Studies On The Effect And Mechanisms Of HMGB1 Mediated Autophagy In Glycyrrhizin Acid -Protected Cadiotoxicity Induced By Sunitinib

Objectives:As the world’s first multi-targeted tyrosine kinase inhibitor, Sunitinib is clinically used for the treatment of Gastrointestinal Stromal Tumors (GIST) and advanced renal cell carcinoma (ARCC). The cardiotoxicity caused by off-target effects is one of the serious adverse reactions of Sunitinib, which limits the clinical application of Sunitinib. Further study of the molecular mechanisms of Sunitinib-induced cardiotoxicity and interferential targets has far-reaching significance. In the previous studies, we found that Sunitinib could cause self-offensive autophagy in cardiomyocytes leading to a myocardial injury. Aiming to make the mechanisms of Sunitinib-induced autophagy clear, we start our study to find out the key protein and its activators or inhibitors to protect the cardiomyocytes against the Sunitinib-caused injury. Therefore, this study could provide experimental evidence for protection of Sunitinib-induced cardiotoxicity, expand the depth of its clinical applications.Methods:Using Sprague-Dawley Rat model, nude mice xenografted tumor model, the rat primary cardiomyocytes to confirm the cardiotoxicity caused by Sunitinib in clinical treatment doses, accompanied with self-offensive autophagy. (1) SD rats were given Sunitinib with a clinical dose in treatment of GIST for 30 days, heart injury was assessed by Myocardial enzymes test in serum including creatine kinase MB (CKMB), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities. (2) The ECG and Echocardiography was measured after Sunitinib treatment to evaluate the cardiotoxicity clinically. (3) 786-0 xenografted nude mice were given Sunitinib with a dose in treatment of GIST for 31 days. The anti-tumor efficacy of Sunitinib was assessed by tumor weight, tumor volumn (TV), relative tumor volumn (RTV). (4) Body weight curve was drafted to evaluate the organ index of heart. Hematoxylin and eosin (H&E) staining of heart sections was done to certificate the coexisted cardiotoxicity and anti-tumor effects. (5) Autophagy was detected by immune transmission electron microscopy (TEM) and Western Blot in rats’ myocardial tissues. (6) TEM was used again to confirm the state of autophagy in primary cardiomyocytes.Using Sprague-Dawley Rat model, the rat primary cardiomyocytes model to detect the protective effects of glycyrrhizin acid (GA) on Sunitinib-induced cardiotoxicity. (1) The survival rate of primary cardiomyocytes was examed after GA and Sunitinib treatment. (2) Using SD rat model to detect the change of serum markers (CKMB, AST, LDH) after given clinical doses of Sunitinib and GA for 30days. According to the date, evaluate the protective effect of GA on sunitinib-induced cardiotoxicity. (3) Heart functional change with the intervention of GA were assessed by ECG and echocardiography. (4)HE staining of heart tissue was observed to evaluate the protective ability of GA on sunitinib-induced heart pathological changes.Using Sprague-Dawley Rat model, the rat primary cardiomyocytes model to explore the effect and mechanisms of HMGBl-mediated Autophagy in Glycyrrhizin Acid -Intervened Cadiotoxicity Induced by Sunitinib.(1) Using Sprague-Dawley Rat model, the rat primary cardiomyocytes model to test the effect of GA in sunitinib-induced myocardial autophagy by Western Blot and TEM. (2) the influence of Sunitinib on the expression and subcellular distribution of HMGB1 in myocardio tissue and cardiomyocytes.(3) Primary cardiomyocytes was treated with ethyl pyruvate (EP), an HMGB1 nuclear translocation inhibitor, to evaluate the subcellular distribution of HMGB1 by immunofluorescence. The interaction between HMGB1 nuclear translocational funcition and sunitinib-induced myocardio autophagy was examed on the same model by Western Blot, and finally MTT method was used to detect the survival rate transformation of cardiomyocytes. (4) siRNA of HMGB1 could be helpful to silence the expression of HMGB1 and test the change of sunitinib-induced autophagy by Western Blot and TEM. (5) we selected RT-PCR, Western Blot and fluorescence confocal microscopy to search for the influence of GA in sunitinib-upregulatted HMGB1 expression and its subcellular distribution.(6)Using the Western Blot and immunoprecipitation to detect the protein expression levels of Beclinl after sunitinib treatment, and to verify the binding of HMGBland Beclinl.(7) Using the Western Blot and immunoprecipitation to detect the protein phosphorylation levels of Bc12 after sunitinib treatment, and to verify the binding of Bc12 and Beclinl.(8) Using the Western Blot and immunoprecipitation to detect the influence of GA in sunitinib-induced promotion of HMGBl/Beclinl binding and the decline of Bc12/Beclin1 binding.Results:Cardiotoxicity was induced after clinical dose Sunitinib treatment with an activated autophagy.SD rats were treated with 100 mg/kg of Sunitinib for 30days, cardiomyocytes dysfunction was detected. Therefore, the serum cardio enzyme activity of CKMB, AST and LDH were obviously increased compared with control. R wave reduction was detected by ECG in Sunitinib-treated rats. The decrease of LVEF and FS was measured by echocardiography to confirm the left ventricular dysfunction and cardiotoxicity after Sunitinib treatment.786-O xenografted nude mice was given a clinical-conversed dose of Sunitinib (100 mg/kg) for 31 days, and finally performed an obvious body weight reduction with an excellent anti-tumor effect. Compared with control, the Sunitinib-treated rats showed a minor organ index of heart. Hematoxylin and eosin (H&E) staining of heart sections was done to confirm the pathological changes like myocardial edema, blur grain, etc. the autophagosomes in double membrane were found in Sunitinib-treated rats by TEM. LC3 results showed the occurrence of autophagy in Sunitinib-affected myocardial tissues by Western Blot. Cardiomyocytes were treated in different concentrations of Sunitinib which showed the formation of autophagosomes by TEM, accompanied with a final broken nuclear membrane indicating that autophagy was induced in Sunitinib-treated cardiomyocytes and finally autophagy turned into advanced necrosis.GA intervented Sunitinib-induced cardiotoxicity.MTT methods showed that GA could significantly protect the primary cardiomyocytes against sunitinib-raised cardiomyocytes death. After given 100 mg/kg of Sunitinib and 10mg/kg GA for 30days, the results of myocardial enzymes showed that GA can protect rats against sunitinib-raised CKMB, AST and LDH levels. HE staining of myocardio tissue indicate that GA could significantly reduce the sunitinib-induced pathological cardiac performance. ECG tell the truth that GA could effectively raise the sunitinib-induced R-wave decline, while the left ventricular ejection fraction and fractional shortening decline could be significantly alleviated under echocardiographic analysis.GA could inhibit the sunitinib-induced HMGB1 expression enhancement to decrease the combination of HMGB1 and Beclin1, and to inhibit the phohsphorylation of Bc12 to increase the binding between Bc12 and Beclin1. In this way, GA could intervented the sunitinib-triggered cardiomyocytes autophagy.The sunitinib-induced autophagy and necrosis was down regulated by GA under Western Blot and TEM test and accompanied with the phenomenon of blocked autophagosome formation. Thus, we proved that GA could inhibit the sunitinib-induced autophagy to preotect the heart away from the cardiotoxicity. RT-PCR, Western Blot and fluorescence confocal microscopy test showed that sunitinib could upregulate the HMGB1 transcript and protein levels, and it could promote the translocation of HMGB1 from nucleus to the cytoplasm. EP could inhibit the nuclear translocation of HMGB1 to. With the silence of HMGB1 in cardiomyocytes, siRNA of HMGB1 was proved to inhibit the sunitinib-induced autophagy, and finally eased the heart damage. HMGB1 expression enhancement at the transcriptional level urged by sunitinib could be declined by GA. Cytoplasmic HMGB1 could a) bind to Beclinl to activate autophagy; b) activate the phosphorylation of Bc12 to inhibit the combination of Bcl2 and Beclin1 and finally activate the autophagy. GA could inhibit the transcript level of HMGB1 to a) block the binding of Beclin1 and Bc12; b) down regulate the phosphorylation of Bc12 to promote the Beclin1/Bc12 combination formation, and to intervent the sunitinib-induced myocardio autophagy.Conclusions:In this paper, we proved that with an anti-tumor dose of Sunitinib treatment, the cardiotoxicity could be induced effectively in vivo. Glycyrrhizin Acid (GA), a compounds from natural sources, could intervene sunitinib-induved cardiotoxicity. It is the mechanism that GA inhibit the HMGB1 expression in transcript level which raised by sunitinib. In this way, HMGB1 expression and nuclear translocation was blocked, thus, leading to the end that HMGB1/Beclinl formation and Bc12 phosphorylation restrained. As an termination, GA inhibit sunitinib-induced myocardio autophagy, deepen the depth of sunitinib research and increase the theoretical basis for sunitinib clinical application.