Salmonella enterica serovar Typhimurium, an intracellular pathogen, evades the host immune response mechanisms to cause gastroenteritis in animals and humans. After invading the host cells, the bacteria proliferate in Salmonella-containing vacuole (SCV) and escapes from antimicrobial therapy. Moreover, Salmonella Typhimurium develops resistance to various antimicrobials including, fluoroquinolones. Treating intracellular bacteria and combating drug resistance is essential to limit the infection rate. One way of overcoming these challenges is through combination therapy. In this study, Pyrogallol (PG), a polyphenol, is combined with marbofloxacin (MAR) to investigate its effect on Salmonella Typhimurium invasion and intracellular survival inhibition. The Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PG against Salmonella Typhimurium were 128 and 256 μg/mL, respectively. The lowest fractional inhibitory concentration (FIC) index for a combination of PG and MAR was 0.5. The gentamycin protection assay revealed that PG (30 μg/mL) alone and in combination with sub-MIC of MAR inhibited 72.75 and 76.18% of the invading bacteria in Caco-2 cells, respectively. Besides, the intracellular survival of Salmonella Typhimurium was reduced by 7.69 and 74.36% in treatment with PG alone and combined with sub-MIC of MAR, respectively, which was visualized by the confocal microscopy. PG has also shown to increase the intracellular accumulation of fluoroquinolone by 15.2 and 34.9% at 30 and 100 μg/mL concentration, respectively. Quantitative real-time PCR demonstrated PG suppressed the genetic expression of hilA, invF, sipB, and acrA by 14.6, 15.4, 13.6, and 36%, respectively. However, the downregulation of hilA, invF, sipB, and acrA increased to 80, 74.6, 78, and 70.1%, in combination with sub-MIC of MAR, respectively. Similarly, PG combined with MAR inhibited the expression of sdiA, srgE, and rck genes by 78.6, 62.8, and 61.8%, respectively. In conclusion, PG has shown antimicrobial activity against Salmonella Typhimurium alone and in combination with MAR. It also inhibited invasion and intracellular survival of the bacteria through downregulation of quorum sensing, invading virulence, and efflux pump genes. Hence, PG could be a potential antimicrobial candidate which could limit the intracellular survival and replication of Salmonella Typhimurium.

Chronic alcohol consumption can cause hepatic injury and alcohol-induced toxicities. Extracts from Smilax china root have been widely used in traditional medicine and for their potential pharmacological benefits. We aimed to determine if fermented Smilax china extract (FSC) regulates alcoholic fatty liver and liver injury using two in vivo experiments. Sprague-Dawley rats were administered ethanol (3 g/kg b.w.; po) with or without FSC pretreatment to induce an acute hangover. In another experiment, rats were fed either a normal or Lieber-DeCarli ethanol (6.7%) diet with or without FSC pretreatment (125, 250, and 500 mg/kg b.w.; po) for 28 days. Serum biomarkers, liver histopathology, and the mRNA levels of anti-inflammatory, antioxidant, lipogenic, and lipolytic genes were analyzed. FSC pretreatment significantly reduced blood alcohol and acetaldehyde concentrations, upregulated the mRNA expression of alcohol dehydrogenase, aldehyde dehydrogenase, and superoxide dismutase, and decreased the activities of liver enzymes in a dose-dependent manner. It also downregulated SERBP-1c and upregulated PPAR-α and reduced the gene expression of the anti-inflammatory cytokine IL-6 in the liver. The final extract after fermentation had increased GABA content. Furthermore, FSC was found to be safe with no acute oral toxicity in female rats. Thus, FSC increases alcohol metabolism and exhibits antioxidant and anti-inflammatory effects to induce hepatoprotection against alcohol-induced damage. It may be used as a functional food ingredient after excess alcohol consumption.

In the present study, we investigated the potential immunomodulatory effects of heat-killed (hLR) and live Limosilactobacillus reuteri PSC102 (LR; formerly Lactobacillus reuteri PSC102) in RAW264.7 macrophage cells and Sprague-Dawley rats. RAW264.7 murine macrophage cells were stimulated with hLR and LR for 24 h. Cyclophosphamide (CTX)-induced immunosuppressed Sprague-Dawley rats were orally administered with three doses of hLR (L-Low, M-Medium, and H-High) and LR for 3 weeks. The phagocytic capacity, production of nitric oxide (NO), and expression of cytokines in RAW264.7 cells were measured, and the different parameters of immunity in rats were determined. hLR and LR treatments promoted phagocytic activity and induced the production of NO and the expression of iNOS, TNF-α, IL-1β, IL-6, and Cox-2 in macrophage cells. In the in vivo experiment, hLR and LR treatments significantly increased the immune organ indices, alleviated the spleen injury, and ameliorated the number of white blood cells, granulocytes, lymphocytes, and mid-range absolute counts in immunosuppressive rats. hLR and LR increased neutrophil migration and phagocytosis, splenocyte proliferation, and T lymphocyte subsets (CD4⁺, CD8⁺, CD45RA⁺, and CD28⁺). The levels of immune factors (IL-2, IL-4, IL-6, IL-10, IL-12A, TNF-α, and IFN-γ) in the hLR and LR groups were upregulated compared with those in the CTX-treatment group. hLR and LR treatments could also modulate the gut microbiota composition, thereby increasing the relative abundance of Bacteroidetes and Firmicutes but decreasing the level of Proteobacteria. hLR and LR protected against CTX-induced adverse reactions by modulating the immune response and gut microbiota composition. Therefore, they could be used as potential immunomodulatory agents.

Pyrus ussuriensis Maxim (Korean pear) has been used for hundreds of years as a traditional herbal medicine due to its strong phytochemical profile and pharmacological efficacy. In this study, we evaluated the anti-obesity potential of Pyrus ussuriensis Maxim extracts (PUE) and investigated the underlying mechanisms using a combination of in vitro, in vivo, and microbiota regulation approaches. In an adipogenesis assay, the fermented (F)PUE and non-fermented (NF)PUE significantly reduced the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC₅₀ of 85.33 and 96.67 µg/mL, respectively. In a high-fat diet (HFD)-induced obese rat model (n = 8 animals/group), oral administration of FPUE additionally reduced the total body weight gain significantly. No difference in food intake was observed, however, between the control-chow diet, FPUE, and NFPUE-treated HFD rats. Adipose tissue mass and systemic insulin resistance were markedly reduced in FPUE-treated HFD rats, in a dose-dependent manner. Treatment with FPUE also greatly improved obesity-related biomarkers, including total cholesterol, leptin, active ghrelin, Total GIP, adiponectin, and proinflammatory cytokines. Moreover, FPUE significantly suppressed HFD-induced adipogenic genes expression, while increasing fatty acid oxidation-related genes expression. Additionally, FPUE treatment attenuated the HFD-induced Firmicutes proportion within the intestinal microbiota by regulating key metabolic pathways, thus enhancing microbial population diversity (e.g., increasing Bacteroides, Bifidobacterium, Prevotella, Eubacterium, and Clostridium). Together, these results reveal a strong anti-obesity potential of FPUE through adipogenesis, lipid metabolism, weight reduction, and microbiota regulation, raising the possibility of developing FPUE as a novel therapeutic agent to control obesity and obesity-associated metabolic disorders.

The kidney proximal tubule is responsible for reabsorbing water and NaCl to maintain the homeostasis of the body fluids, electrolytes, and nutrients. Thus, abnormal functioning of the renal proximal tubule can lead to life-threatening imbalances. Bisphenol A (BPA) has been used for decades as a representative chemical in household plastic products, but studies on its effects on the kidney proximal tubule are insufficient. In this study, immunocytochemical and cytotoxicity tests were performed using two- and three-dimensional human renal proximal tubular epithelial cell (hRPTEC) cultures to investigate the impact of low-dose BPA (1-10 μM) exposure. BPA was found to interfere with straight tubule formation as observed by low filamentous actin formation and reduced Na⁺/K⁺-ATPase expression in the tubules of hRPTEC 3D cultures. Similar results were observed in rat pup kidneys following oral administration of 250 mg/kg BPA. Moreover, the expression of HO-1 and 8-OHdG, key markers for oxidative stress, was increased in vitro and in vivo following BPA administration, whereas that of OAT1 and OAT, important transporters of the renal proximal tubules, was not altered. Overall, no-observed-adverse-effect-level (NOAEL)-dose BPA exposure can decrease renal function by promoting abnormal tubular formation both in vitro and in vivo. Therefore, we propose that although it does not exhibit life-threatening toxicity, exposure to low levels of BPA can negatively affect homeostasis in the body by means of long-term deterioration of renal proximal tubular function in humans.

Background: Trifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Prolonged consumption of herbal extract has been shown to regulate the composition of gut microbiota. This study was designed to elucidate the influence of TPEE on the gut microbiota composition in the ovariectomized (OVX) rats. Methods: OVX rats were treated with TPEE at 125, 250, 500 mg/kg/day, or controls (pomegranate extract, 500 mg/kg/day; estradiol, 25 μg/kg/day) for 12 weeks. Gut microbiota analysis was conducted by extracting the microbial DNA from fecal samples and microbiome taxonomic profiling was carried out by using next-generation sequencing. The levels of serum biomarkers were analyzed using enzyme-linked immunosorbent assay (ELISA) kit. The prediction of functional biomarker of microbiota was performed using PICRUSt to investigate the potential pathways associated with gut health and serum lipid profile regulation. To study the correlation between gut microbiota composition and serum lipid levels, Spearman’s correlation coefficients were defined and analyzed. Additionally, gas chromatography–mass spectrometry analysis was conducted to uncover additional physiologically active ingredients. Results: TPEE-treated OVX rats showed significant reduction in serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDL levels but increase in HDL level. The alteration in the pathways involve in metabolism was the most common among the other KEGG categories. Particularly, TPEE also significantly reduced the relative abundance of sequences read associated with inflammatory bowel disease (IBD) and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. TPEE intervention was seen to reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlation analysis at the phylum level revealed that Bacteriodetes and Proteobacteria were strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlate with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels. Conclusions: TPEE treatment showed therapeutic benefits by improving the intestinal microbiota composition which strongly correlated with the serum lipid and cholesterol levels in the OVX rats.