Chitosan nanoparticle encapsulation increased the prophylactic efficacy of Lactobacillus plantarum RM1 against AFM1-induced hepatorenal toxicity in rats

Abstract

Aflatoxin M₁ (AFM₁) is a significant contaminant of food, particularly dairy products and can resist various industrial processes. Several probiotic strains like Lactobacillus plantarum are known to reduce aflatoxin availability in synthetic media and some food products. The current work investigated the possible chitosan coating prophylactic efficacy of Lactobacillus plantarum RM1 nanoemulsion (CS-RM1) against AFM₁-induced hepatorenal toxicity in rats. Twenty-eight male Wistar rats were divided into four groups (n = 7) as follows: group 1 received normal saline, group 2 received CS-RM1 (1mL contains 6.7 × 10¹⁰ CFU), group 3 received AFM₁ (60 µg/kg bwt), and group 4 received both CS-RM1(1 mL contains 6.7 × 10¹⁰ CFU) and AFM₁ (60 µg/kg bwt). All receiving materials were given to rats daily via oral gavage for 28 days. AFM₁ caused a significant elevation in serum levels of ALT, AST, ALP, uric acid, urea, and creatinine with marked alterations in protein and lipid profiles. Additionally, AFM₁ caused marked pathological changes in the liver and kidneys, such as cellular necrosis, vascular congestion, and interstitial inflammation. AFM₁ also increased the MDA levels and decreased several enzymatic and non-enzymatic antioxidants. Liver and kidney sections of the AFM₁ group displayed strong caspase-3, TNF-α, and iNOS immunopositivity. Co-treatment of CS-RM1 with AFM₁ significantly lowered the investigated toxicological parameter changes and markedly improved the microscopic appearance of liver and kidneys. In conclusion, AFM₁ induces hepatorenal oxidative stress damage via ROS overgeneration, which induces mitochondrial caspase-3-dependent apoptosis and inflammation. Furthermore, CS-RM1 can reduce AFM₁ toxicity in both the liver and kidneys. The study recommends adding CS-RM1 to milk and milk products for AFM₁-elimination.

Keywords: Aflatoxins M1; Chitosan nanoparticles; Hepatorenal toxicity; Lactobacillus planterum; Oxidative stress.

Fig. 1

The size distribution curve (A) and HR-TEM image (B) of the prepared nanoemulsion

Fig. 2

Hematoxylin and eosin stained hepatic photographs representing different experimental groups. A The control group exhibits normal histology. B–F The AFM₁ group exhibits severe histological abnormalities. Note: oval cell hyperplasia (black arrow), focal coagulative necrosis (black star), hepatocellular vacuolization (black triangle) and necrosis (blue triangle), portal fibroplasia (blue star), portal inflammation (blue arrow). G–H The AFM₁ + CS-RM1 group displayed sparse cell necrosis (blue triangle) with moderate portal inflammatory cell infiltration (blue arrow)

Fig. 3

Hematoxylin and eosin-stained kidney photographs representing different experimental groups. A The control group exhibits normal histology. B–C The AFM₁ group exhibits moderate histological abnormalities. Note: vascular congestion (red star), RBC extravasation (red triangle), cytoplasmic vacuolization (black triangle), necrosis (back arrow) and desquamation (blue arrows) of tubular epithelium, glomerular damage (circle), widening of bowman’s space (black star). D The AFM₁ + CS-RM1 group displayed sparse vacuolar degeneration (black arrow) of tubular epithelium

Fig. 4

Bar chart representing the microscopic lesion scoring in the liver (A) and kidneys (B). Values were expressed as median (n = 35 random microscopic fields/group). Different uppercase letters (a, b, c, d) mean significant difference at P ≤ 0.05

Fig. 5

A–I Photographs representing the protein expression of caspase-3, iNOS, and TNF-α in the liver of different groups. A–C The control group showed negative expression of the studied immune markers. D–F) The AFM₁ group displayed strong immunopositivity of the above-mentioned immune markers. G–I The AFM₁ + CS-RM1 group displayed weak reaction for all immune markers. J Bar chart representing the mean percentage area of caspase-3, iNOS, and TNF-α immunostaining in the liver of different groups. Values were expressed as mean ± SEM (n = 35 random microscopic fields/group). The lowercase letter (a) means a significant difference from the AFM₁ group, while (b) means a significant difference from the control group at P ≤ 0.05

Fig. 6

A–I Photographs representing the protein expression of caspase-3, iNOS, and TNF-α in kidneys of different groups. A–C The control group showed negative expression of the studied immune markers. D–F The AFM₁ group displayed strong immunopositivity of the above-mentioned immune markers. G–I The AFM₁ + CS-RM1 group displayed weak reaction for all immune markers. J Bar chart representing the mean percentage area of caspase-3, iNOS, and TNF-α immunostaining in the kidneys of different groups. Values were expressed as mean ± SEM (n = 35 random microscopic fields/group). The lowercase letter (a) means a significant difference from the AFM₁ group, while (b) means a significant difference from the control group at P ≤ 0.05