Pakistan Journal of Pharmaceutical Sciences

Abstract: Background: Ferroptosis-related oxidative injury has been implicated in the progression of heart failure (HF). Rosmarinic acid (RA) has antioxidant and anti-lipid peroxidation properties, but its role in HF-associated ferroptosis-related cardiomyocyte injury remains unclear. Objectives: This study aimed to investigate whether RA attenuates Ang II-induced cardiomyocyte injury by modulating ferroptosis-related oxidative injury and GPX4/xCT-associated defense responses. Methods: RA-related targets were retrieved from TCMSP, while HF-related and ferroptosis-related genes were obtained from GeneCards/OMIM and FerrDB. Intersection targets were analyzed using PPI network construction, GO and KEGG enrichment analyses, RA-target-disease-pathway network construction and molecular docking. An Ang II-induced H9c2 cardiomyocyte injury model was established for validation. Cell viability was assessed by CCK-8 assay; ANP and BNP mRNA levels were measured by RT-qPCR; MDA content was measured to evaluate lipid peroxidation; and GPX4 and xCT protein expression was examined by Western blotting. Results: Twenty-six RA-related targets, 3311 HF-related genes and 1393 ferroptosis-related genes were identified. Intersections yielded 397 HF-associated ferroptosis targets and 9 RA-related candidate targets. Network analysis highlighted PPARG, PTGS2, MAPK1, CDKN1A and CASP3 as important targets. Enrichment analyses indicated associations with oxidative stress, apoptotic regulation, iron ion response, inflammatory signaling, lipid and atherosclerosis, TNF, IL-17 and AGE-RAGE pathways. Molecular docking suggested favorable binding of RA to these targets, especially PPARG and PTGS2. In Ang II-treated H9c2 cells, RA restored cell viability, reduced ANP and BNP expression, decreased MDA accumulation and increased GPX4 and xCT expression; these effects were partially reversed by erastin. Conclusion: RA may attenuate Ang II-induced cardiomyocyte injury by modulating ferroptosis-related oxidative injury and GPX4/xCT-associated defense responses. These findings provide preliminary computational and cellular evidence supporting RA as a candidate compound for further investigation in HF-related cardiomyocyte injury.

Abstract: Background: Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) can deliver microRNAs to recipient cells and modulate their functions; however, investigations into their molecular effects have frequently relied on surrogate cancer cell lines, whose relevance to benign CAG pathology remains uncertain and must be explicitly acknowledged. Objectives: To explore whether BMSC-derived exosomal miR-618-3p influences proliferation, apoptosis and epithelial-mesenchymal transition (EMT). Methods: Twelve CAG tissues and six normal gastric mucosa samples were obtained from the laboratory tissue bank. The human gastric adenocarcinoma cell line SGC-7901 was used as an in-vitro malignant surrogate for exploratory mechanistic evaluation, not as a model of benign CAG. BMSC-derived exosomes (BMSC-exo) were isolated by ultracentrifugation and characterized. CAG cells were treated with control exosomes or exosomes enriched with miR-618-3p mimic. miR-618-3p and TFF3 expression were detected by RT-qPCR and Western blot. Cell proliferation was assessed by CCK-8, apoptosis by caspase-3 activity and epithelial-mesenchymal transition (EMT) markers by RT-qPCR. A dual-luciferase reporter assay validated the interaction between miR-618-3p and the TFF3 3′UTR. Results: miR-618-3p was decreased and TFF3 increased in CAG tissues (both p < 0.05). The dual-luciferase assay suggested a direct interaction between miR-618-3p and the TFF3 3′UTR. Unexpectedly, unmodified BMSC-exo treatment reduced miR-618-3p expression in recipient SGC-7901 cells, increased TFF3 protein levels, enhanced proliferation, reduced caspase-3 activity, decreased E-cadherin and increased vimentin expression (all p < 0.05. In contrast, BMSC-exo enriched with miR-618-3p mimic increased miR-618-3p levels, suppressed TFF3, reduced proliferation, increased caspase-3 activity, upregulated E-cadherin and downregulated vimentin (all p < 0.05). Conclusion: In this preliminary in-vitro study using a gastric adenocarcinoma cell line, BMSC-exo miR-618-3p was associated with reduced TFF3 expression and altered proliferation, apoptosis and EMT markers. These findings describe a potential regulatory axis but remain strictly exploratory.

Abstract: Background: Moderate hamstring strain is a common sports injury impairing daily and athletic function. While topical nonsteroidal anti-inflammatory drugs (NSAIDs) offer localized pain relief with reduced systemic effects, high-quality evidence specific to hamstring injury is limited. Objectives: To evaluate the effect of a short-course topical diclofenac epolamine patch combined with rehabilitation on pain and functional recovery in moderate hamstring strain. Methods: In this single-center, double-blind, placebo-controlled trial, 120 patients were randomized to an experimental group (diclofenac patch twice daily for 14 days plus rehabilitation) or a control group (placebo patch plus identical rehabilitation). Outcomes assessed included pain measured by the Visual Analogue Scale (VAS), as the primary endpoint, muscle strength, knee range of motion (ROM), functional back-kick test, time to rehabilitation criteria and safety (adverse events, liver/renal function). Results: The diclofenac group demonstrated significantly greater pain reduction at days 14, 21 and 28 post-intervention (all p < 0.01). Muscle strength recovery was superior at days 21 and 28, knee ROM improved more at day 28 and functional back-kick performance was better at day 28 (all p < 0.05). Time to meet rehabilitation criteria was significantly shorter in the experimental group (p = 0.002). Adverse event incidence and changes in liver/renal function were similar between groups (all p > 0.05). Conclusion: A short-course topical diclofenac epolamine patch, combined with rehabilitation training, effectively alleviates pain, accelerates functional recovery, and is safe for treating moderate hamstring strain, supporting its use in clinical and sports rehabilitation.

Abstract: Background: Pseudomonas aeruginosa is widely recognized as a ubiquitous, drug-resistant, biofilm-forming bacterium that produces numerous virulence factors and plays a prominent role in hospital-acquired infections. Objectives: This study aimed to determine the prevalence of multidrug resistance (MDR), biofilm formation and biofilm-associated genes among P. aeruginosa isolates recovered from burn wound infections. Methods: A prospective cross-sectional observational study was conducted in which total of 176 burn wound samples were collected and processed for bacterial isolation. Identification of isolates was confirmed by polymerase chain reaction (PCR) targeting the oprL gene. Antimicrobial susceptibility testing was performed using the Kirby–Bauer disc diffusion method. Biofilm-forming ability was assessed using the microtiter plate assay. Furthermore, PCR was used to detect biofilm-associated genes, including pelA, pelD, cupA, algD, pelF, pslA, pslB and pslD, in biofilm-producing isolates. Results: Out of 176 burn wound samples, 74 (42%) were positive for P. aeruginosa, among which 58 isolates were identified as MDR while all isolates remained susceptible to polymyxin B. Biofilm formation was observed in all MDR isolates, with 13.79%, 44.82% and 41.37% categorized as weak, moderate and strong biofilm producers, respectively, whereas only a limited proportion of non-MDR isolates demonstrated biofilm-forming ability (P = 0.001). Among MDR biofilm-forming isolates, biofilm-associated genes such as pslA and algD were each detected in 96.55% of isolates while cupA were identified in 94.82% of isolates. Notably, cupA showed a statistically significant association with biofilm-forming intensity (P = 0.020), suggesting its potential role in enhanced biofilm establishment among MDR isolates. Conclusion: Overall, the high prevalence of multidrug-resistant, biofilm-forming P. aeruginosa isolates recovered from burn wounds is an important microbiological concern. The frequent coexistence of multidrug resistance and biofilm-forming capacity observed in this study suggests a possible association that may complicate infection management and emphasizes the importance of continuous surveillance and effective infection control strategies.

Abstract: Background: Hepatocyte nuclear factor 4α (HNF-4α) is a liver-enriched transcription factor that is downregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms by which it regulates the malignant phenotype of HCC cells remain unclear. Objectives: To investigate whether HNF-4α affects HCC cell proliferation, invasion and apoptosis by regulating the IGFBP-2/Notch1 signaling pathway. Methods: HNF-4α was overexpressed and silenced in HepG2 cells using a pcDNA3.1-HNF-4α plasmid and HNF-4α-specific siRNA, respectively. RT-qPCR and Western blotting verified transfection efficiency. Cell proliferation and invasion were assessed by MTT and Transwell assays, respectively. Apoptosis was evaluated by Caspase 3 activity assay. IGFBP-2 secretion was measured by ELISA. Protein expression of Bcl-2, Bax, IGFBP-2 and Notch1 was detected by Western blotting. Results: HNF-4α overexpression significantly inhibited HepG2 cell proliferation (by approximately 35% at 48 h and 45% at 72 h) and invasion (by approximately 55%), while markedly increasing Caspase 3 activity (approximately 2.8-fold) and the Bax/Bcl-2 ratio (from 0.42 to 2.18). Moreover, HNF-4α overexpression significantly suppressed IGFBP-2 secretion (by approximately 60%) and downregulated IGFBP-2 (by approximately 55%) and Notch1 NICD (by approximately 50%) protein expression (all P < 0.001). Conversely, HNF-4α knockdown produced opposite effects, significantly promoting proliferation (by approximately 40% at 48 h and 55% at 72 h) and invasion (by approximately 70%), decreasing Caspase 3 activity (by approximately 55%) and the Bax/Bcl-2 ratio (from 0.45 to 0.19) and upregulating IGFBP-2 (approximately 2.2-fold) and Notch1 NICD (approximately 1.8-fold) expression (all P < 0.01). Conclusion: HNF-4α inhibits HCC cell proliferation and invasion and promotes apoptosis through negative regulation of the IGFBP-2/Notch1 signaling pathway, providing new experimental evidence for the tumor-suppressive mechanism of HNF-4α in HCC.