The protective effect of Chlorella vulgaris against diclofenac toxicity in Clarias gariepinus: haemato-immunological parameters and spleen histological features as outcome markers

Abstract

Introduction: Diclofenac (DCF) is a commonly utilized medication in the non-steroidal anti-inflammatory drug category that is released into aquatic systems in significant amounts. Chlorella vulgaris (C. vulgaris) is rich in active phytochemicals known for their haemato-immunological boosting properties.

Methods: Our objective was to investigate the haemato-immunological protective properties of Chlorella in mitigating the toxic effects of DCF. Five groups of Clarias gariepinus, each comprising 36 fish, were assigned over a two-week period. The groups were assigned as follows: control group, which received a basal diet only; DCF1 group, which received a basal diet and was exposed to 20 μg/L of DCF; DCF2 group, which received a basal diet and was exposed to 10 mg/L of DCF; and Chlorella +DCF1 and Chlorella+DCF2 groups, which were exposed to the same DCF doses as Groups 2 and 3, respectively, while also being fed a diet containing 25% Chlorella.

Results: Exposure to both doses of DCF significantly decreased erythrocyte count, hemoglobin content, white blood cell count, phagocytic index, and lysozyme activity, while increased eosinophil and neutrophil % in an equipotent manner. The low dose caused a more pronounced reduction in packed cell volume (PCV)% and large lymphocyte% compared to the high dose. A significant decline in platelet count was observed only with the low DCF dose, while the high dose led to a decrease in monocyte%. DCF intoxication led to a dose-related decrease in small lymphocyte% and an increase in erythrocyte morphological alterations and interleukin (IL)-6 levels. The DCF2 group exhibited a higher increase in apoptotic RBCs than the DCF1 group. Intervention with Chlorella alongside the two DCF doses significantly normalized RBC count and eosinophil %, increased PCV% and small lymphocyte%, and decreased erythrocyte abnormalities to an equal extent. Large lymphocyte% in the Chlorella+DCF1 group was successfully restored to normal levels. Phagocytic index and lysozyme activity in the supplemented groups were lower, while IL-6 levels were higher than in the DCF groups. The percentage of apoptotic cells decreased with Chlorella administration, with the Chlorella+DCF1 group showing fewer apoptotic cells than the Chlorella+DCF2 group. Histopathological deterioration and excessive collagen deposition were observed in the spleen of DCF groups, while notable improvements were seen following C. vulgaris supplementation.

Conclusion: These findings suggest that dietary inclusion of C. vulgaris may antagonize the haemato-cytological abnormalities induced by DCF intoxication.

Keywords: african catfish; haemato-immunology; microalgae; non-steroidal anti-inflammatory drug; physiology; spleen.

Figure 1

Represented blood smears from of C. gariepinus showing (a) the normal erythrocytes, (b) the deformed ones after exposure to DCF1, (c) the deformed ones after exposure to DCF2, (d) the deformed ones after exposure to Chorella+ DCF1, (e) the deformed ones after exposure to Chorella+ DCF2, and (f) The percentage of cell alteration and nuclear abnormalities of RBCs in C. gariepinus exposed to the effects of different doses of DCF for 14 days; Er, normal erythrocytes; Sch, schistocytic; Cr, crenated cell; Ac, acanthocyte; Va, Vacuolated cells; Hyc, hymolyzed cells (H & E stain, scale bar: 100 μm). Bars represent means ± SE of 6 fish/group. Different letters indicate significant differences among treatments (p < 0.05).

Figure 2

Apoptosis detection in red blood cells of C. gariepinus showing (a) control, (b) the apoptosis detection after exposure to DCF1, (c) apoptosis detection after exposure to DCF2, (d) apoptosis detection after exposure to Chorella+ DCF1, (e) apoptosis detection after exposure to Chorella+ DCF2, and (f) The percentage of apoptosis of RBCs in C. gariepinus exposed to the effects of different doses of DCF for 14 days; Bars represent means ± SE of 6 fish/group. Different letters indicate significant differences among treatments (p < 0.05).

Figure 3

Transverse section of fish spleen satin’s with (H&E) in all experimental groups. (A) Control, (B) fish exposed to DS1, (C) Fish exposed to DS1 + Chlorella, (D) Fish exposed to DS2, and (E) Fish exposed to DS2 + Chlorella. Notice: white pulp (WP), red pulp (RP) melanomacrophage cell (MC), melanomacrophage centers (MMC), ellipsoid bodies (EB), Edema (E), dilated blood vessel (DBV). X-400, Scale Bar= 50µm.

Figure 4

Transverse section of spleen of all experimental groups stained by Massion trichome stain for collagen fibers. (A) Control, (B) fish exposed to DS1, (C) Fish exposed to DS1 + Chlorella, (D) Fish exposed to DS2, and (E) Fish exposed to DS2 + Chlorella. Noticed: elebsoide bodes (EB), Ground substance (black arrow). X-400, Scale Bar= 50µm.