This study aims to explore the effects and action mechanisms of the active ingredients in Buyang Huanwu Decoction(BYHWD), namely tetramethylpyrazine(TMP) and hydroxy-safflor yellow A(HSYA), on oxygen-glucose deprivation/reglucose-reoxygenation(OGD/R)-induced inflammation and oxidative stress of microglia(MG). Network pharmacology was used to screen the effective monomer ingredients of BYHWD and determine the safe concentration range for each component. Inflammation and oxidative stress models were established to further screen the best ingredient combination and optimal concentration ratio with the most effective anti-inflammatory and antioxidant effects. OGD/R BV2 cell models were constructed, and BV2 cells in the logarithmic growth phase were divided into a normal group, a model group, an HSYA group, a TMP group, and an HSYA + TMP group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory cytokines such as interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6). Oxidative stress markers, including superoxide dismutase(SOD), nitric oxide(NO), and malondialdehyde(MDA), were also measured. Western blot was used to analyze the protein expression of both inflammation-related pathway [Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)] and oxidative stress-related pathway [nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)]. Immunofluorescence was used to assess the expression of proteins such as inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). The most effective ingredients for anti-inflammatory and antioxidant effects in BYHWD were TMP and HSYA. Compared to the normal group, the model group showed significantly increased levels of IL-1β, TNF-α, IL-6, NO, and MDA, along with significantly higher protein expression of NF-κB, TLR4, Nrf2, and HO-1 and significantly lower SOD levels. The differences between the two groups were statistically significant. Compared to the model group, both the HSYA group and the TMP group showed significantly reduced levels of IL-1β, TNF-α, IL-6, NO, and MDA, lower expression of NF-κB and TLR4 proteins, higher levels of SOD, and significantly increased protein expression of Nrf2 and HO-1. Additionally, the expression of the M1-type MG marker iNOS was significantly reduced, while the expression of the M2-type MG marker Arg-1 was significantly increased. The results of the HSYA group and the TMP group had statistically significant differences from those of the model group. Compared to the HSYA group and the TMP group, the HSYA + TMP group showed further significant reductions in IL-1β, TNF-α, IL-6, NO, and MDA levels, along with significant reductions in NF-κB and TLR4 protein expression, an increase in SOD levels, and elevated Nrf2 and HO-1 protein expression. Additionally, the expression of the M1-type MG marker iNOS was reduced, while the M2-type MG marker Arg-1 expression increased significantly in the HSYA + TMP group compared to the TMP or HSYA group. The differences in the results were statistically significant between the HSYA + TMP group and the TMP or HSYA group. The findings indicated that the combined use of HSYA and TMP, the active ingredients of BYHWD, can effectively inhibit OGD/R-induced inflammation and oxidative stress of MG, showing superior effects compared to the individual use of either component.
Oxidative Stress/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Glucose/metabolism* ; Cell Line ; Inflammation/genetics* ; Oxygen/metabolism* ; Pyrazines/pharmacology* ; Microglia/metabolism* ; NF-E2-Related Factor 2/immunology* ; NF-kappa B/immunology* ; Toll-Like Receptor 4/immunology* ; Anti-Inflammatory Agents/pharmacology* ; Humans

BACKGROUND: The sirtuin family is well recognized for its crucial involvement in various cellular processes. Nevertheless, studies on its role in the human endometrium are limited. This study aimed to explore the expression and localization of the sirtuin family in the human endometrium, focusing on sirtuin 3 (SIRT3) and its potential role in the oxidative imbalance of the endometrium in polycystic ovary syndrome (PCOS). METHODS: Endometrial specimens were collected from both patients with PCOS and controls undergoing hysteroscopy at the Center for Reproductive Medicine, Peking University Third Hospital, from July to August 2015 and used for cell culture. The protective effects of SIRT3 were investigated, and the mechanism of SIRT3 in improving endometrial receptivity of patients with PCOS was determined using various techniques, including cellular bioenergetic analysis, small interfering ribonucleic acid (siRNA) silencing, real-time quantitative polymerase chain reaction, Western blot, immunofluorescence, immunohistochemistry, and flow cytometry analysis. RESULTS: The sirtuin family was widely expressed in the human endometrium, with SIRT3 showing a significant increase in expression in patients with PCOS compared with controls ( P <0.05), as confirmed by protein and gene assays. Concurrently, endometrial antioxidant levels were elevated, while mitochondrial respiratory capacity was reduced, in patients with PCOS ( P <0.05). An endometrial oxidative stress (OS) model revealed that the downregulation of SIRT3 impaired the growth and proliferation status of endometrial cells and reduced their receptivity to day 4 mouse embryos. The results suggested that SIRT3 might be crucial in maintaining normal cellular state by regulating antioxidants, cell proliferation, and apoptosis, thereby contributing to enhanced endometrial receptivity. CONCLUSIONS Our findings proposed a significant role of SIRT3 in improving endometrial receptivity in patients with PCOS by alleviating OS and regulating the balance between cell proliferation and apoptosis. Therefore, SIRT3 could be a promising target for predicting and improving endometrial receptivity in this patient population.
Humans ; Female ; Polycystic Ovary Syndrome/metabolism* ; Endometrium/metabolism* ; Sirtuin 3/genetics* ; Oxidative Stress/genetics* ; Adult ; Animals ; Mice ; Apoptosis/physiology* ; Immunohistochemistry ; Cell Proliferation/physiology*

Buyang Huanwu Decoction(BYHWD), as one of the classic formulas in traditional Chinese medicine(TCM) for the treatment of cerebral ischemic stroke(CIS), has demonstrated definite effects in clinical practice. However, the material basis and mechanism of treatment have not been systematically elucidated. This study employed network pharmacology and molecular docking to analyze the potential targets and mechanisms of blood-and brain-penetrating active components of BYHWD in reducing cell apoptosis in CIS. Cell experiments were then carried out to validate the prediction results. In the experiments, five active components including hydroxysafflor yellow A( HSYA), tetramethylpyrazine( TMP), astragaloside Ⅳ( AS-Ⅳ), amygdalin( AMY), and paeoniflorin(PF) were selected to explore the pharmacological effects of BYHWD. HT22 cells were treated with BYHWD, and the cell counting kit-8(CCK-8) method was employed to examine the toxic and side effects of BYHWD. A cell model of oxygen-glucose deprivation/reoxygenation( OGD/R) was constructed, with apoptosis and pyroptosis as the main screening indicators. The levels of lactate dehydrogenase(LDH) and glutathione(GSH) were measured to assess the cell membrane integrity. Flow cytometry was employed to detect apoptosis, and the activities of caspase-3 and caspase-1 were measured to clarify the status of apoptosis and pyroptosis. ELISA was employed to determine the levels of interleukin(IL)-1β and IL-18 to confirm pyroptosis. HSYA and AMY were identified in this study as the active components regulating apoptosis and pyroptosis. TUNEL was employed to detect the apoptosis rate, and Western blot was employed to determine the expression levels of apoptosis-related proteins B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and caspase-3, which confirmed that the anti-apoptotic effect of the combined component group was superior to that of the single component groups. The molecular docking results revealed strong binding affinity of HSYA and AMY with SDF-1α and CXCR4.AMD3100, a selective antagonist of CXCR4, was then used for intervention. The results of Western blot showed alterations in the expression levels of apoptosis-associated proteins, SDF-1α, and CXCR4. In conclusion, HSYA and AMY influence cellular apoptosis by modulating the SDF-1α/CXCR4 signaling cascade.
Drugs, Chinese Herbal/chemistry* ; Apoptosis/drug effects* ; Animals ; Neurons/cytology* ; Mice ; Molecular Docking Simulation ; Cell Line ; Glucose/metabolism* ; Humans ; Neuroprotective Agents/pharmacology*

Spermatogenesis is a fundamental process that requires a tightly controlled epigenetic event in spermatogonial stem cells (SSCs). The mechanisms underlying the transition from SSCs to sperm are largely unknown. Most studies utilize gene knockout mice to explain the mechanisms. However, the production of genetically engineered mice is costly and time-consuming. In this study, we presented a convenient research strategy using an RNA interference (RNAi) and testicular transplantation approach. Histone H3 lysine 9 (H3K9) methylation was dynamically regulated during spermatogenesis. As Jumonji domain-containing protein 1A (JMJD1A) and Jumonji domain-containing protein 2C (JMJD2C) demethylases catalyze histone H3 lysine 9 dimethylation (H3K9me2), we firstly analyzed the expression profile of the two demethylases and then investigated their function. Using the convenient research strategy, we showed that normal spermatogenesis is disrupted due to the downregulated expression of both demethylases. These results suggest that this strategy might be a simple and alternative approach for analyzing spermatogenesis relative to the gene knockout mice strategy.
Spermatogenesis/physiology* ; Animals ; Male ; Mice ; Epigenesis, Genetic ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Histones/metabolism* ; RNA Interference ; Testis/metabolism* ; Methylation ; Mice, Knockout ; Histone Demethylases

Extensive studies have identified potential adverse effects on semen quality of obesity, based on body mass index, but the association between body fat distribution, a more relevant indicator for obesity, and semen quality remains less clear. We conducted a longitudinal study of 4304 sperm donors from the Guangdong Provincial Human Sperm Bank (Guangzhou, China) during 2017-2021. A body composition analyzer was used to measure total and local body fat percentage for each participant. Generalized estimating equations were employed to assess the association between body fat percentage and sperm count, motility, and morphology. We estimated that each 10% increase in total body fat percentage (estimated change [95% confidence interval, 95% CI]) was significantly associated with a 0.18 × 10 6 (0.09 × 10 6 -0.27 × 10 6 ) ml and 12.21 × 10 6 (4.52 × 10 6 -19.91 × 10 6 ) reduction in semen volume and total sperm count, respectively. Categorical analyses and exposure-response curves showed that the association of body fat distribution with semen volume and total sperm count was stronger at higher body fat percentages. In addition, the association still held among normal weight and overweight participants. We observed similar associations for upper limb, trunk, and lower limb body fact distributions. In conclusion, we found that a higher body fat distribution was significantly associated with lower semen quality (especially semen volume) even in men with a normal weight. These findings provide useful clues in exploring body fat as a risk factor for semen quality decline and add to evidence for improving semen quality for those who are expected to conceive.
Humans ; Male ; Adult ; Semen Analysis ; China ; Body Fat Distribution ; Longitudinal Studies ; Sperm Count ; Sperm Motility ; Body Mass Index ; Tissue Donors ; Obesity/complications* ; Spermatozoa ; Young Adult ; Middle Aged ; East Asian People

OBJECTIVE: To investigate the genetic causal relationship of leukocyte telomere length (LTL) with prostatitis, orchitis and epididymitis by two-sample Mendelian randomization (MR). METHODS: Using LTL as the exposure factor and prostatitis, orchitis and epididymitis as outcome factors, we mined the Database of Genome-Wide Association Studies (GWAS). Then, we analyzed the causal relationship of LTL with prostatitis, orchitis and epididymitis by Mendelian randomization using inverse variance weighting (IVW) as the main method and weighted median and MR-Egger regression as auxiliary methods, determined the horizontal multiplicity by MR-Egger intercept test, and conducted sensitivity analysis using the leaving-one-out method. RESULTS: A total of 121 related single nucleotide polymorphisms (SNPs) were identified in this study. IVW showed LTL to be a risk factor for prostatitis (OR = 1.383, 95% CI: 1.044－1.832, P = 0.024), and for orchitis and epididymitis as well (OR = 1.770, 95% CI: 1.275－2.456, P = 0.000 6). CONCLUSION Genetic evidence from Mendelian randomized analysis indicates that shortening of LTL reduces the risk of prostatitis, orchitis and epididymitis.
Humans ; Male ; Mendelian Randomization Analysis ; Epididymitis/genetics* ; Prostatitis/genetics* ; Polymorphism, Single Nucleotide ; Leukocytes ; Orchitis/genetics* ; Genome-Wide Association Study ; Telomere ; Risk Factors

OBJECTIVE: It has been reported that the mRNA expression of serine protease 23 (PRSS23) was increased in skin fibroblasts from systemic sclerosis patients (SSc). The purpose of this study is to explore the pathogenetic effect and mechanism of PRSS23 in skin fibrosis of SSc. METHODS: The expression of PRSS23 in skin tissues from the SSc patients and healthy controls was detected by immunohisto-chemistry. Fibroblasts isolated from fresh skin tissue were used to detect the expression of PRSS23 by real-time quantitative PCR (RT-qPCR) and Western blot. Overexprssion of PRSS23 in BJ, the fibroblasts cell line of skin, was constructed by lentivirus. After stimulation with 400 μmol/L hydrogen peroxide for 12 h, Annexin V/7-AAD staining was used to detect apoptosis of fibroblasts; flow cytometry and Western blot were used to detect the expression of apoptosis-related protein cleaved Caspase-3. The expression of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) in fibroblasts was detected by RT-qPCR and enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with the healthy controls, the expression of PRSS23 in skin tissues of the SSc patients was significantly increased [4.952 (3.806-5.439) vs. 0.806 (0.395-1.173), P < 0.001], and fibroblast was the main cell that expressed PRSS23. The mRNA [27.59 (25.02-30.00) vs. 1.00, P < 0.001] and protein [0.675 (0.587-0.837) vs. 0.451 (0.342-0.502), P=0.029] of PRSS23 in skin fibroblasts isolated from the SSc patients were significantly up-regulated. Compared with the control group, the anti-apoptotic ability of skin fibroblasts overexpressing PRSS23 was enhanced, and the proportion of apoptotic cells was significantly reduced after hydrogen peroxide induction [(5.043±1.097)% vs. (17.480±3.212)%, P=0.022], the expression of apoptosis-related protein cleaved Caspase-3 was also markedly reduced [(0.718±0.022) vs. (1.422±0.105), P=0.003]. In addition, the mRNA [(99.780±1.796) vs. (1.000±0.004), P < 0.001] and protein [(211.600±2.431) ng/L vs. (65.930±1.768) ng/L, P < 0.001] of IL-6 in the fibroblasts overexpressing PRSS23 were significantly up-regulated; the mRNA[(3.555±0.555) vs. (1.000±0.004), P < 0.001] and protein levels [(41.190±0.949) ng/L vs. (31.150±0.360) ng/L, P < 0.001] of TNF-α in the fibroblasts overexpressing PRSS23 were also significantly up-regulated. CONCLUSION The expression of PRSS23 is increased in skin fibroblasts of SSc patients. PRSS23 can inhibit cell apoptosis, promote the secretion of inflammatory factors such as IL-6 and TNF-α, and regulate the process that skin fibroblasts transform into pro-inflammatory type. So, PRSS23 is associated with the development of skin fibrosis.
Humans ; Scleroderma, Systemic/enzymology* ; Fibroblasts/pathology* ; Apoptosis ; Skin/metabolism* ; Fibrosis ; Interleukin-6/metabolism* ; Caspase 3/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Male ; Female ; Cells, Cultured ; RNA, Messenger/metabolism* ; Middle Aged ; Adult ; Serine Endopeptidases/genetics*

Accurate timing of myelination is crucial for the proper functioning of the central nervous system. Here, we identified a de novo heterozygous mutation in TMEM63A (c.1894G>A; p. Ala632Thr) in a 7-year-old boy exhibiting hypomyelination. A Ca²⁺ influx assay suggested that this is a loss-of-function mutation. To explore how TMEM63A deficiency causes hypomyelination, we generated Tmem63a knockout mice. Genetic deletion of TMEM63A resulted in hypomyelination at postnatal day 14 (P14) arising from impaired differentiation of oligodendrocyte precursor cells (OPCs). Notably, the myelin dysplasia was transient, returning to normal levels by P28. Primary cultures of Tmem63a^-/- OPCs presented delayed differentiation. Lentivirus-based expression of TMEM63A but not TMEM63A_A632T rescued the differentiation of Tmem63a^-/- OPCs in vitro and myelination in Tmem63a^-/- mice. These data thus support the conclusion that the mutation in TMEM63A is the pathogenesis of the hypomyelination in the patient. Our study further demonstrated that TMEM63A-mediated Ca²⁺ influx plays critical roles in the early development of myelin and oligodendrocyte differentiation.
Animals ; Cell Differentiation/physiology* ; Oligodendroglia/metabolism* ; Mice, Knockout ; Mice ; Male ; Myelin Sheath/metabolism* ; Humans ; Child ; Cells, Cultured ; Oligodendrocyte Precursor Cells/metabolism*

Nano/microplastics(NMPs),due to their environmental persistence and resistance to degradation,have emerged as a major contributor to global pollution.NMPs are capable of adsorbing various hazardous chemicals and heavy metals,thereby posing threats to aquatic ecosystem health,which may ultimately cause potential risks to human health.Conventional analytical methods suffered from limited resolution,insufficient chemical information,or destruction of sample,invalidating these assays for on-site detection of NMPs.Surface-enhanced Raman scattering(SERS)offers distinct advantages such as high sensitivity,superior specificity,rich fingerprint information,and non-destructive analysis,thus facilitating the on-site analysis of NMPs in complex matrices.This review summarized recent advances in SERS substrates for detection of NMPs,discussed the construction and applications of SERS-based multimodal detection strategies,and introduced the research progress of SERS detection of NMPs in food safety,environmental pollution,and bioanalysis.Moreover,the main challenges and future directions of SERS-based NMP detection were outlined.

Non-cystic fibrosis bronchiectasis(referred to as bronchiectasis)is a common chronic airway disease.Neutrophils have traditionally been considered to play a dominant role in the airway inflammation of bronchiectasis.However,it has been discovered that approximately 20％to 30％of bronchiectasis patients exhibited elevated eosinophil counts(Sputum eosinophil ratio＞3％or blood eosinophil count＞300/μl)in their sputum and blood samples,referred to as eosinophilic bronchiectasis(EB).Based on this novel discovery of the inflammatory phenotype,some scholars have advocated treating bronchiectasis as an"inflammatory disease"rather than an"infectious disease"in a novel therapeutic approach.This article reviews the discovery and the latest research advancements of EB,exploring its pathophysiological basis,clinical characteristics,and treatment,with the aim to facilitate the understanding and management of EB.