Mechanism of Yitangkang in Improving Apoptosis of Skeletal Muscle Cells by Inhibiting AGE/RAGE Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo determine the mechanism of Yitangkang in correcting excessive apoptosis of skeletal muscle cells to improve insulin resistance （IR） by inhibiting the advanced glycation end product （AGE）/receptor for the advanced glycation end product （RAGE） signaling pathway. Method① In vitro experiments. Yitangkang-medicated serum was prepared. C2C12 cells were divided into a blank group， a model group， high-， medium-， and low-dose Yitangkang-medicated serum groups （40， 20， and 10 g·kg-1）， and a RAGE inhibitor group. The IR model was induced by palmitic acid in C2C12 cells except for those in the blank group. After the corresponding intervention methods were conducted，the cell viability and glucose consumption level of each group were determined. In addition，the apoptosis rate was determined using flow cytometry. The mRNA and protein expression levels of the important apoptotic proteins ［B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， p53， cysteinyl aspartate-specific protease-3 （Caspase-3）， and cysteinyl aspartate-specific protease-9 （Caspase-9）］ were determined using Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. ② In vivo experiments. Ninety-six eligible Wistar rats were divided into a blank group， a model group， high-，medium-，and low-dose Yitangkang groups （40， 20， and 10 g·kg-1）， and a western medicine group （pioglitazone hydrochloride，1.35 mg·kg-1）. The IR model was induced using high-glucose and high-fat feed for diabetes combined with intraperitoneal injection of low-dose streptozotocin （STZ） in animals and verified by the hyperinsulinemic-euglycemic clamp （HEC） test. After the model was determined successfully， the rats in each group were given intragastric administration of drugs as required. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay was performed to determine the number of positive apoptotic cells in the skeletal muscle tissues of rats in each group，while Real-time polymerase chain reaction（Real-time PCR） and Western blot were performed to determine the mRNA and protein expression levels of the important apoptotic proteins Bcl-2， Bax， p53， Caspase-3， and Caspase-9. Result① In vitro experiments. compared with the blank group， the model groups showed increased apoptosis rate of C2C12 cells and decreased cell viability and glucose consumption （P<0.01）. Compared with the model group， the Yitangkang-medicated serum groups and the RAGE inhibitor group showed decreased apoptosis rate of C2C12 cells and increased cell viability and glucose consumption （P<0.01）. Compared with the blank group， the model group showed decreased expression levels of Bcl-2 mRNA and protein in C2C12 cells and increased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.01）. Compared with the model group， the Yitangkang-medicated serum groups and the RAGE inhibitor group showed increased expression levels of Bcl-2 mRNA and protein in C2C12 cells （P<0.01） and decreased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.05， P<0.01）. ② In vivo experiments. The number of positive apoptotic cells in the skeletal muscle tissues of rats in the model group significantly increased as compared with that in the blank group （P<0.01）. The number of positive apoptotic cells in the skeletal muscle tissues of rats in the Yitangkang groups and the western medicine group decreased as compared with that in the model group （P<0.01）. Compared with the blank group， the model group showed decreased expression levels of Bcl-2 mRNA and protein in skeletal muscle tissues of rats and increased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.01）. Compared with the model group， the Yitangkang groups and the western medicine group showed increased expression levels of Bcl-2 mRNA and protein in skeletal muscle tissues of rats （P<0.01） and decreased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.05， P<0.01）. The medium-dose Yitangkang showed a similar effect as RAGE inhibitor， and the effect was equivalent to that of pioglitazone hydrochloride. ConclusionYitangkang can inhibit skeletal muscle cell apoptosis by inhibiting the AGE/RAGE signaling pathway.

The Chinese herbal formulae (Yitangkang) exerts an antidiabetic effect through the regulation of substance metabolism and energy metabolism in type 2 diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Type 2 diabetes mellitus (T2DM) regarded as a "hot" disease in traditional Chinese medicine (TCM). Accordingly, TCM uses a cold drug or formula such as the Chinese herbal formulae "Yitangkang" (YTK) as a treatment. YTK exhibited a good clinical antidiabetic effect in several experiments. The correlation between the properties of a TCM drug or formula and its ability to regulate the substance metabolism, the energy metabolism and the endocrine system has been proven. AIM OF THE STUDY: The present study aiming to evaluate the mechanism of antidiabetic action of YTK from the above perspective. MATERIALS AND METHODS: Three groups of streptozotocin (STZ)-diabetic rats have been treated with YTK at oral doses of 56 g/kg/d, 28 g/kg/d and 14 g/kg/d for 28 days using metformin as a reference drug. After treatment, several indices correlated with energy metabolism (superoxide dismutase, glutathione peroxidase, lactic dehydrogenase, adenotriphos, creatine phosphate kinase, AMPK, Na+-K+-ATPase and Respiratory Chain Complex I, II, III, IV), substance metabolism (hepatic glycogen, acetyl-coenzyme A, pyruvic acid, adipose triglyceride lipase, triglycerides, high-density lipoproteins, low-density lipoproteins, malonyldialdehyde), endocrine system (triiodothyronine, thyroxine, 17-hydroxycorticosteroid) and cyclic nucleotide system (cyclic adenosine monophosphate, cyclic guanosine monophosphate) have been determined. The specialty and tendency of YTK's effects were analyzed to elucidate its property and mechanism of action according to the theory of TCM. RESULTS: Our findings showed that the formulae YTK could effectively regulate the levels of blood glucose, HbA1c, glucagon-like peptide-1, and significantly down-regulate the substance metabolism, energy metabolism and endocrine system indices of the diabetic rats. CONCLUSION: These results were consistent with the TCM description of YTK as a "cold" treatment. It could provide an effective way to interpret the scientific connotation and comprehensive system of the Chinese herbal formulae.

The effect of Chinese herbal compound Yitangkang extract on expression of renal AGEs and RAGEs in rats with diabetic nephropathy / 中国生化药物杂志

Objective To observe the effect of Chinese herbal compound Yitangkang on the expressions of advanced glyclation end-products (AGEs) and the receptor of advanced glyclation end-products mRNA(RAGE-mRNA) in rats with diabetic nephropathy(DN),and to discuss the mechanism of the formulas in the treatment of DN. Methods The rat model of DN were established by intraperitoneal injection of strepotozotocin(STZ). After 8 weeks intervention with medications, the expression of renal RAGEs mRNA was detected by Real-time PCR, renal RAGE by immunohistochemistry, and renal content of AGEs by spectrophotometry. Results Compared with normal control group, the expression of renal RAGEs mRNA and RAGEs in diabetic nephropathy rats were increased significantly(P<0.01). Compared with DN group, the expression of renal RAGEs mRNA and RAGEs in three doses Yitangkang groups and western medicine group were decreased significantly(P<0.01). Conclusion Chinese herbal compound Yitangkang could protect kidney of diabetic nephropathy rats,and the mechanism is related to its loweirng renal expression of RAGEs mRNA, RAGEs and AGEs content.