Zhen Yang

Bovine mastitis is a persistent and challenging illness in dairy industry, bringing about devastating economic losses to the sector. The longstanding over-reliance on antibiotic therapy has raised severe public health concerns, highlighting the critical need to develop safe and effective alternative regimens. Pulsatilla saponin B4 (PSB4), a plant-derived triterpenoid saponin, has been shown to regulate inflammation. However, the effect of PSB4 on mastitis in lactating cows has not been elucidated. The aim of this study was to explore the anti-inflammatory property and the molecular mechanisms of PSB4 in bovine mammary epithelial cells (BMECs) and mouse mastitis model. In cultured cells, PSB4 alleviated LPS-induced inflammatory reaction by suppressing the expression of pro-inflammatory mediators in BMECs. Notably, RNA-seq analysis indicated that the anti-inflammatory effect of PSB4 was correlated with IL-17RA and NF-κB signaling, with subsequent Western blot validation. Moreover, BMECs were stimulated with recombinant interleukin-17A (rIL-17A) to induce inflammation and challenged with Taltz to specifically inhibit IL-17RA signaling. The results showed that PSB4 reversed the rIL-17A-induced upregulation of elements within the IL-17RA pathway and its downstream MAPK/NF-κB cascade, including their downstream effectors. Furthermore, Taltz blocked the efficacy of PSB4 in protecting against LPS-induced inflammation. In vivo, PSB4 alleviated the inflammatory damage of mammary gland, pro-inflammatory mediator levels in mammary gland tissue and se-rum, while blocking the activation of IL-17RA signal along with downstream MAPK/NF-κB signal in LPS-induced mouse mastitis model. Taken together, studies in both BMECs and mice demonstrated that PSB4 alleviates mastitis by inhibiting IL-17RA signaling and downstream MAPK/NF-κB pathway, which may be a new strategy and a target for the management of mastitis.

Background and Aim: Calf diarrhea represents a major threat to yak (Bos grunniens) husbandry on the Qinghai-Tibet Plateau, where extreme environmental conditions (high altitude, low oxygen, cold temperatures) and irregular antibiotic use may accelerate the emergence of multidrug-resistant (MDR) bacterial pathogens. This study aimed to isolate and identify the predominant bacterial agents responsible for diarrhea in yak calves, determine their antimicrobial resistance profiles, and investigate the genomic features and pathogenicity of the most resistant strain to provide evidence-based guidance for prevention and control. Materials and Methods: Rectal swabs were collected from 12 naturally diarrheic yak calves across four geographically distinct farms in Huangyuan County, Qinghai Province, during the peak season (June–July). Bacterial isolates were obtained through enrichment in Luria-Bertani broth followed by plating on Luria-Bertani agar, and identified by Gram staining, 16S rRNA gene amplification (primers 27F/1492R), and Sanger sequencing with BLAST comparison (>99.5% identity). Antimicrobial susceptibility was assessed using the Kirby-Bauer disk diffusion method with 17 antibiotics representing eight classes, interpreted according to Clinical and Laboratory Standards Institute VET08-Ed4 breakpoints. The most MDR isolate (HYCQ01) underwent whole-genome sequencing (WGS) on the Oxford Nanopore Technologies MinION platform. Genome assembly quality was evaluated with BUSCO v5.4.7; virulence factors and antibiotic resistance genes were annotated against the Virulence Factor Database and Comprehensive Antibiotic Resistance Database, respectively (BLASTP, e-value ≤ 1e-5, identity ≥ 40%, length ≥ 50 bp). Pathogenicity was tested in 20 male C57BL/6 mice (7–8 weeks, 20 ± 2 g) via intraperitoneal injection of 1.0 × 10⁸ colony-forming units/mL bacterial suspension (100 μl/kg); survival was monitored, and organ histopathology (heart, jejunum, kidneys, liver, lungs, spleen) was examined after hematoxylin-eosin staining. All animal procedures were approved by the Experimental Animal Ethics Committee (No. 2024-030). Data were analyzed using GraphPad Prism 10.1.2 with one-way analysis of variance. Results: Eight Escherichia coli strains were isolated from the 12 samples and confirmed by 16S rRNA sequencing. All isolates displayed MDR phenotypes, showing 100% resistance to penicillin G and clindamycin, 87.5% to sulfafurazole, and 75.0% to erythromycin. WGS of HYCQ01 revealed 32 resistance classes and 152 resistance genes, consistent with its phenotype (including β-lactamases, macrolide-lincosamide resistance determinants, and tetracycline efflux pumps). Virulence genes included Type III secretion system components, alginate biofilm regulators, iron acquisition systems (pvdE), and hemolysins (rck). Phylogenetically, HYCQ01 clustered near enterotoxigenic E. coli O139:H28 but exhibited a hybrid profile combining animal-associated colonization factors (F17, CFA/I) with atypical extraintestinal traits. In the mouse model, HYCQ01 induced 100% mortality within 27 h post-challenge (p < 0.0001) and caused severe histopathological damage in spleen and jejunum, indicating strong systemic invasiveness. Conclusion: MDR E. coli, exemplified by the hybrid strain HYCQ01, predominates as a causative agent of yak calf diarrhea on the Qinghai-Tibet Plateau, shaped by local ecological pressures and horizontal gene transfer. These results highlight the urgent need for region-specific antimicrobial resistance surveillance, rational antibiotic stewardship, and exploration of non-antibiotic alternatives (probiotics, plant-derived antimicrobials) within a One Health framework. WGS data are deposited at NCBI under BioProject PRJNA1289237. Keywords: antimicrobial resistance, diarrhea, Escherichia coli, multidrug-resistant, pathogenicity, Qinghai-Tibet Plateau, whole-genome sequencing, yak calves.

Chronic inflammatory bowel disease, ulcerative colitis (UC), currently lacks specific drugs for clinical treatment, and screening effective therapeutic agents from natural plants represents a critical research strategy. This study aimed to investigate the therapeutic potential of the flavonoid extract of Polygonum viviparum L. (TFPV) against UC. Liquid chromatography-mass spectrometry (LC-MS) was used to identify the chemical components of TFPV, while cell and animal models were employed to evaluate its anti-inflammatory effects on lipopolysaccharide (LPS)-induced inflammation. The mechanism of anti-inflammatory action was further investigated using a mouse model of UC induced by dextran sulfate sodium (DSS). The results revealed the identification of 32 bioactive components in TFPV, with major compounds such as kaempferol, luteolin, galangin, and quercetin. TFPV effectively mitigated inflammatory damage induced by LPS in IPEC-J2 cells and C57BL/6 mice. In the UC modeled by DSS, TFPV attenuated intestinal inflammation by reducing pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; increasing the anti-inflammatory cytokine IL-10; up-regulating tight junction protein expression such as Claudin-1, Occludin, and ZO-1; and inhibiting the expression of PI3K, AKT, NF-κB, and IL-17 proteins. Analysis of mice fecal samples through 16S rRNA gene sequencing demonstrated that TFPV adjusted the equilibrium of gut microbiota by boosting the abundance of Dubosiella and diminishing that of Enterococcus, Romboutsia, and Enterobacter. Untargeted metabolomics analysis further revealed that TFPV reduced inosine and ADP levels while increasing dGMP levels by the regulation of purine metabolism, ultimately resulting in decreased uric acid levels and thereby alleviating intestinal inflammation. Additionally, TFPV safeguarded the intestinal mucosal barrier by enhancing the expression of tight junctions. In conclusion, TFPV alleviates UC by blocking the PI3K/AKT/NF-κB and IL-17 signaling pathways, lessening intestinal inflammation and injury, safeguarding intestinal barrier integrity, balancing gut microbiota, and lowering uric acid levels, suggesting its promise as a therapeutic agent for UC.

Oxidative stress refers to the phenomenon in which the redox balance of the body is disrupted in response to stimuli, leading to an excessive accumulation of reactive oxygen species in vivo, which can lead to a variety of diseases. In contrast to artificial antioxidants, whose safety is controversial, natural antioxidants, which are widely available, pharmacologically active, and have little toxic side effects, are expected to be candidates for the treatment of oxidative stress-related diseases. Polygonum viviparum L. (PV) is a natural herbal medicine with antioxidant properties and is used as a traditional medicine in the Tibetan Plateau region. However, there are few studies that have focused on its antioxidant activity and mechanism of action in vitro and in vivo. Therefore, the present study firstly demonstrated that PV could exert good in vitro antioxidant effects by scavenging DPPH radicals and inhibiting the production of hydroxyl radicals through in vitro experiments. Secondly, PV was proven to attenuate the effects of oxidative stress on body weight gain and thymus development by establishing the Senna leaf-induced diarrhea model in rats, as well as to increase the activity of antioxidant enzymes and the content of non-enzymatic antioxidants in the intestinal tract and to enhance the rats’ own antioxidant defenses, to mitigate the oxidative damage caused by diarrhea. Subsequently, the application of the cellular oxidative stress model evidenced that PV could play a protective role against cellular oxidative stress by inhibiting the overaccumulation of ROS in macrophages. Furthermore, the candidate antioxidant targets of PV were analyzed and screened using a comprehensive network pharmacology method, and their expression were then examined at the mRNA level and protein level. Our results suggest that PV may protect against H₂O₂-induced oxidative damage in macrophages by activating BCL2L1 and inhibiting ESR1, JAK2/STAT3, and MMP2. These findings open new perspectives on the antioxidant mechanism of PV and the prospect of developing it as a novel natural antioxidant drug.

The aim of the present study was to establish a simple and reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method and apply it for the determination of pharmacokinetics of moxidectin-loaded microspheres (MOX-MS) in rats. Plasma samples were processed using a simplified liquid–liquid extraction method and were separated using an Agilent Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of a 10 mM ammonium formate solution with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.4 mL/min for 5 min. Avermectin B1a was used as an internal standard (IS). The sample was injected at a volume of 10 μL with a column temperature of 35 °C and detected in a positive ion mode. A good linear response across the concentration range of 1.00–200 ng/mL (r² > 0.99) and a lower limit of quantification (LLOQ) of 1.00 ng/mL were achieved. The extraction recovery of moxidectin exceeded 94.1%, the matrix effect was between 91.2% and 96.2%, the accuracy ranged from 100.1 to 103.6%, and the relative standard deviation (RSD) did not exceed 15% for the intra- and inter-day accuracy and precision. The pharmacokinetic results showed that MOX-MS significantly decreased C_max, prolonged T_1/2, and improved bioavailability. The developed method significantly reduced the assay volume, shortened detection time, simplified sample processing methods and saved assay costs, which may contribute to the development of the new antiparasitic drug.

The escalating demand for enhanced therapeutic efficacy and reduced adverse effects in the pharmaceutical domain has catalyzed a new frontier of innovation and research in the field of pharmacy: novel drug delivery systems. These systems are designed to address the limitations of conventional drug administration, such as abbreviated half-life, inadequate targeting, low solubility, and bioavailability. As the disciplines of pharmacy, materials science, and biomedicine continue to advance and converge, the development of efficient and safe drug delivery systems, including biopharmaceutical formulations, has garnered significant attention both domestically and internationally. This article presents an overview of the latest advancements in drug delivery systems, categorized into four primary areas: carrier-based and coupling-based targeted drug delivery systems, intelligent drug delivery systems, and drug delivery devices, based on their main objectives and methodologies. Additionally, it critically analyzes the technological bottlenecks, current research challenges, and future trends in the application of novel drug delivery systems.

At present, the occurrence of a large number of infectious and non-communicable diseases poses a serious threat to human health as well as to drug development for the treatment of these diseases. One of the most significant challenges is finding new drug candidates that are therapeutically effective and have few or no side effects. In this respect, the active compounds in medicinal plants, especially flavonoids, are potentially useful compounds with a wide range of pharmacological activities. They are naturally present in nature and valuable in the treatment of many infectious and non-communicable diseases. Flavonoids are divided into fourteen categories and are mainly derived from plant extraction, chemical synthesis and structural modification, and biosynthesis. The structural modification of flavonoids is an important way to discover new drugs, but biosynthesis is currently considered the most promising research direction with the potential to revolutionize the new production pipeline in the synthesis of flavonoids. However, relevant problems such as metabolic pathway analyses and cell synthesis protocols for flavonoids need to be addressed on an urgent basis. In the present review, new research techniques for assessing the biological activities of flavonoids and the mechanisms of their biological activities are elucidated and their modes of interaction with other drugs are described. Moreover, novel drug delivery systems, such as nanoparticles, bioparticles, colloidals, etc., are gradually becoming new means of addressing the issues of poor hydrophilicity, lipophilicity, poor chemical stability, and low bioavailability of flavonoids. The present review summarizes the latest research progress on flavonoids, existing problems with their therapeutic efficacy, and how these issues can be solved with the research on flavonoids.

Background and Aim: Simiao Yong'an decoction (SYD) is a classic traditional Chinese medicine (TCM) prescription that has the effects of clearing heat, detoxifying, promoting blood circulation, and relieving pain. In this study, we investigated the effect of SYD on the diversity of intestinal microbiota after fermentation by Bacillus subtilis. Materials and Methods: SYD was fermented using B. subtilis. Female Sprague-Dawley rats were randomly divided into the following four groups with six rats in each group: Negative sample group (NS), water exaction non-fermentation group (WE), B. subtilis group (BS), and fermentation liquid group (FL). All rats were orally administered for 14 days. High-throughput Illumina sequencing was used to analyze 16S rRNA expression in rat fecal samples. Results: A total of 2782 operational taxonomical units (OTUs) were identified in this study, and 634 OTUs were shared among all samples. Bacteroidetes (28.17%-53.20%) and Firmicutes (48.35%-67.83%) were the most abundant phyla identified among the four groups. The abundance of Escherichia and Alistipes was lower in the FL group than in the NS group, whereas the abundance of Bifidobacteria and Lactobacillus was increased in the FL group (p < 0.05). The abundance of Bifidobacterium was significantly upregulated in the FL group compared with the WE and BS groups (p < 0.05). Conclusion: After fermentation, SYD had a significantly better effect than SYD or B. subtilis. SYD significantly promoted the growth of intestinal probiotics, inhibited the growth of pathogenic bacteria, and maintained the balance of intestinal microbiota in SD rats. This study provides new insights into the development and use of SYD. Keywords: Bacillus subtilis, fermentation, intestinal microbiota, probiotics, Simiao Yong'an Decoction.

Inflammation is the host response of immune cells during infection and traumatic tissue injury. An uncontrolled inflammatory response leads to inflammatory cascade, which in turn triggers a variety of diseases threatening human and animal health. The use of existing inflammatory therapeutic drugs is constrained by their high cost and susceptibility to systemic side effects, and therefore new therapeutic candidates for inflammatory diseases need to be urgently developed. Natural products are characterized by wide sources and rich pharmacological activities, which are valuable resources for the development of new drugs. This study aimed to uncover the alleviating effect and potential mechanism of natural product Limonium aureum (LAH) on LPS-induced inflammatory responses in macrophages. The experimental results showed that the optimized conditions for LAH ultrasound-assisted extraction via response surface methodology were an ethanol concentration of 72%, a material-to-solvent ratio of 1:37 g/mL, an extraction temperature of 73 °C, and an extraction power of 70 W, and the average extraction rate of LAH total flavonoids was 0.3776%. Then, data of 1666 components in LAH ethanol extracts were obtained through quasi-targeted metabolomics analysis. The ELISA showed that LAH significantly inhibited the production of pro-inflammatory cytokines while promoting the secretion of anti-inflammatory cytokines. Finally, combined with the results of network pharmacology analysis and protein expression validation of hub genes, it was speculated that LAH may alleviate LPS-induced inflammatory responses of macrophages through the AKT1/RELA/PTGS2 signaling pathway and the MAPK3/JUN signaling pathway. This study preliminarily revealed the anti-inflammatory activity of LAH and the molecular mechanism of its anti-inflammatory action, and provided a theoretical basis for the development of LAH as a new natural anti-inflammatory drug.

The redox system is closely related to changes in cellular metabolism. Regulating immune cell metabolism and preventing abnormal activation by adding antioxidants may become an effective treatment for oxidative stress and inflammation-related diseases. Quercetin is a naturally sourced flavonoid with anti-inflammatory and antioxidant activities. However, whether quercetin can inhibit LPS-induced oxidative stress in inflammatory macrophages by affecting immunometabolism has been rarely reported. Therefore, the present study combined cell biology and molecular biology methods to investigate the antioxidant effect and mechanism of quercetin in LPS-induced inflammatory macrophages at the RNA and protein levels. Firstly, quercetin was found to attenuate the effect of LPS on macrophage proliferation and reduce LPS-induced cell proliferation and pseudopodia formation by inhibiting cell differentiation, as measured by cell activity and proliferation. Subsequently, through the detection of intracellular reactive oxygen species (ROS) levels, mRNA expression of pro-inflammatory factors and antioxidant enzyme activity, it was found that quercetin can improve the antioxidant enzyme activity of inflammatory macrophages and inhibit their ROS production and overexpression of inflammatory factors. In addition, the results of mitochondrial morphology and mitochondrial function assays showed that quercetin could upregulate the mitochondrial membrane potential, ATP production and ATP synthase content decrease induced by LPS, and reverse the mitochondrial morphology damage to a certain extent. Finally, Western blotting analysis demonstrated that quercetin significantly upregulated the protein expressions of SIRT1 and PGC-1α, that were inhibited by LPS. And the inhibitory effects of quercetin on LPS-induced ROS production in macrophages and the protective effects on mitochondrial morphology and membrane potential were significantly decreased by the addition of SIRT1 inhibitors. These results suggested that quercetin reprograms the mitochondria metabolism of macrophages through the SIRT1/PGC-1α signaling pathway, thereby exerting its effect of alleviating LPS-induced oxidative stress damage.