Experimental Animals Research Articles

Understanding the Relationship Between Vascular Smooth Muscle Cell Function and the Efficacy of Acupuncture in Treating Cerebral Ischemic Stroke: A Preclinical Meta-Analysis and Systematic Review.

Cerebral blood flow and vascular structures serve as the fundamental components of brain metabolism and circulation. Acupuncture, an alternative and complementary medical approach, has demonstrated efficacy in treating cerebral ischemic stroke (CIS). Nevertheless, the mechanisms underlying the impact of acupuncture on vascular smooth muscle cell (VSMC) function remain uncertain. The objective of this systematic review and meta-analysis is to assess the alterations in VSMC function following acupuncture stimulation in CIS models. The databases PubMed, Web of Science, SCOPUS, and EMBASE were queried until November 2022 using a predetermined search strategy. The FORMAT BY SYRCLE guidelines were adhered to, and the risk of bias of the included studies was evaluated using the Risk of Bias tool developed by the Systematic Review Centre for Laboratory Animal Experimentation. The random-effects model was employed to estimate the standardized mean difference (SMD). Eighteen articles are included in this review. Acupuncture showed significant positive effects on the region cerebral blood flow (SMD=8.15 [95% CI, 4.52 to 11.78]) and neurological deficiency (SMD=-3.75 [95% CI, -5.54 to -1.97]). Descriptive analysis showed a probable mechanism of acupuncture stimulation in CIS rats related to VSMC function. Limitations and publication bias were presented in the studies. In this systematic review and meta-analysis, our findings indicate that acupuncture stimulation has the potential to improve regional cerebral blood flow and alleviate neurological deficits, possibly by regulating VSMC function. However, it is important to exercise caution when interpreting these results due to the limitations of animal experimental design and methodological quality.

Fabrication of Human Milk Fat Substitute: Based on the Similarity Evaluation Model and Computer Software.

We aimed to obtain the optimal formula for human milk fat substitute (HMFS) through a combination of software and an evaluation model and further verify its practicability through an animal experiment. The results showed that a total of 33 fatty acid (FA) and 63 triglyceride (TAG) molecular species were detected in vegetable oils. Palmitic acid, oleic acid, linoleic acid, 18:1/16:0/18:1, 18:2/16:0/18:2, 18:1/18:1/18:1 and 18:1/18:2/18:1, were the main molecular species among the FAs and TAGs in the vegetable oils. Based on the HMFS evaluation model, the optimal mixed vegetable oil formula was blended with 21.3% palm oil, 2.8% linseed oil, 2.6% soybean oil, 29.9% rapeseed oil and 43.4% maize oil, with the highest score of 83.146. Moreover, there was no difference in the weight, blood routine indices or calcium and magnesium concentrations in the feces of the mice between the homemade mixed vegetable oil (HMVO) group and the commercial mixed vegetable oil (CMVO) group, while nervonic acid (C24:1) and octanoic acid (C8:0) were absorbed easily in the HMVO group. Therefore, these results demonstrate that the mixing of the different vegetable oils was feasible via a combination of computer software and an evaluation model and provided a new way to produce HMFS.

BACH1 transcriptionally upregulates FOSL2 to induce M2 macrophages phenotype by activating TGFβ/SMAD signaling to promote the transformation of lung fibroblasts into myofibroblasts

Pulmonary fibrosis is a chronic and progressive lung disorder. The pro-fibrosis factors induced by M2 macrophage phenotype promote the differentiation of fibroblasts into myofibroblasts, which is essential for pulmonary fibrosis. We aimed to explore the role and mechanism of BTB domain and CNC homology 1 (BACH1) in pulmonary fibrosis. BACH1 was knocked down in THP‐1 polarized M2 macrophages with or without FOS-like antigen 2 (FOSL2) overexpression, the expression of M2 macrophage markers was detected. Cell viability, migration, invasion and extracellular matrix (ECM) accumulation were estimated by CCK-8, wound healing, transwell, western bot and immunofluorescence staining. Luciferase reporter and chromatin immunoprecipitation assays were used to verify the binding of BACH1 to FOSL2 promotor region. In vivo, a bleomycin (BLM)-induced pulmonary fibrosis mice model was established to evaluate the effect of BACH1 silencing on the histopathological changes, M2 macrophage phenotype and extracellular matrix (ECM) deposition. Expression of proteins was assessed with western blot. Results indicated that BACH1 expression was upregulated in M2 macrophages polarized from THP-1 cells. BACH1 deficiency inhibited the polarization of THP-1 to the M2 macrophage phenotype to promote the transformation of lung fibroblasts into myofibroblasts. Additionally, BACH1 could transcriptionally activate FOSL2 expression in THP-1-derived macrophages to upregulate TGFβ/SMAD signaling in HFL-1 cells. The animal experiments indicated that BACH1 knockdown alleviated BLM-induced pulmonary fibrosis, M2 macrophage polarization and inactivated FOSL2/TGFβ/SMAD signaling in mice lung tissues. Together, this finding suggests BACH1/FOSL2 may be useful therapeutic targets for the treatment of pulmonary fibrosis.

Circular RNA LMBR1 Inhibits Bladder Cancer Progression by Enhancing Expression of the Protein ALDH1A3

BackgroundCircular RNAs (circRNAs) have been identified as playing an integral role in the development of bladder cancer (BC). However, the mechanism by which circRNAs operate in the chemical carcinogenesis of BC remains unclear. MethodsTo explore this mechanism, we used RNA high-throughput sequencing to identify differentially expressed circRNA in bladder epithelial cells and chemically induced malignant transformed BC cells. Subsequently, in vitro experiments were conducted to investigate the biological function and molecular mechanism of circLMBR1 in BC. Finally, animal experiments were conducted to examine the clinical relevance of circLMBR1 in vivo. ResultsOur profiling of circular RNA expression during cellular malignant transformation induced by chemical carcinogens identified a subset of circRNAs associated with cell transformation. We verified that the expression of circLMBR1 in bladder epithelial malignant transformed cells was decreased compared with control cells, as well as in BC tissues and bladder cell lines. Furthermore, circLMBR1 was seen to inhibit the proliferation, invasion, and migration of BC cells both in vitro and in vivo. Mechanistically, circLMBR1 was found to exert its antitumor effect by binding to the protein ALDH1A3. ConclusionsOur findings have revealed that circLMBR1 inhibits the progression of BC cells by binding to ALDH1A3 and upregulating its expression. As such, circLMBR1 serves as a promising predictor of BC and may provide a novel therapeutic target for the treatment of BC.

Exploring the potential molecular intersection of stroke and major depression disorder

Stroke and major depression are common neurological diseases, and a large number of clinical studies have shown that there is a close relationship between the two diseases, but whether the two diseases are linked at the genetic level needs to be further explored. The purpose of this study was to explore the comorbidity mechanism of stroke and major depression by using bioinformatics technology and animal experiments. From the GEO database, we gathered transcriptome data of stroke and depression mice (GSE104036, GSE131712, GSE81672, and GSE146845) and identified comorbid gene set through edgR and WGCNA analyses. Further analysis revealed that these genes were enriched in pathways associated with cell death. Programmed cell death gene sets (PCDGs) are generated from genes related to apoptosis, necroptosis, pyroptosis and autophagy. The intersection of PCDGs and comorbid gene set resulted in two hub genes, Mlkl and Nlrp3. Single-cell sequencing analysis indicated that Mlkl and Nlrp3 are mainly influential on endothelial cells and microglia, suggesting that the impairment of these two cell types may be a factor in the relationship between stroke and major depression. This was experimentally confirmed by RT–PCR and immunofluorescence staining. Our research revealed that two specific genes, namely, Mlkl and Nlrp3, play crucial roles in the complex mechanism that links stroke and major depression. Additionally, we have predicted six possible therapeutic agents and the outcomes of docking simulations of target proteins and drug molecules.

Utilizing network pharmacology, molecular docking, and animal models to explore the therapeutic potential of the WenYang FuYuan recipe for cerebral ischemia-reperfusion injury through AGE-RAGE and NF-κB/p38MAPK signaling pathway modulation

BackgroundStroke is a debilitating condition with high morbidity, disability, and mortality that significantly affects the quality of life of patients. In China, the WenYang FuYuan recipe is widely used to treat ischemic stroke. However, the underlying mechanism remains unknown, so exploring the potential mechanism of action of this formula is of great practical significance for stroke treatment. ObjectiveThis study employed network pharmacology, molecular docking, and in vivo experiments to clarify the active ingredients, potential targets, and molecular mechanisms of the WenYang FuYuan recipe in cerebral ischemia-reperfusion injury, with a view to providing a solid scientific foundation for the subsequent study of this recipe. Materials and methodsActive ingredients of the WenYang FuYuan recipe were screened using the traditional Chinese medicine systems pharmacology database and analysis platform. Network pharmacology approaches were used to explore the potential targets and mechanisms of action of the WenYang FuYuan recipe for the treatment of cerebral ischemia-reperfusion injury. The Middle Cerebral Artery Occlusion/Reperfusion 2 h Sprague Dawley rat model was prepared, and TTC staining and modified neurological severity score were applied to examine the neurological deficits in rats. HE staining and Nissl staining were applied to examine the pathological changes in rats. Immunofluorescence labeling and Elisa assay were applied to examine the expression levels of certain proteins and associated factors, while qRT-PCR and Western blotting were applied to examine the expression levels of linked proteins and mRNAs in disease-related signaling pathways. ResultsWe identified 62 key active ingredients in the WenYang FuYuan recipe, with 222 highly significant I/R targets, forming 138 pairs of medication components and component-targets, with the top five being Quercetin, Kaempferol, Luteolin, β-sitosterol, and Stigmasterol. The key targets included TP53, RELA, TNF, STAT1, and MAPK14 (p38MAPK). Targets related to cerebral ischemia-reperfusion injury were enriched in chemical responses, enzyme binding, endomembrane system, while enriched pathways included lipid and atherosclerosis, fluid shear stress and atherosclerosis, AGE-RAGE signaling in diabetic complications. In addition, the main five active ingredients and targets in the WenYang FuYuan recipe showed high binding affinity (e.g. Stigmasterol and MAPK14, total energy <−10.5 Kcal/mol). In animal experiments, the WenYang FuYuan recipe reduced brain tissue damage, increased the number of surviving neurons, and down-regulated S100β and RAGE protein expression. Moreover, the relative expression levels of key targets such as TP53, RELA and p38MAPK mRNA were significantly down-regulated in the WenYang FuYuan recipe group, and serum IL-6 and TNF-a factor levels were reduced. After WenYang FuYuan recipe treatment, the AGE-RAGE signaling pathway and downstream NF-kB/p38MAPK signaling pathway-related proteins were significantly modulated. ConclusionThis study utilized network pharmacology, molecular docking, and animal experiments to identify the potential mechanism of the WenYang FuYuan recipe, which may be associated with the regulation of the AGE-RAGE signaling pathway and the inhibition of target proteins and mRNAs in the downstream NF-kB/p38MAPK pathway.

The Predictive Value of Serum DAO, HDC, and MMP8 for the Gastrointestinal Injury in the Early Stage of Acute Pancreatitis in an Animal Model and a Clinical Study.

This study was aimed at exploring the use of the acute gastrointestinal injury (AGI) grade and sensitive biomarkers to investigate gastrointestinal (GI) injury in early stage of acute pancreatitis (AP). The AGI grade was used to evaluate intestinal function. Any GI injury above grade I (grades II-IV) was considered as severe. An AP rat model was created by retrograde injection of 4% sodium taurocholate. The pancreatic and intestinal histopathology scores were calculated by hematoxylin-eosin staining. Human and rat sera were assessed using ELISA. Tight junction (TJ) proteins were detected by Western blotting. In clinical study, the GI injury rate in mild acute pancreatitis (MAP), moderate severe acute pancreatitis (MSAP), and severe acute pancreatitis (SAP) groups was 26.8%, 78.4%, and 94.8%, respectively (P < 0.05). Diamine oxidase (DAO), histidine decarboxylase (HDC), and matrix metalloproteinase 8 (MMP8) serum levels were higher in AP patients than in healthy people (P < 0.05). Patients with GI injury had higher serum levels of DAO, HDC, and MMP8 than those without GI injury (P < 0.05). In animal experiments, the serum levels of DAO, HDC, and MMP8 were higher in the AP group than in normal and sham-operated (SO) groups (P < 0.05). The expressions of tricellulin, claudin-1, ZO-1, and occludin were significantly lower in the AP group than in normal and SO groups (P < 0.05). The serum levels of DAO, HDC, and MMP8 are novel biomarkers of GI injury in the early stage of AP; their elevation indicates the development of GI injury in AP. The intestinal TJ disruption may be a primary mechanism of GI injury and requires more in-depth research.

Role of mitochondrial dysfunction in acute traumatic brain injury: evidence from bioinformatics analysis

BackgroundThe intricate regulatory relationship between mitochondrial dysfunction, apoptosis, and immune cells remains largely elusive following traumatic brain injury (TBI). MethodsThe GSE45997 dataset from the Gene Expression Omnibus database and utilized GEO2R to screen for differentially expressed genes (DEGs). Functional enrichment analyses were performed. Mitochondrial gene data from the MitoCarta3.0 database were combined with the DEGs to identify mitochondria-related DEGs (MitoDEGs). The hub MitoDEGs related to apoptosis were further screened. Animal models of TBI were established to investigate the mechanisms underlying mitochondrial dysfunction regulation of apoptosis. Furthermore, we explored the relationship between MitoDEGs/hub MitoDEGs and immune cells using the Spearman correlation method. ResultsFifty-seven MitoDEGs were significantly enriched in pathways related to fatty acid degradation and metabolism. We identified three upregulated hub MitoDEGs, namely Dnm1l, Mcl1 and Casp3, were associated with apoptosis. In the animal experiments, we observed significant expression levels of microtubule-associated protein 1 light chain 3 beta (LC3B) surrounding the injury site. Most LC3B-expressing cells exhibited positive staining for Beclin 1 and colocalization analysis revealed the simultaneous presence of Beclin 1 and caspase-3. The western blot analysis further unveiled a significant upregulation of cleaved caspase-3 levels and LC3B II/LC3B I ratio after TBI. Moreover, the quantity of myeloid cell leukaemia-1 immunoreactive cells was notably higher than that in the control group. Spearman correlation analysis demonstrated strong associations between plasma cells, marginal zone B cells, native CD4 T cells, monocytes, and MitoDEGs/hub MitoDEGs. ConclusionsThis study sheds light on enhanced fatty acid metabolism following mitochondrial dysfunction and its potential association with apoptosis and immune cell activation, thereby providing new mechanistic insights into the acute phase of TBI.

Morphine timing-dependent modulation of TRPV1 phosphorylation correlates with differential morphine effects on myocardial ischemia/reperfusion injury

Opioids are used for pain relief in patients suffering from acute myocardial ischemia or infarction. Clinical and laboratory studies demonstrate that morphine treated patients or the experimental animal model suffering acute myocardial ischemia and reperfusion, may worsen myocardial viability. As transient receptor potential vanilloid 1 (TRPV1) plays important roles in pain sensation and cardio-protection, we query whether opioids may exacerbate myocardial viability via interaction with TRPV1 activity in the pain relief. We found the co-expressions of TRPV1 and opioid μ, δ and κ receptors in adult rat cardiomyocytes. Intravenous injection of morphine (0.3mg/Kg) at 20 min after induction of myocardial ischemia, in the rat model of acute myocardial ischemia and reperfusion, induced significant reduction of phosphorylated TRPV1 (p-TRPV1) in the ventricular myocardium and increase in serum cardiac troponin I (cTnI), compared with the ischemia/reperfusion controls (all P< 0.05). The effects of morphine were completely reversed by selective opioid μ, δ and κ receptor antagonists. While significant upregulation of p-TRPV1 (P<0.05) and improvement of ±dP/dt max (all P<0.05) were detected in the animals giving the same dose of morphine before induction of myocardial ischemia. The changes in p-TRPV1 correlate with the alterations of cTnI (r= -0.5840, P= 0.0283) and ±dP/dt max (r= 0.8084, P=0.0005 and r= -0.8133, P= 0.0004, respectively). The findings of this study may indicate that potentiation and attenuation of TRPV1 sensitivity correlate with the improvement of the cardiac performance and the aggravation of myocardial viability, respectively, by giving morphine before and during myocardial ischemia and reperfusion.

Experimental Animal Models of Periodontal Diseases

Abstract Periodontal diseases involve several conditions that are defined by pathologic loss of the periodontal ligament and alveolar bone. Periodontitis is an inflammatory disease of periodontal tissue caused by a host immune response to dysbiotic microbial biofilm whose progression associated with destruction of tooth supporting tissue is promoted by host proteinases. Periradicular periodontitis is the most commonly diagnosed disease that affects the pathology of periodontal tissue in the area of the tooth root. This disease has also been described as apical periodontitis. Several experimental models of periodontal diseases have been developed in recent years. This paper provides an overview of experimentally induced animal models of periodontal disease (periodontitis and periradicular periodontitis) and the possibility of using different experimental animals to study aspects of periodontal disease, each with advantages and disadvantage.

Microbiota and arthritis: cause or consequence?

The relationship between intestinal microbiota and arthritis has garnered significant attention, with emerging evidence suggesting a potential association between dysbiosis and various forms of inflammatory arthropathies. While observational studies have provided valuable insights into microbiota alterations in patients with arthritis, establishing causality remains challenging. Observational data, influenced by multiple confounders such as environmental factors, medication effects, and dietary habits, are insufficient to conclusively determine whether microbiota changes are somehow causally linked to arthritis. The heterogeneity of results across independent studies further complicates interpretation. To further support this hypothesis, interventional randomised trials are deemed necessary, yet their implementation in this area presents significant technical limitations. Experimental animal models offer insights into potential pathogenic mechanisms linking dysbiosis to arthritis, including compromised intestinal barrier function, the role of microbiota-derived metabolites and molecular mimicry. However, conflicting findings underscore the complexity of hostmicrobiota interactions and the challenges in establishing causality.Efforts to modulate the microbiota for arthritis treatment or prevention have shown promise, yet efficacy and applicability remains uncertain. Antibacterial drugs, dietary interventions, probiotics, and faecal microbiota transplantation have been explored, but their clinical utility awaits further validation. In conclusion, while the association between intestinal microbiota and arthritis is increasingly recognised, establishing causality remains elusive.

A workflow to practically apply true dose considerations to in vitro testing for Next Generation Risk Assessment

With the move away from safety testing assessment based on data generated in experimental animals the concept of Next Generation Risk Assessment (NGRA) has arisen which instead uses data from in silico and in vitro models. A key uncertainty in risk assessment is the actual dose of test chemical at the target site, and therefore surrogate dose metrics, such as nominal concentration in test media are used to describe in vitro effect (or no-effect) doses. The reliability and accuracy of the risk assessment therefore depends largely on our ability to understand and characterise the relationship between the dose metrics used and the actual biologically effective dose at the target site. The objective of this publication is to use 40 case study chemicals to illustrate how in vitro dose considerations can be applied to characterise the “true dose” and build confidence in the understanding of the biologically effective dose in in vitro test systems for the determination e.g. points of departure (PoDs) for NGRA. We propose a workflow that can be applied to assess whether the nominal test concentration can be considered a conservative dose metric for use in NGRA. The workflow examines the implications of volatility, stability, hydrophobicity, binding to plastic and serum, solubility, and the potential use of in silico models for some of these parameters. For the majority of the case study chemicals we found that the use of nominal concentrations in risk assessment would result in conservative decision making. However, for serval chemicals a potential for underestimation of the risk in humans in vivo based on in vitro nominal effect concentrations was identified, and approaches for refinement by characterisation of the actual effect concentration are proposed.

MicroRNA expression profile of chicken liver at different times after Histomonas meleagridis infection

Histomonas meleagridis, an anaerobic intercellular parasite, is known to infect gallinaceous birds, particularly turkeys and chickens. The resurgence of histomonosis in recent times has resulted in significant financial setbacks due to the prohibition of drugs used for disease treatment. Currently, research on about H. meleagridis primarily concentrate on the examination of its virulence, gene expression analysis, and the innate immunity response of the host organism. However, there is a lack of research on differentially expressed miRNAs (DEMs) related to liver infection induced by H. meleagridis. In this study, the weight gain and pathological changes at various post-infection time points were evaluated through animal experiments to determine the peak and early stages of infection. Next, High-throughput sequencing was used to examine the expression profile of liver miRNA at 10 and 15 days post-infection (DPI) in chickens infected with the Chinese JSYZ-F strain of H. meleagridis. A comparison with uninfected controls revealed the presence of 120 and 118 DEMs in the liver of infected chickens at 10 DPI and 15 DPI, respectively, with 74 DEMs being shared between the two time points. Differentially expressed microRNAs (DEMs) were categorized into three groups based on the time post-infection. The first group (L1) includes 45 miRNAs that were differentially expressed only at 10 DPI and were predicted to target 1646 genes. The second group (L2) includes 43 miRNAs that were differentially expressed only at 15 DPI and were predicted to target 2257 genes. The third group (L3) includes 75 miRNAs that were differentially expressed at both 10 DPI and 15 DPI and were predicted to target 1623 genes. At L1, L2, and L3, there were 89, 87, and 41 significantly enriched Gene Ontology (GO) terms, respectively (p<0.05). The analysis of differentially expressed miRNA target genes using KEGG pathways revealed significant enrichment at L1, L2, and L3, with 3, 4, and 5 pathways identified, respectively (p<0.05). This article suggests that the expression of liver miRNA undergoes dynamic alterations due to H. meleagridis and the host. It showed that the expression pattern of L1 class DEMs was more conducive to regulating the development of the inflammatory response, while the L2 class DEMs were more conducive to augmenting the inflammatory response. The observed patterns of miRNA expression associated with inflammation were in line with the liver's inflammatory process following infection. The results of this study provide a basis for conducting a comprehensive analysis of the pathogenic mechanism of H. meleagridis from the perspective of host miRNAs.

Revealing the potential bioactive components and mechanism of Qianhua Gout Capsules in the treatment of gouty arthritis through network pharmacology, molecular docking and pharmacodynamic study strategies

Recent clinical studies have confirmed the effectiveness of Qianhua Gout Capsules (QGC) in the treatment of gouty arthritis (GA). However, the specific regulatory targets and mechanisms of action of QGC are still unclear. To address this gap, we utilized network pharmacology, molecular docking, and pharmacodynamic approaches to investigate the bioactive components and associated mechanisms of QGC in the treatment of GA. By employing UPLC-Q Exactive-MS, we identified the compounds present in QGC, with active ingredients defined as those with oral bioavailability ≥30 % and drug similarity ≥0.18. Subsequently, the targets of these active compounds were determined using the TCMSP database, while GA-related targets were identified from DisGeNET, GeneCards, TTD, OMIM, and DrugBank databases. Further analysis including PPI analysis, GO analysis, and KEGG pathway enrichment was conducted on the targets. Validation of the predicted results was performed using a GA rat model, evaluating pathological changes, inflammatory markers, and pathway protein expression. Our results revealed a total of 130 components, 44 active components, 16 potential shared targets, GO-enriched terms, and 47 signaling pathways related to disease targets. Key active ingredients included quercetin, kaempferol, β-sitosterol, luteolin, and wogonin. The PPI analysis highlighted five targets (PPARG, IL-6, MMP-9, IL-1β, CXCL-8) with the highest connectivity, predominantly enriched in the IL-17 signaling pathway. Molecular docking experiments demonstrated strong binding of CXCL8, IL-1β, IL-6, MMP9, and PPARG targets with the top five active compounds. Furthermore, animal experiments confirmed the efficacy of QGC in treating GA in rats, showing reductions in TNF-α, IL-6, and MDA levels, and increases in SOD levels in serum. In synovial tissues, QGC treatment upregulated CXCL8 and PPARG expression, while downregulating IL-1β, MMP9, and IL-6 expression. In conclusion, this study applied a network pharmacology approach to uncover the composition of QGC, predict its pharmacological interactions, and demonstrate its in vivo efficacy, providing insights into the anti-GA mechanisms of QGC. These findings pave the way for future investigations into the therapeutic mechanisms underlying QGC's effectiveness in the treatment of GA.

Inactivation of cGAS signaling pathway mediated by TDP-43 deficiency protects microglia from hypoxia/reoxygenation induced injury

BackgroundMicroglia are damaged during cerebral ischemia–reperfusion (I/R). This study was performed to investigate the regulatory effect of tAR DNA-binding protein-43 (TDP-43) on microglia after cerebral I/R in vitro and in vivo. MethodThe hypoxia/reoxygenation (H/R) treated microglia and rats with middle cerebral artery occlusion surgery were constructed respectively. The TDP-43 expression in brain tissues and microglia of each group was evaluated by qPCR and western blotting methods. Cell viability and cell apoptosis were combined to evaluate the degree of cell injury. As for animal experiments, neurological score and infarct volume were obtained to evaluate neurological injury. ResultsThe levels of TDP-43 in the brain tissues of I/R group were higher than that in sham group. Both TDP-43 and Iba1, a typical microglia marker, were expressed in the brain tissues. TDP-43 was also elevated in microglia with H/R treatment. Inhibition of TDP-43 significantly down-regulated neurological deficit scores of rats after I/R surgery, and weakened the H/R treatment induced injury by promoting cell viability, inhibiting cell apoptosis, down-regulating IL-6 and iNOS levels, and up-regulating Arg-1 and IL-10 levels. Inactivation of cGAS pathway mediated by TDP-43 knockdown protects microglia from H/R treatment induced injury. ConclusionThe highly expressed TDP-43 level is associated with cerebral I/R, and inhibition of TDP-43 protects microglia from H/R induced injury through cGAS pathway in vitro and in vivo.

The effects of fine particulate matter (SRM 2786) on three different 3D lung models exposed at the air-liquid interface – A comparative study

3D cell culture models exposed at the air-liquid interface (ALI) represent a potential alternative to animal experiments for hazard and risk assessment of inhaled compounds. This study compares cocultures composed of either Calu-3, A549 or HBEC3-KT lung epithelial cells, cultured together with THP-1-derived macrophages and EA.hy926 endothelial cells, in terms of barrier capacity and responses to a standard reference sample of fine particulate matter (SRM 2786). High-content imaging analysis revealed a similar cellular composition between the different cell models. The 3D cell cultures with Calu-3 cells showed the greatest barrier capacity, as measured by transepithelial electrical resistance and permeability to Na-fluorescein. Mucus production was detected in 3D cell cultures based on Calu-3 and A549 cells. Exposure to SRM 2786 at ALI increased cytokine release and expression of genes associated with inflammation and xenobiotic metabolism. Moreover, the presence of THP-1-derived macrophages was central to the cytokine responses in all cell models. While the different 3D cell culture models produced qualitatively similar responses, more pronounced pro-inflammatory responses were observed in the basolateral compartment of the A549 and HBEC3-KT models compared to the Calu-3 model, likely due to their reduced barrier capacity and lower retention of secreted mediators in the apical compartment.

Exploring the pharmacological mechanism of Glycyrrhiza uralensis against KOA through integrating network pharmacology and experimental assessment.

Knee osteoarthritis (KOA), a major health and economic problem facing older adults worldwide, is a degenerative joint disease. Glycyrrhiza uralensis Fisch. (GC) plays an integral role in many classic Chinese medicine prescriptions for treating knee osteoarthritis. Still, the role of GC in treating KOA is unclear. To explore the pharmacological mechanism of GC against KOA, UPLC-Q-TOF/MS was conducted to detect the main compounds in GC. The therapeutic effect of GC on DMM-induced osteoarthritic mice was assessed by histomorphology, μCT, behavioural tests, and immunohistochemical staining. Network pharmacology and molecular docking were used to predict the potential targets of GC against KOA. The predicted results were verified by immunohistochemical staining Animal experiments showed that GC had a protective effect on DMM-induced KOA, mainly in the improvement of movement disorders, subchondral bone sclerosis and cartilage damage. A variety of flavonoids and triterpenoids were detected in GC via UPLC-Q-TOF/MS, such as Naringenin. Seven core targets (JUN, MAPK3, MAPK1, AKT1, TP53, RELA and STAT3) and three main pathways (IL-17, NF-κB and TNF signalling pathways) were discovered through network pharmacology analysis that closely related to inflammatory response. Interestingly, molecular docking results showed that the active ingredient Naringenin had a good binding effect on anti-inflammatory-related proteins. In the verification experiment, after the intervention of GC, the expression levels of pp65 and F4/80 inflammatory indicators in the knee joint of KOA model mice were significantly downregulated. GC could improve the inflammatory environment in DMM-induced osteoarthritic mice thus alleviating the physiological structure and dysfunction of the knee joint. GC might play an important role in the treatment of knee osteoarthritis.

Cetyltrimethylammonium-chloride assisted in situ metabolic incorporation of nano-sized ROS-generating cascade-reaction containers in Gram-positive and Gram-negative peptidoglycan layers for the control of bacterially-induced sepsis

Cascade-reaction containers generating reactive oxygen species (ROS) as an alternative for antibiotic-based strategies for bacterial infection control, require endogenous oxygen-sources and ROS-generation close to or preferably inside target bacteria. Here, this is achieved by cetyltrimethylammonium-chloride (CTAC) assisted in situ metabolic labeling and incorporation of mesoporous SiO2-nanoparticles, dual-loaded with glucose-oxidase and Fe3O4-nanoparticles as cascade-reaction containers, inside bacterial cell walls. First, azide-functionalized d-alanine (D-Ala-N3) was inserted in cell wall peptidoglycan layers of growing Gram-positive pathogens. In Gram-negatives, this could only be achieved after outer lipid-membrane permeabilization, using a low concentration of CTAC. Low concentrations of CTAC had no adverse effect on in vitro blood clotting or hemolysis nor on the health of mice when blood-injected. Next, dibenzocyclooctyne-polyethylene-glycol modified, SiO2-nanoparticles were in situ click-reacted with d-Ala-N3 in bacterial cell wall peptidoglycan layers. Herewith, a two-step cascade-reaction is facilitated inside bacteria, in which glucose-oxidase generates H2O2 at endogenously-available glucose concentrations, while subsequently Fe3O4-nanoparticles catalyze generation of •OH from the H2O2 generated. Generation of •OH inside bacterial cell walls by dual-loaded mesoporous SiO2-nanoparticles yielded more effective in vitro killing of both planktonic Gram-positive and Gram-negative bacteria suspended in 10 % plasma than SiO2-nanoparticles solely loaded with glucose-oxidase. Gram-positive or Gram-negative bacterially induced sepsis in mice could be effectively treated by in situ pre-treatment with tail-vein injected CTAC and d-Ala-N3, followed by injection of dual-loaded cascade-reaction containers without using antibiotics. This makes in situ metabolic incorporation of cascade-reaction containers as described attractive for further investigation with respect to the control of other types of infections comprising planktonic bacteria. Statement of SignificanceIn situ metabolic-incorporation of cascade-reaction-containers loaded with glucose-oxidase and Fe3O4 nanoparticles into bacterial cell-wall peptidoglycan is described, yielding ROS-generation from endogenous glucose, non-antibiotically killing bacteria before ROS inactivates. Hitherto, only Gram-positives could be metabolically-labeled, because Gram-negatives possess two lipid-membranes. The outer membrane impedes direct access to the peptidoglycan. This problem was solved by outer-membrane permeabilization using a quaternary-ammonium compound. Several studies on metabolic-labeling perform crucial labeling steps during bacterial-culturing that in real-life should be part of a treatment. In situ metabolic-incorporation as described, can be applied in well-plates during in vitro experiments or in the body as during in vivo animal experiments. Surprisingly, metabolic-incorporation proceeded unhampered in blood and a murine, bacterially-induced sepsis could be well treated.

Animal Scientism: Making Biology Experimental in Republican China

This essay follows animals from the animal house through the laboratory to their representation as abstracted knowledge circulating the world to trace the emergence of experimental biology in Republican China. Experimental biology was promoted and institutionalized by the Chinese Physiological Society, which British-Chinese experimental physiologist Robert Lim established in 1926, at a time when descriptive taxonomy and morphology were dominant. Proponents argued that experimental research on live animals was more advanced than the collection and observation of specimens, and that experimentation, rather than description, now undergirded international science. In championing experimentalism, they were not only trying to shift the focus of Chinese biology from description to experiment, but also revising the scientism by then entrenched in Republican China. They put forth a scientism of experiment: cultivating experimental biology would help China catch up more quickly to imperial powers. Animals, as the material basis for experimental biology, came to signify the science that they produced; the Buddhist-aligned Chinese Animal Protection Society, established in 1932, chose not to protest animal experimentation in part due to the association between animals and modern experimentalism. In Republican China, animals were thus material and cultural objects that made and embodied a new scientism of experiment.

Targeted screening of an anti-inflammatory polypeptide from Rhopilema esculentum Kishinouye cnidoblasts and elucidation of its mechanism in alleviating ulcerative colitis based on an analysis of the gut microbiota and metabolites

Ulcerative colitis (UC) is a recurrent inflammatory bowel disease that imposes a severe burden on families and society. In recent years, exploiting the potential of marine bioactive peptides for the treatment of diseases has become a topic of intense research interest. This study revealed the mechanism underlying the protective effect of the dominant polypeptide PKKVV (Pro-Lys-Lys-Val-Val) of Rhopilema esculentum cnidoblasts against DSS-induced UC through a combined analysis of the metagenome and serum metabolome. Specifically, the polypeptide composition of R. esculentum cnidoblasts was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Molecular docking showed that the dominant peptide PKKVV could bind better with tumor necrosis factor-α (TNF-α) than the original ligand. Subsequent animal experiments suggested that PKKVV could modulate disorganized gut microorganisms in mice with UC; affect serum metabolites through the arachidonic acid, glycerophospholipid and linoleic acid metabolism pathways; and further alleviate UC symptoms. This study provides a reference for the comprehensive development of marine bioactive substances and nonpharmaceutical treatments for UC.