Enzyme-linked Immunosorbent Assay Analysis Research Articles

Investigating the mechanism of action of Yanghe Pingchuan Granule in the treatment of bronchial asthma based on bioinformatics and experimental validation

BackgroundYanghe Pingchuan Granule (YPG) is a patented Chinese medicine developed independently by the Anhui Provincial Hospital of Traditional Chinese Medicine. For many years, it has been used for the treatment of asthma with remarkable clinical effects. However, the composition of YPG is complex, and its potential active ingredients and mechanism of action for the treatment of asthma are unknown. Materials and methodsIn this study, we investigated the potential mechanism of action of YPG in the treatment of asthma through a combination of bioinformatics and in vivo experimental validation. We searched for active compounds in YPG and asthma targets from multiple databases and obtained common targets. Subsequently, a protein-protein interaction (PPI) network for compound disease was constructed using the protein interaction database for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Finally, hematoxylin and eosin (H&E) staining, Masson staining, enzyme-linked immunosorbent assay (ELISA) analysis, immunofluorescence (IF) experiments, and Western blot (WB) experiments were performed to verify the possible mechanism of action of YPG for asthma treatment. ResultsWe obtained 72 active ingredients and 318 drug target genes that overlap with asthma. Serine/threonine-protein kinase (AKT1), tumor protein p53 (TP53), tumor necrosis factor (TNF), interleukin (IL)-6, IL-1β, vascular endothelial growth factor-A (VEGFA), prostaglandin-endoperoxide synthase 2 (PTGS2), caspase-3 (CASP3), mitogen-activated protein kinase 3 (MAPK3) and epidermal growth factor receptor (EGFR) were the most relevant genes in the PPI network. KEGG analysis showed a high number of genes enriched for the nuclear factor kappa-B (NF-κB) signaling pathway. Animal experiments confirmed that YPG reduced inflammatory cell infiltration and down-regulated the expression of ovalbumin-induced inflammatory factors. Furthermore, YPG treatment decreased the protein expression of NFĸB1, nuclear factor kappa B kinase subunit beta (IKBKB), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR2) in lung tissue. ConclusionYPG has a positive effect on asthma by interfering with multiple targets. Furthermore, YPG may significantly inhibit the follicle-induced inflammatory response through the NF-ĸB signaling pathway.

Atractylenolide III ameliorated reflux esophagitis via PI3K/AKT/NF-κB/iNOS pathway in rats

Reflux esophagitis (RE), an esophageal inflammation caused by reflux of gastric contents, often damages the lower esophagus, seriously affecting the quality of life of patients. This study aims to investigate the therapeutic effects and underlying molecular mechanisms of atractylenolide III (ATL III) on RE model rats. In this research, the RE rat model is established sequentially following hemipyloric ligation, cardia transection, and hydrochloric acid perfusion. Further, the RE-induced rats are intragastrically administrated with ATL III (0.6, 1.2, and 2.4 mg/kg/D) for 28 days to evaluate ATL III therapeutic effects. To study the molecular mechanism, RE rats are treated with a phosphoinositide-3 kinase (PI3K) agonist (740 Y–P) combined with ATL III. The histopathological changes in the esophagus are eventually observed by hematoxylin & eosin (H&E) staining. In addition to changes in gastric pH and levels of reactive oxygen species (ROS), enzyme-linked immunosorbent assay (ELISA) and Western blot analyses are used to detect the expression levels of tumor necrosis factor-α (TNF-α, mmol/L), interleukin (IL)-8, IL-6, IL-1β in the esophageal tissues. As a result, the lesions in the esophageal tissues of RE rats are alleviated, decreasing the macroscopic observation scores of the esophageal mucosa after ATL III treatment,. The experimental results indicated significantly increased pH value of the gastric contents and reduced ROS, thiobarbituric acid reactants (TBARS), TNF-α, IL-8, IL-6, and IL-1β levels, as well as expression levels of p-PI3K, p-AKT, iNOS, and nuclear NF-κB proteins in esophageal tissues. In conclusion, the study indicated that ATL III could efficiently treat RE in rats by inhibiting oxidative stress and inflammatory damage through the PI3K/AKT/NF-κB/iNOS pathway.

Baizhu-Baishao herb pair ameliorates functional constipation and intestinal microflora disorder in rats.

In China, Rhizoma atractylodis macrocephalae-Paeonia lactiflora Pall (Biazhu-Baishao, BZBS) is a classic herb pair used to treat intestinal stress syndrome, ulcerative colitis and other diseases. However, the mechanism of BZBS in the treatment of functional constipation (FC) has been little studied and remains unclear. In this study, a behavioral investigation, colon tissue morphology, enzyme-linked immunosorbent assay (Elisa) and intestinal microflora analysis have been used to illuminate the potential mechanism of the effects of BZBS on FC in a rat model. A FC rat model was constructed and BZBS was given as treatment. Observations and recordings were made of the fecal moisture content, the defecation time of the first black stool, and the rate of intestinal propulsion. Elisa was used to detect the expression levels of substance P (SP), vasoactive intestinal peptide (VIP), 5-hydroxytryptamine (5-HT) in the colon. To ascertain the composition of the microbial community, a high throughput 16S ribosomal RNA (16S rRNA) gene sequencing technique was employed. Oral administration of BZBS significantly ameliorated several key excretion parameters, including the time to first black stool defecation, stool water content, and the propulsion rate in the small intestine in FC rats. It increased the expression of SP, VIP and 5-HT in the colon. 16S rRNA gene sequencing results showed that BZBS changed the microbial community structure, decreased the Bacteroidetes/Firmicutes ratio, increased the relative abundance of Blautia and Fusicatenibacter, and decreased the relative abundance of Ruminococcus and Roseburia. BZBS effectively alleviates FC and improves dysbacteriosis.

Human umbilical cord mesenchymal stem cells alleviated TNBS-induced colitis in mice by restoring the balance of intestinal microbes and immunoregulation

AimsHuman umbilical cord mesenchymal stem cells (HUMSCs) have been documented to be effective for several immune disorders including inflammatory bowel diseases (IBD). However, it remains unclear how HUMSCs function in regulating immune responses and intestinal flora in the trinitrobenzene sulfonic acid (TNBS)-induced IBD model. Materials and methodsWe assessed the regulatory effects of HUMSCs on the gut microbiota, T lymphocyte subpopulations and related immune cytokines in the TNBS-induced IBD model. The mice were divided into the normal, TNBS, and HUMSC-treated groups. The effect of HUMSCs was evaluated by Hematoxylin and Eosin (H&E) staining, fluorescence-activated cell sorting (FACS), and enzyme-linked immunosorbent assay (ELISA) analyses. Metagenomics Illumina sequencing was conducted for fecal samples. Key findingsWe demonstrated that the disease symptoms and pathological changes in the colon tissues of TNBS-induced colitis mice were dramatically ameliorated by HUMSCs, which improved the gut microbiota and rebalanced the immune system, increasing the abundance of healthy bacteria (such as Lactobacillus murinus and Lactobacillus johnsonii), the Firmicutes/Bacteroidetes ratio, and the proportion of Tregs; the Th1/Th17 ratio was decreased. Consistently, the expression levels of IFN-γ and IL-17 were significantly decreased, and transforming growth factor-β1 (TGF-β1) levels were significantly increased in the plasma of colitis mice HUMSC injection. SignificanceOur experiment revealed that HUMSCs mitigate acute colitis by regulating the rebalance of Th1/Th17/Treg cells and related cytokines and remodeling the gut microbiota, providing potential future therapeutic targets in IBD.

M13KO7 bacteriophage enables Potato Virus Y detection.

Potato virus Y (PVY) is one of the foremost plant viruses affecting potato plants and production. Many diagnostic and detection techniques are available to detect and prevent the spread of this virus. Serological analyses, such as enzyme-linked immunosorbent assay (ELISA), are widely used in laboratories as well as in the field. In addition, recently, serological methods using whole or recombinant antibodies have been developed. However, these antibodies are unstable under certain chemical conditions and can be easily degraded by exposure to enzymes. M13KO7, a derivative of bacteriophage M13, with a filamentous structure similar to PVY, was used for antibody screening. We found that PVY and "bald" M13KO7 had binding activity similar to that of antigen-antibody in ELISA analyses. The binding interactions between PVY and M13KO7 were highly specific. A new method for detecting PVY using ELISA analysis with M13KO7 was developed in this study. These results provide new insights into two virus-binding interactions and novel systems for the detection of plant viruses.IMPORTANCEIn this study, we confirmed the binding of M13KO7 to Potato virus Y (PVY) using enzyme-linked immunosorbent assay. M13KO7 is a "bald" bacteriophage in which no recombinant antibody is displayed. M13KO7 is easy to propagate by using Escherichia coli, making this method more reasonable in economic perspective. Based on this study, we suggest that M13KO7 detection system has applicability as a novel biological tool for the detection of PVY.

Enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell related biochemicals in oral submucous fibrosis

Oral submucous fibrosis (OSMF), a potentially malignant disorder, is developed by progressive fibrous tissue deposition in connective tissue along with atrophy of oral mucosa. Histological sections also show the mast cell infiltration in submucosa which may indicate their possible role in this entity. Abundant availability of biochemicals in mast cells like histamine and serine proteases like chymase may be released and play specific pathways in the disease pathophysiology. Possibly, if the histamine release has some part to play, diamine oxidase may also be found to have a relationship as it metabolizes histamine. The present study is proposed to identify the presence of chymase, histamine, and diamine oxidase in both, serum as well as tissue by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) respectively. This study may provide probable insight into the mast cell-related chemicals and their association with OSMF.

Development of dual-functional core-shell electrospun mats with controlled release of anti-inflammatory and anti-bacterial agents for the treatment of corneal alkali burn injuries

In this study, a novel dual-drug carrier for the co-administration of an anti-inflammatory and antibiotic agent consisting of core-shell nanofibers for the treatment of cornea alkali burns was designed. The core-shell nanofibers were prepared via coaxial electrospinning of curcumin-loaded silk fibroin as the core and vancomycin-loaded chitosan/polyvinyl alcohol (PVA) as the shell.Electron microscopy (SEM and TEM) images confirmed the preparation of smooth, bead-free, and continuous fibers that formed clear core-shell structures. For further studies, nanofiber mats were cross-linked by heat treatment to avoid rapid disintegration in water and improve both mechanical properties and drug release. The release profile of curcumin and vancomycin indicated an initial burst release, continued by the extended release of both drugs within 72 hours. Rabbit corneal cells demonstrated high rates of proliferation when evaluated using a cell metabolism assay. Finally, the therapeutic efficiency of core/shell nanofibers in healing cornea alkali burn was studied by microscopic and macroscopic observation, fluorescence staining, and hematoxylin-eosin assay on rabbit eyes. The anti-inflammatory activity of fabricated fibers was evaluated by enzyme-linked immunosorbent assay and Immunofluorescence analysis. In conclusion, using a robust array of in vitro and in vivo experiments this study demonstrated the ability of the dual-drug carriers to promote corneal re-epithelialization, minimize inflammation, and inhibit corneal neovascularization. Since these parameters are critical to the healing of corneal wounds from alkali burns, we suggest that this discovery represents a promising future therapeutic agent that warrants further study in humans.

Midkine characterization in human ovaries: potential new variants in follicles

To characterize the growth factor midkine (MDK) in the human ovary to determine whether MDK is produced locally within the ovary, examine whether different ovarian cell types are more likely to produce MDK, and determine whether there are any stage-specific variations during follicle growth. Previous studies have revealed that MDK potentially affects human follicle growth and oocyte maturation. Proteomic analyses in follicular fluid (FF) have identified MDK to functionally cluster together and follow a similar expression profile to that of well-known proteins involved in ovarian follicle development. Midkine has not yet been characterized in the human ovary. Descriptive study. University Hospital. The study included samples from 121 patients: 71 patients (aged 17-37 years) who underwent ovarian tissue cryopreservation provided granulosa cells (GC), cumulus cells, ovarian cortex, medulla tissue, and FF from small antral follicles (SAF); and 50 patients (aged 20-35 years) receiving invitro fertilization treatment provided FF from preovulatory follicles before and after induction of final follicle maturation. None. MDK relative gene expression was quantified using a real-time quantitative polymerase chain reaction in cumulus cells, GC, and medulla tissue. Additionally, immunostaining and western blotting assays were used to detect MDK protein in the ovarian cortex, which contains preantral follicles, SAF, and medulla tissue. Furthermore, enzyme-linked immunosorbent assay analyses were performed to measure the concentration of MDK in FF aspirated from SAF and preovulatory follicles both before and 36 hours after inducing the final maturation of follicles. Immunostaining and reverse transcription-quantitative polymerase chain reaction revealed a more prominent expression of MDK in GC compared with other ovarian cell types. Intrafollicular MDK concentration was significantly higher in SAF compared with preovulatory follicles. In addition, different molecular weight species of MDK were detected using western blotting in various ovarian sample types: GC and FF samples presented primarily one band of approximately 15 kDa and an additional band of approximately 13 kDa, although other bands with higher molecular weight (between 30 and 38 kDa) were detected in medulla tissue. This is the first time that MDK has been immunolocalized in human ovarian cells at the protein level and that potentially different MDK variants have been detected in human FF, GC, and ovarian medulla tissue. Future studies are needed to sequence and identify the different potential MDK variants found to determine their functional importance for ovary and oocyte competence.

Tolterodine ameliorates inflammatory response and ferroptosis against LPS in human bladder epithelial cells.

Bacterial endotoxin lipopolysaccharide (LPS)-induced inflammatory response and ferroptosis play an important role in urinary tract infections. Tolterodine has been used as a urinary tract antispasmodic and anticholinergic agent. However, the effects of Tolterodine against LPS-induced insults in human bladder epithelial cells (hBECs) have not been reported before. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays to determine the cell viability, reactive oxygen species (ROS) and malondialdehyde level detection were used to determine the level of oxidative stress, enzyme-linked immunosorbent assay and Western blot analysis were used to detect the protein level. In the current study, we found that Tolterodine ameliorated LPS-induced production of ROS and lipid oxidation in hBECs. Interestingly, Tolterodine inhibited the production of interleukin 6, interleukin-1β, and tumor necrosis factor α. Also, Tolterodine reduced the levels of Fe2+ and suppressed ferroptosis by reducing the levels of glutathione peroxidase 4, prostaglandin-endoperoxide synthase 2, and acyl-CoA synthetase long-chain family member 4 in LPS-challenged bladder epithelial cells. Mechanistically, it was shown that Tolterodine restored the nuclear factor E2-related factor 2 (Nrf2)/nuclear factor-κB signaling. Importantly, inhibition of Nrf2 with its specific inhibitor ML385 abolished the protective effects of Tolterodine in the inflammatory response and ferroptosis, suggesting that the effects of Tolterodine are mediated by Nrf2. Based on these findings, we conclude that Tolterodine might serve as a promising agent for the treatment of LPS-induced bladder inflammation.

Review of recently reported Ricin detection techniques focusing on combined immunoassay detection with abrin and saxitoxin in human plasma

Increasing non-traditional threats from biological or chemical weapons, the Organisation for the Prohibition of Chemical Weapons (OPCW) have tried to perform the preliminary analysis of biotoxin sample to standardize analysis methods and strengthen analytical capabilities among OPCW member countries. With the changes of new analysis, ROK CBRN Defense Research Institute (CDRI) established enzyme-linked immunosorbent assay (ELISA) and cytotoxicity analysis methods for ricin, abrin, and saxitoxin through the OPCW exercise on Biotoxin sample analysis. Thus, this study aims to established analytical methods (ELISA and cytotoxicity analysis) for the biological toxins called ricin, abrin and saxitoxin according to recent OPCW Biotoxin detection exercise. In particular, to refine practical and effective methods of biological analysis, we reviewed recent research on scientific analysis of ricin as a potential bioterror weapon, letter with ricin sent in White House, and suggested future agendas for preparedness testing.

Molecular and Serological Survey of Severe Fever with Thrombocytopenia Syndrome Virus in Horses from the Republic of Korea.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic tick-borne disease in East Asia caused by the SFTS virus (SFTSV). It is to investigate the presence of SFTSV RNA and antibodies in horses from a slaughterhouse and equestrian centers in the Republic of Korea (ROK). A prevalence study of SFTSV-specific RNA and antibodies was designed from 889 horses in the ROK. Materials and Methods: Serum samples were collected from horses at a slaughterhouse and equestrian centers from 2018 to 2020. To detect the presence of SFTSV, RNA was extracted from the serum samples, and a nested reverse transcription-polymerase chain reaction (RT-PCR) was conducted. Sequencing data were analyzed, and a phylogenetic tree was constructed using the maximum-likelihood method with Molecular Evolutionary Genetics Analysis Version 7.0 software. The horse sera were also tested for SFTSV-specific immunoglobulin G antibodies using enzyme-linked immunosorbent assay (ELISA). Results: Twelve of 889 (1.3%) horse sera were positive for SFTSV RNA, and 452 of 887 (51.0%) horse sera were seropositive by ELISA. Among the RT-PCR-positive samples, 12 of the SFTSV S-segment sequences were classified as sub-genotypes B-2 (n = 6) and B-3 (n = 6). ELISA analysis was evaluated by comparison with neutralization test. We investigated SFTSV infection in horses over a 3-year period, but sampling was not performed evenly by season; continuous surveillance of SFTSV in horses is needed. Conclusions: We report the detection of SFTSV RNA and provide serological data on SFTSV prevalence in horses in the ROK. The detection of SFTSV-specific RNA and antibodies in horses, which are in close proximity to humans, suggests that SFTS is an emerging and important health issue, indicating that more attention to its relevance for equestrian workers is needed.

Effects of albiflorin on oxidative stress and inflammatory responses in rats with acute spinal cord injury.

Oxidative stress and inflammatory responses are often the predominant detrimental factors associated with spinal cord injury (SCI). This study investigates the potential therapeutic effects of albiflorin (AF) on alleviating inflammation and oxidative stress in the rat model with SCI. Initially, the behavior of SCI-induced rats is examined by Basso-Beattie-Bresnahan score and the inclined plane examination. Then, the immunohistochemical staining of inflammasome-related protein (for instance, NACHT, LRR, and PYD domains-containing protein 3, NLRP3) is performed in combination with enzyme-linked immunosorbent assay (ELISA) of corresponding proinflammatory factors to assess the immunomodulatory effects of AF. Further, the markers involved in oxidative stress are examined by ELISA and western blot analysis analyses. These findings indicated that AF could alleviate motor dysfunction and the loss of neuron cells in SCI-induced rats. Mechanistically, AF could attenuate the inflammatory responses by reducing oxidative stress and activating nuclear erythroid-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in SCI rats. Depleting the antioxidant capacity by inhibiting glutathione biosynthesis could counteract the anti-inflammatory activity of AF in SCI rats. Together, our data suggested that AF could serve as a potential therapeutic agent against the aggravation of SCI in rats.

Intestinal permeability in patients undergoing stem cell transplantation correlates with systemic acute phase responses and dysbiosis.

Intestinal permeability may correlate with adverse outcomes during hematopoietic stem cell transplantation (HSCT), but longitudinal quantification with traditional oral mannitol and lactulose is not feasible in HSCT recipients because of mucositis and diarrhea. A modified lactulose:rhamnose (LR) assay is validated in children with environmental enteritis. Our study objective was to quantify peri-HSCT intestinal permeability changes using the modified LR assay. The LR assay was administered before transplant, at day+7 and+30 to 80 pediatric and young adult patients who received allogeneic HSCT. Lactulose and rhamnose were detected using urine mass spectrometry and expressed as an L:R ratio. Metagenomic shotgun sequencing of stool for microbiome analyses and enzyme-linked immunosorbent assay analyses of plasma lipopolysaccharide binding protein (LBP), ST2, REG3α, claudin1, occludin, and intestinal alkaline phosphatase were performed at the same timepoints. L:R ratios were increased at day+7 but returned to baseline at day+30 in most patients (P= .014). Conditioning regimen intensity did not affect the trajectory of L:R (P= .39). Baseline L:R ratios did not vary with diagnosis. L:R correlated with LBP levels (r2= 0.208; P= .0014). High L:R ratios were associated with lower microbiome diversity (P= .035), loss of anaerobic organisms (P= .020), and higher plasma LBP (P= .0014). No adverse gastrointestinal effects occurred because of LR. Intestinal permeability as measured through L:R ratios after allogeneic HSCT correlates with intestinal dysbiosis and elevated plasma LBP. The LR assay is well-tolerated and may identify transplant recipients who are more likely to experience adverse outcomes.

PM2.5 exposure promotes asthma in aged Brown-Norway rats: Implication of multiomics analysis

Children are disproportionately represented among those who suffer asthma, which is a kind of chronic airway inflammation. Asthma symptoms might worsen when exposed to the air pollutant particulate matter 2.5 (PM2.5). However, it is becoming more prevalent among older adults, with more asthma-related deaths occurring in this pollution than in any other age group, and symptoms caused by asthma can reduce the quality of life of the elderly, whose asthma is underdiagnosed due to physiological factors. Therefore, in an effort to discover a therapy for older asthma during exposure to air pollution, we sought to ascertain the effects of pre-exposure (PA) and persistent exposure (PAP) to PM2.5 in aged asthma rats. In this study, we exposed aged rats to PM2.5 at different times (PA and PAP) and established an ovalbumin-mediated allergic asthma model. The basic process of elderly asthma caused by PM2.5 exposure was investigated by lung function detection, enzyme-linked immunosorbent assay (ELISA), histopathology, cytology, cytokine microarray, untargeted metabolomics, and gut microbiota analysis. Our findings demonstrated that in the PA and PAP groups, exposure to PM2.5 reduced lung function and exacerbated lung tissue damage, with varying degrees of effect on immunoglobulin levels, the findings of a cytological analysis, cytokines, and chemokines. The PA and PAP rats had higher amounts of polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, 2-methylNaphthalene, 1-methylNaphthalene and flourene. Moreover, exposure to PM2.5 at different times showed different effects on plasma metabolism and gut microbiota. Bioinformatics analysis showed a strong correlation between PAHs, cytokines, and gut microbiota, and PAHs may cause metabolic disorders through the gut microbiota. These findings point to a possible mechanism for the development of asthma in older people exposure to PM2.5 that may be related to past interactions between PAHs, cytokines, gut microbiota, and plasma metabolites.

Effect of Ampelopsis brevipedunculata (Maxim.) Trautv extract on a model of atopic dermatitis in HaCaT cells and mice.

Ampelopsis brevipedunculata (Maxim.) Trautv. has been used for a long time as a folk remedy. According to studies, it possesses anti-inflammatory, antioxidant, and antibacterial properties. However, its effects on atopic dermatitis (AD) are poorly studied. Thus, we investigated the therapeutic effect of A. brevipedunculata (Maxim.) Trautv. extract (ABE-M) on 2,4-dinitrochlorobenzene (DNCB)-induced AD. For in vitro analysis, keratinocytes cell lines (HaCaT cells) were used. To evaluate the gene and protein expression levels of cytokines and chemokines, TNF-α/IFN-γ-stimulated HaCaT cells were treated with ABE-M. The cells and the supernatant were collected, then gene and protein levels were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay analysis. For in vivo analysis, BALB/c mice (6 weeks) were randomly separated into five groups (n = 5). The mice were applied DNCB and phosphate-buffered saline, dexamethasone (DX) or ABE-M (50, 100, and 200 mg/kg) was orally administrated for 28 days. At the end, ear tissues and blood were collected for histological analysis and evaluation of cytokines and chemokines. In keratinocytes, ABE-M inhibited the protein and mRNA levels of chemokines, and cytokines exposed by TNF-α/IFN-γ. Similarly, the expression of chemokines was suppressed by ABE-M in AD animal model induced by DNCB and the level of pro-inflammatory cytokines was decreased in a dose-dependent manner. Our research indicates that ABE-M could be a candidate material that can be used to improve skin immunity enhancement, health, and beauty.

Electroacupuncture alleviates neuropathic pain caused by SNL by promoting M2 microglia polarization through PD-L1

As a common clinical disease, neuropathic pain is difficult to be cured with drugs. The occurrence and progression of pain is closely related to the response of spinal microglia. Aspartof the regulation of microglialactivity,PD-L1 playsacriticalrole. Loss of PD-L1 promoted the polarization of M1-like microglia. Increased expression of PD-L1 promoted M2-like polarization. Electroacupuncture has a significant analgesic effect in clinical practice, but its specific mechanism remains to be further explored. In this study, we verified the role of PD-L1 in EA analgesia and the underlying molecular mechanism through spinal nerve ligation (SNL) in rats and lipopolysaccharide (LPS)-treated BV2 microglial cells. Forbehavioralstudiesofrats,mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured, and spinal cord neuros were examined under transmission electron microscopyto determine changes to their myelin structure. The expression levels of PD-L1 and M1/M2-specific markers in rat spinal cord and BV2 microglial cells were measured by enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence staining and Western blot analysis. Our study showed that EA increased the pain threshold, reduced the destruction of myelin structure, promoted the expression of PD-L1 and PD-1, inhibited the MAPK signaling pathway, and promoted the conversion of microglial polarization from the M1 phenotype to the M2 phenotype in SNL rats. PD-L1 knockdown reversed these effects of EA. In addition, PD-L1 knockdown activated the MAPK signaling pathway, promoted microglial polarization to the M1 phenotype, decreased the expression of anti-inflammatory mediators and increased the expression of proinflammatory factors in LPS-stimulated BV2 microglial cells. Our results showed that EA may regulate the excitability of primary afferent neurons through PD-L1 and then inhibit the MAPK signaling pathway to promote the transformation of activated M1 microglia into M2 microglia, reduce inflammatory reactions, and finally achieve analgesic effects. A therapy targeting PD-L1 may be an effective strategy for treating neuropathic pain.

Abstract P1060: Correlation Of Plasma Glutathione Peroxidase 4 Concentration With Coronary Heart Disease And The Severity Of Coronary Artery Stenosis

Introduction: Selenium-dependent glutathione peroxidase (GPX4) is an antioxidative, monomeric enzyme that can protect cells from membrane lipid oxidation-linked damage. Given the potential influence of GPX4 concentration on cardiovascular risk, we investigated how GPX4 levels change in patients with coronary heart disease (CHD), and whether GPX4 concentration correlates with the severity of coronary artery stenosis (CAS). Methods: Patients admitted at our institution between August 2022 and January 2023 for suspected CHD and completed coronary angiography were included. Patients were stratified according to their coronary angiography results: CHD group (positive) and control group (negative). The severity of CAS in patients with CHD was evaluated by dividing patients into low (<43 points) or high (≥43 points) Gensini score groups. Plasma GPX4 concentration was measured by enzyme-linked immunosorbent assay (ELISA). Results: Eighty-one patients were included based on our eligibility criteria, 41 patients in the CHD group and 40 patients in the control group. Patients with CHD were more commonly male and hypertensive, and presented with significantly greater levels of triglycerides and LDL-cholesterol at baseline. ELISA analysis indicates that patients with CHD have significantly lower plasma GPX4 concentrations as compared to patients in the control group. Furthermore, Spearman’s correlation analysis shows an inverse association between plasma GPX4 concentration and Gensini score in patients with CHD. Conclusion: Our results suggest that plasma GPX4 concentration was significantly lower in patients with CHD and is negatively correlated with the severity of CAS.

Recent advances in the preventative strategies for postoperative adhesions using biomaterial-based membranes and micro/nano-drug delivery systems

Postoperative adhesions are one of the clinically significant complications of almost all surgeries, leading to lifelong problems. To date, three strategies have been introduced to prevent or reduce the formation of these fibrous bands, including implementation of physical barriers, local or systemic administration of pharmacological agents, or a combination of both at the site of postsurgical traumatized tissue. This so far unsolved challenge has prompted scientists to focus on developing more effective preventative therapies. In this review, we aimed to provide a detailed overview of drug-free and drug-incorporated anti-adhesion barrier membranes and micro/nano-drug delivery systems implemented for postoperative adhesion prevention. The formulation components, the mean particle sizes of the particulate-based systems, release behavior of pharmacologic agents, and various evaluation methods determining the in vivo efficacy of the prepared systems in animal models including scoring, gross evaluation, Western blot, polymerase chain reaction (PCR), tissue scanning electron microscopy (SEM), enzyme-linked immunosorbent assay (ELISA), histological and immunohistochemical analyses were identified and discussed. Although a great number of studies have focused on the preparation and evaluation of the anti-adhesion efficacy of drug-free or drug-loaded anti-adhesion systems, further sophisticated research is required to develop effective and safe anti-adhesion products for clinical application.

Ultrasound-assisted intravesical botulinum toxin A delivery attenuates acetic acid-induced bladder hyperactivity in rats.

Background: Intradetrusor injection of botulinum toxin A (BTX-A) is an effective treatment for overactive bladder (OAB). However, the occurrence of adverse events associated with BTX-A injection therapy hinders its acceptance among patients and its clinical promotion. Intravesical instillation of BTX-A offers a promising alternative to injection therapy for treating OAB. Nevertheless, due to the presence of the bladder permeability barrier (BPB) and the high molecular weight of BTX-A, direct instillation is unable to penetrate the bladder urothelium. Purpose: This study aims to investigate the safety and feasibility of ultrasound-assisted intravesical delivery of BTX-A and its potential benefits in a rat model of bladder hyperactivity induced by acetic acid instillation. Methods: Hengli BTX-A and microbubbles (MB) were mixed and prepared as a novel complex. The size distribution and zeta potentials of the complex were measured. On day 1, rats' bladders were instilled with 1mL of saline, BTX-A (20U in 1mL), MB, or MB-BTX-A (20U in 1mL) complex with or without ultrasound (US) exposure (1MHz, 1.5W/cm2, 50% duty cycle, sonication for 10s with a 10-s pause for a total of 10min). The instillations were maintained for 30min. After 7days, cystometry was performed by filling the bladder with saline and 0.3% acetic acid (AA). Bladders were collected, weighed, and processed for immunoblotting, enzyme-linked immunosorbent assay (ELISA), histologic, and immunofluorescence analyses. Expression and distribution of SNAP-25 and SNAP-23 were assessed using Western blot and immunofluorescence. Calcitonin gene-related peptide (CGRP) in the bladder was detected using ELISA. Results: Intercontraction intervals (ICI) decreased by 72.99%, 76.16%, and 73.96% in rats pretreated with saline, BTX-A, and US + MB, respectively. However, rats treated with US + MB + BTX-A showed a significantly reduced response to AA instillation (57.31% decrease in ICI) without affecting amplitude, baseline pressure, or threshold pressure. Rats treated with US + MB + BTX-A exhibited increased cleavage of SNAP-25 and CGRP expression compared to the control group. Conclusion: Ultrasound-assisted intravesical delivery of BTX-A, with the assistance of MB cavitation, led to cleavage of SNAP-25, inhibition of calcitonin gene-related peptide release from afferent nerve terminals, and amelioration of acetic acid-induced bladder hyperactivity. These results support ultrasound-assisted intravesical delivery as an efficient non-injection method for administering BTX-A.

Development of a high-throughput centrifugal microsystem for enzyme-linked immunosorbent assay to detect SARS-CoV-2

Enzyme-linked immunosorbent assay (ELISA) is a commonly used diagnostic and quantification tool for protein biomarkers in biomedical research and industrial quality control. However, this technique has prolonged assay durations, along with requiring expensive equipment and trained technicians, which limits its on-site applications. In this study, we report a novel centrifugal microfluidic device that can perform the conventional ELISA assay in a high-throughput (HTP) and automatic manner. The centrifugal microfluidic microsystem incorporates four innovative features: (1) a rapid and easy dispenser structure with aliquoting microchannels, which can accurately fill the solution in 40 reaction chambers with just one injection, replacing the labor-intensive aliquoting process, (2) ultra-fast reagent loading into and discharging from 40 reaction chambers simultaneously within 15 sec through the combination of a siphon valve and centrifugal force control, (3) the exact and automatic handling of a small volume (only 30 µL per reaction) of the designated solution such as a sample, a washing solution, an HRP-antibody solution, and a TMB solution, (4) a miniaturized ELISA diagnostic system, consisting of a solution injector, a simple spindle motor, and a UV–vis absorbance detector for the point-of-care testing (POCT). Using the HTP centrifugal microfluidic device with the in-house POCT ELISA analyzer, we could automatically detect 40 samples of SARS-CoV-2 per run in 75 min, significantly reducing the time, cost, and labor compared with the conventional ELISA assay. SARS-CoV-2 was diagnosed with a limit-of-detection (LOD) of 10.2 ng/mL. In addition, clinical samples as well as the SARS-CoV-2 variants were successfully confirmed on the POCT ELISA analyzer.