Ameliorative effect of citicoline on cyclophosphamide-induced lung injury

Abstract

Background: Cyclophosphamide (CP) is known to cause pulmonary injury through free radical production and proinflammatory cytokines activation. Lung damage is associated with significant mortality, primarily due to the development of severe inflammation and pulmonary edema. On the other hand, citicoline, a key intermediate in the phosphatidylcholine biosynthesis pathway, has neurovascular protective and reparative properties. Numerous studies have highlighted the potential antioxidant and antiinflammatory effects of this agent in the context of various pathological conditions.

Aim: Our study aimed to explore the potential protective effects of citicoline against oxidative stress, inflammation, and tissue damage caused by CP in an experimental model.

Methods: Rats were given 200 mg/kg of CP as a single dose, either alone or in combination with citicoline (500 or 250 mg/kg), administered orally once daily for 14 days, beginning 7 days prior to CP administration. On the final day of the experiment, all animals were euthanized, and lung tissues were collected for further analysis.

Results: CP administration led to a significant elevation in the lung-to-body weight ratio, inflammatory cell infiltration, and elevated levels of lactate dehydrogenase, total protein, nuclear factor kappa B, Interleukin-1β, NOD-like receptor protein 3, and caspase-1 in bronchoalveolar lavage fluid. Furthermore, CP treatment increased the concentrations of malondialdehyde and nitrate/nitrite and reduced glutathione levels in the lungs. Additionally, mRNA of Interleukin 6 and tumor necrosis factor-α levels were significantly elevated. These biochemical alterations were corroborated by histopathological findings, which revealed significant lung tissue damage. However, treatment with citicoline significantly reduced the pulmonary pathological changes induced by CP.

Conclusion: These findings imply that citicoline's antiinflammatory and antioxidant properties provide protection against CP-induced lung damage.

Keywords: Citicoline; Cyclophosphamide; Lung injury; NF-kB; NLRP-3.

Fig. 1.. Impact of citi on CP-induced alteration of total and differential leucocytic counts in the BALF and lung wt/body wt ratio. The impact of oral daily citi treatment (250, 500 mg/kg) for 14 days with a single i.p. injection of CP (200 mg/kg) on day 7 on: A. Total cell count; B. Neutrophils; C. Eosinophils; D. macrophages; E. lymphocytes; F. lung wt/body wt ratio. Values are the mean ± SEM. n = 6. *** p < 0.001, ** p < 0.01, * p < 0.05 versus the control; ### p < 0.001, ## p < 0.01 versus the CP group; $$ p < 0.01, $ p < 0.05 versus the citi 250 group. CP: cyclophosphamide, citi: citicoline, BALF: bronchoalveolar lavage fluid.

Fig. 2.. Influence of citi on CP-induced changes in LDH levels and protein content in BALF: The impact of oral daily citi treatment (250, 500 mg/kg) for 14 days with a single i.p. injection of CP (200 mg/kg) on day 7 on: A. LDH level; B. protein content. The mean ± SEM is used to express data. n = 6. ***p < 0.001, ** p < 0.01 versus the control; ### p < 0.001, # p < 0.01 versus the CP group. CP: cyclophosphamide, citi: citicoline, LDH: lactate dehydrogenase, BALF: bronchoalveolar lavage fluid.

Fig. 3.. Impact of citi on oxidative injury and antioxidant suppression caused by CP in lung tissue homogenates: The impact of oral daily citi treatment (250, 500 mg/kg) for 14 days with a single i.p. injection of CP (200 mg/kg) on day 7 on: A. MDA; B. NOx; C. GSH. The mean ± SEM is used to express data. n = 6. ***p < 0.001, ** p < 0.01, * p < 0.05 versus the control; ## p < 0.01, # p < 0.05 versus the CP group; $ p < 0.05 versus the citi 250 group. CP: cyclophosphamide, citi: citicoline, MDA: malondialdehyde, NOx: total nitrate/nitrite, GSH: reduced glutathione.

Fig. 4.. Impact of citi on CP-induced alteration of proinflammatory cytokines and NF-kB: The impact of oral daily citi treatment (250, 500 mg/kg) for 14 days with a single i.p. injection of CP (200 mg/kg) on day 7 on: A. Relative mRNA expression of TNF-α in lung tissue; B. Relative mRNA expression of IL-6 in lung tissue; C. IL-1B level in BALF; D. NF-kB level in BALF. The mean ± SEM is used to express data. n = 6. ***p < 0.001, ** p < 0.01 versus the control; ### p < 0.001, ## p < 0.01 versus the CP group; $ p < 0.05 versus the citi 250 group. CP: cyclophosphamide, citi: citicoline, TNF-α: tumor necrosis factor-α, IL-6: Interleukin-6, IL-1B: Interleukin-1B, NF-kB: nuclear factor kappa B, BALF: bronchoalveolar lavage fluid.

Fig. 5.. Impact of citi on CP-induced activation of the NLRP3 inflammasome pathway: The impact of oral daily citi treatment (250, 500 mg/kg) for 14 days with a single i.p. injection of CP (200 mg/kg) on day 7 on: A. NLRP3 level in BALF; B. Caspase-1 level in BALF. The mean ± SEM is used to express data. n = 6. ***p < 0.001, ** p < 0.01, * p < 0.05 versus the control; ### p < 0.001, ## p < 0.01 versus the CP group; $ p < 0.05 versus the citi 250 group. CP: cyclophosphamide, citi: citicoline, NLRP3: NOD-like receptor protein 3, BALF: bronchoalveolar lavage fluid.

Fig. 6.. Effect of citi on CP-induced histopathological lung damage: CP induced thickening of the alveolar wall (black arrow) with numerous inflammatory cell infiltrations (blue arrows), together with hemorrhage and congestion. This condition is associated with arterial hypertrophy (red arrow) and emphysema (*). Meanwhile, the deleterious manifestations of CP were dose-dependently mitigated by citi treatment. Lung sections were stained with H&E. Low magnification ×:100 bar 100 and high magnification ×:400 bar 50. CP: cyclophosphamide, citi: citicoline, H&E: Hematoxylin and eosin.