Gypenosides Improve High Glucose-induced Myocardial Injury By Inhibiting NLRP3 Inflammasome Activation

BackgroundDiabetes is a metabolic disease which threatens human life and health seriously, whose feature is high blood glucose. For the middle-late patients with diabetes, they are often accompanied by multiple organ damage complications, such as heart, kidney, brain and blood vessels, which are the main reasons leading the deteriorate of life and even death. Moreover, among these complications, the damage of heart is particularly serious. As one common kind of diabetic angiocardiopathy complications, diabetic cardiomyopathy(DCM) has been the major cause of heart failure and death. Previuosly researches show that: the dysfunction of vascular endothelial cell or myocardial cell plays an important role on the genesis and development of DCM. Moreover, high glucose can induce the oxidative stress, anomalous change of other cell signaling, the activation of rennin-angiotensin system and the increase in free fatty acid, which participate in the development of DCM. In recent years, more and more domestic and international studies reveal that inflammation also plays an important role in myocardial injury induced by diabetes. Inflammation can lead to injury of cell and tissue, which induces the death. NLRP3 inflammasome, as a significant form of inflammatory activation, plays an important role in tissue damage. Thus, inhibition of NLRP3 inflammasome activation may be an effective strategy to improve high glucose-induced myocardial injury. As a kind of traditional Chinese medicine, Gypenosides contribute in anti-inflammatory, and improve cardiovascular disease. However, the mechanism of Gypenosides is not clear. In this study, we intend to explore whether Gypenosides can inhibit the activation of NLRP3 inflammasome to improve high glucose-induced myocardial injury.PurposeEstablish high glucose-induced myocardial damage model in vitro and vivo, evaluate whether the NLRP3 inflammasome is involved in high glucose-induced myocardial injury and investigate the mechanism. And then explore the effect and mechanism of Gypenosides in the process of improving high glucose-induced myocardial injury.Methods In vitro studies: rat myocardial cell line(H9C2) were respectively treated with different concentrations(5.5 mM, 25 mM and 35 mM) of glucose for 6 h, 12 h, 24 h, 36 h and 48 h. The cell injury and activity of H9C2 were assessed by lactate dehydrogenase(LDH) release and tetrazolium salt colorimetry assay(MTT assay); The methods of TdT mediated dUTP nick end labeling(TUNEL) and flow cytometry were used to detect apoptotic level of H9C2; The gene expressions of ASC, caspase-1 and IL-1? were detected by real-time fluorescent polymerase chain reaction(RT-PCR), and the protein expressions of NLRP3, caspase-1, ASC and IL-1? were detected by Western Blot and fluorescence microscopy. With the application of caspase-1 inhibitor(Z-YVAD-FMK), the cell injury of H9C2 was detected by LDH release, while the protein levels of inflammation-related factors were detected by Western Blot; The release of cytochrome c was detected by mitochondrial separation. Then with the application of cytochrome c and ROS inhibitors(Cyclosporin A and NAC), LDH assay was used to detect myocardial cell injury, and Western Blot was applied to detect protein levels of inflammation-related factors. With the application of Gypenosides, cell apoptosis, injury and activity were detected by flow cytometry, LDH release and MTT assay. At the same time, Western Blot was used to detect the release of cytochrome c and activation of NLRP3 inflammasome. Type II diabetes rat model was established, and the blood glucose and body weight change of rats were detected. After the model was established, the Z-YVAD-FMK(240 ng/time, a total of 6 times, tail intravenous injection) and Gypenosides(400 mg/kg/time, a total of 14 times, irrigation stomach) were administrated. At the end of the experiment, the rats were sacrificed, and myocardium tissues were obtained. The expressions of NLRP3, ASC, caspase-1 and IL-1? were detected by Western Blot and RT-PCR. The level of serum IL-1? was detected by enzyme-linked immunoabsorbent assay(ELISA). The paraffin sections of myocardium tissues were obtained and the pathology analysis of myocardium was detected by HE stain. The expression and distribution of inflammasome in the myocardial tissues were detected by immunohistochemical staining.ResultsHigh glucose-induced myocardial injury model and type II diabetes rat model were established. LDH release and MTT assay showed that: compared to normal group(5.5 mM glucose), medium glucose group(25 mM) and high glucose group(35 mM) did not significantly induce cell damage at 6 h and 12 h. However, as time goes on, cell injury was aggravated, and the viability was gradually reduced with dose and time dependent. TUNEL and flow cytometry showed that high glucose could induce the apoptosis of myocardial cells. RT-PCR showed that high glucose significantly upregulated gene levels of inflammasome-related factors. Moreover, Western Blot and immunofluorescence demonstrated that high glucose upregulated related protein expression. After the application of Z-YVAD-FMK, the expression of inflammasome-related protein was reduced, and then the injury of cell was improved; Isolated mitochondria showed that high glucose induced cytochrome c release from mitochondria to cytoplasm, which was particularly obvious at 48 h. Cyclosporin A could reduce the release of cytochrome c, inhibit activation of inflammasome and improve cell injury. At the same time, NAC(one ROS inhibitor) significantly decreased the release of cytochrome c from mitochondrial and inflammasome associated protein expression, and then improved the cell injury. Besides these, Gypenosides inhibited the release of cytochrome c from mitochondria to the cytoplasm, reduced NLRP3 inflammasome-related proteins' expressions, and ameliorated the cellular injury. The gene and protein levels of NLRP3, ASC, caspase-1 and IL-1? were significantly increased in myocardium of diabetic rats. After being administrated with Z-YVAD-FMK or Gypenosides, related gene and protein expression in rats were decreased. ELISA showed that the level of IL-1? in serum of diabetic rats was significantly higher than that in serum of normal rats. Moreover, the level of IL-1? was significantly decreased in rats treated with Z-YVAD-FMK and Gypenosides, which has statistical difference. HE staining showed that: compared to normal rats, the injury of myocardial tissue in diabetic rats was more serious, and the severity of injury was significantly reduced in Z-YVAD-FMK or Gypenosides group. Immunohistochemistry showed that the myocardium of diabetic rats obviously expressed positive granules, which meant inflammatory protein, and the positive granules in Z-YVAD-FMK or Gypenosides group were significantly decreased.ConclusionsAbove experiments in vitro and vivo demonstrate that the activation of NLRP3 inflammasome by high glucose induces myocardial injury. The activated mechanism is that: high glucose induces damage of mitochondrial, release abundant ROS which promotes cytochrome c release into the cytoplasm that activates NLRP3 inflammasome, and the activated NLRP3 inflammasome promotes mature IL-1? release, and futher induce myocardial injury. Meanwhile, as a traditional Chinese medicine, Gypenosides improve high glucose-induced myocardial injury by inhibiting the release of cytochrome c, regulating NLRP3 inflammasome activation, which provides theoretical support for clinical applications.