Cyclophosphamide: Potential Hepatorenal Toxicity and the Possible Therapeutic Role of Mesenchymal Stem Cell-Derived Exosomes in Wistar Rats.

This study aimed to examine therapeutic impact of exosomes derived from adipose tissue- mesenchymal stem cells (AD-MSCs-Exos) on a rat model of hepatorenal toxicity. 32 Wistar male rats were grouped into 4 groups: Control group, rats received intraperitoneally (i.p.) phosphate buffered saline (PBS). Cyclophosphamide (CTX) group, rats injected i.p. with a single dose of CTX (50 mg/kg) followed by rotating doses of 8 mg/kg of CTX daily for 2 weeks. CTX + AD-MSCs group, rats infused with (1 × 106 AD-MSCs cells/rat) dissolved in PBS intravenously (i.v.) day after day for 1 week starting from second day of CTX last dose. CTX + AD-MSCs-Exos group, rats injected with 100 μg of Exos derived from AD-MSCs in 1 ml PBS by i.v. injection for 1 week starting from second day of CTX last dose. 5 weeks following initial CTX dose, blood, liver, and kidneys extracted. Serum alanine transaminase (ALT), aspartate transaminase (AST), creatinine and urea levels; hepatic malate dehydrogenase (MDH) and glutamate dehydrogenase (GLDH); renal kidney injury molecule-1 (KIM-1) and clusterin measured. Tumor necrosis factor alpha (TNF-α) and malonialdehyde (MDA) were estimated in hepatic and renal tissues. Furthermore, nuclear factor kappa B/toll like receptor-4 (NF-κB/TLR-4), nuclear factor erythroid 2- related factor 2/heme oxygenase-1 (Nrf-2/HO-1) and BCL-2-associated X protein/B-cell lymphoma 2 (Bax/Bcl-2) signaling pathways were analyzed by qRT-PCR. Immunohistochemical staining for cyclooxygenase-2 "COX-2" and inducible nitric oxide synthase "iNOS" performed in hepatic and renal tissues. Finally, histopathological investigation of both liver and kidney tissue carried out. Treatment with MSCs and their derived exosomes markedly improved antioxidant and anti-inflammatory markers while reducing apoptotic gene expression compared to CTX group. Specifically, in liver tissues; Bax expression decreased 2.6-fold and 3.3-fold, while Bcl-2 increased 3.1-fold and 1.8-fold in AD-MSCs- and AD-MSCs-Exos groups, respectively. Similarly, NF-κB was reduced 1.5-fold and 2.8-fold; Nrf2 and HO-1 were upregulated by approximately 7.4- and 5.2-fold (AD-MSCs) and 7.4- and 6.2-fold (AD-MSCs Exos), respectively compared to CTX group. In kidney tissues, Compared to CTX group, Bax expression decreased by approximately 1.5-fold and 3.6-fold, whereas Bcl-2 increased 3.0-fold and 4.4-fold in AD-MSCs- and AD-MSCs Exos groups, respectively. NF-κB and TLR-4 both were downregulated by ~2.6 (AD-MSCs) and 7.9, 2.1-fold (AD-MSCs Exos), while Nrf2 and HO-1 were upregulated by 12.1 and 2.2- fold (AD-MSCs); 12.8- and 3.0-fold (AD-MSCs Exos), respectively. These findings confirm that both treatments mitigate CTX induced hepatorenal injury through modulation of apoptosis, inflammation, and oxidative stress pathways, with exosomes exhibiting slightly superior therapeutics efficacy. Also, immunological and histopathological investigation verified curative effect of MSCs-Exos against CTX-induced hepatorenal toxicity. These findings highlight that both AD-MSCs and their exosomes confer significant therapeutic effect against CTX-induced hepatorenal toxicity through modulation of apoptosis, inflammation, and oxidative stress pathways. Importantly, AD-MSCs-Exos demonstrated superior therapeutic efficacy compared to AD-MSCs in restoring antioxidant defenses and attenuating inflammatory and apoptotic responses in both liver and kidney tissues.