Pakistan Journal of Pharmaceutical Sciences

Abstract: Background: Liver fibrosis is a chronic wound healing process of liver injury and gut–liver axis dysregulation has been recently identified as an important contributor to liver fibrosis progression. Quercetin has been reported to have anti-inflammatory activity and microbiota-modulating effects. However, the potential targets of quercetin remain unclear, as does the relationship between quercetin-associated targets and changes in the gut microbiota in liver fibrosis. Objectives: The purpose of this study was to use network pharmacology to predict potential targets of quercetin associated with liver fibrosis and to explore changes in gut microbiota following treatment with quercetin using a CCl₄-induced rat model. Methods: Eighteen male Sprague–Dawley rats were randomized into CON group, MOD group and QR group (n = 6 per group). Liver fibrosis was induced by weekly subcutaneous injection of carbon tetrachloride (CCl₄) for eight weeks. Oral gavage of quercetin (50 mg/kg/day) was performed in QR group. Histopathological changes were assessed by hematoxylin–eosin staining, Masson staining and Ishak scoring. Potential targets of quercetin were screened using network pharmacology, followed by protein–protein interaction (PPI) network construction and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Cecal microbiota composition was determined by 16S rRNA gene sequencing. Parametric or non-parametric tests were used accordingly, including one-way ANOVA or Kruskal–Wallis test. Results: Histopathology revealed significant liver injury and fibrosis in the MOD group relative to the CON group. The QR group showed lower Ishak fibrosis scores and milder histopathological features compared with the MOD group. Network pharmacology analysis identified UBC, UBE2I, RBX1, PSMB7 and UBA1 as hub targets, with KEGG pathways of interest including the TNF signaling pathway, p53 signaling pathway, Toll-like receptor signaling pathway and ubiquitin-mediated proteolysis. Alpha diversity of the cecal microbiota differed significantly among groups, whereas beta diversity did not. Relative abundance of Proteobacteria decreased and Firmicutes increased in the QR group relative to the MOD group. Taxa enrichment analysis identified enrichment of Muribaculaceae and Ligilactobacillus in the QR group. Conclusion: Histopathology and microbiome analyses indicated that quercetin intervention was associated with altered liver histopathological features and shifts in gut microbiota composition in rats with CCl₄-induced liver fibrosis. Network pharmacology predicted several hub targets and pathways potentially linked to these observations. As the present analyses are purely correlational and lack mechanistic validation at the molecular level, no causal relationships are implied between the predicted targets/pathways and the observed histological or microbiome changes. These findings should be regarded as hypothesis-generating and warrant further validation studies.

Abstract: Background: Gallic acid is a natural phenolic compound with excellent antioxidant, antimicrobial and anti-inflammatory activity and good skin penetration. Gallic acid demonstrates both stability and solubility problems in topical formulations. A nanoemulgel combines an emulsion for enhanced solubility with a gel base for improved physical stability. Objectives: The main objective of this study is to characterize and assess the stability of a gallic acid-containing nanoemulgel. Methods: Various gallic acid nanoemulsions (GNE) were prepared using optimized ratios of oil, surfactants, gallic acid and water and evaluated for stability. Finally, stable GNE-19 was characterized for particle size and converted to nanoemulgel (GNEG-19). Nanoemulgel was subjected to short-term and accelerated stability studies and characterized for pH, viscosity, spreadability, occlusion effect, FTIR, in-vitro drug release and in-vitro pharmacological activities. Results: The results showed that formulation GNEG-19 showed optimum thermodynamic stability, having no phase separation and color change. The droplet size of freshly prepared GNE and after 28 days was 175.43nm and 183.23nm, respectively. The pH was slightly acidic but still within the human skin range (3.9–4.5). No interaction was found between the gallic acid and other components of nanoemulgel following FTIR analysis. The viscosity of freshly prepared GNEG-19 was 2127.1 cP and after 90 days, it was 2102.3 cP, which showed its stability. The percent drug release of gallic acid from nanoemulgel after 24 hrs was 93.12%. GNEG-19 also showed effective occlusion properties when compared with gel base and petroleum jelly. In-vitro pharmacological activities showed that GNEG-19 had antioxidant activity of 63.77% and a zone of inhibition (ZOI) of 11.4 mm compared to pure gallic acid (9.5 mm). Conclusion: In conclusion, nanoemulgel containing gallic acid showed good physical and chemical stability and was pharmacologically active.

Abstract: Background: HPV-associated cervical cancer remains a leading cause of cancer-related death in women worldwide. The role of bone marrow mesenchymal stem cells (BMSCs) and miR-129 in tumor progression remains unclear. Objectives: To investigate miR-129 expression and its clinical significance in HPV-associated cervical cancer and to explore whether BMSCs regulate malignant phenotypes through miR-129. Methods: miR-129 expression was measured by qRT-PCR in 77 cervical cancer and 30 normal cervical tissues. Associations with clinicopathological features and overall survival (OS) were analyzed. Diagnostic value was assessed by ROC curve. Rat BMSCs were co-cultured with SiHa and HeLa cells. Following miR-129 overexpression or knockdown, cell proliferation (CCK-8, colony formation), invasion (Transwell) and expression of E-cadherin, N-cadherin and MRP1 (Western blot) were examined. Results: miR-129 expression was significantly higher in cervical cancer tissues than in normal controls (3.24±0.56 vs. 1.00±0.32, P<0.05). High miR-129 expression correlated with tumor size ≥3 cm (P=0.009), FIGO stage III-IV (P=0.0034) and lymph node metastasis (P=0.038). High expression was associated with shorter OS (median: 28.4 vs. 46.2 months; HR=2.944, 95%CI:1.484-5.842, P=0.002). The AUC was 0.788 (95%CI:0.693-0.884), with a cut-off of 1.85 (sensitivity:76.9%, specificity:70.4%). BMSC co-culture upregulated miR-129 expression (SiHa: 3.52±0.42 vs. 1.00±0.08, P<0.01). miR-129 overexpression promoted proliferation, colony formation and invasion, while knockdown suppressed these phenotypes. Overexpression of miR-129 reduced E-cadherin (0.42±0.05 vs. 1.00±0.08, P<0.01) and increased N-cadherin (2.85±0.22 vs. 1.00±0.10, P<0.01) and MRP1 (2.46±0.18 vs. 1.00±0.09, P<0.01). Knockdown of miR-129 partially reversed BMSC-induced changes in these proteins. Conclusion: miR-129 is significantly upregulated in HPV-associated cervical cancer and shows potential as a diagnostic and prognostic biomarker. BMSCs upregulate miR-129 in cervical cancer cells and miR-129 modulates proliferation, invasion, EMT marker expression and MRP1 expression. These findings suggest that the BMSCs/miR-129 axis may contribute to cervical cancer progression, but further in-vivo and mechanistic studies are needed.

Abstract: Background: Dysregulated expression of prolyl 4-hydroxylase subunit alpha 3 (P4HA3) has been identified in Colon cancer, breast cancer, head and neck cancers, etc. and is closely associated with tumor progression. However, its specific roles in gastric cancer (GC), particularly regarding alterations in metabolic pathways, are remain unknown. Objectives: This study investigated how P4HA3 influences glycolytic metabolic reprogramming and the proliferation and invasion of gastric cancer cells via the PI3K/AKT/mTOR signaling pathway. Methods: To determine P4HA3 levels in GC tissues and corresponding cell lines, bioinformatics methods, immunohistochemical (IHC) staining and Western blotting (WB) were conducted. Furthermore, gain- and loss-of-function experiments, including cell viability assays (CCK-8), colony-forming tests, scratch wound assays and Transwell invasion experiments, were utilized to explore the influence of P4HA3 on GC cell growth, motility and invasive capacity. Changes in cellular metabolism were assessed using the Seahorse system (under normoxic conditions), including measurements of basal metabolism, glycolytic capacity and mitochondrial respiration (oxidative phosphorylation), as well as Western blot analysis of key glycolytic proteins. Additionally, the involvement of the PI3K/AKT/mTOR signaling axis was examined by gene set enrichment analysis (GSEA), WB analysis and pharmacological inhibition studies. Results: GC tissues and cell lines exhibited markedly increased levels of P4HA3, correlating significantly with decreased overall survival (OS). Experimental data indicated that P4HA3 facilitated GC cell proliferation, migration and invasion. In addition, P4HA3 enhanced glycolytic activity and increased the expression of key glycolysis-related proteins. Unexpectedly, P4HA3 also increased mitochondrial respiration under the assay conditions, suggesting that P4HA3 may induce a broader metabolic activation rather than an exclusively glycolytic shift. Mechanistically, the glycolysis-enhancing effects of P4HA3 were found to require PI3K/AKT/mTOR signaling (as shown by inhibitor studies). Conclusion: P4HA3 promotes glycolysis via the PI3K/AKT/mTOR signaling pathway, thereby promoting the progression of gastric cancer.

Abstract: Background: Bacteria have become more resistant to commonly used antibiotics. Among the many reasons, biofilm-mediated protection plays an indispensable role. To overcome microbial resistance, alternative antibiofilm therapies may be developed to address this challenge and they should be cost-effective and readily available. Objectives: To evaluate the antibiofilm activities of natural compounds Allium Cepa (Onion), Menthe spicata (mint) and Zingiber officinale (Ginger) against Escherichia coli and Pseudomonas Aeruginosa Biofilms. Methods: This cross-sectional study includes 150 patients from various clinics in Karachi, Pakistan. Samples of Escherichia coli and Pseudomonas aeruginosa were collected and the study was carried out to determine the antibiofilm-forming activity of natural product extracts of Allium cepa (Onion) G.H.NO 100929, Mentha spicata (mint) G.H.NO100933 and Zingiber officinale (Ginger) G.H.NO100928, evaluated separately and in different combinations. 96 well plate method was used to evaluate biofilm forming activity of microorganisms and antibiofilm forming activity of Allium Cepa (Onion) Menthe Spicata( mint) Zingiber Officinale (Ginger Peels) Results: Natural compounds Allium Cepa (Onion) Menthe Spicata( mint) Zingiber Officinale (Ginger) has shown antibiofilm activity against Escherichia coli and Pseudomonas aeruginosa biofilms separately but when used in combination only synergistic activity shows ginger and mint combination with FIC 0.45 against Pseudomonas Aeruginosa. Whereas both organisms have shown resistance to conventional antimicrobial agents. Conclusion: This study concludes that Allium Cepa (Onion) Menthe Spicata (mint) Zingiber Officinale (Ginger) having antibiofilm activity when used separately but in combination Zingiber Officinale (Ginger) And Menthe Spicata (mint) against Pseudomonas Aeruginosa only shows synergistic activity where as other combination shows indifferent activity but shows lower concentration of extract as compare to when used separately but not match the criteria of synergism but importantly none of combination shows antagonist activity. These findings emphasize the need to incorporate these natural agents, after drug authority approval, with already available antimicrobial agents.