[Objective] To investigate the alleviating effects and mechanisms of Lacticaseibacillus paracasei CCFM1538 on inflammatory pain in mice. [Methods] A mouse model of inflammatory pain was induced by complete Freund’s adjuvant, and blank, model, dexamethasone, culture medium, fermentation supernatant, and high-dose/low-dose bacterial groups were set up. The ability of CCFM1538 to alleviate inflammatory pain was evaluated based on paw thickness, thermal/mechanical withdrawal threshold, HE staining of paw skin, inflammatory cytokines, glial cell immunofluorescence, and oxidative stress indicators. [Results] The fermentation supernatant of CCFM1538 significantly alleviated mouse paw swelling, increased the mechanical withdrawal threshold after modeling, reduced paw inflammatory cell infiltration, and decreased the expression of pro-inflammatory cytokines (IL-6 and CCL2). High-dose CCFM1538 cells significantly reduced the expression of pro-inflammatory cytokines (IL-1β and CCL2), decreased serum malondialdehyde levels, and inhibited abnormal activation of spinal cord glial cells. In addition, both significantly increased the thermal withdrawal threshold and serum superoxide dismutase activity in mice after modeling. [Conclusion] The fermentation supernatant and high-dose cells of CCFM1538 can alleviate inflammatory pain by reducing inflammation, alleviating oxidative stress, and regulating spinal glial cell activation. This study provides experimental evidence for the development of related functional foods.

[Objective] To investigate the mechanism by which Camphora kanahirae leaves alleviate alcoholic liver disease through the regulation of the gut microbiota-short-chain fatty acid-intestinal barrier pathway. [Methods] Sixty mice were randomly allocated into six groups [normal, model, silymarin (100 mg/kg), and low-, medium-, and high-dose (100, 200, and 400 mg/kg, respectively) C. kanahirae leaves], with 10 mice in each group. After 14 consecutive days of administration, a mouse model of alcoholic liver disease was established. The liver index, alanine aminotransferase (ALT) and aspartate transferase (AST) levels, colon histopathological changes, short-chain fatty acid content, and alterations in gut microbiota structure were measured. [Results] Compared with the model group, medium- and high-dose C. kanahirae leaves reduced ALT and AST levels (P<0.05). Pathological evaluation showed that C. kanahirae leaves at all doses alleviated alcohol-induced colon mucosal damage and inflammatory cell infiltration to varying degrees. Alcohol damage resulted in decreases in levels of short-chain fatty acids, such as acetic acid, propionic acid, butyric acid, and valeric acid (P<0.05). C. kanahirae leaves promoted the proliferation of beneficial bacteria, increased short-chain fatty acid levels, and enhanced intestinal barrier function. Furthermore, 16S rRNA gene analysis of the gut microbiota indicated that alcohol intake led to dysbiosis, and C. kanahirae leaves effectively reversed this microbial imbalance, restoring the composition and diversity of the gut microbiota. [Conclusion] C. kanahirae leaves exert hepatoprotective effects by regulating the gut microbiota balance, promoting short-chain fatty acid production, and maintaining intestinal barrier integrity.

Early life is a critical window for the establishment of the infant gut microbiome and the shaping of its metabolic functions, and gestational age and feeding mode are considered key determinants of this process. [Objective] To compare the fecal gut microbiome and metabolome between preterm and full-term infants under breastfeeding and non-breastfeeding conditions, thus exploring the effects of feeding mode and gestational age on early-life gut microbial ecology and metabolic features. [Methods] Infants were assigned into four groups: breastfeeding preterm infants (BPI), non-breastfeeding preterm infants (NBPI), breastfeeding full-term infants (BTI), and non-breastfeeding full-term infants (NBTI), with 10 infants per group. The 16S rRNA gene sequencing and untargeted metabolomics analysis were performed. Alpha/beta diversity analyses, differential abundance testing, and linear discriminant analysis effect size (LEfSe) were performed to identify key microbial taxa. Partial least squares-discriminant analysis (PLS-DA), volcano plots, and KEGG pathway enrichment were employed to determine differential metabolites and functional pathways, followed by microbiome-metabolite association network analysis. [Results] The 16S rRNA gene sequencing showed that the gut microbiome in the BTI group was dominated by Actinomycetota, Bifidobacterium (45.98%), and Bifidobacterium breve, forming a typical “breastfeeding-type” structure. The BPI group showed enrichment of Pseudomonadota and Streptococcus, with attenuated dominance of Bifidobacterium. Among non-breastfed infants, the NBTI group was dominated by Enterococcus (59.20%), whereas the NBPI group showed a fluctuating gut microbiome. Untargeted metabolomics further revealed functional differences consistent with these compositional patterns. KEGG enrichment indicated that differential metabolites were mainly involved in amino acid metabolism, lipid metabolism, bile acid-related pathways, and carbohydrate digestion and absorption. At the level of key metabolites, the BPI group showed significant enrichment of metabolites related to antioxidant and immune support, such as glutathione and vitamin D sulfate conjugates; the BTI group enriched long-chain polyunsaturated fatty acids and indole-derived metabolites; and non-breastfeeding groups generally exhibited accumulation of fermentable carbohydrates and specific bile acid derivatives. Correlation analysis confirmed a strong positive association between Bifidobacterium and indole-related metabolite outputs in breastfeeding groups, whereas microbiome–metabolite networks were looser in non-breastfeeding groups. [Conclusion] Both gestational age and feeding mode are related to differences in the early gut microbiome structure and related metabolic characteristics of infants, with the association between feeding mode and metabolic profile differences being more prominent. Gestational age may affect the establishment pattern of dominant microbial taxa in the context of breastfeeding. As a pioneering exploratory study, this study preliminarily reveals the specificity of gut microbiome structure and functional metabolism in different populations of infants, providing a basis for subsequent prospective cohort validation, mechanism research, and early nutritional intervention optimization.

[Objective] To investigate the efficacy of postbiotics prepared with Bifidobacterium animalis CCFM1155, a strain previously identified for its ability to enhance nuclear factor erythroid 2-related factor 2 (Nrf2) secretion in human immortalized epidermal cells, in alleviating skin aging. [Methods] A mouse model of skin aging was established by daily subcutaneous injection of D-galactose (500 mg/kg) for eight weeks (n=6 per group). The model mice were then orally administered either heat-inactivated cells (1155B) or concentrated fermentation supernatant (1155T) of CCFM1155 cells at a dose equivalent to 5×10⁹ CFU of live bacteria. [Results] The 1155B was the primary postbiotic component exerting anti-skin aging effects, whereas the 1155T group showed no significant effect. Compared with the model group, the 1155B treatment ameliorated skin aging phenotypes by improving the skin tissue structure, increasing the epidermal thickness (P<0.05) and dermal collagen fiber density (P<0.01), and decreasing the expression of the senescence marker protein p21 and the activity of senescence-associated β-galactosidase (P<0.001). In addition, the 1155B treatment enhanced the antioxidant defense system of the skin by elevating the activities of total superoxide dismutase (P<0.05) and catalase (P<0.01), as well as the content of reduced glutathione (P<0.01), while lowering the malondialdehyde level (P<0.01). Mechanism studies revealed that the 1155B upregulated both the mRNA (P<0.05) and protein (P<0.001) levels of Nrf2 in the skin. Consequently, it promoted the transcription of key downstream antioxidant molecules, including heme oxygenase-1, peroxiredoxin, thioredoxin 1, and the glutamate-cysteine ligase catalytic subunit, thereby activating the downstream signaling pathway of Nrf2. [Conclusion] The inactivated cells of B. animalis CCFM1155 alleviates skin aging by activating the Nrf2 signaling pathway. These findings offer a novel perspective on postbiotic regulation of the Nrf2 pathway and lay a theoretical foundation for developing safe and effective anti-skin aging products.

[Objective] To investigate the effects of diets with different concentrate to roughage ratios on the growth performance, rumen fermentation parameters, and serum metabolites of Yajiang snow cattle. [Methods] Twenty-seven healthy individuals of Yajiang snow cattle with similar body weights were selected and randomized into three groups (n=9). Cattle in the three groups were fed diets with the concentrate to roughage ratios being 40:60 (C40 group), 50:50 (C50 group), and 60:40 (C60 group). The pre-experimental period lasted for 7 days, and the experimental period lasted for 45 days. The growth performance, nutrient digestibility, volatile fatty acid content and microbiota structure in the rumen fluid, and serum metabolite content of the Yajiang snow cattle were measured. [Results] With the increase in the dietary concentrate to roughage ratio, the average daily gain and average daily feed intake of Yajiang snow cattle increased, while the apparent digestibility of crude protein and acid detergent fiber decreased (all P<0.05). As the dietary concentrate to roughage ratio increased, the pH value, acetate content, and acetate/propionate ratio in the rumen fluid decreased, whereas the propionate content increased (all P<0.05). Different concentrate to roughage ratios altered the microbiota structure in the rumen fluid of Yajiang snow cattle (P<0.05 or P<0.01). Specifically, the C60 group had lower relative abundance of Bacteroidota, Verrucomicrobia, Ruminococcus, Clostridium, Butyrivibrio, Pseudobutyrivibrio, and Bibersteinia but higher relative abundance of Bacillota, Prevotella, Succiniclasticum, and Selenomonas than the C40 group. Furthermore, different concentrate to roughage ratios changed the serum metabolite content of Yajiang snow cattle (P<0.05 or P<0.01). Specifically, the C40 group had significantly higher content of 5,6-dihydrouridine, 2-hydroxybutyric acid, and nitrilotriacetic acid but significantly lower content of 6-hydroxy-2-methylindole, N-acetylneuraminic acid, 2,6-dihydroxybenzoic acid, and taurodeoxycholic acid in the serum than the C50 and C60 groups. [Conclusion] Under the conditions of this experiment, different dietary concentrate to roughage ratios had significant effects on the production performance, rumen fermentation parameters, and serum metabolites of Yajiang snow cattle. Compared with the high roughage diet, the high concentrate diet significantly improved the production performance and promoted the rumen fermentation, while reducing the diversity of the rumen microbiota in Yajiang snow cattle.

[Objective] To investigate the specific role and molecular mechanism of the host protein protein disulfide isomerase A4 (PDIA4) during bovine viral diarrhea virus (BVDV) infection, thus providing a theoretical basis for elucidating the role of this protein in viral replication and developing novel prevention and control strategies. [Methods] Western blotting and qPCR were employed to analyze PDIA4 expression in Madin-Darby bovine kidney (MDBK) cells following BVDV infection. The expression levels of autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (p62) were measured by Western blotting and laser confocal microscopy. MDBK cell lines with pdia4 knockdown and overexpression were constructed. The effects of pdia4 on LC3 and p62 expression were examined, and the autophagic flux was evaluated by a tandem GFP-mRFP-LC3 reporter system. Cells were subjected to starvation or treated with bafilomycin A1 (BafA1), followed by BVDV infection. Viral replication was assessed by measuring BVDV mRNA levels through qPCR and double-stranded RNA (dsRNA) levels through immunofluorescence staining. [Results] BVDV infection significantly upregulated the endogenous expression of PDIA4. The LC3 level increased progressively with infection time, whereas the p62 level showed an initial increase followed by a decrease. The cell lines with pdia4 knockdown and overexpression were successfully established. pdia4 knockdown significantly increased both LC3 and p62 levels, whereas pdia4 overexpression increased the LC3 level and decreased the p62 level. Under starvation or BafA1 treatment, pdia4 knockdown inhibited BVDV replication, whereas pdia4 overexpression promoted BVDV replication. [Conclusion] BVDV infection upregulates host PDIA4 expression. PDIA4 promotes BVDV replication by activating the autophagic flux through regulation of LC3 and p62. These findings provide new insights into the pathogenic mechanism of BVDV and offer a theoretical basis for developing targeted antiviral strategies.

Porcine deltacoronavirus (PDCoV), a major porcine intestinal coronavirus, has caused enormous economic losses to the pig industry. Up to now, there is no commercial vaccine available. [Objective] To obtain the epidemic strain of PDCoV and study its biological characteristics and pathogenicity, thereby providing biological materials for the development of an effective vaccine against PDCoV as soon as possible. [Methods] LLC-PK1 cells were used to isolate the virus from PDCoV-positive samples from a pig farm in Gansu Province. The virus was identified by observation of cytopathic effect (CPE), indirect immunofluorescence assay (IFA), and whole genome sequencing. [Results] After blind passage of the PDCoV-positive LLC-PK1 cells to P6, the cells became larger and rounded, and then shrunk and shed into single or clustered dense particles, typical features of CPE. The IFA results confirmed successful isolation of the PDCoV strain CHN/GSYD/2023. The whole genome sequencing and phylogenetic analysis showed that CHN/GSYD/2023 isolated in this study was more closely related to the strain from China and was located on a different clade from the strain CHN/XJYN/2016 preserved in our laboratory. After 5-day-old piglets were infected with CHN/GSYD/2023, 100% (5/5) of piglets became diseased. Autopsy and histopathology of piglets showed thinning small intestinal wall, a large amount of effusion in the intestinal cavity, ileum intestinal villus necrosis and other pathological damage. [Conclusion] A PDCoV strain CHN/GSYD/2023 was successfully isolated from PDCoV-positive samples in a pig farm in Gansu. The strain can be stably proliferated and passaged in LLC-PK1 cells, and it is highly pathogenic to suckling piglets.

[Objective] To investigate the genomic phylogeny as well as the in vivo and in vitro infection and replication characteristics of the tick-borne parainfluenza virus type 5 (strain PIV5-JC12) recently isolated from Yunnan Province. [Methods] The strain PIV5-JC12 was identified through cytopathic effect (CPE) observation, negative staining electron microscopy, and an indirect immunofluorescence assay (IFA) based on the P protein. We determined its optimal culture temperature by comparing viral proliferation efficiency at 33 ℃ and 37 ℃ in Vero cells. A phylogenetic tree based on the whole genome was constructed via the maximum likelihood method to elucidate the molecular evolutionary characteristics of this strain. After infection of six representative cell lines of different origins (Vero, MDCK, HeLa, Huh7.5, MRC-5, and BHK-21), CPE observation, RT-qPCR, and tissue culture infectious dose 50% (TCID₅₀) assays were employed to evaluate the virus replication kinetics. The in vivo pathogenicity of PIV5-JC12 was evaluated in Kunming (KM) and C57BL/6J mice. Mice were infected intranasally with high and low doses (2×10⁷ TCID₅₀ and 2×10⁶ TCID₅₀) and monitored for body weight and survival rate. The viral loads in various tissue and organ samples were measured at the time points of 1, 4, 7, and 10 days post-infection (dpi), and histopathological changes were examined. [Results] PIV5-JC12 induced CPEs, as manifested by the rounding and detachment of Vero cells. Irregular spherical particles with diameters of 50-200 nm were observed, and the specific P protein was identified by immunofluorescence. The tick-borne strain PIV5-JC12 showed the highest sequence identity with the tiger-borne strain PIV5 (OQ236149.1). Viral replication and proliferation in Vero cells were more efficient at 37 ℃ than at 33 ℃. PIV5-JC12 infected all the six cell lines of human, murine, simian, and canine origins. However, the CPEs varied among the cell lines, being more pronounced in Vero, MDCK and BHK-21 cells. Higher RNA replication efficiency was observed in Vero, MDCK, and HeLa cells. In vivo infection studies revealed differential susceptibility of KM and C57BL/6J mice to PIV5-JC12. The body weight loss (5%) was only observed in the high-dose infected C57BL/6J mice at 10 dpi. Lung viral loads in both mouse lines reached 10⁴-10⁵ copies/g, while no infectious virus was detected in the lung tissue at any of the time points examined. Histopathological staining analysis of lung tissue at the early stage (4 dpi) and late stage (10 dpi) of infection showed no significant pathological damage. [Conclusion] PIV5-JC12 isolated from tick samples from Yunnan Province shows broad cellular tropism and low pathogenicity in mice. The findings provide a basis for research on the pathogenic characteristics of PIV5 and its potential application as a vaccine vector.

[Objective] To investigate the ability of enterovirus A71 (EV-A71) to traverse the blood-brain barrier (BBB) and infect human cerebral organoids, as well as the impact of EV-A71 on BBB integrity following retrograde invasion into the central nervous system via neural routes, and to characterize the infection profiles of EV-A71 in the BBB and human cerebral organoids and the associated inflammatory responses. [Methods] An invitro BBB model was established by seeding human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes into a Transwell system. Human cerebral organoids were generated from human induced pluripotent stem cells (hiPSCs), and a BBB-human cerebral organoid co-culture model was subsequently established. EV-A71 at three titers (10², 10⁴, and 10⁶ TCID₅₀) was used to infect each cell type of the BBB to evaluate viral replication kinetics and cytopathic effects (CPE) across different cell types. Subsequently, the BBB-human cerebral organoid co-culture model was infected with 10⁴ TCID₅₀ EV-A71 using two distinct routes: (1) an apical inoculation strategy, in which EV-A71 was directly applied to the human brain microvascular endothelial cell layer; and (2) a retrograde infection strategy, in which human cerebral organoids were first infected and subsequently co-cultured with the BBB model. Viral RNA copy numbers in culture supernatants and cells were quantified by RT-qPCR. BBB integrity was assessed by measuring transendothelial electrical resistance, and inflammatory responses were evaluated by determining the expression levels of inflammatory cytokines in human cerebral organoids using RT-qPCR. [Results] EV-A71 efficiently replicated in human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes, inducing pronounced CPE. Among these cell types, astrocytes were the most susceptible to EV-A71 infection, showing the most rapid progression of cytopathic changes, followed by brain vascular pericytes. Human brain microvascular endothelial cells were relatively less susceptible, requiring higher viral titers to establish effective infection. Under apical inoculation conditions, EV-A71 replicated within the BBB, compromised BBB integrity, traversed the barrier to infect human cerebral organoids, and significantly activated inflammatory responses. In the retrograde infection model, EV-A71 replicated within human cerebral organoids, preferentially targeted neurons and astrocytes, triggered cellular inflammatory responses, and subsequently invaded the BBB in a retrograde manner, resulting in compromised structural integrity of the BBB. [Conclusion] EV-A71 can traverse the BBB to infect human cerebral organoids and activate inflammatory responses, and can also infect human cerebral organoids first and subsequently disrupt BBB integrity in a retrograde manner. This study systematically characterizes the infection dynamics of EV-A71 in an invitro BBB-human cerebral organoid model, provides a robust experimental model for in-depth analysis of the mechanisms underlying EV-A71 invasion of the central nervous system, and lays a research foundation for future investigations into viral pathogenesis and antiviral therapeutic development.

Pseudomonas aeruginosa is a widely distributed Gram-negative opportunistic pathogen that poses a serious threat to immunocompromised individuals, often leading to severe clinical infections. Sodium houttuyfonate (SH), the main active derivative of the Chinese medicinal herb Houttuynia cordata, has been widely used for anti-infection therapy. Recent studies suggest that the efficacy of SH against antibiotic-resistant pathogens extends beyond direct antibacterial effects to include delicate regulation of the host immune system. [Objective] To investigate the role of SH in promoting M1 polarization of alveolar macrophages against P. aeruginosa infection. [Methods] A mouse model of acute pneumonia caused by P. aeruginosa was established via intratracheal intubation. Hematoxylin and eosin (HE) staining was used to evaluate the histopathological changes in the lung tissue. Bacterial load in the lung tissue was determined by the plate counting method, and the expression of Toll-like receptor 4 (TLR-4) in the lung tissue was analyzed by immunohistochemistry (IHC). In the cell experiment, MH-S alveolar macrophages were co-cultured with SH for 6 h to induce M1 polarization. Polarization markers CD86 and CD206 were analyzed by immunofluorescence and flow cytometry, and phagocytic capacity was evaluated with P. aeruginosa at 1×10⁵ CFU/mL. The mRNA levels and inflammation levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) were measured by RT-qPCR and ELISA, respectively. The expression of proteins in the TLR-4/MyD88/NF-κB signaling pathway was analyzed by Western blotting. To decipher the mechanism, we constructed TLR-4 overexpression and knockdown cell models to confirm SH regulation of this pathway and related inflammatory factors via TLR-4. [Results] The in vivo experiment results indicated that SH significantly reduced P. aeruginosa proliferation in the mouse lung tissue. Both HE staining and IHC results demonstrated that pulmonary inflammation in the treatment group was alleviated compared with the model group, suggesting that SH may enhance host immune clearance and thereby mitigate acute pulmonary infection. The cell experiment showed that SH promoted early M1 polarization of MH-S cells and enhanced their phagocytosis of P. aeruginosa. Western blotting further confirmed activation of the TLR-4/MyD88/NF-κB pathway with upregulated protein expression after SH treatment. [Conclusion] SH can activate the TLR-4/MyD88/NF-κB pathway to induce M1 polarization of alveolar macrophages during early infection, thereby exerting significant phagocytic and anti-infective effects.