The effects of shilajit (mumio) extract and mesenchymal stem cells on experimental chronic obstructive pulmonary disease (COPD) induced in rats

Introduction: Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by chronic inflammation, oxidative stress, immune dysregulation, and irreversible tissue remodeling. Current therapeutic interventions are effective in mitigating symptoms; however, they are unable to halt the progression of the disease. Shilajit, a naturally occurring compound with antioxidant and immunomodulatory properties, and mesenchymal stem cells (MSCs), which possess regenerative and anti-inflammatory potential, are emerging as promising candidates. This study aimed to evaluate the therapeutic effects of shilajit, mesenchymal stem cells, and their combined administration in a porcine pancreatic elastase (PPE)-induced COPD rat model. Materials and Methods: A total of 38 female Wistar rats were used in the study. In all groups except the control group, the COPD model was created by intratracheal administration of porcine pancreatic elastase (PPE). The animals were divided into five groups: control, sham (COPD, untreated), shilajit (oral, 21 days), mesenchymal stem cells (intravenous, once a week, two doses in total), and shilajit + mesenchymal stem cells. The therapeutic interventions were initiated 10 days following the elastase administration and continued for a duration of 21 days. Body weights were monitored on a weekly basis throughout the experiment; blood gas parameters (pH, pCO₂, pO₂, Hct, ctHb, lactate, HCO₃⁻, and sO₂) were measured on day 3 after elastase application and at the end of the experiment. Following the termination, of the experiment, peripheral blood samples were analyzed by flow cytometry using fluorescently labeled monoclonal antibodies to determine the CD3⁺, CD4⁺, and CD8⁺ T lymphocyte subsets. ELISA method was used to assess serum IL-33, HMGB-1, and IL-1α levels; lung tissues were examined histopathologically; and also, MMP-9, TIMP-1, and F-actin protein expression levels were determined by Western blot analysis. Statistical analyses were performed using ANOVA and appropriate post-hoc tests, and p ≤ 0.05 was considered significant. Results: In the COPD model created with PPE, decreases in pH and pO₂ levels, and increases in pCO₂ and lactate levels were detected in the early period, and impaired gas exchange was confirmed. At the end of the experiment, pO₂ levels in the sham group were significantly lower than in the control group (254.13 ± 130.30 vs. 412.67 ± 30.32 mmHg; p = 0.015). Hematocrit values were considerably higher in the sham group (p = 0.006), and oxygen saturation was markedly decreased (94.47 ± 4.32%; p = 0.018). Lactate levels were significantly higher in the sham group compared to all other groups (p < 0.001). In analyses of serum proinflammatory cytokine, no significant difference was observed between groups in IL-33 levels (p = 0.213). However, HMGB-1 (p = 0.001) and IL-1α (p = 0.003) levels were significantly increased in the sham group. In all treatment groups, these markers decreased to levels close to those in the control group. In flow cytometric analyses, the ratios of CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ T cells were found to be highest in the sham group (p < 0.001 for all); a significant decrease was observed in all subpopulations in the Shilajit and Shilajit + MSC treatment groups. Histopathological examination revealed interstitial inflammation, lymphoid accumulation, and emphysema in 100% of the sham group (p < 0.001). The findings demonstrated a substantial reduction in the treatment groups, with the most effective histological protection observed in the combined treatment group. In Western blot analyses, the MMP-9/TIMP-1 ratio increased dramatically in the sham group (p < 0.001), whereas it decreased significantly in the MSC treatment group; a significant decrease was also observed in the combined group compared to the sham group. F-actin expression increased substantially in the combined treatment group (p < 0.001). Conclusion: This study demonstrated that shilajit and MSC applications, both as monotherapy and in combination, in a porcine pancreatic elastase-induced COPD rat model improved impaired gas exchange, suppressed systemic and pulmonary inflammatory responses, regulated immune cell imbalance, and significantly modulated molecular mechanisms associated with extracellular matrix degradation. The combined treatment approach was found to have a more pronounced effect than single applications in terms of controlling inflammation, preserving lung tissue architecture, and supporting cytoskeletal integrity. The findings suggest that the combination of shilajit and MSC may be effective across multiple biological processes involved in the pathophysiology of COPD; this approach should be validated across different experimental models and further investigated at the mechanistic level. Keywords: Immune modulation; COPD; Mesenchymal stem cells; MMP-9/TIMP-1 balance; Shilajit