Phosphate-buffered Saline Group Research Articles

A cleaved adhesin DNA vaccine targeting dendritic cell against Porphyromonas gingivalis-induced periodontal disease.

Arg-gingipain A (RgpA) is the primary virulence factor of Porphyromonas gingivalis and contains hemagglutinin adhesin (HA), which helps bacteria adhere to cells and proteins. Hemagglutinin's functional domains include cleaved adhesin (CA), which acts as a hemagglutination and hemoglobin-binding actor. Here, we confirmed that the HA and CA genes are immunogenic, and using adjuvant chemokine to target dendritic cells (DCs) enhanced protective autoimmunity against P. gingivalis-induced periodontal disease. C57 mice were immunized prophylactically with pVAX1-CA, pVAX1-HA, pVAX1, and phosphate-buffered saline (PBS) through intramuscular injection every 2 weeks for a total of three administrations before P. gingivalis-induced periodontitis. The DCs were analyzed using flow cytometry and ribonucleic acid sequencing (RNA-seq) transcriptomic assays following transfection with CA lentivirus. The efficacy of the co-delivered molecular adjuvant CA DNA vaccine was evaluated in vivo using flow cytometry, immunofluorescence techniques, and micro-computed tomography. After the immunization, both the pVAX1-CA and pVAX1-HA groups exhibited significantly elevated P. gingivalis-specific IgG and IgG1, as well as a reduction in bone loss around periodontitis-affected teeth, compared to the pVAX1 and PBS groups (p<0.05). The expression of CA promoted the secretion of HLA, CD86, CD83, and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) in DCs. Furthermore, the RNA-seq analysis revealed a significant increase in the chemokine (C-C motif) ligand 19 (p<0.05). A notable elevation in the quantities of DCs co-labeled with CD11c and major histocompatibility complex class II, along with an increase in interferon-gamma (IFN-γ) cells, was observed in the inguinal lymph nodes of mice subjected to CCL19-CA immunization. This outcome effectively illustrated the preservation of peri-implant bone mass in rats afflicted with P. gingivalis-induced peri-implantitis (p<0.05). The co-administration of a CCL19-conjugated CA DNA vaccine holds promise as an innovative and targeted immunization strategy against P. gingivalis-induced periodontitis and peri-implantitis.

Differentially expressed genes involved in immune pathways following Vibro parahaemolyticus challenge: A study on red swamp crayfish Procambarus clarkii

This study conducted a comprehensive analysis of the damage mechanism of Vibrio parahaemolyticus (V. parahaemolyticus) on crayfish, utilizing pathological section observations, biochemical indicator detection, and transcriptomics analysis. Histopathological examination showed fragmentation of the gill lamellae, spotted pigment deposition on the branchial membrane, degeneration of epithelial cells, and disruption of the normal structure of gill tissues. Biochemical tests indicated a significant decrease in the activity of antioxidant-related enzymes in crayfish injected with V. parahaemolyticus compared with the phosphate-buffered saline group. Transcriptomics analysis identified 2307 upregulated and 1721 downregulated differentially expressed genes (DEGs). Gene ontology enrichment analysis highlighted that DEGs were primarily involved in the regulation of lipid metabolic processes, cellular nitrogen compound biosynthesis and metabolic processes, and the negative regulation of neuron death. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed significant enrichment in N-glycan biosynthesis, fatty acid metabolism, and the mTOR signaling pathway. Gene set enrichment analysis indicated pathways related to immunity and oxidative stress, including oxidative phosphorylation and tyrosine metabolism. Quantitative reverse transcription–polymerase chain reaction results for selected genes involved in the oxidative phosphorylation pathway and immune-related genes corroborated the RNA-Seq trends, confirming the transcriptomics data's reliability. The findings suggest that infection with V. parahaemolyticus leads to excessive oxidative stress due to bacterial proliferation, overwhelming the crayfish's antioxidative defense. This causes a continuous increase in reactive oxygen species levels, leading to irreversible damage to the oxidative phosphorylation and innate immune systems. This study elucidated the mechanism of oxidative stress damage in crayfish following V. parahaemolyticus infection, providing valuable insights for reducing bacterial and viral diseases in aquaculture. A deeper understanding of pathogen affects the physiology and immune systems of aquatic organisms can help develop more effective prevention and control strategies, thereby improving the efficiency and sustainability of aquaculture.

Effect of plasminogen activator urokinase receptor gene on the activation and apoptosis of neutrophil

Objective: To investigate the effect of plasminogen activator urokinase receptor (PLAUR) gene on neutrophil activation and apoptosis in neutrophil-like cell model. Methods: Human acute myeloid leukemia cell line HL60 was cultured in vitro and induced to differentiate into neutrophil-like cells by all-trans retinoic acid (ATRA). Lentiviral vectors interfering with human PLAUR gene was constructed and transfected into neutrophil-like cells (siRNA group). The phosphate buffer saline (PBS) group (untransfected neutrophil-like cells) and normal blank control group (NC group) (neutrophil-like cells transfected with blank plasmid) were used as controls (n=3). After starvation culture and addition of interleukin-17 afterwards in these 3 groups, the expression of CD11b on the cell membrane was detected by flow cytometry, and the levels of myeloperoxide (MPO) and extracellular neutrophil traps (NETs) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) to investigate the activation of neutrophil-like cells. The apoptosis was detected by flow cytometry with annexin V/propidium iodide (PI) double staining and the expressions of apoptosis-related proteins caspase-3, bax and bcl-2 were detected by Western blotting. Results: The expression of CD11b in siRNA group (32.37±8.17) was lower than that in PBS group (46.27±1.54) and NC group (53.07±8.14) (P<0.05) by flow cytometry. The levels of MPO and NETs (33.37±1.11, 57.69±3.03) in the supernatant of siRNA group were significantly lower than those in PBS group (41.64±2.20, 77.60±4.33) and NC group (40.84±5.11, 76.15±2.10) (P<0.05). Flow cytometry with annexin V/PI showed that the expression of apoptosis in siRNA group (20.42%±2.45%) was significantly higher than that in PBS group (11.91%±2.23%) and NC group (11.13%±2.56%) (P<0.05). The relative expression of caspase-3 protein and bax protein (0.84±0.05, 0.83±0.04) in siRNA group was significantly higher than that in PBS group (0.68±0.02, 0.63±0.08) and NC group (0.71±0.01, 0.66±0.10) (P<0.05), and the relative expression of anti-apoptosis protein bcl-2 decreased in siRNA group (0.38±0.02) than in PBS group (0.73±0.05) and NC group (0.69±0.06) (P<0.05). Conclusion: PLAUR promotes the activation of neutrophil-like cells and inhibits the apoptosis.

Evidence for chronic headaches induced by pathological changes of myodural bridge complex

Clinical studies have shown that there may be a certain relationship between pathological changes of the myodural bridge complex (MDBC) and chronic headaches of unknown cause. But there is still a lack of experimental evidence to explain the possible mechanism. This study aims to further confirm this relationship between MDBC and chronic headaches and explore its potential occurrence mechanism in rats. Bleomycin (BLM) or phosphate-buffered saline (PBS) was injected into the myodural bridge fibers of rats to establish the hyperplastic model of MDBC. After 4 weeks, the occurrence of headaches in rats was evaluated through behavioral scores. The immunohistochemistry staining method was applied to observe the expression levels of headache-related neurotransmitters in the brain. Masson trichrome staining results showed that the number of collagen fibers of MDBC was increased in the BLM group compared to those of the other two groups. It revealed hyperplastic changes of MDBC. The behavioral scores of the BLM group were significantly higher than those of the PBS group and the blank control group. Meanwhile, expression levels of CGRP and 5-HT in the headache-related nuclei of the brain were increased in the BLM group. The current study further confirms the view that there is a relationship between pathological changes of MDBC and chronic headaches of unknown cause. This study may provide anatomical and physiological explanations for the pathogenesis of some chronic headaches of unknown cause.

Comparative evaluation of rhFGF18 and rhGDF11 treatment in a transient ischemia stroke model.

Pharmacological treatments for ischemic stroke remain limited to thrombolysis, which is associated with increased risk of potentially fatal hemorrhage. Treatments with Recombinant Human Fibroblast Growth Factor 18 (rhFGF18) and Growth and Differentiation Factor 11 (rhGDF11) appear promising based on different preclinical models. The goal of this study was to compare the effects of rhFGF18 and rhGDF11 directly on survival, behavioral deficits, and histological fingerprint of cerebral ischemia in the Wistar rat middle cerebral artery occlusion (MCAO) model of stroke. Ischemia-reperfusion injury was induced using a 2-hour transient MCAO. Animals were administered rhFGF18 (infusion), rhGDF11 (multi-injection), or Phosphate Buffered Saline (PBS) vehicle control and followed for 42 days. Motor-Cognitive deficits were evaluated using the Morris Water Maze at Days 0 (pre-MCAO), 7, 21, and 42. Histopathological assessments were performed on Days 21 and 42. Day 7 post-ischemia water maze performance times increased 38.3%, 2.1%, and 23.1% for PBS, rhFGF18, and rhGDF11-treated groups, respectively. Fraction of neurons with abnormal morphology (chromatolysis, pyknotic nuclei, somal degeneration) decreased in all groups toward Day 42 and was lowest for rhFGF18. AChE-positive fiber density and activity increased over time in the rhFGF18 group, remained unchanged in the rhGDF11 treatment arm, and declined in the PBS control. Metabolic increases were greatest in rhGDF11 treated animals, with both rhFGF18 and rhGDF11 achieving improvements over PBS, as evidenced by increased succinate dehydrogenase and lactate dehydrogenase activity. Finally, rhFGF18 treatment exhibited a trend for reduced mortality relative to PBS (5.6%, 95% CI [27.3%, 0.1% ] vs. 22.2%, 95% CI [47.6%, 6.4% ]). rhFGF18 treatment appears promising in improving survival and promoting motor-cognitive recovery following cerebral ischemia-reperfusion injury.

Platelet RNA sequencing reveals profile of caffeic acid affecting hemostasis in mice

BackgroundCaffeic acid (CA) is a naturally occurring phenolic compound with diverse pharmacologic properties. CA plays a crucial role in hemostasis by increasing platelet count. However, the mechanism by which CA regulates platelets to promote hemostasis remains unclear. ObjectivesWe aim to identify the potential target pathways and genes by which CA regulates platelets to promote hemostasis. MethodsWe performed RNA sequencing (RNA-seq) analysis of mouse platelet pools in both the CA-gavaged group and phosphate-buffered saline–gavaged group. ResultsThe 12,934 expressed transcripts had been annotated after platelet RNA-seq. Compared with the phosphate-buffered saline group, 987 differentially expressed genes (DEGs) were identified, of which 466 were downregulated and 521 were upregulated in CA group. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Reactome gene set enrichment analysis demonstrated that upregulated DEGs were enriched in the pathways of hemostasis, platelet activation, signaling, aggregation, and degranulation. Moreover, Kyoto Encyclopedia of Genes and Genomes and Reactome gene set enrichment analysis revealed that 5 of the 25 cosignificantly upregulated DEGs were essential in CA-mediated platelet regulation to promote hemostasis. ConclusionOur findings of platelet RNA-seq analysis demonstrate that CA regulates the gene expression of hemostasis and platelet activation–related pathways to increase platelet count and promote hemostasis. It will also provide reference molecular resources for future research on the function and mechanism by which CA regulates platelets to promote hemostasis.

Knockdown of programmed cell death factor 4 restores erectile function by attenuating apoptosis in rats with bilateral cavernous nerve crush injury.

Apoptosis is an important pathologic mechanism of erectile dysfunction after radical prostatectomy. Studies have shown that programmed cell death factor 4 is connected to the modulation of apoptosis in many cells. However, the programmed cell death factor 4 function in the cavernous nerve injury erectile dysfunction is unclear. This investigation aimed to explore the programmed cell death factor 4 function in erectile dysfunction in rats with bilateral cavernous nerve crush. The experiment used 30 male Sprague Dawley rats (18 months old) that were screened for normal erectile function by the apomorphine test. Ten rats were randomized into Sham and bilateral cavernous nerve crush groups to detect changes in programmed cell death factor 4 expression. The remaining 20 rats were distributed at random to four groups: the Sham group treated by sham surgery, the phosphate-buffered saline group, the lentivirus containing negative control short hairpin RNA group, and the lentivirus containing short hairpin RNA targeting programmed cell death factor 4 group underwent bilateral cavernous nerve crush and were afterward administered intracavernous injections of phosphate-buffered saline, lentivirus containing negative control short hairpin RNA, or lentivirus containing short hairpin RNA targeting programmed cell death factor 4. Electrical stimulation of the cavernous nerve was conducted 2 weeks later for penile erectile function assessment. The cavernous tissue was collected for histological analysis and western blotting. The apoptosis level in rat corpus cavernosum was elevated, and programmed cell death factor 4 expression was increased after bilateral cavernous nerve crush. Knockdown of programmed cell death factor 4 significantly improved erectile function in bilateral cavernous nerve crush rats. Furthermore, lentivirus containing short hairpin RNA targeting programmed cell death factor 4 treatment raised smooth muscle content and attenuated cavernous fibrosis and apoptotic levels. Additionally, programmed cell death factor 4 was found to mediate the PI3K/AKT pathway. Elevated programmed cell death factor 4 expression may be an important pathogenetic mechanism for erectile dysfunction after bilateral cavernous nerve crush, and the knockdown of programmed cell death factor 4 enhanced erectile function in 18-month-old rats after cavernous nerve damage. The potential mechanism may be the stimulation of the PI3K/AKT pathway to attenuate the cavernous apoptosis level.

Evasion of maternal antibody protection by an IBDV Argentine variant

Infectious bursal disease (IBD) is a viral disease that affects the ability of chickens to produce humoral immune responses. One way to prevent the disease is the passage of maternally derived antibodies (MDA) from dams to offsprings via the yolk. Despite sanitary measures, which include immunization with genogroup 1 (G1) vaccines, infections with IBDV genogroup 4 (G4) in young animals have been detected. The aim of this study was to determine whether a local IBDV isolate belonging to G4 could evade the immunity generated by MDAs. Twelve-day-old animals positive for MDA, were inoculated with G1 or G4 isolates or phosphate buffered saline (PBS) as a control. After 1 wk, the animals were sacrificed and the following parameters were evaluated: bursa-body (BB) ratio, viral load, and histologic damage in the bursa of Fabricius. Results showed that G4-infected animals had significant differences in the BB ratio compared to the PBS group. In addition, viral load was significantly higher in the G4 group than in the G1 group. Histologic damage in the bursa of Fabricius was detected only in G4-infected MDA chickens. Our results suggest that infection with G4 local isolate can circumvent the immunity generated by MDA and, furthermore, that G4 isolate does not differ in its pathogenicity from G1 isolate, which underlines the need to include variant strains in vaccine formulations to reduce potential losses caused by these viruses.

RgpA-Engineered/Functionalized DNA Vaccine as a Novel Prophylactic Vaccination to Prevent Porphyromonas gingivalis-Induced Periodontitis: An in Vivo Study.

Arg-gingipain A (rgpA) and Arg-gingipain B (rgpB) are crucial virulence factors associated with Porphyromonas gingivalis (P. gingivalis) and have been recognized as promising targets for antibacterial vaccines. Although vaccines containing rgpA have shown efficacy, the incorporation of rgpB, which lacks the haemagglutinin adhesin (HA) domain, diminishes the vaccine's effectiveness. This study aims to assess the immunogenicity of the functional HA domain of rgpA in mouse periodontitis models. A total of 24 mice were randomly divided into four groups, each receiving different immune injections: group A received phosphate-buffered saline (PBS) as an empty control; group B received pVAX1 as a negative control (NC); group C received pVAX1-HA; and group D received pVAX1-rgpA. The mice were subjected to intramuscular injections every two weeks for a total of three administrations. Prior to each immunization, blood samples were collected for antibody detection under isoflurane anesthesia. Following the final immunization, periodontitis was induced two weeks later by using sutures soaked in a P. gingivalis solution. The mice were euthanized after an additional two-week period. To assess the safety of the procedure, major organs were examined through hematoxylin-eosin (HE) staining. Subsequently, the levels of IgG, IgG1, and IgG2a in the serum were quantified via enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of inflammatory factors in the gingiva, including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor alpha (TNF-α), was determined using quantitative real-time reverse transcript PCR (qRT-PCR). The extent of bone loss in periodontal tissues was evaluated using micro-computed tomography (micro-CT) and HE staining. HE staining of the organs confirmed the absence of vaccine-induced toxicity in vivo. After the second immunization, both the rgpA and HA groups displayed significantly higher specific IgG titers in comparison to the NC and PBS groups (p < 0.05). Furthermore, the rgpA and HA groups exhibited a noteworthy predominance of IgG1 antibodies after three immunization doses, while there was a noticeable reduction in IgG2a levels observed following ligation with P. gingivalis sutures, as opposed to the NC and PBS groups (p < 0.05). Additionally, both the HA and rgpA groups showed a significant decrease in the expression of inflammatory factors such as IL-6, IL-1β, and TNF-α, as well as a reduction in bone loss around periodontitis-affected teeth, when compared to the NC and PBS groups (p < 0.05). The results of this study demonstrate that the rgpA-engineered/functionalized HA gene vaccine is capable of eliciting a potent prophylactic immune response against P. gingivalis-induced periodontitis, effectively serving as an immunogenic and protective agent in vivo.

Human adipose tissue-derived stem cells inhibit coronary artery vasculitis in a mouse model of Kawasaki disease.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are used for the treatment of various diseases because of their rapid proliferation and high anti-inflammatory and tissue repair properties. Kawasaki disease is a systemic vasculitis with coronary arteritis and aneurysms occurring in pediatric patients. In this study, we examined serologically and pathologically whether the administration of human ADSCs (hADSCs) to a mouse model of Kawasaki disease could suppress vasculitis. Candida albicans water-soluble fractions were intraperitoneally injected into DBA/2 mice for 5 consecutive days to generate a mouse model of Kawasaki disease. The model mice were intravenously administered hADSCs or phosphate-buffered saline (PBS). Serum samples collected on days 15 and 29 were used to compare cytokine levels. Mouse hearts dissected on day 29 were subjected to hematoxylin and eosin and immunohistological staining using Galectin-1 (Gal-1), a protein involved in cardiovascular homeostasis, and CD44, a cell-surface marker of hADSCs. Comparison of inflammation-related cytokines showed a significant decrease in IL-1α expression at day 15 (P<0.05) and IL-6 expression at day 29 (P<0.01) in the hADSCs-treated group compared to the PBS group. Evaluation by hematoxylin and eosin staining showed decreased inflammatory cell infiltration and a tendency towards increased Gal-1 expression in the hADSCs group. CD44 expression was not observed in both the groups. The survival curve showed that the hADSCs group had a significantly longer survival time (P<0.05). The present experimental results indicate that hADSCs have an early anti-inflammatory effect, and that Gal-1 may be involved in preventing inflammation and reducing tissue damage.

Establishment of a juvenile mouse asthma model induced by postnatal hyperoxia exposure combined with early OVA sensitization

ObjectiveTo establish a juvenile mouse asthma model by postnatal hyperoxia exposure combined with early ovalbumin (OVA) sensitization. MethodsFemale C57BL/6J newborn mice were exposed to hyperoxia (95 % O2) from postnatal day-1 (PND1) to PND7; intraperitoneally injected with OVA suspension on PND21, PND28; and stimulated by nebulized inhalation of 1 % OVA from PND36 to PND42. Within 48 h of the last challenge, we observed their activity performance and evaluated airway responsiveness (AHR). All mice were executed at PND44. Female (n = 32) were divided into four groups as follows: room air（RA）+phosphate-buffered saline (PBS) group; O2 (hyperoxia, 95 % O2) + PBS group; RA + OVA group; O2+OVA group. We obtained the serum, bronchoalveolar lavage fluid (BALF), and lung tissues. The Wright-Giemsa staining was performed for leukocyte classification in BALF and HE staining for pathological examination. The levels of IL-2, IL-5, IL-13, IL-17A and IL-10 in BALF and tIgE and sIgE in serum were detected by ELISA. ResultsCompared with OVA sensitization or hyperoxia exposure alone, the mice in the model group (O2+OVA) showed asthma-like symptoms and increased airway hyperreactivity,The levels of IL-5,IL-13 IL-17A were increased in BLAF,and total leukocyte and eosinophil counts were also significant increasesed. The levels of tIgE and sIgE in serum were increased. ConclusionPostnatal hyperoxia exposure combined with early OVA sensitization might establish a juvenile mouse asthma model.

Toxicity study of rats treated by plasma-activated solution

BackgroundCold atmospheric plasma (CAP) is an effective treatment for various skin diseases. Plasma-activated solution (PAS) is an indirect method of CAP treatment that produces biological effects similar to those of direct treatment with plasma devices. The anticancer and bacteriostatic effects of PAS have been demonstrated in vitro experiments; however, on the basis of the lack of toxicological studies on PAS, its effects on living mammals when administered by subcutaneous injection is poorly known. PurposeThe purpose of this study was to evaluate the effects of PAS on local skin tissue cells, blood system, heart, liver, lungs, kidneys and other vital organs of the rat when injected subcutaneously. MethodsPAS was prepared by CAP irradiation of phosphate-buffered saline (PBS). PBS and different PBS groups (CAP irradiation for 1, 3, or 5 min) were injected subcutaneously once every 48 h. The rats were euthanized immediately after 10 cycles of therapy. ResultsNo adverse effects were observed during the entire period of the experiment. Histopathological examination of organs and tissues revealed no structural changes. Moreover, no obvious structural changes were observed in skin tissue. DNA damage and cancerous proliferative changes were not detected in skin tissue treated with PAS. Subsequently, RNA sequencing and western blotting were performed. The results showed that PAS increased the expression of growth factors like transforming growth factor beta (TGF-β) and vascular endothelial growth factor A (VEGFA). These results might be directly linked to the role of PAS in stimulating TGF-β receptor signaling pathway and angiogenesis. ConclusionThe results showed that multiple subcutaneous injections of PAS did not show significant toxic side effects on local skin tissues and some vital organs in rats, providing a scientific basis to support the future treatment of skin diseases with PAS.

Intratracheal administration of cross-linked water-soluble acrylic acid polymer is associated with inducible bronchi-related lymphoid tissue formation and allergic inflammation.

In a Japanese chemical factory, lung diseases such as pneumoconiosis have been reported among workers handling cross-linked water-soluble acrylic acid polymers (CWAAP). Our previous study reported that a single intratracheal administration of CWAAP induces acute inflammation and fibrosis. In this study, we investigated the effects of multiple intratracheal administrations of CWAAP on inflammatory responses and pulmonary fibrosis along with inducible bronchus-associated lymphoid tissues (iBALT) formation, which is involved in allergic inflammation. Male F344 rats (190-200 g) received single or multiple intratracheal administrations of phosphate-buffered saline (PBS) or CWAAP. To assess inflammatory responses and pulmonary fibrosis, immunohistochemical and histological staining was performed. CD68, CD163, CD169, TGF-β, and collagen I positive cells/areas in the lungs of the CWAAP-group rats were significantly increased than those in the PBS group. Furthermore, the number of iBALT structures, CD4 + T cells, along with CD19, PAX5, IL-4, GATA-3, T-bet, and IgE-positive cells in the terminal bronchioles and blood vessels of the lungs were significantly increased in the CWAAP group. Moreover, pulmonary fibrosis, iBALT formation, and levels of specific IgG were significantly increased in rats who received multiple intratracheal administrations of CWAAP compared to those with single intratracheal administration. Multiple intratracheal administrations of CWAAP potentiated the classical fibrotic pathway (M2 macrophage-TGF-β-collagen I) more potently than single intratracheal administration. Furthermore, it was possible that iBALT was formed around terminal bronchioles and blood vessels and the number of immune cells was increased, resulting in enhanced allergic inflammation and pulmonary fibrosis.

Peripheral nerve regeneration by bioabsorbable nerve conduits filled with platelet-rich fibrin

PurposeTo repair peripheral nerve defects and seek alternatives for autografts, nerve conduits with various growth factors and cells have been invented. Few pieces of literature report the effect of nerve conduits plus platelet-rich fibrin (PRF). This study aimed to investigate the effectiveness of nerve conduits filled with PRF. MethodsThe model of a 10 mm sciatic nerve gap in a rat was used to evaluate peripheral nerve regeneration. The thirty rats were randomly divided into one of the following three groups (n = 10 per group). Autogenous nerve grafts (autograft group), conduits filled with phosphate-buffered saline (PBS) (PBS group), or conduits filled with PRF group (PRF group). We assessed motor and sensory functions for the three groups at 4, 8, and 12 weeks postoperatively. In addition, axon numbers were measured 12 weeks after repair of the peripheral nerve gaps. ResultsSignificant differences in motor function were observed between the autograft group and the other two groups at 12 weeks postoperatively. In the test to evaluate the recovery of sensory function, there were significant differences between the PBS group and the other two groups at all time points. The most axon number was found in the autograft group. The axon number of the PRF group was significantly more extensive than that of the PBS group. ConclusionsThe nerve conduit filled with PRF promoted the axon regeneration of the sciatic nerve and improved sensory function.

Adipose-derived stem cells repair radiation-induced chronic lung injury via inhibiting TGF-β1/Smad 3 signaling pathway.

To investigate the effect of adipose-derived stem cells (ASCs) transplantation on radiation-induced lung injury (RILI), Sprague-Dawley rats were divided into phosphate-buffered saline (PBS) group, ASCs group, Radiation + PBS group, and Radiation + ASCs group. Radiation + PBS and Radiation + ASCs groups received single dose of 30 Gy X-ray radiation to the right chest. The Radiation + PBS group received 1 mL PBS suspension and Radiation + ASCs group received 1 mL PBS suspension containing 1 × 107 CM-Dil-labeled ASCs. The right lung tissue was collected on Days 30, 90, and 180 after radiation. Hematoxylin-eosin and Masson staining were performed to observe the pathological changes and collagen fiber content in the lung tissue. Immunohistochemistry (IHC) and western blot (WB) were used to detect levels of fibrotic markers collagen I (Collal), fibronectin (FN), as well as transforming growth factor-β1 (TGF-β1), p-Smad 3, and Smad 3. Compared with the non-radiation groups, the radiation groups showed lymphocyte infiltration on Day 30 after irradiation and thickened incomplete alveolar walls, collagen deposition, and fibroplasia on Days 90 and 180. ASCs relieved these changes on Day 180 (Masson staining, P = 0.0022). Compared with Radiation + PBS group, on Day 180 after irradiation, the Radiation + ASCs group showed that ASCs could significantly decrease the expressions of fibrosis markers Collal (IHC: P = 0.0022; WB: P = 0.0087) and FN (IHC: P = 0.0152; WB: P = 0.026) and inhibit the expressions of TGF-β1 (IHC: P = 0.026; WB: P = 0.0152) and p-Smad 3 (IHC: P = 0.0043; WB: P = 0.0087) in radiation-induced injured lung tissue. These indicated that ASCs could relieve RILI by inhibiting TGF-β1/Smad 3 signaling pathway.

The effect of cardiosphere-derived cell extracellular vesicles embedded in cardiac matrix hydrogel on cardiac remodeling in a porcine model of ischemic cardiomyopathy

Abstract Background Numerous preclinical studies have confirmed that stem cell-derived extracellular vesicles (EVs) can prevent from negative cardiac remodeling after an ischemic injury, reduce the scar and preserve heart function. It is believed that by increasing the engraftment of the therapeutic product at the target site may bust its efficacy. The use of bioengineering materials such as hydrogels as delivery scaffolds for intramyocardial EVs administration may constitute a minimally invasive therapeutic strategy that improves the efficacy of EVs by increasing their permanence in the tissue. Objective To evaluate the therapeutic efficacy of a new regenerative product constituted by the combination of EVs secreted by cardiosphere-derived cell (CDC-EVs) embedded in a hydrogel-based scaffold made from decellularized cardiac matrix (HDM). Methods Twelve pigs (50% females) with induced ischemic cardiomyopathy were randomly assigned to receive: phosphate-buffer saline (PBS) as control group (n=4), CDC-EVs alone (n=4) and combination of CDC-EVs and HDM (EV-HDM, n=4). Treatment was administered via percutaneous intramyocardial injection in the scar border zone 1-month after myocardial infarction with NOGA system. Animals were followed-up another month and sacrificed. Hearts were excised for histological analysis performed according to the cardiac area: scar (anterior wall), scar border zone and remote zone (inferior wall). Functional evaluation was performed with cardiac magnetic resonance imaging immediately before- and 1-month after treatment. Results Cardiomyocytes size was significantly lower in EV-HDM group compared to control PBS both at scar border (4660± 2088 vs. 5254±2294 pixels; p=0.02) and remote areas (3381±1259 vs. 4237±1256 pixels; p&amp;lt;0.001), while no changes were observed in CDC-EV alone group compared to controls. Interstitial fibrosis was significantly lower in EV-HDM group compared to PBS controls both at scar epicardial side (34% vs. 44%; p=0.01) and remote areas (16% vs. 20%; p&amp;lt;0.01), similarly to CDC-EV vs. PBS (34% at the scar; p&amp;lt;0.01 and 17% at the remote zone; p=0.01). While left ventricular ejection fraction (LVEF) tended to decrease in PBS group from 36% to 32%, it showed an opposite trend in CDC-EV (from 35 % to 38%) and EV-HDM (from 31% to 37%). Conclusion The combined product of EV-HDM reduce cardiac fibrosis and hypertrophy, favorably affecting the remodeling process and may thus increase LVEF in ischemic heart disease. The results of our study fail to clearly demonstrate the superiority of EV-HDM over CDC-EVs alone, although it may exist.

Adjuvant effect of allogeneic blood in vaccines against edwardsiellosis in ginbuna crucian carp Carassius auratus langsdorfii

Edwardsiella tarda (E. tarda), an intracellular pathogen, has caused severe economic losses in aquaculture. Effective vaccine development for E. tarda prevention is urgently needed. A previous study indicates that cell-mediated immunity (CMI) might play an important role in E. tarda infection. We believe that the involvement of allograft rejection and CMI has now been well documented in mammals and some fishes. However, there is still little research on the application of blood allograft rejection in vaccine development. In the current study, we investigate the immune response and vaccine effect in fish vaccinated with allogeneic blood + formalin-killed cells vaccine (FKC), allogeneic blood + phosphate-buffered saline (PBS), PBS + FKC and PBS + PBS. In the challenge test, the relative percentage survival (RPS) of the allogeneic + FKC, the allogeneic blood + PBS and the PBS + FKC group was 61.46, 35.41, and 30.63 % respectively. The up-regulated expression of Th1-related genes IFN-γ 1, IFN-γ 1rel2, IL-12p35 and T-bet suggests the protection is via CMI induction. Only in the allogeneic + FKC group, gene expression of IFN-γ 1, IL-12p35 and T-bet is significantly higher, indicating synergy between the two substances. Furthermore, among the fish injected with the allogeneic blood cells, syngeneic blood cells and PBS group, only in the fish of the allogenic blood cells injection group, did expression of IFN-γ 1, IFN-γ 2 and IFN-γ rel2 gene expression significantly increased. The results indicate that the rejection was induced by allogeneic components. Thus, our findings might provide essential information and insights into vaccine development in aquaculture.

α2-Macroglobulin Promotes Chondrocyte Proliferation and Cartilage Matrix Synthesis via Inducing PCNA.

α2-Macroglobulin (A2M) can prevent cartilage degeneration by blocking many types of cartilage-degrading enzymes, but the mechanism remains to be clarified. This study aimed to test that A2M protects against cartilage degeneration by promoting chondrocyte proliferation and cartilage matrix synthesis via inducing proliferating cell nuclear antigen (PCNA). The cartilage degeneration of the anterior cruciate ligament transection (ACLT) model was evaluated by Safranin O-fast green staining, and articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. The chondrocyte proliferation was detected by 5-Bromodeoxyuridinc (BrdU), MTT, and Cell Counting Kit-8 (CCK8) methods. The chondrocyte apoptosis was detected by lactate dehydrogenase (LDH) assay and Annexin PI staining with the flow cytometer. The glycosaminoglycan (sGAG) and aggrecan in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the type II collagen and aggrecan mRNA expression. The PCNA protein expression was analyzed by western blot and immunofluorescent staining. A2M can attenuate cartilage degeneration in ACLT rats. The OARSI scores for cartilage degeneration in the A2M group were lower than those in the phosphate-buffered saline (PBS) group. A2M can promote chondrocyte proliferation and inhibit chondrocyte apoptosis, promote the cartilage matrix synthesis in chondrocytes (type II collagen and aggrecan), and culture supernatant (sGAG and aggrecan). At the same time, it also up-regulated the PCNA protein expression in chondrocytes. A2M can promote chondrocyte proliferation and cartilage matrix synthesis via inducing PCNA expression.

Suppressive effect of nitric oxide synthase (NOS) inhibitor L-NMMA acetate on choroidal fibrosis in experimental myopic guinea pigs through the nitric oxide signaling pathway

Myopia is one of the most prevalent eye diseases that seriously threaten the eyesight of children and adolescents worldwide. However, the pathogenesis is still unclear, and effective drugs are still scarce. In the present study, the guinea pigs were randomly divided into a normal control (NC) group, a lens-induced myopia (LIM) group, a NOS inhibitor (L-NMMA) injection group, and a NOS inhibitor solvent phosphate-buffered saline (PBS) group and the animals received relevant treatments. After 2- and 4-week different treatments, we noted that the refraction and choroidal thickness in the LIM group decreased compared with the NC group, whereas the ocular axial length increased significantly, and the choroid showed a fibrotic trend. The expression of NOS1, NOS3, TGF-β1, COLI, and α-SMA at gene and protein levels was increased significantly in the choroid (all P < 0.05). After intravitreal injection of NOS inhibitor L-NMMA, we found that compared with the LIM group, the refraction and the choroidal thickness significantly increased, whereas the axial length reduced significantly, accompanied by an increase of choroidal thickness and an improvement of choroidal fibrosis. The expression levels of choroidal NOS1, NOS3, TGF-β, COLI, and α-SMA were significantly reduced (all P < 0.05). In conclusion, the trend of choroidal fibrosis in LIM guinea pigs is positively correlated with the increase in axial length. The NOS inhibitor L-NMMA can alleviate the process of choroidal fibrosis in myopic guinea pigs by inhibiting NO signaling pathway.

Ultrasound-guided periadventitial administration of rapamycin-fibrin glue attenuates neointimal hyperplasia in the rat carotid artery injury model

IntroductionArterial restenosis caused by intimal hyperplasia (IH) is a serious complication after vascular interventions. In the rat carotid balloon injury model, we injected phosphate buffer saline (PBS), rapamycin-phosphate buffer saline suspension (RPM-PBS), blank fibrin glue (FG) and rapamycin-fibrin glue (RPM-FG) around the injured carotid artery under ultrasound guidance and observed the inhibitory effect on IH. MethodsThe properties of RPM-FG in vitro were verified by scanning electron microscopy (SEM) and determination of the drug release rate. FG metabolism in vivo was observed by fluorescence imaging. The rat carotid balloon injury models were randomly classified into 4 groups: PBS group (control group), RPM-PBS group, FG group, and RPM-FG group. Periadventitial administration was performed by ultrasound-guided percutaneous puncture on the first day after angioplasty. Carotid artery specimens were analyzed by immunostaining, Evans blue staining and hematoxylin-eosin staining. ResultsThe RPM particles showed clustered distributions in the FG block. The glue was maintained for a longer time in vivo (> 14 days) than in vitro (approximately 7 days). Two-component liquid FG administered by ultrasound-guided injection completely encapsulated the injured artery before coagulation. The RPM-FG inhibited IH after carotid angioplasty vs. control and other groups. The proliferation of vascular smooth muscle cells (VSMCs) was significantly inhibited during neointima formation, whereas endothelial cell (EC) repair was not affected. ConclusionPeriadventitial delivery of RPM-FG contributed to inhibiting IH in the rat carotid artery injury model without compromising re-endothelialization. Additionally, FG provided a promising platform for the future development of a safe, effective, and minimally invasive perivascular drug delivery method to treat vascular disease.