Hematoxylin-eosin Research Articles

Macroanatomy, Histomorphometry, and Androgen Receptor Expression in the Epididymis of Kacang Goats Aged 4, 8, and 12 Months

The epididymis is a crucial component of the goat's reproductive structure. The epididymis is responsible for the transportation, concentration, and maturation of sperm. This study aimed to examine the differences in the structure of macro and microanatomy, tissue composition, and the existence of androgen receptors in the epididymis of Kacang goats aged 4, 8, and 12 months. The assessment of macroscopic organ growth was done immediately after sampling, whereas microscopic measurements were carried out following histological preparations using hematoxylin-eosin (HE) and immunohistochemical (IHC) procedures. The results on the macroscopic anatomy of the epididymis indicated a significant association between age with the width of the caput dexter and sinister and the circumference of the right cauda. However, no significant relationship was found between age with the corpus length and the left cauda's circumference. Significant variations were observed in the diameter and concentration of the agglutinated spermatozoa in the lumen during histomorphometry of the epididymis in three age groups of Kacang goats. There were no statistically significant variations in the expression of androgen receptors among the three age groups. This study showed that the correlation coefficient test reveals a positive relationship between age and the caput width and corpus length dimensions, indicating that these measurements tend to grow as age increases. On the other hand, the diameter of the agglutinated spermatozoa in the epididymal lumen exhibits significant variations between the ages of 4 months, with the ages of 8 months and 12 months, suggesting that the sperm becomes fully matured by the age of 8 months.

Modification of transvaginal polypropylene mesh with co-axis electrospun nanofibrous membrane to alleviate complications following surgical implantation

BackgroundSurgeries for treating pelvic organ prolapse involving the utilization of synthetic mesh have been associated with complications such as mesh erosion, postoperative pain, and dyspareunia. This work aimed to reduce the surgical implantation-associated complications by nanofibrous membranes on the surface of the polypropylene mesh. The nanofiber of the nanofibrous membrane, which was fabricated by co-axial electrospinning, was composed of polyurethane as fiber core and gelatin as the fiber out layer. The biocompatibility of the modified mesh was evaluated in vitro by cell proliferation assay, immunofluorescence stain, hematoxylin-eosin (HE) staining, and mRNA sequencing. Polypropylene mesh and modified mesh were implanted in a rat pelvic organ prolapse model. Mesh-associated complications were documented. HE and Picro-Sirius red staining, immunohistochemistry, and western blotting were conducted to assess the interactions between the modified mesh and vaginal tissues.ResultsThe modified mesh significantly enhanced the proliferation of fibroblasts and exerted a positive regulatory effect on the extracellular matrix anabolism in vitro. When evaluated in vivo, no instances of mesh exposure were observed in the modified mesh group. The modified mesh maintained a relatively stable histological position without penetrating the muscle layer or breaching the epidermis. The collagen content in the vaginal wall of rats with modified mesh was significantly higher, and the collagen I/III ratio was lower, indicating better tissue elasticity. The expression of metalloproteinase was decreased while the expression levels of tissue inhibitor of metalloproteinase were increased in the modified mesh group, suggesting an inhibition of collagen catabolism. The expression of TGF-β1 and the phosphorylation levels of Smad3, p38 and ERK1/2 were significantly increased in the modified mesh group. NM significantly improved the biocompatibility of PP mesh, as evidenced by a reduction in macrophage count, decreased expression levels of TNF-α, and an increase in microvascular density.ConclusionsThe nanofibrous membrane-coated PP mesh effectively reduced the surgical implantation complications by inhibiting the catabolism of collagen in tissues and improving the biocampibility of PP mesh. The incorporation of co-axial fibers composed of polyurethane and gelatin with polypropylene mesh holds promise for the development of enhanced surgical materials for pelvic organ prolapse in clinical applications.

Gelatinase MMP Expression Is Correlated with Muscle Fiber Growth in Maiwa Yak Gluteus Maximus.

Yak (Bos grunniens) is the only large mammal species in the Qinghai-Tibet Plateau. The most of the studies in yak remain confined for the main contributor of meat, which requires a good understanding of muscle growth. Matrix metalloproteinases-2 (MMP-2) and MMP-9 are widely expressed in mammal tissues they mainly degrade collagen in the extracellular matrix for muscle development. However, the influence of MMPs on yak muscle remains unclear. Hence, we assessed the expression of MMP-2, MMP-9, and related factors with ages in Maiwa yak for study the correlation between MMPs expression and yak muscle growth. The mRNA expression of MMP-2, MMP-9, MMP-14, and collagen III increased with age, except collagen I by quantitative real-time PCR. Moreover, muscle fiber diameter increased with age, whereas the density decreased, which showed that fiber grew thicker with age using hematoxylin-eosin staining. Interestingly, MMP and collagen expression significantly decreased with age using western blotting. Pearson correlation method showed that both mRNA and protein expression of MMP-14 and collagen were strongly correlated with muscle fiber growth, but MMP-2 protein and MMP-9 mRNA expression were moderately correlated with muscle fiber growth. Overall, the expression of MMPs and collagen significantly changed with age, which means that MMPs and their function related genes could correlate with Maiwa yak muscle fiber growth.

Se-Methylselenocysteine Ameliorates DEHP-Induced Ferroptosis in Testicular Sertoli Cells via the Nrf2/GPX4 Axis.

Di (2-ethylhexyl) phthalate (DEHP) is an important plasticizer in industrial production, and its toxic effects on testes are widely recognized. Se-methylselenocysteine (SMC) is a major selenium compound found in selenium-rich plants, which possesses unique biological properties such as antioxidants. However, the effect of SMC on DEHP-induced testicular injury and the specific mechanism remains unknown. In this study, 50 mice were randomly divided into 5 groups and were given corn oil (Control), DEHP, low-dose SMC (L-SMC), moderate-dose SMC (M-SMC), or high-dose SMC (H-SMC). The sperm quality of the mice in each group was determined, and HE staining and transmission electron microscopy (TEM) were applied to observe testicular morphology, and testicular tissues were collected for the subsequent molecular biological analyses. The TM4 cell line was applied in vitro for mechanism validation. Our results showed that DEHP could lead to decreased sperm quality and blood-testis barrier damage in mice, which could be alleviated by SMC. Mitochondrial damage accompanied by accumulation of total iron content, MDA, and 4-HNE, as well as downregulation of antioxidants SOD, GSH, and GSH-Px were observed after DEHP treatment, which exhibited a typical ferroptosis feature. In vitro experiments confirmed that SMC promoted upregulation of GPX4 in TM4 cells and was able to alleviate DEHP metabolite MEHP-induced ferroptosis and promote the expression of cell junction key proteins ZO-1, Occludin, and Connexin 43, which could be inhibited by the GPX4 inhibitor RSL3 or the Nrf2 inhibitor ML385. Overall, the above results suggest that SMC ameliorates the DEHP-induced ferroptosis in testicular Sertoli cells, protects the blood-testis barrier, and prevents sperm aberrations via the Nrf2/GPX4 axis.

Schistosoma japonicum infection-mediated downregulation of lncRNA Malat1 contributes to schistosomiasis hepatic fibrosis by the Malat1/miR-96/Smad7 pathway

BackgroundSchistosoma japonicum infection causes hepatic fibrosis, a primary cause of morbidity and mortality associated with the disease, and effective treatments are still lacking. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenic process of various tissue fibroses. However, the role of lncRNAs in schistosomiasis hepatic fibrosis (HF) is poorly understood. Understanding the role of lncRNAs in schistosomiasis HF will enhance knowledge of disease processes and aid in the discovery of therapeutic targets and diagnostic biomarkers.MethodsDifferentially expressed lncRNA profiles in primary hepatic stellate cells (HSCs) of mice infected with S. japonicum were identified using high-throughput lncRNA sequencing. Primary HSCs were isolated from infected mice using collagenase digestion and density-gradient centrifugation, cultured in DMEM with 10% fetal bovine serum. Dual-luciferase reporter assays, nuclear cytoplasm fractionation and RIP assays were employed to assess the relationship between Malat1 and miRNA-96. Malat1 lentivirus and ASO-Malat1 were constructed for forced expression and downregulated expression of Malat1. The Malat1-KO mouse was constructed by CRISPR/Cas9 technology. Pathological features of the liver were evaluated by hematoxylin-eosin (HE), Masson’s trichrome staining and immunohistochemistry (IHC). The expression levels of fibrosis-related genes were determined by quantitative real-time PCR (qRT-PCR) and Western blot.ResultsA total of 1561 differentially expressed lncRNAs were identified between infected and uninfected primary HSCs. Among the top altered lncRNAs, the downregulated Malat1 was observed in infected HSCs and verified by qPCR. Treatment of infected mice with praziquantel (PZQ) significantly increased the Malat1 expression. Elevated Malat1 expression in infected primary HSC reduced the expressions of profibrogenic genes, whereas Malat1 knockdown had the opposite effect. Moreover, Malat1 was found to interact with miR-96, a profibrotic miRNA, by targeting Smad7. Forced Malat1 expression reduced miR-96 levels in infected primary HSCs, attenuating fibrogenesis and showing negative correlation between Malat1 expression and the expression levels of miR-96 and profibrogenic genes α-SMA and Col1α1. Notably, in Malat1-KO mice, knockout of Malat1 aggravates schistosomiasis HF, while restored Malat1 expression in the infected HSCs reduced the expression of profibrogenic genes.ConclusionsWe demonstrate that lncRNA is involved in regulation of schistosomiasis HF. Elevated lncRNA Malat1 expression in infected HSCs reduces fibrosis via the Malat1/miR-96/Smad7 pathway, thus providing a novel therapeutic target for schistosomiasis HF. Furthermore, Malat1 expression is sensitive to PZQ treatment, thus offering a potential biomarker for assessing the response to chemotherapy.Graphical abstract

NUPR1 modulates pulmonary embolism progression via smooth muscle cells phenotypic transformation

ObjectiveThis study aimed to investigate the role of Nuclear Protein 1 (NUPR1) in pulmonary embolism (PE) and its impact on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs). MethodsA PE model was established via autologous pulmonary emboli infusion into the jugular vein. Partial Pressure of Oxygen (PaO2), Oxygenation Index (OI), Brain Natriuretic Peptide (BNP), and Troponin I (TnI) were measured, and lung tissue was subjected to hematoxylin-eosin (HE) staining. NUPR1 expression was assessed through Immunofluorescence and Western blot analyses. To investigate role of NUPR1, PE rats were treated with lentiviral vectors for NUPR1 knockdown (si-NUPR1) or overexpression (ov-NUPR1), and the effects on lung pathology were examined. NUPR1 expression was evaluated in human PASMCs. Additionally, PASMCs from SD rats were cultured under normoxic and hypoxic conditions to evaluate NUPR1 expression. Transfection of NUPR1 expression vectors into PASMCs allowed monitoring of phenotypic transformation-associated protein changes and PASMCs activity. ResultsIncreased NURP1 was observed in human-derived PASMCs. In PE rats, histological examination revealed ruptured pulmonary alveoli, exudate accumulation, interstitial edema, and infiltration of inflammatory cells, concomitant with elevated NUPR1 expression levels. Knockdown of NUPR1 in PE rats significantly improved lung tissue structure, reducing alveolar rupture and interstitial edema. Conversely, NUPR1 overexpression exacerbated lung damage, leading to increased inflammatory infiltration. NUPR1 expression in rat PASMCs remained stable under normoxic conditions; however, under hypoxic conditions, NUPR1 protein expression increased progressively over time. Subsequent upregulation of NUPR1 expression led to a decrease in the levels of contractile phenotype markers α-SMA and SM22α in PASMCs, accompanied by increased expression of synthetic phenotype markers Vimentin and OPN. This phenotypic shift was associated with enhanced cellular proliferation, invasion, and migration. ConclusionsElevated NUPR1 expression in PE exacerbates abnormal PASMCs proliferation by promoting their phenotypic transformation, thereby fostering the pathological progression of PE.

Integrating network pharmacology and transcriptomics to study the potential mechanism of Jingzhi Niuhuang Jiedu tablet in rats with accumulation of heat in the lungs and stomach

Ethnopharmacological relevanceAccumulation of heat in the lungs and stomach (AHLS) is an important syndrome within the realm of traditional Chinese medicine (TCM). It is the fundamental reason behind numerous illnesses, including mouth ulcers, dermatological conditions, acne, and pharyngitis. Jingzhi Niuhuang Jiedu tablet (JN) serves as the representative prescription for treatment of AHLS clinically. However, the effective components and mechanism of JN's impact on AHLS remain unexplored. Aim of the studyThe objective of this research was to analyze the effective components of JN and investigate the therapeutic effect and potential mechanism of JN on AHLS. Materials and methodsThe effective compounds of JN extract were analyzed and identified using UHPLC-Q-Exactive/HRMS. Utilizing network pharmacology to investigate JN's multi-target, multi-pathway process in treating AHLS. Subsequently, anti-inflammatory activities of JN extract were evaluated in the RAW264.7 cells stimulated by lipopolysaccharide (LPS). In addition, a rat AHLS model induced by LPS and dried ginger was established. Pathological changes in rat lung and stomach tissues observed by HE staining and Masson's trichrome staining. Additionally, the expression of TNF-α, IL-6, and IL-1β in bronchoalveolar lavage fluid (BALF) was identified through the ELISA assay. For a deeper understanding of how JN might affect AHLS, transcriptomics was utilized to examine differential genes and their underlying mechanisms. Concurrently, techniques like quantitative polymerase chain reaction (q-PCR), immunofluorescence, and western blotting (WB) were employed to confirm various mRNA and protein expression, including Il17ra, Il17re, IL-17A, IL-1β, IL-6, PPARγ, PGC1-α and UCP1. ResultsWe identified 178 potential effective components in the JN extract. Network pharmacology analysis showed that the 144 components in JN act on 200 key targets for the treatment of AHLS by suppressing inflammation, regulating energy metabolism, and gastric function. In addition, JN suppressed the LPS-stimulated generation of NO, TNF-α, IL-1β, and IL-6 in RAW264.7 cells. And JN treatment effectively alleviated lung and stomach injury and reduced inflammation in rats. Analysis of RNA-seq from lung tissues revealed JN's substantial control over crucial genes in the IL-17 signaling pathway, including Il1b and Il17ra. Likewise, RNA sequencing of stomach tissues revealed that JN markedly decreased crucial genes in the Thermogenesis pathway, including Ppargc1a and Ppara. Additional experimental findings confirmed that treatment with JN significantly reduced the expression levels of mRNA (Il17ra, Il17re, Il1b, Ppargc1a and Ucp1), and the expression levels of protein (IL-17A, IL-1β, IL-6, PPARγ, PGC1-α and UCP1). ConclusionThis study not only analyzes the effective components of JN but also reveals that JN could effectively ameliorate AHLS by inhibiting IL-17 signaling pathway and Thermogenesis pathway, which provides evidence for subsequent clinical studies and drug development.

Exploring the bioactive ingredients of three traditional Chinese medicine formulas against age-related hearing loss through network pharmacology and experimental validation.

Traditional Chinese medicine (TCM) formulas, including the Er-Long-Zuo-Ci pill, Tong-Qiao-Er-Long pill, and Er-Long pill, have long been utilized in China for managing age-related hearing loss (ARHL). However, the specific bioactive compounds, pharmacological targets, and underlying mechanisms remain elusive. This study aims to find the shared bioactive ingredients among these three formulas, uncover the molecular pathways they regulate, and identify potential therapeutic targets for ARHL. Furthermore, it seeks to validate the efficacy of these major components through both in vivo and in vitro experiments. Common bioactive ingredients were extracted from the TCMSP database, and their putative target proteins were predicted using the Swiss Target Prediction database. ARHL-related target proteins were collected from GeneCards and OMIM databases. Our approach involved constructing drug-target networks and drug-disease-specific protein-protein interaction networks and conducting clustering, topological property analyses, and functional annotation through GO and KEGG enrichment analysis. Molecular docking analysis was utilized to delineate interaction mechanisms between major bioactive ingredients and key target proteins. Finally, in vivo and in vitro experiments involving ABR recording, immunofluorescent staining, HE staining, and quantitative PCR were conducted to validate the treatment effects of flavonoids on the declining auditory function in DBA/2J mice. We identified 11 common chemical compounds across the three formulas and their associated 276 putative targets. Additionally, 3350 ARHL-related targets were compiled. As an intersection of the putative targets of the common compounds and ARHL-related proteins, 145 shared targets were determined. Functional enrichment analysis indicated that these compounds may modulate various biological processes, including cell proliferation, apoptosis, inflammatory response, and synaptic connections. Notably, potential targets such as TNFα, MAPK1, SRC, AKT, EGFR, ESR1, and AR were implicated. Flavonoids emerged as major bioactive components against ARHL based on target numbers, with molecular docking demonstrating diverse interaction models between these flavonoids and protein targets. Furthermore, baicalin could mitigate the age-related cochlear damage and hearing loss of DBA/2J mice through its multi-target and multi-pathway mechanism, involving anti-inflammation, modulation of sex hormone-related pathways, and activation of potassium channels. This study offers an integrated network pharmacology approach, validated by in vivo and in vitro experiments, shedding light on the potential mechanisms, major active components, and therapeutic targets of TCM formulas for treating ARHL.

UPLC-Q-TOF-MS/MS Combined with Network Pharmacology, Molecular Docking, and Animal Verification Reveals the Mechanism of Insomnia Treatment by Shen Qi Wu Wei Zi Capsules.

Shen Qi Wu Wei Zi capsules (SQWWZ) are often used to treat insomnia; however, the potential therapeutic mechanism is still unclear. This study aimed to investigate the mechanism underlying the therapeutic effects of the Shen Qi Wu Wei Zi capsules on insomnia. The components of SQWWZ were identified using the UPLC-Q-TOF-MS/MS technique in conjunction with relevant literature. Insomnia-related targets were searched in the Gene- Cards and DisGeNET databases, and the intersection targets were obtained using a Venn diagram. A component-target-insomnia network diagram was constructed using Cytoscape 3.7.2 software. Core targets underwent GO and KEGG enrichment analyses. Molecular docking techniques were employed to verify the key proteins involved in the pathway and their corresponding compounds. Insomnia was induced in SD rats through the intraperitoneal injection of pchlorophenylalanine (DL-4-chlorophenylalanine, PCPA). The rats were treated orally with SQWWZ, and the serum levels of 5-HT and GABA in each group were determined using ELISA. Histological analysis of hippocampal tissue sections from the rats was performed using HE staining. Using UPLC-Q-TOF-MS/MS and reviewing relevant literature, we identified 49 components of SQWWZ. Additionally, we obtained 1,043 drug targets and 367 insomnia-related targets. Among these, 82 targets were found to be common to both drug and insomnia targets. Following drug administration, rats in the treatment group exhibited a significant increase in the serum levels of 5-HT and GABA. Moreover, histological analysis using HE staining revealed neatly arranged hippocampal neuronal cells in the treated rats. The active components of SQWWZ had good inhibition of insomnia. This study provides a reference and guidance for the in-depth study of SQWWZ for the treatment of insomnia.

Preparation of decellularized bone graft material with supercritical carbon dioxide extraction technique.

To evaluate the immunogenicity and osteogenic ability of animal-derived bone graft material decellularized with supercritical carbon dioxide. Porcine femurs were randomly divided into two groups after preliminary treatment, and decellularized with conventional method (conventional control group) or supercritical carbon dioxide (experimental group). Clearance rate of galactose-α-1, 3-galactose (α-Gal) and residual DNA of the two groups were analyzed to assess the immunogenicity of the xenogenic materials. Clearance rate of α-Gal was determined by enzyme-linked immunosorbent assay and residual DNA was detected by fluorescence method. Nine SPF-grade male athymic nude mice of 6 weeks old were randomly divided into experimental group, conventional control group and positive control group. Samples were implanted over biceps femoris muscle of athymic nude mice, the explants were collected 4 weeks post implantation, hematoxylin and eosin (HE) staining and immunohistochemistry were applied to determine the osteogenic ability and bone tissue-associated protein expressions of the implants. The clearance rates of α-Gal antigen in the experimental group and the conventional control group were (99.09±0.26)% and (30.18±2.02)%, respectively (t=58.67, P<0.01). The residual DNA of the experimental group, the conventional control group and the positive control group were (13.49±0.07) ng/mg, (15.20±0.21) ng/mg and (14.70±0.17) ng/mg, the residual DNA in the experimental group was significantly lower than that in the conventional control group (t=－13.41, P<0.01) and the positive control group (t=－11.30, P<0.01). HE staining showed that multiple bone formation centers with active osteogenesis and rich bone marrow were observed in experimental group 4 weeks after implantation, only a small number of bone formation centers were observed in the conventional control group and the positive control group, with no obvious osteoblasts present. Immunohistochemistry results indicated that the expressions of alkaline phosphatase, Runt-related transcription factor 2, typeⅠcollagen and osteocalcin in the experimental group showed an increasing trend compared with those in the conventional control group and the positive control group. Compared with clinically used demineralized bone matrix and bone graft material decellularized with conventional method, bone graft material decellularized with supercritical carbon dioxide exhibits lower immunogenicity and better osteogenic ability.

Quercetin inhibits ferroptosis through the SIRT1/Nrf2/HO-1 signaling pathway and alleviates asthma disease.

Quercetin (QCT) is a bioflavonoid derived from vegetables and fruits that has anti-inflammatory and anti-ferroptosis effects against various diseases. Previous studies have shown that QCT modulates the production of cellular inflammatory factors in asthma models and delays the development of chronic airway inflammation. However, the regulatory mechanism of QCT, a traditional Chinese medicine, in the treatment of asthma has not been elucidated. The aim of the present study is to investigate whether QCT can inhibit ferroptosis via the SIRT1/Nrf2 pathway and play a therapeutic role in asthma. An ovalbumin-induced mouse asthma model was established, and its function was verified by hematoxylin eosin staining, enzyme linked immunosorbent assay, ferric ion assay, malondialdehyde and superoxide dismutase assays, dihydroethidium staining, immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction. Our results indicated that an ovalbumin-induced asthma mouse model had been successfully established and that QCT inhibited inflammation, reduced serum levels of inflammatory factors IL-4, IL-5 and IL-13, increased superoxide dismutase levels in lung tissue homogenates, and reduced malondialdehyde and ferric ion production in asthmatic mice. In addition, we found that QCT was able to reverse the expression of SIRT1, Nrf2 and HO-1 in an in vivo asthma mouse model. The data from this study indicate that QCT can alleviate asthma, and its mechanism is related to the regulation of ferroptosis, oxidative stress, and the expression of SIRT1 protein.

The Activation of PKM2 Induces Pyroptosis in Hippocampal Neurons via the NLRP3/Caspase-1/GSDMD Pathway in Neonatal Rats With Hypoxic-Ischemic Brain Injury.

The presence of hypoxic-ischemic brain damage (HIBD) in neonates triggers a strong neuroinflammatory reaction. Pyroptosis, a programmed cell death mechanism associated with inflammation, plays a crucial role in HIBD. Pyruvate kinase M2 (PKM2) plays a significant role in connecting metabolic processes and inflammatory responses, but whether it affects hippocampus pyroptosis in HIBD is unclear. The aim of this study is to elucidate the role of PKM2 in HIBD and to propose a novel therapeutic approach for neonatal ischemic-hypoxic encephalopathy. In this study, we employed neonatal 7-day-old Sprague Dawley rats to establish a model of HIBD using the Rice-Vannucci surgical technique and a hypoxia device. To inhibit the elevation of PKM2, we utilized the PKM2 inhibitor shikonin. The rats were categorized into four groups: Sham, Shikonin, HIBD, and Shikonin+HIBD. Behavioral tests, hematoxylin eosin staining, immunofluorescence staining, ELISA (IL-1β, IL-18), and LDH were conducted in each group to evaluate neurological function, hippocampal damage, the occurrence of neuronal pyroptosis, and the neuroinflammation. Western blot was used to assess the expression levels of PKM2, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD, GSDMDN, and IL-1β. The expression of PKM2 elevated in hippocampal tissues of the HIBD model and the localization of PKM2 in the hippocampus was activated in neurons instead of microglia during the HIBD. Meanwhile, the inhibition of PKM2 improved the behavioral test scores and the body weight of rats, the neuronal damage in the CA1 region of hippocampal tissue was also attenuated. In addition, inhibiting PKM2 alleviated neuronal pyroptosis by decreasing the expression of PKM2, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD, GSDMDN. Furthermore, serum levels of LDH and inflammatory factors IL-1β and IL-18 decrease with PKM2 inhibition. Based on these findings, we can conclude that PKM2 plays a crucial role in regulating hippocampal neuronal pyroptosis of HIBD rats via NLRP3/Caspase-1/GSDMD pathway. Therefore, inhibiting PKM2 could be a promising therapeutic strategy for the treatment of neonatal ischemic-hypoxic encephalopathy.

Regulatory effects of the p38 mitogen-activated protein kinase-myosin light chain kinase pathway on the intestinal epithelial mechanical barrier and the mechanism of modified Pulsatilla decoction in the treatment of ulcerative colitis.

To investigate the mechanism of the protective effect of modified Pulsatilla decoction (, MPD) on the mechanical barrier of the ulcerative colitis (UC) intestinal epithelium in vitro and in vivo. We established an intestinal epithelial crypt cell line-6 cell barrier injury model by using lipopolysaccharide (LPS). The model was then treated with p38 mitogen-activated protein kinase-myosin light chain kinase (p38MAPK-MLCK) pathway inhibitors, p38MAPK-MLCK pathway silencing genes (si-p38MAPK, si-NF-κB, and si-MLCK), and MPD respectively. Transepithelial electronic resistance (TEER) measurements and permeability assays were performed to assess barrier function. Immunofluorescence staining of tight junctions (TJ) was performed. In in vivo experiment, dextran sodium sulfate-induced colitis rat model was conducted to evaluate the effect of MPD and mesalazine on UC. The rats were scored using the disease activity index based on their clinical symptoms. Transmission electron microscopy and hematoxylin-eosin staining were used to examine morphological changes in UC rats. Western blotting and real-time quantitative polymerase chain reaction were performed to examine the gene and protein expression of significant differential molecules. In in vitro study, LPS-induced intestinal barrier dysfunction was inhibited by p38MAPK-MLCK pathway inhibitors and p38MAPK-MLCK pathway gene silencing. Silencing of p38MAPK-MLCK pathway genes decreased TJ expression. MPD treatment partly restored the LPS-induced decreased in TEER and increase in permeability. MPD increased the gene and protein expression of TJ, while down-regulated the LPS-induced high expression of p-p38MAPK and p-MLC. In UC model rats, MPD could ameliorate body weight loss and disease activity index, relieve colonic pathology, up-regulate TJ expression as well as decrease the expression of p-p38MAPK and p-MLC in UC rat colonic mucosal tissue. The p38MAPK-MLCK signaling pathway can affect mechanical barrier function and TJ expression in the intestinal epithelium. MPD restores TJ expression and attenuates intestinal epithelial barrier damage by suppressing the p38MAPK-MLCK pathway.

Qisheng wan decoction alleviates the inflammation of CCI rats via TRP channels

Qisheng wan decoction (QWD), a traditional Chinese medicine, has promising potential anti-inflammatory effects against neuropathic pain (NP). However, its valid ingredients and specific anti-inflammatory mechanisms are still unclear. This study aimed to identify the active ingredients of QWD responsible for its anti- inflammatory effect by combining liquid chromatography with network pharmacology, and to explore its anti- inflammatory mechanism by chronic constriction injury (CCI) model rats. The UHPLC-Q Exactive Orbitrap-MS technique was used to identify the active ingredients of QWD. The potential ingredients of QWD, which targeted to the pathways of treating NP, were performed by network pharmacology, molecular docking and molecular dynamics simulations. After CCI rats-induced NP model operation, QWD (5.6 g/kg/d, 11.2 g/kg/d, 22.4 g/kg/d) and Pregabalin (10g/kg/d) as positive controls, were administered to the rats for 7 days. The behaviors of the different groups were tested at 0, 1, 3, 5, 7, 12 days, respectively. And the inflammatory factor including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was detected by ELISA. Meantime, the inflammation of the sciatic nerve was evaluated by the hematoxylin-eosin staining. Ionized calcium-binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. Moreover, the expressions of TRPA1, TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, and P38 mitogen-activated protein kinase (MAPK) were tested by RT-PCR, western blot, and immunohistochemistry. After screening by the liquid chromatography and network pharmacology approach, seventy ingredients of QWD were identified, and seven core targets including oncogene tyrosine-protein kinase (SRC), mitogen-activated protein kinase 3 (MAPK3), signal transducer and activator of transcription 1 (STAT1), protein-serine-threonine kinase 1 (AKT1), mitogen-activated protein kinase 1 (MAPK1), TNF-α, and IL-6 were confirmed. Six active ingredients exhibited binding energies less than -5 kcal/mol, and the complexes were structurally stable within 50 ns. Pathway analysis indicated that transient receptor potential (TRP) channels were mainly responsible for anti-inflammatory mediator regulation. Compared with the CCI group, the behavioral tests showed that QWD-L, QWD-M, and QWD-H group alleviated mechanical, thermal and cold hyperalgesia (p<0.05). HE staining results found out QWD-L, QWD-M, and QWD-H group decreased the inflammation of the sciatic nerve (p<0.05). Similarly, compared with the CCI group, the serum level of TNF-α and IL-6 of QWD groups decreased conformably (p<0.05). This reduction was downtrend with the inhibition of Iba-1, GFAP, and the TRP channel signaling pathway and p38 MAPK. This study provides a primary investigation of the composition of QWD for its anti- inflammation effect and its molecular mechanism in CCI model rats. And this therapeutic efficacy of QWD was achieved by decreasing the inflammation. QWD also inhibited the level TNF-α and IL-6 and decreasing the activation of Iba-1 and GFAP in glia. And this anti-inflammation mechanism involved in inhibiting the TRPA1, TRPV1, TRPV2, TRPV4, and TRPM8 and p38 MAPK signaling pathways. These findings provide a scientific and theoretical basis for the prevention and treatment of NP with QWD.

Integrated Small Intestine Microbiota and Serum Metabolomics Reveal the Potential Mechanisms of Wine Steaming in Alleviating Rhubarb-Induced Diarrhea.

Long-term use of rhubarb (RH) commonly leads to diarrhea, which can be alleviated by steaming with wine. However, the specific mechanism by which wine steaming alleviates RH-induced diarrhea remains unknown. This study aims to reveal the underlying mechanisms of wine steaming in alleviating RH-induced diarrhea by examining small intestinal flora and serum metabolomics. Major anthraquinone and anthrone components were detected using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Eighty-four ICR mice were randomly divided into control, RH, and RH steamed with wine (PRH) groups and were administered RH and PRH (1, 4, and 8 g/kg, i.g). for 14 consecutive days. Histopathological analysis was performed using hematoxylin-eosin staining. Levels of inflammatory factors and tight junction proteins, zonula occludens-1 (ZO-1) and occludin, in the small intestine were measured. The small intestine content was analyzed using 16S rRNA sequencing, and UPLC-MS was used to analyze endogenous metabolites. Levels of major anthraquinone and anthrone components decreased in PRH. Both RH and PRH groups showed varying degrees of loose stools and increased fecal water rates; the RH group exhibited more severe effects. Compared with the control group, RH caused small intestine injuries, increased levels of inflammatory cytokines, downregulated the expression of ZO-1 and occludin, and induced gut microbiota (GM) imbalance. The relative abundance of Lactobacillus decreased, while the relative abundance of Shigella and Streptococcus increased. However, PRH had a milder impact than RH. The glycerophospholipid metabolic pathway was involved in this effect. The levels of inflammatory cytokines and potential metabolites (sn-glycero-3-phosphoethanolamine) were positively correlated with Streptococcus infection, while the levels of ZO-1 and occludin were negatively correlated with Streptococcus infection. GM imbalance and abnormal glycerophospholipid metabolism contributed to impaired intestinal barrier function and inflammatory factor release, which may underlie RH-induced diarrhea, though PRH had a weaker effect. PRH alleviated RH-induced diarrhea by recovering GM balance, reducing ZO-1 and occludin expression, and decreasing the release of inflammatory factors. This mechanism may be linked to the reduced anthraquinone content. This study is the first to explore the mechanism of wine steaming in alleviating RH-induced diarrhea through small intestinal flora and serum metabolomics. It provides data to support the broader clinical use of RH and its safer application.

Total alkaloids in Fritillaria cirrhosa D. Don alleviate OVA-induced allergic asthma by inhibiting M2 macrophage polarization

Fritillaria cirrhosa D. Don (FCD) is a traditional Chinese medicine used to treat respiratory disorders, known for its effects in clearing heat, moistening the lungs, resolving phlegm, and relieving cough. Additionally, the total alkaloids extracted from FCD can alleviate asthma symptoms and reduce airway inflammation. However, no studies have investigated the effects of total alkaloids on lung macrophages. This study explored whether the total alkaloids of FCD (TAs-FCD) reduce M2 macrophage polarization and, consequently, attenuate airway remodeling in asthmatic mice. This study further elucidated its mechanism of action in treating allergic asthma. The extracted TAs-FCD was analyzed for its composition using UPLC-Q-TOF/MS. Network pharmacology was employed to identify the active ingredients and potential mechanisms of TAs-FCD in the treatment of allergic asthma. A mouse model of ovalbumin-induced allergic asthma was established, adopted, and validated through in vivo experiments. Hematoxylin-eosin staining (H&E), immunohistochemistry (IHC), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), and real-time fluorescence quantitative polymerase chain reaction (q-PCR) were used to investigate the role of TAs-FCD in inhibiting M2 macrophage polarization in the context of allergic asthma. A total of 66 active ingredients were screened from 116 compounds using SWISSADME. The targets of these 66 compounds were predicted by SwissTargetPrediction, resulting in 808 unique drug targets after excluding duplicates. Additionally, 1,756 targets related to allergic asthma were identified from the DisGeNET, Genecard, and OMIM databases. This led to 267 cross-targets between the active ingredient targets and allergic asthma targets, including interleukin (IL)-1β, tumor necrosis factor (TNF), and STAT3. Animal experiments demonstrated that TAs-FCD improved histopathological injury in mouse lungs, reduced peri-airway collagen fiber accumulation, airway mucus secretion, and airway smooth muscle proliferation. TAs-FCD also lowered IL-1β, TNF-α and IL-4 levels in lung tissues and alleviated airway inflammation. Furthermore, TAs-FCD significantly reduced levels of Arg1 and CD206, which are closely associated with M2 macrophages, and downregulated the expression of p-STAT3 and p-JAK2. TAs-FCD may inhibit M2 macrophage polarization by regulating the JAK2/STAT3 pathway, thereby alleviating airway remodeling and inflammation in allergic asthmatic mice.

The Regulatory Effect of Remifentanil on JNK Signaling during Remission of Flap Ischemia-Reperfusion Injury.

The impact of remifentanil on hypogastric flap function following ischemia-reperfusion (I/R) injury remains largely unknown, limiting its potential clinical application in flap surgery. This study investigated the therapeutic effects of remifentanil on hypogastric flap I/R injury. Aortic endothelial cells were extracted from the hypogastric flap I/R injury models established in-house using Sprague-Dawley rats, and were treated under hypoxic conditions. The cells were treated with 0.1, 1, 10 and 100 ng/mL remifentanil and 10 ng/mL anisomycin (the activator of c-Jun N-terminal kinase [JNK]). Histopathological changes and tumor necrosis factor alpha (TNF-α) content of the flaps were observed after hematoxylin-eosin staining and immunohistochemistry. Immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry were employed for apoptosis evaluation. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were utilized to assess the protein and gene expression levels of TNF receptor 1 (TNFR1), JNK1, phosphorylated (p)-JNK1, malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO) and TNF-α in the flaps and cells. The endothelial necrosis and cell apoptosis of rat flaps induced by I/R injury were ameliorated by remifentanil, and declining aortic endothelial cell viability and augmented apoptosis induced by hypoxia were reversed by remifentanil (10, 100 ng/mL) (p < 0.05). Remifentanil reversed the increased expressions of TNFR1, JNK1, p-JNK1, MDA and TNF-α induced by I/R injury or hypoxia in the flaps and cells (p < 0.05), and counteracted the decreased levels of NO and SOD induced by I/R injury in the flaps (p < 0.05). Anisomycin reversed the effects of remifentanil on suppressing TNFR1, JNK1 and p-JNK1 levels and apoptosis in the cells (p < 0.05). Remifentanil ameliorates cell apoptosis and vascular endothelial necrosis induced by I/R injury in the hypogastric flap, likely by downregulating the TNF-α/TNFR1 pathway and JNK1 signaling. These findings suggest that remifentanil may be a promising therapeutic agent for improving hypogastric flap survival in clinical settings.

Carvacrol acts on the larval midgut of Spodoptera frugiperda by destroying the tissue structure and altering the bacterial community

The fall armyworm, Spodoptera frugiperda, has become a global invasive pest in recent years, threatening agricultural production. Carvacrol (CAR) is a multifunctional plant-derived compound and exhibited significant bioactivities against many pests. Our previous study found that CAR inhibited growth and development in S. frugiperda. However, the effects of CAR on the midgut of this pest remain unknown. In this study, the actions of CAR on the larval midgut of S. frugiperda were investigated. The results found that 1.0 and 2.0 g/kg CAR treatments destroyed the structure of the larval midgut based on hematoxylin-eosin staining and transmission electron microscope observation. In addition, 2.0 g/kg CAR exposure significantly altered the larval midgut bacterial community composition and structure. The results of the linear discriminant analysis effect size (LEfSe) analysis suggest that six bacterial genera contributed to the different structures of the larval bacterial community. Furthermore, PICRUSt2 analysis found that 16 bacterial function categories were altered by CAR treatment, of which 8 were significantly increased. Our results provided new insight into the toxicological mechanisms of CAR against S. frugiperda larvae and laid the foundation for the field application of S. frugiperda control.

CCL2 may contribute to the damage of fallopian tube epithelium in women with tubal endometriosis

Research QuestionHow does tubal endometriosis (TEM) impact the morphology and ultrastructure of the fallopian tube epithelium? What are the underlying pathophysiological mechanisms of TEM? DesignEpithelial samples of the fallopian tubes from patients with (TEM group) and without (non-TEM group) TEM were collected. The morphological characteristics, ultrastructure, ciliated cell percentage, and ciliary beat frequency (CBF) were assessed via hematoxylin-eosin staining, electron microscopy, and high-speed video microscopy. mRNA microarray analysis was conducted to identify differentially expressed genes (DEGs) in the fallopian tube epithelium, which were further validated using qPCR, immunohistochemistry, and Western blotting. The effects of recombinant CCL2 protein on primary human fallopian tube epithelial cells (FTEC) were examined in vitro. ResultsPatients with TEM exhibited significant abnormalities in the morphology and ultrastructure of the fallopian tube epithelium with reduced percentage of ciliated cells and CBF, compared to the non-TEM patients. Among the 765 DEGs identified in the fallopian tube epithelium, 512 genes were upregulated and 253 genes were downregulated in the TEM group. qPCR, immunohistochemistry, and Western blotting validation confirmed a significant upregulation of CCL2 expression in the TEM group. In vitro experiments revealed that recombinant CCL2 protein significantly reduced ciliated cell differentiation, length of cilia, and CBF in FTEC, suggesting a role for CCL2 in the development of the TEM-related pathological phenotype. ConclusionsThese findings indicate that CCL2 may contribute to the pathological phenotype associated with TEM and could be a crucial signaling molecule in the damage of fallopian tube epithelium in patients with TEM.

Regulatory role of electroacupuncture on satellite glial cell activity in the colon and dorsal root ganglion of rats with irritable bowel syndrome.

To investigate the role of satellite glial cells in irritable bowel syndrome (IBS) and the effect of electroacupuncture (EA) at the Tianshu (ST25) and Shangjuxu (ST37) combination. A model for visceral hypersensitivity in IBS was induced through colorectal distension (CRD) stimulation. Clean-grade male Sprague-Dawley (SD) rats were randomly divided into four groups: a normal group (NG), a model group (MG), an electroacupuncture group (EA), and a glial cell inhibitor group (FCA). Bilateral EA (2/100 Hz, 1 mA, 30 min) was administered at the Tianshu (ST25) and Shangjuxu (ST37) in week 6. Abdominal withdrawal reflex (AWR) scores were used to assess the behavioral response associated with visceral hyperalgesia, while hematoxylin-eosin staining was employed to evaluate pathological changes in the colon. The protein and mRNA levels of glial fibrillary acidic protein (GFAP) in the colon and colon-related dorsal root ganglion (DRG) were analyzed using immun-ofluorescence, immun-ohistochemistry, Western blotting, real-time polymerase chain reaction. The impact of EA on electrophysiological properties of colon-related DRG neurons was observed through whole-cell patch clamp analysis. EA significantly reduced the visceral pain behavior scores in rats with IBS in response to graded (20, 40, 60, 80 mm Hg) CRD stimulation. Additionally, EA downregulated the protein and mRNA expression levels of GFAP in the colon and colon-related DRG of rats with IBS. EA also regulated the resting membrane potential, rheobase and action potential of colon-related DRG neurons in rats with IBS. EA can regulate the excitatory properties of colon-related DRG neurons by downregulating the protein and mRNA expression of GFAP in the colon and colon-related DRG, indicating a potential neurobiological mechanism by which EA relieves visceral hypersensitivity in rats with IBS.