multifunctional Nanosystem Based on Bacterial Cell-Penetrating Photosensitizer for Fighting Periodontitis Via Combining Photodynamic and Antibiotic Therapies.

Photodynamic therapy (PDT), an emerging approach that involves photosensitizers, light, and molecular oxygen, has shown promise for fighting periodontitis. However, PDT does not always acquire the desired therapeutic outcomes since some photosensitizers have strong hydrophobic properties and are difficult to absorb efficiently by periodontal pathogenic bacteria. Here, a hydrophobic photosensitizer chlorin e6 (Ce6) was hydrophilically modified via conjugation with TAT peptide, a cationic cell-penetrating peptide, to improve its solubility and enhance its bacterial adsorption by promoting its interaction with the negatively charged cell walls and penetration through the cell membranes. The obtained TAT-Ce6 conjugate (TAT-Ce6) was used to prepare self-assembled nanoparticles (NPs) for loading tinidazole (TDZ), a clinically used antibiotic agent, thus hoping to achieve synergistic antiperiodontitis effects through combining PDT and antibiotic therapy. Compared to free Ce6, TAT-Ce6 nanoparticles (TAT-Ce6 NPs) had greatly enhanced adsorption and penetration abilities for periodontal pathogen bacteria and also exhibited significantly increased PDT efficiencies in both periodontal pathogen bacteria and monocyte macrophages. Upon 635 nm laser irradiation, TDZ-loaded TAT-Ce6 (TAT-Ce6/TDZ) NPs exerted remarkable synergistic antiperiodontitis effects of PDT and antibiotic therapy, reflecting in the effective killing of periodontal pathogenic bacteria in vitro and the reduced adsorption of alveolar bone in the Sprague-Dawley rat model of periodontitis. Altogether, this study develops a novel photosensitizer that can be efficiently absorbed by the periodontal pathogenic bacteria and also provides a potent combination strategy of PDT with antibiotic therapy for clinical periodontitis treatment.

DNA methylation and histone acetylation are involved in Wnt10b expression during the secondary hair follicle cycle in Angora rabbits.

Secondary hair follicles (SHFs) in the Angora rabbit exhibit classic cyclic hair development, but the multiple molecular signals involved in hair cycling are yet to be explored in detail. In the present study, we investigated the expression pattern, methylation and histone H3 acetylation status of Wnt10b, as a molecular signal participating in hair cycling, during the SHF cycle in the Angora rabbit. Expression of Wnt10b at the anagen phase was significantly higher than that at both the telogen and catagen phases, suggesting that Wnt10b might serve as a critical activator during cyclic transition of SHFs. Methylation frequency of the fifth CpG site (CpG5-175 bp) in CpG islands at the anagen phase was lower than that at both the catagen and telogen phases. The methylation status of the CpG5 site was negatively correlated with Wnt10b expression. This indicated that the methylation of CpG5 might participate in Wnt10b transcriptional suppression in SHFs. Furthermore, histone H3 acetylation status in the regions-256~-11 bp and 98 ~ 361 bp were significantly lower at both the catagen and telogen phases than at the anagen phase. The histone H3 acetylation level was significantly positively correlated with Wnt10b expression. This confirmed that histone acetylation was likely involved in upregulating Wnt10b transcription in SHFs. Additionally, potential binding to the transcription factors ZF57 and HDBP was predicted within the CpG5 site. In conclusion, our findings reveal the epigenetic mechanism of Wnt10b transcription and provide a new insight into epigenetic regulation during the SHF cycle in the Angora rabbit.

Isolation, culture and growth characteristics of dermal papilla cells from Rex rabbits.

Dermal papilla cells (DPCs) is the key dermal component of the hair follicle that directly regulates hair follicle development, growth and regeneration. Successfully isolated and cultured DPCs of Rex rabbit could provide a good model for the study of hair follicle development mechanism in vitro. Skin samples were collected from 30-day old Rex rabbits and separated by combination of Dispase II and Collagenase D, separation, culture, and purification of DPCs. The morphology of DPCs in vitro was observed and the growth curve was drawn, the number of DPCs presented progressive increase in a logarithmic model between the 4th day and the 7th day. The results of immune chemical and immune fluorescence shown that α smooth muscle actin (α-SMA) and versican were positive in cells. Growth character of the passages 3 (P3), P6, P9 and P12 DPCs were observed using MTT at 24 h, 48 h, 72 h, 96 h, 120 h and 144 h. The cell density of P12 was lower than P3 (P < 0.05); the flow cytometric analysis showed that DPCs at resting state/first gap (G0/G1) stage of P3 was higher than P12 (P < 0.05), and second gap/mitosis (G2/M) stage of P3 was lower than P12 (P < 0.05). However, the DPCs of P12 present triangular or short fusiform, retaining their unique aggregative growth characteristics. This results shown that the DPCs properties of P12 from Rex rabbits, still fit functional research in vitro. In conclusion, we successfully established the culturing condition of DPCs from Rex rabbits, and provide a material for studying the molecular mechanism of hair follicle development.

The functions of ocu-miR-205 in regulating hair follicle development in Rex rabbits.

BACKGROUND: Hair follicles are an appendage of the vertebrate epithelium in the skin that arise from the embryonic ectoderm and regenerate cyclically during adulthood. Dermal papilla cells (DPCs) are the key dermal component of the hair follicle that directly regulate hair follicle development, growth and regeneration. According to recent studies, miRNAs play an important role in regulating hair follicle morphogenesis and the proliferation, differentiation and apoptosis of hair follicle stem cells. RESULTS: The miRNA expression profile of the DPCs from Rex rabbits with different hair densities revealed 240 differentially expressed miRNAs (|log2(HD/LD)| > 1.00 and Q-value≤0.001). Among them, ocu-miR-205-5p was expressed at higher levels in DPCs from rabbits with low hair densities (LD) than in rabbits with high hair densities (HD), and it was expressed at high levels in the skin tissue from Rex rabbits (P < 0.05). Notably, ocu-miR-205 increased cell proliferation and the cell apoptosis rate, altered the progression of the cell cycle (P < 0.05), and modulated the expression of genes involved in the PI3K/Akt, Wnt, Notch and BMP signalling pathways in DPCs and skin tissue from Rex rabbits. It also inhibited the phosphorylation of the CTNNB1 and GSK-3ß proteins, decreased the level of the noggin (NOG) protein, and increased the level of phosphorylated Akt (P < 0.05). A significant change in the primary follicle density was not observed (P > 0.05), but the secondary follicle density and total follicle density (P < 0.05) were altered upon interference with ocu-miR-205-5p expression, and the secondary/primary ratio (S/P) in the ocu-miR-205-5p interfered expression group increased 14 days after the injection (P < 0.05). CONCLUSIONS: In the present study, ocu-miR-205 promoted the apoptosis of DPCs, altered the expression of genes and proteins involved in the PI3K/Akt, Wnt, Notch and BMP signalling pathways in DPCs and skin from Rex rabbits, promoted the transition of hair follicles from the growth phase to the regression and resting phase, and altered the hair density of Rex rabbits.

Effects of Wnt10b on dermal papilla cells via the canonical Wnt/ß-catenin signalling pathway in the Angora rabbit.

Wnt10b is a member of Wnt family that plays a variety of roles in biological functions, including those in the development of hair follicles. To investigate the effect of Wnt10b on hair growth in the Angora rabbit and to determine the underlying molecular mechanism, we cultured dermal papilla (DP) cells with exogenous Wnt10b in vitro. We observed the expressions of downstream critical gene ß-catenin and lymphoid enhancer-binding factor 1 (LEF1) in Wnt/ß-catenin pathway. The levels of ß-catenin mRNA and protein were higher in the Wnt10b group of DP cells than in the Control group, and the mRNA level of LEF1 in the Wnt10b group was higher than in the Control group. Moreover, translocation of ß-catenin from cytoplasm to nucleus was activated in the Wnt10b group. Furthermore, the mRNA levels of the hair follicle-regulatory genes, insulin-like growth factor-1 (IGF-1) and alkaline phosphatase (ALP), and the protein activity of ALP was also upregulated in the Wnt10b group compared to their corresponding levels in the Control group. These data suggest that Wnt10b could activate the canonical Wnt/ß-catenin signalling pathway to induce DP cells in the Angora rabbit. In addition, the proliferation of DP cells was significantly promoted when cultured with Wnt10b for 48 and 72 hr, suggesting that Wnt10b plays a pivotal role in the proliferation and maintenance of DP cells in vitro. In conclusion, this study demonstrates that Wnt10b may promote hair follicle growth in Angora rabbit through the canonical Wnt/ß-catenin signalling pathway that promotes the proliferation of DP cells.

Heat stress decreased hair follicle population in rex rabbits.

The aim of this study was conducted to investigate the effect of heat stress on the hair follicle population and related signalling pathways in rex rabbits. Forty-eight rabbits were randomly divided into two groups: one group in a high ambient environment (32 ± 2°C, heat stress) and the other group with normal temperature (20 ± 2°C, control). The results show that heat stress decreased the body weight gain and feed conversion rate, rabbit hair length and hair follicle density (p < 0.05). Besides, heat stress suppressed the gene expression of noggin, insulin-like growth factor 1 (IGF-1) and IGF-1 receptor and protein expression of phosphorylated mechanistic target of rapamycin (mTOR) in rabbit skin (p < 0.05), while stimulated significantly the gene expression of bone morphogenetic protein 2 (BMP2) and BMP4 (p < 0.05). Heat exposure did not alter significantly the gene expression of alkaline phosphatase, versican and hepatocyte growth factor compared with the control (p > 0.05). In conclusion, noggin-BMP, IGF-1 and mTOR signalling pathways may be associated with the process of heat stress-repressing hair follicle development.

A comparison of methods for effective differentiation of the frozen-thawed 3T3-L1 cells.

The differentiation efficiency of 3T3-L1 preadipocytes is an essential factor affecting studies on cellular mechanisms associated with obesity, diabetes, and related disorders. Differentiation of 3T3-L1 cells is commonly induced by an adipogenic cocktail containing insulin, dexamethasone (DEX), and 3-isobutyl-1-methylxanthine (IBMX). However, 3T3-L1 cells after freezing and thawing for many times always have a low differentiation efficiency. To solve this problem, we compared the differentiation efficiency of six commonly used adipogenic cocktails and protocols published in 2017. On this basis, we further compared 18 adipogenic cocktails with 2 µM rosiglitazone added and/or with a prolonged treatment with IBMX. The results revealed that the adipogenic cocktail containing 0.5 mM IBMX, 1 µM DEX, and 10 µg/mL insulin was the most effective for the frozen-thawed 3T3-L1 cells differentiation. Rosiglitazone, and IBMX under a prolonged treatment, could improve the differentiation efficiency of the frozen-thawed 3T3-L1 cells. However, the effect was closely related to concentrations of agents in the adipogenic cocktails.

ß-Cryptoxanthin uptake in THP-1 macrophages upregulates the CYP27A1 signaling pathway.

SCOPE: Mitochondrial sterol 27-hydroxylase (CYP27A1), a mediator of cholesterol homeostasis, is reported to exhibit antiatherogenic properties. Many studies suggested that all-trans retinoic acid can be used to treat atherosclerosis through retinoic acid receptor (RAR)-mediated upregulation of CYP27A1 expression. In this study, we hypothesized that ß-cryptoxanthin (ß-cry), as a natural ligand of RAR, might act as antiatherogenic agent by upregulating CYP27A1. METHODS AND RESULTS: We found that ß-cry treatment significantly upregulated genes involved in the uptake, transport, and metabolism of retinoids and the signaling pathway of CYP27A1 expression in THP-1 macrophages as detected by microarray analysis. Meanwhile, intracellular levels of ß-cry were correlated to the concentration and exposure time of the treatment. The expression of genes, involved in signaling pathway of CYP27A1, was dramatically decreased due to repressed activity of RAR. Higher level of 27-hydroxycholesterol was detected in ß-cry-treated macrophages by HPLC. Docking simulation showed that ß-cry could interact with cellular retinoic acid binding protein 2. These findings were further confirmed through microarray results. CONCLUSION: Our results provide strong evidence that ß-cry can be actively taken up by THP-1 macrophages and exhibits antiatherogenic effect on THP-1 macrophages by inducing CYP27A1 expression via RAR.

Characterization of the mechanism of inhibin α-subunit gene in mouse anterior pituitary cells by RNA interference.

Inhibin, a member of the transforming growth factor-ß [TGF-ß] superfamily, is a suppressor of follicle-stimulating hormone [FSH] release through pituitary-gonadal negative feedback loop to regulate follicular development. In this study, Inhibin α-subunit [Inha] gene was knocked down successfully in mice primary anterior pituitary cells at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors. The results indicated that inhibin silencing significantly promoted apoptosis by up-regulating Caspase-3, Bax and Bcl-2 genes without affecting p53 both at transcriptional and translational levels. Furthermore, it markedly impaired the progression of G1 phase of cell cycle and decreased the amount of cells in S phase [as detected by flow cytometry]. Inhibin silencing resulted in significant up-regulation of mRNA and protein expressions of Gondotropin releasing hormone receptors [GnRHR] and down-regulated mRNA levels of ß-glycans with parellel change in the amount of its protein expression. Silencing of inhibin-a significantly increased [P<0.05] activin-ß concentration without affecting FSH and LH levels in anterior pituitary cells. These findings revealed that up regulation of GnRH receptors by silencing inhibin a-subunit gene might increase the concentration of activin-ß in the culture medium. Inhibin a silencing resulted in increased mRNA and protein expressions of inhibinß which may demonstrate that both inhibin subunits co-participate in the regulation of reproductive events in anterior pituitary cells. This study concludes that inhibin is a broad regulatory marker in anterior pituitary cells by regulating apoptosis, cellular progression and simultaneously by vital fluctuations in the hormonal signaling.