Rat Nasal Mucosa Research Articles

Ozone Induces Oxidative Stress and Inflammation in Nasal Mucosa of Rats

Background: The development of the global economy has led to changes in air pollution patterns. The haze phenomenon characterized by high concentrations of particulate matter 2.5 (PM2.5) has changed to complex pollution, and photochemical pollution characterized by ozone (O3) has become increasingly prominent. Ozone pollution and its impact on human health has become an important topic that needs to be studied urgently. Objective: To investigate the effects of ozone on oxidative stress and inflammation in the nasal mucosa of a rat model. Methods: Thirty-two healthy female Sprague–Dawley rats, eight in each group, were divided into four groups using the randomized numeric table method: normal control group (NC group), normal rats with a low level of ozone inhalation exposure (NEL group, 0.5 ppm), medium ozone inhalation exposure (NEM group, 1 ppm), and high ozone inhalation exposure (NEH group, 2 ppm). The ozone inhalation exposure groups were placed in the ozone inhalation exposure system and exposed to different concentrations of ozone for 2 h each day for 6 weeks. Nasal secretion was measured, and nasal lavage and nasal mucosa were collected. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were measured by colorimetric assay, and the nasal mucosa was analyzed by Western blot. Western blot (WB) was used to detect the expression of NF-κB p65 nuclear protein in nasal mucosa. The mRNA expression of NF-κB target genes IL-6 and IL-8 and tumor necrosis factor-α (TNF-α) was detected by real-time quantitative PCR (qRT-PCR), and the protein content of pro-inflammatory factors IL-6, IL-8, and TNF-α was detected by ELISA in serum and nasal lavage fluid. The nasal mucosa of rats was stained with hematoxylin-eosin (HE) to observe the pathological changes in the nasal mucosa. The data were analyzed by SPSS 20.0 software. Results: The amount of nasal secretion increased significantly in all groups after ozone exposure compared with that in the NC group. The MDA content of the nasal mucosa was significantly increased in the ozone-exposed group compared with the NC group, and the activity levels of SOD and GSH-Px in the nasal mucosa were lower in the ozone-exposed group than in the NC group. The mRNA expression of IL-6, IL-8, and TNF-α in the nasal mucosa of the ozone-exposed group was elevated, and the protein content of TNF-α, IL-6, and IL-8 in the nasal lavage fluid was elevated, and the content increased with the increase in ozone concentration. The expression of NF-κB p65 intracellular protein in the nasal mucosa of each ozone-exposed group was higher than that of the normal group, and the content increased with the increase in ozone concentration. Conclusions: Ozone inhalation exposure promotes oxidative stress and the release of inflammatory factors TNF-α, IL-6, and IL-8, leading to pathological damage of the nasal mucosa, the degree of which increases with increasing concentration. This pathological process may be related to the activation of the transcription factor NF-κB by ozone in the nasal mucosa of rats, which increases the expression of its target genes.

Randomly methylated β-cyclodextrin improves water – solubility, cellular protection and mucosa permeability of idebenone

Neurodegenerative diseases such as Alzheimer’s are very common today. Idebenone (IDE) is a potent antioxidant with good potential for restoring cerebral efficiency in cases of these and other medical conditions, but a serious drawback for the clinical use of IDE in neurological disorders lies in its scarce water solubility, which greatly inhibits its bioavailability. In this work, we prepared the inclusion complex of IDE with randomly methylated β-cyclodextrin (RAMEB), resulting in improved water solubility of the included drug; then its in vitro biological activity and ex vivo permeability was evalutated. The solid complex was characterized through FT-IR spectroscopy, Thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC). A 78-fold improvement of the solubility of IDE in water resulted, together with a strong 1:1 host–guest interaction (association constant of 12630 M−1), and dissolution of the complex within 15 min, all evidenced during the in-solution studies. Biological in vitro studies were then performed on differentiated human neuroblastoma cells (SH-SY5Y) subjected to oxidative stress. Pretreatment with IDE/RAMEB positively affected cell viability, promoted the nuclear translocation of Nrf2, and increased the levels of GSH as well as those of the endogenous antioxidant enzymes Mn-SOD and HO-1. Lastly, the complexation significantly improved the permeation of IDE through isolated rat nasal mucosa.

Fibrin-konjac glucomannan-black phosphorus hydrogel scaffolds loaded with nasal ectodermal mesenchymal stem cells accelerated alveolar bone regeneration

BackgroundEffective treatments for the alveolar bone defect remain a major concern in dental therapy. The objectives of this study were to develop a fibrin and konjac glucomannan (KGM) composite hydrogel as scaffolds for the osteogenesis of nasal mucosa-derived ectodermal mesenchymal stem cells (EMSCs) for the regeneration of alveolar bone defect, and to investigate the osteogenesis-accelerating effects of black phosphorus nanoparticles (BPNs) embedded in the hydrogels.MethodsPrimary EMSCs were isolated from rat nasal mucosa and used for the alveolar bone recovery. Fibrin and KGM were prepared in different ratios for osteomimetic hydrogel scaffolds, and the optimal ratio was determined by mechanical properties and biocompatibility analysis. Then, the optimal hydrogels were integrated with BPNs to obtain BPNs/fibrin-KGM hydrogels, and the effects on osteogenic EMSCs in vitro were evaluated. To explore the osteogenesis-enhancing effects of hydrogels in vivo, the BPNs/fibrin-KGM scaffolds combined with EMSCs were implanted to a rat model of alveolar bone defect. Micro-computed tomography (CT), histological examination, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were conducted to evaluate the bone morphology and expression of osteogenesis-related genes of the bone regeneration.ResultsThe addition of KGM improved the mechanical properties and biodegradation characteristics of the fibrin hydrogels. In vitro, the BPNs-containing compound hydrogel was proved to be biocompatible and capable of enhancing the osteogenesis of EMSCs by upregulating the mineralization and the activity of alkaline phosphatase. In vivo, the micro-CT analysis and histological evaluation demonstrated that rats implanted EMSCs-BPNs/fibrin-KGM hydrogels exhibited the best bone reconstruction. And compared to the model group, the expression of osteogenesis genes including osteopontin (Opn, p < 0.0001), osteocalcin (Ocn, p < 0.0001), type collagen (Col , p < 0.0001), bone morphogenetic protein-2 (Bmp2, p < 0.0001), Smad1 (p = 0.0006), and runt-related transcription factor 2 (Runx2, p < 0.0001) were all significantly upregulated.ConclusionsEMSCs/BPNs-containing fibrin-KGM hydrogels accelerated the recovery of the alveolar bone defect in rats by effectively up-regulating the expression of osteogenesis-related genes, promoting the formation and mineralisation of bone matrix.

Rat nasal mucosa-derived ectodermal mesenchymal stem cells: A new therapeutic option for chronic rhinosinusitis.

To investigate the effect of nasal mucosa-derived ectodermal mesenchymal stem cells (NM-EMSCs) on the inflammatory state of rats with chronic rhinosinusitis (CRS) and the underlying therapeutic mechanism. NM-EMSCs were isolated and extracted to construct a rat model of CRS. Fifteen Sprague‒Dawley (SD) rats were randomly divided into threegroups: CK + NS group rats were injected locally with saline in the nasal mucosa; CRS + NS group rats were injected locally with saline in the nasal mucosa; and CRS + EMSCs group rats were injected locally with NM-EMSCs in the nasal mucosa. One rat from the CRS + EMSCs group was randomly euthanized at 2, 4, and 6 days after injection, and the nasal mucosa tissues were collected for HE staining, Masson's trichrome staining, and periodic acid-Schiffstaining. NM-EMSCs specifically expressing CD73, CD105, and CD90 were successfully isolated from the nasal mucosa of rats and were able to differentiate into adipocytes, osteoblasts, and chondrocytes. After saline and NM-EMSC injection, compared with those in the blank control CK + NS group, the nasal mucosa in the CRS + NSand CRS + EMSC groups exhibited obvious thickening, a large amount of inflammatory cell infiltration, and increased collagen and mucin distribution. Four days post-NM-EMSC injection, the thickening of the nasal mucosa in the CRS group was gradually alleviated, the inflammatory cell infiltration gradually decreased, and the distribution of collagen and mucin and the collagen-positive area gradually decreased. Moreover, only a small number of inflammatory cells were visible, and the distribution of mucins was limited to 6 days post-NM-EMSC injection. NM-EMSCs effectively attenuated inflammation in the nasal mucosa of CRS model rats.

Acupuncture at "Die E acupoint" alleviates inflammatory reaction via inhibiting TLR4/MyD88/NF-κB signaling in rats with allergic rhinitis.

To observe effects of acupuncture at "Die E acupoint" on the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear transcription factor κB (NF-κB), transcription factor T-bet (T-bet), and GATA-binding protein-3 (GATA-3) in the nasal mucosa and the serum contents of related inflammatory cytokines in rats with allergic rhinitis, so as to explore the mechanism of acupuncture in treating allergic rhinitis. Twenty-four healthy SD rats were randomly divided into blank, model, acupuncture, and sham acupuncture groups, with 6 rats in each group. The rat model of allergic rhinitis was established by using ovalbumin induction. The rats in the acupuncture group received bilateral acupuncture at the "Die E acupoint" with a depth of 15-20 mm, while the rats in the sham acupuncture group received only sham acupuncture (light and shallow acupunture of the skin at the "Die E acupoint" ). Both interventions were performed once daily for a total of 6 days. Behavioral scores of rats in each group were recorded. Pathological changes of nasal mucosa were observed by H.E. staining. Serum contents of IgE, ovalbumin-specific IgE (OVA-sIgE), interferon(IFN)-γ, interleukin(IL)-4, IL-10 and IL-17 were measured by ELISA and the protein expression levels of T-bet, GATA-3, TLR4, MyD88 and NF-κB p65 in the nasal mucosa were detected by Western blot. After modeling, compared with the blank group, rats in the model group showed increased behavioral scores, serum IgE, OVA-sIgE, IL-4, and IL-17 contents, and nasal mucosal GATA-3, TLR4, MyD88, and NF-κB p65 protein expression levels (P<0.05), whereas the contents of serum IFN-γ, IL-10 and the protein expression level of T-bet in the nasal mucosa were decreased (P<0.05). Comparison between the EA and model groups showed that acupuncture intervention can decrease the behavioral scores of rats with allergic rhinitis, the contents of serum IgE, OVA-sIgE, IL-4, IL-17, and the protein expression levels of GATA-3, TLR4, MyD88, and NF-κB p65 in the nasal mucosa (P<0.05), and up-regulate the contents of serum IFN-γ, IL-10, and the nasal mucosal T-bet protein expression level. Sham acupuncture did not have a significant modulating effect on the above indicators. Inflammatory infiltration of nasal mucosa was seen in the model group and sham acupuncture, and the inflammatory reaction was milder in the acupuncture group. Acupuncture at "Die E acupoint" can alleviate the symptoms of allergic rhinitis and suppress the inflammation of nasal mucosa in rats, which may be related to inhibiting the TLR4/MyD88/NF-κB signaling and balancing the levels of cytokines of Th1/Th2 and Treg/Th17, and T-bet/GATA-3.

Exosomes from ectoderm mesenchymal stem cells inhibits lipopolysaccharide-induced microglial M1 polarization and promotes survival of H2O2-exposed PC12 cells by suppressing inflammatory response and oxidative stress

To investigate the potential value of exosomes derived from rat ectoderm mesenchymal stem cells (EMSCs-exo) for repairing secondary spinal cord injury. EMSCs-exo were obtained using ultracentrifugation from EMSCs isolated from rat nasal mucosa, identified by transmission electron microscope, nanoparticle tracking analysis (NTA), and Western blotting, and quantified using the BCA method. Neonatal rat microglia purified by differential attachment were induced with 100 μg/L lipopolysaccharide (LPS) and treated with 37.5 or 75 mg/L EMSCs-exo. PC12 cells were exposed to 400 μmol/L H2O2 and treated with EMSCs-exo at 37.5 or 75 mg/L. The protein and mRNA expressions of Arg1 and iNOS in the treated cells were determined with Western blotting and qRT- PCR, and the concentrations of IL- 6, IL-10, and IGF-1 in the supernatants were measured with ELISA. The viability and apoptosis of PC12 cells were detected using CCK-8 assay and flow cytometry. The isolated rat EMSCs showed high expressions of nestin, CD44, CD105, and vimentin. The obtained EMSCs-exo had a typical cup-shaped structure under transmission electron microscope with an average particle size of 142 nm and positivity for CD63, CD81, and TSG101 but not vimentin. In LPS-treated microglia, EMSCs-exo treatment at 75 mg/L significantly increased Arg1 protein level and lowered iNOS protein expression (P < 0.05). EMSCs-exo treatment at 75 mg/L, as compared with the lower concentration at 37.5 mg/L, more strongly increased Arg1 mRNA expression and IGF-1 and IL-10 production and decreased iNOS mRNA expression and IL-6 production in LPS-induced microglia, and more effectively promoted cell survival and decreased apoptosis rate of H2O2-induced PC12 cells (P < 0.05). EMSCs-exo at 75 mg/L can effectively reduce the proportion of M1 microglia and alleviate neuronal apoptosis under oxidative stress to promote neuronal survival, suggesting its potential in controlling secondary spinal cord injury.

Study on the Mechanism of Allergic Rhinitis Based on the Expression of FIB, PCT, hs-CRP, and Th17/Treg-IL10/IL-17 Axis Balance.

The pathogenesis of allergic rhinitis (AR) is ambiguous, while it is clear that various immune cells and cytokines play crucial roles in its occurrence and development. To investigate the effect of exogenous interleukin-10 (IL-10) on the expression of fibrinogen (FIB), procalcitonin (PCT), hypersensitive C-reactive protein (hs-CRP), and Th17/Treg-IL10/IL-17 axis balance in the nasal mucosa of rats with AR. In this study, 48 female-specific pathogen-free Sprague-Dawley rats were randomly divided into 3 groups: blank control group, AR group, and IL-10 intervention group. The AR model was established in the AR group and IL-10 group. The rats in the control group were treated with normal saline; the rats in the AR group were given 20 μL of saline containing 50 μg of ovalbumin (OVA) every day. The rats in the IL-10 intervention group were intraperitoneally injected with 1 mL of 40 pg/kg IL-10 and provided with OVA. The IL-10 intervention group was composed of mice with AR that received IL-10. The behavior of nasal allergic symptoms (such as nasal itching, sneezing, and runny nose) and the hematoxylin and eosin staining of nasal mucosa were observed. The levels of FIB, PCT, hs-CRP, IgE, and OVA sIgE in serum were determined by enzyme-linked immunosorbent assay. The levels of Treg and Th17 cells in serum were detected by flow cytometry. The protein levels of TGF-β, IL-10, and IL-17 in nasal mucosa were detected by the Western-blot method. The scores of snots, nasal itching, and sneezing in the AR group were significantly higher than those in the control group, while the scores of the above symptoms in the IL-10 intervention group were lower than those in the AR group. The levels of FIB, PCT, hs-CRP, IgE, and OVA sIgE in serum and the protein levels of IL-10 and IL-17 in the nasal mucosa in the AR group were higher than those in the blank control group. Meanwhile, the levels of FIB, PCT, hs-CRP, IgE, and OVA sIgE in serum and IL-10 and IL-17 protein in the nasal mucosa in the IL-10 group were lower than those in the AR group. IL-10 can relieve the allergy of AR rats by affecting the expression of FIB, PCT, and hs-CRP, as well as the balance of the Th17/Treg-IL10/IL-17 axis in the nasal mucosa of AR rats.

Investigation of the Relationship Between Trefoil Factor Family Peptides and Sinonasal Inflammation.

The trefoil factor family (TFF) is a relatively new family of peptides. In some studies, an association between trefoil factors and inflammatory diseases of the nasal and paranasal sinuses has been suggested. However, it is still not clear whether there is a relationship between trefoil peptides and inflammation of the respiratory tract. The aims of this study are to determine the presence of TFF1, TFF2, and TFF3 in the nasal mucosa and investigate their relationships with inflammation by using rat models of various sinonasal inflammations. Nasal tampon, lipopolysaccharide, and ovalbumin were used to generate rat models of sinonasal inflammation, i.e., rhinosinusitis and allergic rhinitis. The study was conducted on seventy rats in seven groups, each with ten rats: four groups with rhinosinusitis, two groups with allergic rhinitis, and a control group. Histological evaluation of sinonasal mucosa from all rats was performed, and Trefoil factors were investigated using immunohistochemical methods. All three TFF peptides were detected in rat nasal mucosa by histological evaluation. No significant differences in the trefoil factor scores were observed among the study groups. A significant correlation between the TFF1 and TFF3 scores and loss of cilia was identified (p < 0.05). In conclusion, no direct relationship between sinonasal inflammation and TFF scores was observed. However, a possible association between the TFF and epithelial damage or regeneration in sinonasal inflammation can be suggested based on the correlation observed between the TFF1 and TFF3 scores and scores of cilia loss.

Human placental extract regulates polarization of macrophages via IRGM/NLRP3 in allergic rhinitis

Allergic rhinitis (AR) is globally prevalent and its pathogenesis remains unclear. Alternative activation of macrophages is suggested in AR and thought to be involved in natural immunoregulatory processes in AR. Aberrant activation of Nod-like receptor protein 3 (NLRP3) inflammasome is linked with AR. Human placenta extract (HPE) is widely used in clinics due to its multiple therapeutic potential carried by diverse bioactive molecules in it. We aim to investigate the effect of HPE on AR and the possible underlying mechanism. Ovalbumin (OVA)-induced AR rat model was set up and treated by HPE or cetirizine. General manifestation of AR was evaluated along with the histological and biochemical analysis performed on rat nasal mucosa. A proteomic analysis was performed on AR rat mucosa. Mouse alveolar macrophages (MH-S cells) were cultured under OVA stimulation to investigate the regulation of macrophages polarization. The morphological changes and the expression of NLRP3 inflammasome and immunity-related GTPase M (IRGM) in nasal mucosa as well as in MH-S cells were evaluated respectively. The results of our study showed the general manifestation of AR along with the histological changes in nasal mucosa of AR rats were improved by HPE. HPE suppresses NLRP3 inflammasome and the decline of IRGM in AR rats and MH-S cells. HPE regulates macrophage polarization through IRGM/NLRP3. We demonstrated that HPE had protection for AR and the protection is achieved partly through suppressing M1 while promoting M2, the process which is mediated by IRGM via inhibiting NLRP3 inflammasome in AR.

The Effect of Chinese Agarwood Essential Oil with Cyclodextrin Inclusion against PCPA-Induced Insomnia Rats.

To study the extraction process of agarwood active ingredients (AA) and investigate the safety and effectiveness of AA in the treatment of insomnia rats by nasal administration. A β-cyclodextrin (β-CD) inclusion compound (a-β-CD) was prepared from agarwood essential oil (AEO), and the preparation process was optimized and characterized. The safety of AA in nasal mucosa was evaluated through Bufo gargarizans maxillary mucosa and rat nasal mucosa models. Insomnia animal models were replicated by injecting p-chlorophenylalanine (PCPA), conducting behavioral tests, and detecting the expression levels of monoamine neurotransmitters (NE and 5-HT) and amino acids (GABA/Glu) in the rat hypothalamus. The optimum inclusion process conditions of β-CD were as follows: the feeding ratio was 0.35:1.40 (g:g), the inclusion temperature was 45 °C, the inclusion time was 2 h, and the ICY% and IEO% were 53.78 ± 2.33% and 62.51 ± 3.21%, respectively. The inclusion ratio, temperature, and time are the three factors that have significant effects on the ICY% and IEO% of a-β-CD. AA presented little damage to the nasal mucosa. AA increased the sleep rate, shortened the sleep latency, and prolonged the sleep time of the rats. The behavioral test results showed that AA could ameliorate depression in insomnia rats to a certain extent. The effect on the expression of monoamine neurotransmitters and amino acids in the hypothalamus of rats showed that AA could significantly reduce NE levels and increase the 5-HT level and GABA/Glu ratio in the hypothalamus of insomnia rats. The preparation of a-β-CD from AEO can reduce its irritation, improve its stability, increase its curative effect, and facilitate its storage and transport. AA have certain therapeutic effects on insomnia. The mechanism of their effect on rat sleep may involve regulating the expression levels of monoamine neurotransmitters and amino acids in the hypothalamus.

Relative contributions of endogenous and exogenous formaldehyde to formation of deoxyguanosine monoadducts and DNA-protein crosslink adducts of DNA in rat nasal mucosa.

Understanding the dose-response for formaldehyde-induced nasal cancer in rats is complicated by (1) the uneven distribution of inhaled formaldehyde across the interior surface of the nasal cavity and, (2) the presence of endogenous formaldehyde (endoF) in the nasal mucosa. In this work, we used computational fluid dynamics (CFD) modeling to predict flux of inhaled (exogenous) formaldehyde (exogF) from air into tissue at the specific locations where DNA adducts were measured. Experimental work has identified DNA-protein crosslink (DPX) adducts due to exogF and deoxyguanosine (DG) adducts due to both exogF and endoF. These adducts can be considered biomarkers of exposure for effects of endoF and exogF on DNA that may be part of the mechanism of tumor formation. We describe a computational model linking CFD-predicted flux of formaldehyde from air into tissue, and the intracellular production of endoF, with the formation of DPX and DG adducts. We assumed that, like exogF, endoF can produce DPX. The model accurately reproduces exogDPX, exogDG, and endoDG data after inhalation from 0.7 to 15 ppm. The dose-dependent concentrations of exogDPX and exogDG are predicted to exceed the concentrations of their endogenous counterparts at about 2 and 6 ppm exogF, respectively. At all concentrations examined, the concentrations of endoDPX and exogDPX were predicted to be at least 10-fold higher than that of their DG counterparts. The modeled dose-dependent concentrations of these adducts are suitable to be used together with data on the dose-dependence of cell proliferation to conduct quantitative modeling of formaldehyde-induced rat nasal carcinogenicity.

The Effect of Nasal Irrigation with Dewandaru (Eugenia Uniflora L) Leaves Ethanol Extract on Il-4 Levels, Thymic Stromal Lymphopoiethin (TSLP) Expression and Expression of H4 Mucosa Receptors of Wistar Rats with Allergic Rhinitis

Nasal irrigation with Dewandaru leaf extract can reduce inflammatory cell infiltration in the nasal mucosa of rats, and is effective in reducing RA symptoms and has an effect no different from topical corticosteroids. This study aims to prove whether nasal irrigation with ethanolic extract of Dewandaru leaves can reduce the infiltration of allergic inflammatory mediators in the nasal mucosa of male white rats of Wistar strain suffering from RA after ovalbumin nasal spray was induced, by assessing IL-4 levels, TSLP expression and H4 receptor expression. The study was conducted with a randomized post test only control group design, using an animal model of allergic rhinitis, involving 34 male white rats of the Wistar strain, divided into 2, namely: 17 treatment groups and 17 control groups. After 4 weeks of exposure, termination and sampling of rat nasal mucosa was performed. Statistical analysis using the independent sample T test showed that the levels of IL-4 in the nasal mucosa of rats and the expression of H4 receptors on the nasal mucosa of rats were significantly lower than in the control group (p&lt;0.05). Meanwhile, the TSLP expression of the nasal mucosa of rats in the treatment group (2.0154) was not significantly different from the control group (2.1891) with p=0.478, possibly associated with less severe damage to the epithelial barrier cells of the nasal mucosa of TSLP-producing in the group control. The ethanolic extract of Dewandaru leaves has the ability to suppress IL-4 cytokine levels through inhibition of the activity of RH4, so it can be used as a supporting therapy for nasal irrigation that can suppress allergic inflammatory mediators in the nasal mucosa of rats.

Synergistic effect of acupoint injection and moxibustion or catgut embedding or acupuncture on symptoms and expression of Th1/Th2 related cytokines in nasal mucosa of rats with allergic rhinitis

To observe the effect of moxibustion, catgut embedding and acupuncture on allergic symptoms and expression of interferon-γ (IFN-γ) and interleukin-4 (IL-4) of nasal mucosa in rats with allergic rhinitis (AR) based on acupoint injection, so as to explore their synergistic effect and related mechanism in relieving AR. SD rats (half male half female) were randomly divided into normal control, model, acupoint injection (AI), AI+moxibustion, AI+catgut embedding and AI+acupuncture groups, with 8 rats in each group. The AR model was established by intraperitoneal injection of ovalbumin suspension (once every other day for 7 times), and intranasal drop of 0.5% ovalbumin solution (once daily for 7 days). After successful modeling, rats of the AI group received injection of a mixture solution of equal proportion of 1% lidocaine, dexamethasone and transfer factor into "Yingxiang" (LI20) and "Yintang" (EX-HN3) once every 4 days, 4 times altogether. Mild moxibustion or catgut embedment or manual acupuncture was applied to bilateral "Feishu" (BL13) and "Zusanli" (ST36). Both moxibustion (20 min every time) and acupuncture (with the needles retained for 30 min every time) were conducted once daily for 14 times, and catgut embedding was conducted once a week, twice altogether based on acupoint injection. The rats' nasal allergic reaction score (symptom score, 1-3 points) was given according to the times of nose scratching and sneezing, and the running nose state in 30 min, and histopathological changes of nasal mucosa were observed by H.E. staining. The expression levels of IFN-γ and IL-4 in the nasal mucosa were detected by immunohistochemistry and Western blot, separately. Compared with the normal group, the symptom score and the expression of IL-4 positive cells and protein in nasal mucosa were significantly increased (P<0.05), while the expression of IFN-γ positive cells and protein were considerably decreased in the model group (P<0.05). In comparison with the model group, the symptom score and the expression of IL-4 positive cells and protein were obviously decreased (P<0.05), while the expression of IFN-γ positive cells and protein were remarkably increased in the 4 treatment groups (P<0.05). The effects of AI+moxibustion, AI+catgut embedment, AI+acupuncture were signi-ficantly superior to those of simple AI in up-regulating the expression of IFN-γ positive cells and protein and in down-regulating the expression of IL-4 positive cells and protein (P<0.05). Both the symptom score and the expression of IL-4 were notably lower in the AI+moxibustion group than in the AI+catgut embedment and AI+acupuncture groups (P<0.05), whereas the expression of IFN-γ was apparently higher in the AI+moxibustion group than in the other 3 treatment groups (P<0.05). No significant differences were found between the AI+catgut embedment and AI+acupuncture groups in the levels of symptom score, IFN-γ and IL-4 expressions (P>0.05). Moxibustion or catgut embedment or acupuncture and AI have a synergistic effect in relieving symptoms of AR rats, which may be related to their function in regulating the expression levels of nasal IFN-γ and IL-4 proteins. The therapeutic effect of moxibustion is obviously superior to those of both acupuncture and catgut embedment.

Effect of Different Absorption Enhancers on the Nasal Absorption of Nalmefene Hydrochloride.

The purpose of this work is to explore the effects of novel absorption enhancers on the nasal absorption of nalmefene hydrochloride (NMF). First, the influence of absorption enhancers with different concentrations and types and drug concentrations on the nasal absorption of NMF was investigated in vivo in rats. The absorption enhancers studied include n-dodecyl-β-D-maltoside (DDM), hydroxypropyl-β-cyclodextrin (HP-β-CD), and polyethylene glycol (15)-hydroxy Stearate (Solutol®HS15). At the same time, the in situ toad palate model and rat nasal mucosa model were used to assess the cilia toxicity. The results showed that all the absorption enhancers investigated significantly promote the nasal absorption of NMF, but with different degrees and trends. Among them, the 0.5% (w/v) DDM had the strongest enhancement effect, followed by 0.5% (w/v) Solutol®HS15, 0.25% (w/v) DDM, 0.25% (w/v) Solutol®HS15, 0.1% (w/v) Solutol®HS15, 0.1% (w/v) DDM, and 0.25% (w/v) HP-β-CD, with absolute bioavailability of 76.49%, 72.14%, 71.00%, 69.46%, 60.41%, 59.42%, and 55.18%, respectively. All absorption enhancers exhibited good safety profiles in nasal ciliary toxicity tests. From the perspective of enhancing effect and safety, we considered DDM to be a promising nasal absorption enhancer. And in addition to DDM, Solutol®HS15 can also promote intranasal absorption of NMF, which will provide another option for the development of nalmefene hydrochloride nasal spray.

Periodic Heat Stress Licenses EMSC Differentiation into Osteoblasts via YAP Signaling Pathway Activation.

Background The repair and regeneration of large bone defects represent highly challenging tasks in bone tissue engineering. Although recent studies have shown that osteogenesis is stimulated by periodic heat stress, the thermal regulation of osteogenic differentiation in ectomesenchymal stem cells (EMSCs) is not well studied. Methods and Results In this study, the direct effects of periodic heat stress on the differentiation of EMSCs into osteoblasts were investigated. EMSCs derived from rat nasal respiratory mucosa were seeded onto culture plates, followed by 1 h of heat stress at 41°C every 7 days during osteogenic differentiation. Based on the results of the present study, periodic heating increases alkaline phosphatase (ALP) activity, upregulates osteogenic-related proteins, and promotes EMSC mineralization. In particular, increased YAP nuclear translocation and YAP knockdown inhibited osteogenic differentiation induced by heat stress. Furthermore, the expression and activity of transglutaminase 2 (TG2) were significantly increased after YAP nuclear translocation. Conclusion Together, these results indicate that YAP plays a key role in regulating cellular proteostasis under stressful cellular conditions by modulating the TG2 response.

Development and in vivo Evaluation of Hydroxy-α-Sanshool Intranasal Liposomes as a Potential Remedial Treatment for Alzheimer's Disease.

PurposeHydroxy-α-sanshool (HAS) improves cognitive dysfunction, but its structural instability has limited its clinical application. The present study was conducted to investigate the optimal formulation of hydroxy-α-sanshool liposomes (HAS-LPs) and its effect on ameliorating learning and memory disorders in an Alzheimer’s disease (AD) model.MethodsIn this study, HAS was prepared as HAS-LP using a thin film dispersion method. After selecting the optimal preparation conditions, HAS-LP was characterized using transmission electron microscopy (TEM) and by measuring the zeta potential, particle size, and in vitro drug release. Next, evaluated the effect of HAS-LP on the rat nasal mucosa and then applied it to AD mice. By performing behaviour experiments, pathological test and related pharmacokinetic parameters, we explored its effect on attenuating learning and memory impairment in mice.ResultsWhen the mass ratio of HAS:cholesterol:soybean lecithin was 1:4:16 and 15 mL of ultrapure water were added, the highest encapsulation efficiency and drug loading were obtained. HAS-LP had a particle size of 181.77 nm, a polydispersity index of 0.207 and a zeta potential of −53.8 mV, and it remained stable at 25 °C for 1 week and 4 °C for 8 weeks. Moreover, HAS-LP exhibited slow drug release and was highly consistent with the Higuchi release model. HAS-LP was not significantly toxic to the nasal mucosa and effectively alleviated D-galactose-induced learning memory deficits and protected mouse hippocampal neuronal cells. HAS-LP was highly enriched in plasma and brain tissue after administration via the nasal route and obtained some ability to target the brain.ConclusionHAS encapsulated in soybean lecithin and cholesterol was successfully developed, suggesting that treatment with the nanoparticles might reverse some AD symptoms. Therefore, these nanoparticles might be used as promising new candidates for the delivery of HAS to treat AD.

Studies of Mucosal Irritation and Cellular Uptake Mechanisms of Xingnaojing Nanoemulsion

Xingnaojing (XNJ) injection was used to treat pneumonia and stroke in clinic in China, but with poor patient compliance. Xingnaojing nanoemulsion for intranasal delivery was developed to improve it. This article tried to evaluate the mucosal irritation of Xingnaojing nanoemulsion and investigate cellular uptake mechanism of its encapsulated lipophilic drugs. The toad palate model and rat nasal mucosa model were used to study the nasal ciliotoxicity and nasal mucosal irritation of nanoemulsion to evaluate its safety intranasally. The cellular uptake mechanism was studied by Calu-3 cell model. Coumarin 6 was encapsulated in nanoemulsion and the endocytic pathways were studied by cellular uptake experiments after being treated with different inhibitors. In toad palate model, the cilia movement of Xingnaojing nanoemulsion group last for 467.40 ± 39.02 min, which was obviously longer than deoxycholate group (90.60 ± 15.40 min). Studies on rats showed that the damage caused by nanemulsion is capable of being recovered. Nanoemulsion uptake was reduced obviously when cells were treated with wortmannin, and it also decreased about 13% when the temperature reduced from 37ºC to 4ºC. Mucosal irritation caused by nanoemulsion is low and the damage is recoverable. The cellular uptake of Xingnaojing nanoemulsion is energy-dependent, and macropinocytosis was the most important pathway for cellular uptake.

Low Molecular Weight Heparin Improves the Inflammatory State of Acute Sinusitis Rats Through Inhibiting the TLR4-MyD88-NF-κB Signaling Pathway.

Introduction: Low molecular weight heparin (LMWH), a natural sulfated glycosaminoglycan with an affinity for proangiogenic factors, is produced by chemical or enzymatic depolymerization of unfractionated heparin (UFH). Known for its anticoagulant effects, LMWH has recently been reported to have a strong anti-inflammatory effect on colitis, myocarditis, and airway inflammation. However, as a newly-developed drug, its anti-inflammatory mechanism in upper respiratory tract inflammation has not been well-studied. Methods: SD rats were randomly divided into control and experimental groups. The experimental group was established by building an acute nasal sinusitis model with expansion sponges mixed with Streptococcus pneumoniae. Then the experimental group rats were subcutaneously injected with different concentrations of LMWH. After seven consecutive days of injection, some rats were sacrificed, and blood and nasal mucosa samples were taken to determine their inflammation status. The remaining acute sinusitis rats were randomly selected for a week of nasal irrigation with normal saline or saline mixed with different concentrations of LMWH. One week later, rats were sacrificed, and samples of blood and nasal mucosa were taken to determine the inflammation status. Results: Rat nasal mucosa in the model group had obvious inflammation. The degree of nasal mucosa inflammation damage in the experimental group was lower than in the experimental control group, proving that LMWH has a protective effect on the nasal mucosa and that the effect correlates with dosage. Irrigation of the nose with saline mixed with LMWH can improve the anti-inflammatory effect. Protein related to the TLR4-MyD88-NF-κB signaling pathway was activated in the acute sinusitis rat model, and LMWH can significantly inhibit its expression. Conclusion: This is the first report of the anti-inflammatory effect of LMWH in acute upper respiratory tract inflammation, together with an explanation of its anti-inflammatory mechanism. The findings contribute a theoretical basis for its potential anti-tumor effect.

Comparative analysis of ACE2 protein expression in rodent, non-human primate, and human respiratory tract at baseline and after injury: A conundrum for COVID-19 pathogenesis.

Angiotensin converting enzyme 2 (ACE2) is the putative functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current literature on the abundance and distribution of ACE2 protein in the human respiratory tract is controversial. We examined the effect of age and lung injury on ACE2 protein expression in rodent and non-human primate (NHP) models. We also examined ACE2 expression in human tissues with and without coronavirus disease 19 (COVID-19). ACE2 expression was detected at very low levels in preterm, but was absent in full-term and adult NHP lung homogenates. This pattern of ACE2 expression contrasted with that of transmembrane protease serine type 2 (TMPRSS2), which was significantly increased in full-term newborn and adult NHP lungs compared to preterm NHP lungs. ACE2 expression was not detected in NHP lungs with cigarette smoke-induced airway disease or bronchopulmonary dysplasia. Murine lungs lacked basal ACE2 immunoreactivity, but responded to hyperoxia, bacterial infection, and allergen exposure with new ACE2 expression in bronchial epithelial cells. In human specimens, robust ACE2 immunoreactivity was detected in ciliated epithelial cells in paranasal sinus specimens, while ACE2 expression was detected only in rare type 2 alveolar epithelial cells in control lungs. In autopsy specimens from patients with COVID-19 pneumonia, ACE2 was detected in rare ciliated epithelial and endothelial cells in the trachea, but not in the lung. There was robust expression of ACE2 expression in F344/N rat nasal mucosa and lung specimens, which authentically recapitulated the ACE2 expression pattern in human paranasal sinus specimens. Thus, ACE2 protein expression demonstrates a significant gradient between upper and lower respiratory tract in humans and is scarce in the lung. This pattern of ACE2 expression supports the notion of sinonasal epithelium being the main entry site for SARS-CoV-2 but raises further questions on the pathogenesis and cellular targets of SARS-CoV-2 in COVID-19 pneumonia.

Change in expression of NFκb and MUC5AC in nasal mucosa during pregnancy

Aim: There is very limited information concerning gestational course of chronic inflammatory diseases of the upper airway. In the current study, we tried to reveal biomolecular changes in the nasal mucosa during pregnancy regarding NFκb and MUC5AC. This way, we may have the opportunity to suggest new treatment strategies for pregnancy induced nasal disorders. Methods: Thirty adult female rats were separated into pregnant and control groups. Serum estradiole (E2) and progesterone (PG) levels were evaluated by ELISA. Nasal expression of NFκb and MUC5AC were analyzed by polymerase chain reaction testing. Relative expression of NFκb and MUC5AC was compared between two groups. In addition, the influences of PG and E2 levels on NFκb and MUC5AC were determined. Results: NFκb was significantly low (P=0.015), while MUC5AC was significantly high in pregnant rats (P=0.029). Serum PG had a significantly negative effect on NF-κB expression (P=0.027) and a significantly positive effect on MUC5AC expression (P=0.017). There was a positive correlation between estradiol and MUC5AC (P=0.017). Conclusions: In the current study, the physiological expression of NFκb and MUC5AC was shown for the first time by PCR in rat nasal mucosa. The effect of PG and E2 on the expression of these biomolecules in the context of pregnancy was also revealed. These findings have the potential to elucidate gestational inflammatory alterations in the upper airway.