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Treg-NASHcm and HFMCDm increased the inflammasome and inhibited the proliferative H9c2 cells.

Flow cytometry-assessed relative intracellular levels (/buffer group) of (A) FXR, IL-10R, IL-10, (B) NLRP3, caspase-1 among various CM pre-treated H9c2. Bar graph of (C) immunofluorescence-assessed Ki-67+cells (red color), TUNEL+ cells (green color), and (D) Annexin-5+PI+ (orange color) cells in all H9c2 cells. Scale bars in all images are 100 mm; CD4+CD25+ Treg-NASH+OCA [supernatant of Treg cells from NASH mice as condition medium (cm) plus acute OCA incubation]; field of view (FOV).</p

Graphical summary of the pathogenic mechanisms and effects of chronic OCA treatments on mice with NASH-related cardiac dysfunction.

FM: Fat mass; FS: Fractional shortening; FXR: Farnesoid X receptor; SHP2: Small heterodimer partner 2; Treg: T regulatory cell; Treg-NASH: Treg cells of NASH mice; cm: Condition medium; HFMCD: High-fat and methionine and choline deficiency.</p

OCA treatment-decrease in the collagen deposition was associated with the increase in the fraction of shortening in NASH hearts.

Correlation between (A) OCA-induced decrease in cardiac fibrosis (% of sirius red stained positive area) and (B) OCA-induced increase in fraction of shortening (FS) with OCA-induced increase in expressions of cardiac FXR/IL-10 levels, and infiltrated IL-10R+ Treg cells (CD4+CD25+) in NASH mice heart. (C) Flow cytometry assessed intracellular FXR and NLRP3 levels in Ctrl and NASH group as well as percentile change in FXR and NLRP3 levels by OCA treatment. (D) Immunofluorescence-measured caspase-1+ cells (green color) in primary isolated splenic regulatory T (Treg cells) of Crtl and NASH group as well as percentile decrease in caspase-1+ and IL-1β+ cells by OCA treatment. 20x, Scale bar: 100μm; **,***: pvs. Ctrl or NASH group.</p

Hemodynamic and cardiac injury parameters of mice in different groups.

Hemodynamic and cardiac injury parameters of mice in different groups.</p

Cardiac fibrosis of NASH mice was associated with increased infiltrated CD3⁺ T cells but decreased cardiac Treg-related anti-inflammatory cytokines (IL-10/IL-10R).

(A) Sirius red-assessed cardiac fibrosis. (B) Various cardiac pathogenic protein markers expression. (C) Cardiac levels of caspase-1 and IL-10. (D) Mean degree of cardiac fibrosis and fraction of shortening among mice with mild, moderate, and severe cardiac inflammation. (E) Cardiac CD3+T cells (green color) and IL-10R+ Treg cells (red color) infiltration. **,***: pvs. Ctrl or NASH group.</p

Chronic OCA treatment suppresses NASH-related cardiac dysfunction.

(A) hepatic NAFLD score. (B-C) fraction of shortening (FS). (D) LV mass. (E-F) cardiac H&E staining for inflammation. 20x, Scale bar: 100μm; **,***: pvs. Ctrl or NASH group. FS=(LVDd-LVDs)/LVDd×100%; LVDd: LV diastolic dimension; LVDs: lLV systolic dimension; LVEF=(LVEDV-LVESV)/LVEDV×100% (LVEDV, LV end-diastolic volume; LVESV, LV end-systolic volume). The degree of cardiac inflammation was classified as mild (mononuclear cell infiltration 50%).</p

Acute OCA incubation normalised the HFMCDm and Treg-NASHcm suppressed differentiation and contraction of H9c2 cells.

(A) Expression of the differentiation markers [cardiac myosin heavy chain (MYH), myogenin, TNNI] levels (/Buffer group) in supernatant of H9c2 cells. (B) The collagen-1 and αSMA positive among H9c2 cells. (C) Collagen gel contraction assay for evaluating the function of cardiomyocyte after various pretreatment. Scale bar = 20 μm. CD4+CD25+ Treg-NASH+OCA [supernatant of Treg cells from NASH mice as condition medium (cm) plus acute OCA incubation].</p

OCA-treatment increases the splenic proliferative/activate Treg cells and decreased the apoptotic Treg cells among primary CD4⁺CD25⁺ splenocytes of NASH mice.

IF-based assessments of (A) Foxp3+Ki-67+ (orange color), (B) Foxp3+HLADR+ (orange color), (C) TUNEL+ (green color) (D) Annexin-5+Pi+ (green color) cells among primary CD4+CD25+ Treg cells from Ctrl, Ctrl-OCA, or NASH, NASH-OCA mice. (E) Foxp3+Ki-67+, Foxp3+HLADR+, TUNEL+, and Annexin-5+Pi+ cells among primary CD4+CD25+ Treg cells from Ctrl and NASH mice. (F) percentile increase in Foxp3+Ki-67+, Foxp3+HLADR+, and percentile decrease in TUNEL+, Annexin-5+Pi+ cells in primary CD4+CD25+ Treg cells by OCA treatment. **,***: pvs. Ctrl or NASH group.</p