Ameliorating effect of probiotic on nonalcoholic fatty liver disease and lipolytic gene expression in rabbits

**Background:** Nonalcoholic fatty liver disease (NAFLD) is a growing health concern, with prevalence expected to increase by 18% by 2030 and a 56% increase in NASH patients in the US. NAFLD is associated with increased mortality from heart disease, hepatocellular cancer, and liver-related events. Several genes have been linked to NAFLD development, including PNPLA3, GCKR, MBOAT7, and TM6SF2. Lipolytic genes such as lipoprotein lipase (LPL), low-density lipoprotein receptor (LDLr), hepatic lipase (HL), and cholesteryl ester transfer protein (CETP) play important roles in lipid metabolism. Probiotic treatment, particularly Lactobacillus species, has been shown to decrease NAFLD symptoms in animal models by improving liver function, restoring gut flora, and improving lipid profile. This study aimed to determine the effect of a high cholesterol diet (HCD) inducing NAFLD on lipolytic gene expression, liver function, lipid profile, and antioxidant enzymes in rabbits and the modulatory effects of probiotic Lactobacillus acidophilus.

**Methods:** Forty-five male New Zealand white rabbits (8 weeks old, ~1200 g) were divided into three groups: Group I received a basal diet, Group II received a high cholesterol diet (HCD, 2% cholesterol), and Group III received HCD with probiotic Lactobacillus acidophilus (1 g/L water). The trial lasted 8 weeks after a 2-week adaptation period. Liver samples were collected for histopathological examination and quantitative real-time PCR analysis of LPL, LDLr, HL, and CETP gene expression. Blood samples were analyzed for ALT, AST, ALP, LDH, cholesterol, triglyceride, HDL, LDL, total protein, albumin, glucose, and total bilirubin. Liver antioxidants (GPx, CAT, GSH, SOD) were determined in liver tissue homogenate. Statistical analysis was performed using SPSS with independent sample T-test (P ≤ 0.05).

**Key Results:** Histopathological examination showed that the control group had normal liver structure, the HCD group exhibited pronounced hepatic vacuolation with fat cytoplasmic vacuoles (grade 2, 50 ± 1.58% fat), and the HCD + probiotic group showed significantly reduced hepatic fatty changes (grade 1, 20 ± 2.89% fat). The HCD group showed a significant (P ≤ 0.05) increase in LPL, HL, and CETP gene expression and a significant decrease in LDLr gene expression compared to the control and probiotic-supplemented groups. Probiotic supplementation reversed these effects, with no significant differences between the control and probiotic-supplemented groups for all genes studied. Liver enzymes (ALT, AST, ALP, LDH) were significantly higher in the HCD group than in other groups, with no significant differences between the control and HCD + probiotic groups. The HCD group showed a significant increase in cholesterol, triglycerides, and LDL with a significant decrease in HDL compared to the control and probiotic-supplemented groups. The HCD group also showed a marked increase in glucose and total bilirubin and a marked decrease in total protein and albumin. Liver antioxidants (GPx, CAT, GSH, SOD) were significantly (P ≤ 0.05) lower in the HCD group compared to the control and probiotic-supplemented groups, with no significant differences between the probiotic-supplemented and control groups.

**Clinical Implications:** This study demonstrates that Lactobacillus acidophilus supplementation can ameliorate HCD-induced NAFLD in rabbits by reversing dysregulated lipolytic gene expression, improving liver function tests, normalizing lipid profile, and restoring antioxidant enzyme levels. The cholesterol-fed rabbit model shares many physiopathological characteristics of human NAFLD without exhibiting insulin resistance or obesity, making it useful for studying hyperlipidemia-related NAFLD mechanisms. These findings support the potential therapeutic role of probiotics in managing NAFLD, though clinical trials in humans are needed to confirm these effects. The study suggests that probiotics may work through multiple mechanisms including modulation of bile salt deconjugation, regulation of lipolytic gene expression, and reduction of oxidative stress.