Lar end-systolic dimension; IVS (mm): intraventricular septal wall; LVPW (mm): left ventricular posterior wall thickness; FS: fractional shortening; A wave (cm/s): peak late diastolic flow velocity, E/A: ratio of peak early diastolic filling velocity to peak velocity at atrial contrations. **P,0.01, *P,0.05 vs. NC group; ## P,0.01, # P,0.05 vs. MS group. doi:10.1371/journal.pone.0067530.tlevel was decreased by 36 in aspirin group and 26 in HLJDT group (Fig. 5).HLJDT Improves Ultrastructure of CardiomyocytesTEM analysis showed the derangement of myofibers, and swollen mitochondria in cardiomyocytes in MS rats compared with the NC rats (Fig. 3). Moreover, after aspirin or HLJDT treatment, the cardiac ultrastructure was obviously improved (Fig. 3).HLJDT Decreases NF-kB p65 and ICAM-1 Levels in the HeartThe expression of NF-kB p65 and ICAM-1 in the rat hearts was evaluated by immunohistochemistry staining. In the MS rats, the expression of NF-kB p65 and ICAM-1 was significantly increased in the myocardial tissues compared to the NC group. However, both NF-kB p65 and ICAM-1 staining were decreased in either aspirin or HLJDT-treated rats compared to MS rats (Fig. 6).HLJDT Affects Collagen ContentsSirius red staining revealed an increase of Benzimidazole (DRB)] in nuclear extracts [11]. Thus, the presence of W049 protein interstitial fibrosis in the myocardium of MS rats compared with the NC group. Furthermore, a fraction of interstitial fibrosis was prevented by both aspirin and HLJDT (Fig. 4).HLJDT Downregulates the Expression of Inflammatory Factors in the HeartThe mRNA expression of IL-6, TNF-a, ICAM-1, collagen types I and III, TGF-b1and IKKb were significantly increased in MS rats compared to NC rats (P,0.05, Fig. 7). However, aspirin and HLJDT significantly attenuated the increased expression of IL-6, TNF-a, ICAM-1, collagen I, collagen III and TGF-b1 mRNA (P,0.05, Fig. 7). The interclass analysis showed that theseHLJDT Decreases Serum TNF-a LevelAt baseline, the mean value of TNF-a was similar in the obesefed and normal-fed rats. Serum TNF-a level of MS rats was 4 fold higher than in the controls at 16 weeks (Fig. 5). The difference persisted till the end of the study. However, the elevated TNF-aFigure 2. Transmitral inflow patterns (E and A wave) for MS rat at 22 and 34 weeks. Note increased A waves and decreased E/A in the MS group. doi:10.1371/journal.pone.0067530.gHuan-Lian-Jie-Du-Tang for Cardiac Damages in RatsFigure 3. Cardiac lesions measured by transmission electron microscopy. A: NC group (n = 10). Note the regular membrane and intercalated disc Time point of the experiment. Values are expressed as g 57 Fe between adjacent myocytes. B: MS group (n = 10). Interrupted capillary membrane, and derangement and increase of myofibers and mitochondria in cardiomyocytes were visible. Swollen mitochondria were shown by the arrows. C: MS+A group (n = 11). D: MS+H group (n = 11). Compared with MS group, the ultrastructural changes of drug-fed groups were obviously improved. Original magnification: 610, 000. doi:10.1371/journal.pone.0067530.gvalues were not significantly different between aspirin-treated and HLJDT-treated groups (P.0.05, Fig. 7).HLJDT Downregulates the Phosphorylation of IRS-1 in the HeartWestern blot analysis revealed a profound up-regulation of the levels of SOCS3 and phospho-JNK by inflammation. At the same time, serine phospho-IRS1 was increased and phospho-AKT was decreased in the MS rats compared to the NC group (P,0.05, Fig. 8). Treatment with aspirin markedly attenuated the increases of phospho-JNK and phospho-IRS1 levels (P,0.05, Fig. 8), while the lev.Lar end-systolic dimension; IVS (mm): intraventricular septal wall; LVPW (mm): left ventricular posterior wall thickness; FS: fractional shortening; A wave (cm/s): peak late diastolic flow velocity, E/A: ratio of peak early diastolic filling velocity to peak velocity at atrial contrations. **P,0.01, *P,0.05 vs. NC group; ## P,0.01, # P,0.05 vs. MS group. doi:10.1371/journal.pone.0067530.tlevel was decreased by 36 in aspirin group and 26 in HLJDT group (Fig. 5).HLJDT Improves Ultrastructure of CardiomyocytesTEM analysis showed the derangement of myofibers, and swollen mitochondria in cardiomyocytes in MS rats compared with the NC rats (Fig. 3). Moreover, after aspirin or HLJDT treatment, the cardiac ultrastructure was obviously improved (Fig. 3).HLJDT Decreases NF-kB p65 and ICAM-1 Levels in the HeartThe expression of NF-kB p65 and ICAM-1 in the rat hearts was evaluated by immunohistochemistry staining. In the MS rats, the expression of NF-kB p65 and ICAM-1 was significantly increased in the myocardial tissues compared to the NC group. However, both NF-kB p65 and ICAM-1 staining were decreased in either aspirin or HLJDT-treated rats compared to MS rats (Fig. 6).HLJDT Affects Collagen ContentsSirius red staining revealed an increase of interstitial fibrosis in the myocardium of MS rats compared with the NC group. Furthermore, a fraction of interstitial fibrosis was prevented by both aspirin and HLJDT (Fig. 4).HLJDT Downregulates the Expression of Inflammatory Factors in the HeartThe mRNA expression of IL-6, TNF-a, ICAM-1, collagen types I and III, TGF-b1and IKKb were significantly increased in MS rats compared to NC rats (P,0.05, Fig. 7). However, aspirin and HLJDT significantly attenuated the increased expression of IL-6, TNF-a, ICAM-1, collagen I, collagen III and TGF-b1 mRNA (P,0.05, Fig. 7). The interclass analysis showed that theseHLJDT Decreases Serum TNF-a LevelAt baseline, the mean value of TNF-a was similar in the obesefed and normal-fed rats. Serum TNF-a level of MS rats was 4 fold higher than in the controls at 16 weeks (Fig. 5). The difference persisted till the end of the study. However, the elevated TNF-aFigure 2. Transmitral inflow patterns (E and A wave) for MS rat at 22 and 34 weeks. Note increased A waves and decreased E/A in the MS group. doi:10.1371/journal.pone.0067530.gHuan-Lian-Jie-Du-Tang for Cardiac Damages in RatsFigure 3. Cardiac lesions measured by transmission electron microscopy. A: NC group (n = 10). Note the regular membrane and intercalated disc between adjacent myocytes. B: MS group (n = 10). Interrupted capillary membrane, and derangement and increase of myofibers and mitochondria in cardiomyocytes were visible. Swollen mitochondria were shown by the arrows. C: MS+A group (n = 11). D: MS+H group (n = 11). Compared with MS group, the ultrastructural changes of drug-fed groups were obviously improved. Original magnification: 610, 000. doi:10.1371/journal.pone.0067530.gvalues were not significantly different between aspirin-treated and HLJDT-treated groups (P.0.05, Fig. 7).HLJDT Downregulates the Phosphorylation of IRS-1 in the HeartWestern blot analysis revealed a profound up-regulation of the levels of SOCS3 and phospho-JNK by inflammation. At the same time, serine phospho-IRS1 was increased and phospho-AKT was decreased in the MS rats compared to the NC group (P,0.05, Fig. 8). Treatment with aspirin markedly attenuated the increases of phospho-JNK and phospho-IRS1 levels (P,0.05, Fig. 8), while the lev.
