Chlorella vulgaris and Lactobacillus casei Combination Therapy Attenuates Testicular Endoplasmic Reticulum Stress and Dysfunction in a C57BL/6 Mouse Model of NAFLD

Nonalcoholic fatty liver disease (NAFLD) is a common metabolic disorder closely associated with increased oxidative stress and a range of chronic conditions, including male infertility. Previous studies have indicated that Chlorella vulgaris (CV) and Lactobacillus casei (LBC) exert beneficial effects on NAFLD. This study aimed to evaluate the impact of CV and LBC supplementation on testicular dysfunction in a high-fat diet (HFD)-induced NAFLD mouse model, with a focus on the unfolded protein response (UPR) signaling pathway. Male C57BL/6 mice were randomly assigned to five groups: control, NAFLD (HFD for 8 weeks), NAFLD + CV (8 weeks of HFD, followed by 35 days of normal diet supplemented with CV 150 mg/kg), NAFLD + LBC (HFD followed by 35 days of LBC 10⁸ CFU/ml), and NAFLD + CV+LBC (combined treatment). Total antioxidant capacity (TAC), malondialdehyde (MDA), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels were assessed. Expression of UPR- and apoptosis-related genes was quantified, and histopathological evaluations, including the modified Johnson scoring system, were conducted. CV and LBC supplementation significantly increased serum TAC and testosterone, while reducing MDA, LH, and FSH levels (p < 0.05). Treated groups showed upregulation of UPR genes (GRP78, IRE1α, PERK, ATF6) and downregulation of pro-apoptotic markers (Caspase-3, BAX/Bcl-2 ratio). Histopathological analyses revealed improved testicular structure and sperm parameters, particularly in the combined treatment group. CV and LBC ameliorated the testicular dysfunction in NAFLD by mitigating oxidative stress and modulating UPR and apoptosis pathways. These findings suggest a promising therapeutic strategy warranting further clinical investigation.