This study aims to investigate the synergistic protective effects and underlying mechanisms of the combined application of Puerariae Lobatae Radix polysaccharide 1(PLP1) and betaine in alcohol-induced liver and intestinal injuries in mice and AML-12 hepatocytes. An alcoholic liver disease(ALD) mouse model was established according to the NIAAA protocol using chronic ethanol feeding combined with acute ethanol gavage. Mice were randomly divided into six groups: control, model, PLP1 monotherapy, betaine monotherapy, low-dose combination(LD-Comb), and high-dose combination(HD-Comb). Complementary in vitro studies involved ethanol-injured AML-12 mouse hepatocytes, divided into eight groups: control, model, PLP1, betaine, LD-Comb, medium-dose(MD-Comb), HD-Comb, and LD-Comb + lipopolysaccharide(LPS, 1 μg·mL~(-1)). Hepatic and intestinal histopathological changes were evaluated using hematoxylin-eosin(HE) staining, Sirius red staining, and immunohistochemistry. Alanine aminotransferase(ALT), aspartate aminotransferase(AST), alcohol dehydrogenase(ADH), and aldehyde dehydrogenase(ALDH) were quantified by biochemical assays. Enzyme-linked immunosorbent assays(ELISA) measured tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), interleukin-1β(IL-1β), and LPS levels. Protein expressions of cytochrome P450 2E1(CYP2E1), zonula occludens-1(ZO-1), Occludin, mucin-2(MUC-2), Toll-like receptor 4(TLR4), nuclear factor-κB(NF-κB), and myeloid differentiation primary response 88(MyD88) were analyzed using Western blot, immunohistochemistry, and immunofluorescence. The mRNA expression of TLR4, NF-κB, and MyD88 was analyzed by quantitative PCR(qPCR). The results showed that, compared with the model group, PLP1 or betaine monotherapy significantly alleviated hepatic edema and inflammatory infiltration, reduced collagen deposition, increased intestinal mucus thickness, restored goblet cell count, and decreased serum levels of IL-6, TNF-α, IL-1β, LPS, ALT, and AST, while enhancing ADH and ALDH activity. Concurrently, CYP2E1, TLR4, NF-κB, and MyD88 expression levels were downregulated, and the expression of ZO-1, occludin, and MUC-2 in colon tissues was upregulated. The combined treatment of PLP1 and betaine further enhanced these improvements compared with monotherapies, although the difference between the LD-Comb and HD-Comb groups was not statistically significant. In AML-12 cells, PLP1 or betaine monotherapy significantly reduced ALT, AST, IL-6, and TNF-α levels in the culture supernatant and downregulated the expression of TLR4, NF-κB, and MyD88. The combined treatment showed stronger effects than monotherapy, while co-treatment with the TLR4 agonist LPS partially reversed these benefits. In conclusion, the combination of PLP1 and betaine exerts synergistic protective effects by enhancing ethanol metabolism, promoting tight junction protein synthesis, repairing intestinal barrier function, reducing LPS translocation, and suppressing the activation of the TLR4 signaling pathway, thereby lowering inflammatory cytokine levels. This multi-targeted synergy effectively alleviates liver and intestinal injury in ALD mice and ethanol-injured AML-12 hepatocytes.
