To study the chemical constituents from Leonuri Fructus (the fruits of Leonurus japonicus Houtt.) and their hepatocellular protective effects and mechanisms.The chemical compounds were isolated from the n-butanol extract of Leonuri Fructus by macroporous resin,silica gel,HW-40F column chromatography,and semi-preparative HPLC.Their structures were determined by physicochemical properties and spectroscopic data.Finally,eight lignans were isolated and identified as (+)-isolariciresinol-9′-O-β-D-glucopyranoside (1),(-)-isolariciresinol-9′-O-β-D-glucopyranoside (2),densispicoside (3),(-)-isolariciresinol-4-O-β-D-glucopyranoside (4),isolariciresinol (5),phyllanglaucin B (6),(-)-7R,8S-7′,8′-dihydroxy-dihydrodehydroconiferyl alcohol-9-O-β-D-glucopyranoside (7),and (+)-7S,8R-7′,8′-dihydroxy-dihydrodehydroconiferyl alcohol-9-O-β-D-glucopyranoside (8).Compounds 1-8 were isolated from Leonuri Fructus for the first time.Next,MTT assay was used to detect the protective effects of the isolates on acetaminophen (APAP)-induced hepatocyte injury.The results showed that compounds 1 and 3 significantly improved the survival rate of HL-7702 cells injured by APAP.Furthermore,Hoechst 33342 fluorescence staining and Western blot were carried out to observe cell apoptosis in HL-7702 cells and the expression level of Bcl-2 and Bax of compound 3 which has the best hepatoprotective effects.The results showed that compound 3 reduced the nuclear density of HL-7702 cells and the numbers of apoptotic bodies,significantly promoted the expression of Bcl-2 while inhibited the expression of Bax,and increased the ratio of Bcl-2/Bax expression compared with model group.It indicated that compound 3 played a role in suppressing apoptosis in HL-7702 cells by regulating the expression of Bcl-2 and Bax proteins.