Effects And Mechanisms Of Triptolide On Cardiac Inflammation In Diabetic Rats

Objective:Given the importance of inflammation in the onset and progression of diabetic cardiomyopathy, we investigated the potential protective effects of triptolide, an anti-inflammatory agent, in streptozotocin-induced diabetic rat model and in H9c2rat cardiac cells exposed to high glucose (HG).Methods:1. In vitro study:H9c2rat cardiac cells were divided into three groups (n=6per group) and cultured for48hours:(1) D-glucose (5.5mmol/1);(2) high D-glucose (33mmol/1);(3) co-stimulated with high D-glucose (33mmol/1) and triptolide (20ng/ml).Then, H9c2cells were harvested and processed for the downstream assays. Cellular morphology was observed under inverted phase contrast microscope. Cell viability was determined by MTT assay. The mRNA and protein expressions of TNF-a and NF-κB p65were analysed by quantitative real-time RT-PCR and western blot, respectively. The activity of NF-κB p65was assessed by TransAMTM NF-κB p65ELISA.2. In vivo study:Diabetes mellitus (DM) was induced in8-week-old male Sprague-Dawley (SD) rats by a single intraperitoneal injection of streptozocin. SD rats injected with sodium citrate buffer only were referred to as controls. The1-week diabetic rats and controls were randomly divided into six groups (n=10per group), and treatment was given by daily gastric gavage for6weeks:(1) Control group:1ml/day saline;(2) Control+triptolide group:400μg/kg/day triptolide;(3) DM group:lml/day saline;(4) DM+low-dose triptolide group:100μg/kg/day triptolide;(5) DM+medium-dose triptolide group:200μg/kg/day triptolide; (6) DM+high-dose triptolide group:400μg/kg/day triptolide.At the end of this study, after cardiac function measurements were obtained, rats were sacrificed and their hearts were harvested for further histologic and molecular biologic analysis. In addition, blood from the aorta was collected to assess the safety of triptolide treatment. Hematoxylin-eosin staining was applied to observe the pathologic changes of cardiac tissue. Sirius red staining was used to assess total cardiac collagen content. For immunohistochemistry, we observed collagen deposition (collagen Ⅰ and collagen Ⅲ), and infiltration of inflammatory cells (macrophages/CD68+and T lymphocytes/CD3+) in the rat hearts. The mRNA and protein expressions of TNF-α, IL-1β, ICAM-1, VCAM-1and NF-κB p65were analysed by quantitative real-time RT-PCR and western blot, respectively. The activity of NF-κB p65was assessed by TransAMTM NF-κB p65ELISA.Results:1. In vitro study:(1) Compared with controls, H9c2cells incubated with HG for48hours displayed overt inflammatory response, as shown by the enhanced activity of NF-κB p65, as well as increased mRNA and protein expressions of TNF-α and NF-κB p65.(2) Triptolide markedly inhibited HG-induced inflammation.2. In vivo study:(1) Compared with controls, the untreated and triptolide-treatment diabetic groups displayed severe hyperglycemia, decreased body weight, and higher heart weight to body weight ratio. Triptolide treatment did not affect the above parameters in diabetic groups.(2) Compared with controls, the left ventricular (LV) dimension indices were significantly higher in diabetic groups, indicating LV dilatation in diabetic rats. Accordingly, the LV function was impaired. Importantly, triptolide treatment significantly improved LV dysfunction in diabetic rats, as shown by increased LVEF.(3) Total collagen content was significantly increased in both interstitial and perivascular sites in diabetic rats compared with controls. Accordingly, collagen I and collagen III were increased in these rats. Triptolide treatment attenuated diabetic-induced cardiac fibrosis.(4) Compared with controls, diabetic rats displayed overt intramyocardial inflammation, as evidenced by enhanced activity and expression of NF-κB p65, thus leading to increased levels of cardiac pro-inflammatory cytokines (TNF-α, IL-1β), enhanced expressions of cell adhesion molecules (ICAM-1, VCAM-1), and activated invading immunocompetent cells (macrophages, T lymphocytes). Triptolide treatment significantly attenuated cardiac inflammation through suppressing the activity and the expression of NF-κB.(5) Triptolide treatment (100μg/kg/day～400μg/kg/day) did not show liver, kidney or cardiac toxicity. However, besides its prominent anti-inflammatory effect, triptolide as an immunoregulatory agent also showed the immunosuppressive activity (400μg/kg/day), thus leading to reduced expression of inflammatory mediators in non-diabetic rats.Conclusion:1. NF-κB signaling pathway plays an important role in hyperglycemia-induced inflammation both in vitro and in vivo, and participates in the onset and progression of diabetic cardiomyopathy.2. Triptolide treatment significantly attenuates cardiac inflammation and fibrosis through suppressing the activity and the expression of NF-κB, resulting in improved LV function under diabetic conditions, suggesting triptolide treatment might be beneficial in diabetic cardiomyopathy.