Integrating network pharmacology, molecular docking, and experimental validation to elucidate the functional mechanism of Betulin in treating diabetic neuropathy

Betulin (BET) is a naturally occurring triterpenoid, primarily found in the bark of the Betula species. Although it has demonstrated antidiabetic and neuroprotective properties, the underlying mechanism by which it alleviates diabetic neuropathy (DN) remains unclear. The present study investigates the therapeutic efficacy of BET (40 mg/kg) in a high-fat diet-fed, streptozotocin (HFD/STZ)-induced rat model of DN through an integrated strategy combining network pharmacology, molecular docking, and experimental validation. Antioxidant capacity was assessed using ORAC (AUC, BET: 15.54; better than Trolox: 12.06) and CAP-e tests (IC 50 : 17.22 ± 0.39 µg/mL). BET exhibited mixed-competitive inhibition of α-amylase (IC 50 44.64 ± 1.25 µg/mL) and competitive inhibition of α-glucosidase (9.52 ± 0.70 µg/mL). BET demonstrated potent inhibition of acetylcholinesterase (0.93 ± 0.12 µg/mL) and butyrylcholinesterase activities (5.02 ± 0.52 µg/mL). BET improved markers of hyperglycemia, hepatic, and dyslipidemia. It improved oxidative and inflammatory markers, increased cholinesterase and BDNF levels, decreased Aβ 42 -aggregation, inhibited abnormal MAP-τ phosphorylation, lowered BACE1-expression, and raised ATP1A3 in DN rats. Histological examination showed improved pancreatic tissue integrity and neuronal cell morphology in DN rats. Network pharmacology revealed 67 potential targets between BET and DN, such as EGFR, STAT3, ESR1, MTOR, GSK3B, NFKB1, HSP90AA1, JAK2, PTGS2, ACHE, BCHE, INSR, NTRK1, and VEGF in the PPI network. KEGG analysis revealed IR, AGE-RAGE, PI3K-Akt, and insulin signalling pathways. Strong binding affinities between BET and pathogenic targets (AChE: −7.1, BChE: −10.4, TNF-α: −8.6, IL-6: −7.0, hs-CRP: −7.3, BACE1: −8.2, Atp1a3: −7.3 kcal/mol). BET combated hyperglycemia-induced oxidative stress, improved neurotransmitters, alleviated neuropeptides, and inflammation in DN rats due to the antioxidant capacity offered by the triterpenoid skeleton possessing OH-groups at C-3 and C-28 positions.