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Manjiao Zhuang,1 Guang Xin,1 Zeliang Wei,1 Shiyi Li,1 Zhihua Xing,1 Chengjie Ji,1,2 Junrong Du,1 Hai Niu,1,3 Wen Huang11Laboratory of Ethnopharmacology, West China School of Pharmacy, West China Hospital, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 2Department of Laboratory Medicine, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, Sichuan, People’s Republic of China; 3College of Mathematics, Sichuan University, Chengdu, Sichuan, People’s Republic of ChinaBackground: Our previous studies have demonstrated that diosgenin and diosgenin derivatives exhibit excellent antithrombotic activity via regulating platelet function and coagulation factor level. Platelets and blood coagulation system are highly associated with tumor hematogenous metastasis. Therefore, the purpose of this study was to evaluate whether dihydrodiosgenin (dydio) mediated-platelet inhibition or coagulation factor level modulation is involved in hepatocellular carcinoma cell (HCC) metastasis.Methods: Cell viability was examined by MTT and colony formation assays. Platelet aggregation text and morphology were used to assess dydio’s role on tumor cell-induced platelet activation (TCIPA). Scratch assay, adhesion assay and Western blot were used to evaluate dydio’s role on platelet-mediated metastasis. Western blot and fluorescence detection were performed to clarify dydio’s role on endothelial cell (EC) function. The mice lung metastasis model was constructed to investigated dydio’s function on coagulation factor and platelet-mediated metastasis.Results: This study found that pretreatment with dydio caused a significant inhibition of TCIPA. Platelets exposed to dydio significantly inhibited their adhesion to tumor cells, meanwhile, releasates of platelets that pretreated with dydio led to diminished cancer cell proliferation and migration along with the increase of epithelial markers E-cadherin and loss of mesenchymal phenotype. Additionally, ECs pretreated with dydio suppressed factor VIII (FVIII) level which in turn restrained the activation of platelets and the adhesion of cancer cells or platelets to ECs. Interestingly, our study demonstrated that FVIII could promote HCC proliferation. In vivo study revealed that mice intragastrical (i.g.) administration with dydio significantly inhibited the lung metastasis of hepal-6 cells which is highly correlated with the altered platelet function and coagulation level.Conclusion: Taken together, these results demonstrated that dydio altered platelet function and coagulation FVIII level, resulting in decreased metastatic potential of HCC. Thus, our study reveals that dydio exerts novel mechanisms of antitumor action beside its direct antitumor activity.Keywords: dihydrodiosgenin, hepatocellular carcinoma, platelet, endothelial cell, tumor microenvironment, FVIII

Manjiao Zhuang,1 Honghong Qiu,2 Ping Li,2 Lihua Hu,2 Yayu Wang,3 Lei Rao2 1Department of Pharmacy, Sichuan University, Chengdu, People’s Republic of China; 2Collaborative Innovation Center of Sichuan for Elderly Care and Health, Chengdu Medical College, Chengdu, China; 3Department of Cell Biology, Institute of Biological Medicine, Jinan University, Guangzhou, People’s Republic of China Background and purpose: The traditional Chinese medicine – the flower of Edgeworthia gardneri – is reported as an effective therapeutic for type 2 diabetes mellitus (T2DM). Nevertheless, most constituents of the flowers of E. gardneri have not yet been studied. This study was conducted to investigate the effect of quercetin extracted from the flowers of E. gardneri on islet protection and amelioration in T2DM and explore its mechanism. Method: Quercetin was extracted from the flowers of E. gardneri and verified by high-performance liquid chromatography. Quercetin or crude extract’s effect on insulin secretion was investigated. ERK1/2 and phospho-ERK1/2 were detected by Western blot analysis, and fluo-3 AM was used to detect intracellular Ca2+. The anti-apoptosis effect of quercetin or crude extract on MIN-6 cells was investigated by thiazolyl blue tetrazolium bromide (MTT) assay and flow cytometry analysis. Activation of caspases and expression of Bcl-2 and BAX were tested by Western blot analysis. In addition, the mitochondrial membrane potential was determined by JC-1 probe. Moreover, in vivo activity was also tested in db/db mice. Results: A quercetin level of >10 μmol/L could induce insulin secretion. Intracellular Ca2+ and ERK1/2 were involved in the signaling pathway of quercetin-induced insulin secretion. We also observed that quercetin could inhibit palmitic acid-induced cell apoptosis via suppressing the activation of caspase-3, -9, -12; increasing the ratio of Bcl-2/BAX and reversing the impaired mitochondrial membrane potential. Crude extract’s effect on insulin secretion was similar to that of pure extracted quercetin, while it possessed higher anti-apoptosis activity. Additionally, intraperitoneal glucose tolerance, plasma insulin level, hepatic triglyceride, hepatic glycogen and the pathological histology of both pancreatic islet and liver in db/db mice were significantly improved by the administration of the extracted quercetin. Conclusion: Our study indicated that quercetin extracted from the flowers of E. gardneri exerted excellent properties in islet protection and amelioration. Keywords: quercetin, Edgeworthia gardneri, insulin secretion, anti-apoptosis, type 2 diabetes mellitus