Inhibitory Effect of Cinobufotalin on Macrophage Inflammatory Factor Storm and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism.@*METHODS@#THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot.@*RESULTS@#1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1β, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway.@*CONCLUSION@#Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.

Effect and Mechanism of Cinobufotalin Against Gastrointestinal Malignant Tumors： A Review / 中国实验方剂学杂志

Digestive tract diseases， especially digestive tract tumors， including liver cancer， pancreatic cancer， and colorectal cancer， have high incidence in China. Digestive tract tumor is one of the top 10 cancers in terms of the number of new cases and deaths in the world， and the incidence and mortality of tumor diseases have been increasing year by year. Therefore， the prevention and treatment of tumors is particularly important. With the application and promotion of traditional Chinese medicine in the medical field and the rapid development of molecular biology and pharmacology， more and more potential active components of Chinese medicinal materials have been extracted and studied. These active components can inhibit tumor cells in a multi-target and multi-pathway manner. Cinobufotalin is an effective component extracted from the skin of Bufo bufo gargarizans. It has been prepared into a variety of agents with anti-tumor， immunomodulatory， cardiac boosting， pain-easing， anti-inflammatory， and swelling-relieving activities. In clinical practice， cinobufotalin is mainly used to assist the treatment of liver cancer， lung cancer， colorectal cancer， gastric cancer and other malignant tumors， which can reduce the adverse reactions of patients in the middle and late stages and improve the quality of life and five-year survival rate of patients. The available studies of molecular mechanism have demonstrated that cinobufotalin can play a therapeutic role by inducing cell apoptosis， regulating cell cycle， inhibiting cell proliferation and angiogenesis， modulating immune response， reversing multidrug resistance， enhancing radiochemotherapy sensitivity， inhibiting tumor inflammation， invasion， and metastasis， etc. This review focuses on the clinical application and mechanism of cinobufotalin against digestive tract tumors in recent years， aiming to provide a theoretical basis for the anti-tumor research of cinobufotalin， promote the application of cinobufotalin in tumor treatment， and facilitate the further research and development of this compound.

Cinobufotalin regulating the proliferation and apoptosis of ovarian cancer cells SKOV3 through wee1 protein / 西安交通大学学报(医学版)

【Objective】 To observe whether cinobufotalin （CINO） inhibits the proliferation and improves apoptosis of ovarian cancer SKOV3 cells by inhibiting the expression of wee1. 【Methods】 PcDNA-wee1 was transfected into SKOV3 cells. SKOV3 cells that overexpressed with wee1 were constructed and identified by Real-time PCR and Western blotting. After that， 10 μg/mL CINO was used for intervention treatment and grouping. CCK8 assay was used to detect cell proliferation. Cell apoptosis was detected by flow cytometry after 48 h of treatment. Western blotting detected the expressions of Caspase-3， Bax and Bcl-2 in each group. 【Results】 Real-time PCR and Western blotting showed that the expression of wee1 was significantly increased in the pcDNA-wee1 group， while the expression of wee1 decreased in the CINO + pcDNA-wee1 group compared with the pcDNA-wee1 group. Compared with the pcDNA-wee1 group， the proliferation of SKOV3 cells in CINO + pcDNA-wee1group was inhibited （P<0.05）. Compared with pcDNA-wee1 group， the apoptosis rate of SKOV3 cells in CINO+pcDNA-wee1 group was increased （P<0.05）. Western blotting results showed that Bcl-2 protein expression in CINO+pcDNA-wee1 group was inhibited compared with that in pc-DNA-wee1 group， while Bax and Caspase-3 protein expressions were enhanced. 【Conclusion】 CINO can inhibit the proliferation and improve the apoptosis of ovarian cancer cells by reducing the expression of wee1.

Anti-tumor effect and its related mechanisms of cinobufotalin combined with cisplatin on H22 liver cancer mice / 中国中药杂志

In order to observe the anti-tumor effect of cinobufotalin on H22 liver cancer mice and to explore its regulatory mechanism, 50 Kunming mice were subcutaneously inoculated with H22 intraperitoneal passage cells under the armpit to establish H22 hepatocellular carcinoma model. They were then randomly divided into model group, cinobufotalin low dose group, cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, which received 0.01% ethanol solution, 1 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cisplatin, 5 mg·kg~(-1)cisplatin + 5 mg·kg~(-1) cinobufotalin respectively for 10 days. The general condition of mice during the intervention was observed, and the inhibition rate, tumor mass, thymus index, histopathological changes of the tumors, apoptotic rate of the tumors, the expressions of phosphatidylinositol 3-kinase(PI3 K), protein kinase B(Akt), apoptosis related gene(Fas), Fas ligand(FasL) mRNA and protein phosphorylated Akt(pAkt) protein in the tumors of each group were compared. The results showed that during the modeling period, the mice showed a decline in food intake, dark fur, poor mental status, and gradually worsened over time. The mental status of mice in each intervention group was improved gradually, especially in the cisplatin+cinobufotalin group. As compared with the model group, the tumor mass of each intervention group was lower(P<0.05). As compared with the cinobufotalin low dose group, the tumor mass was lower and inhibition rate was higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, the tumor mass was lower and the inhibition rate was higher in cisplatin+cinobufotalin group(P<0.05). As compared with the model group, the thymus index was higher in cinobufotalin high dose group and cisplatin + cinobufotalin group, while was lower in cisplatin group(P<0.05). As compared with the cinobufotalin low dose group, the thymus index was higher in the cinobufotalin high dose group and lower in the cisplatin group(P<0.05). As compared with the cinobufotalin high dose group, the thymus index was lower in cisplatin group(P<0.05). As compared with cisplatin group, the thymus index was higher in cisplatin+cinobufotalin group(P<0.05). Pathological staining showed that a large number of heterogeneous cells and mitotic phenomena were observed in the model group. Cell fragments and neutrophils were observed in the tumor tissues of the intervention groups, showing diffuse necrosis, and the diffuse necrosis was more obvious in the cisplatin+cinobufotalin group. As compared with the model group, the apoptotic rate of the tumors and the relative expressions of Fas mRNA and protein were higher in the intervention groups, while the relative expressions of PI3 K, FasL mRNA and protein and the relative expression of pAkt protein were lower in the intervention groups(P<0.05). As compared with the cinobufotalin low dose group, the apoptotic rate of the tumors and relative expression of Fas and protein were higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, apoptotic rate of the tumors and the relative expression of Fas mRNA and protein were higher in the cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower in the cisplatin+cinobufotalin group(P<0.05). In summary, cinobufotalin has significant anti-tumor effect on H22 liver cancer mice, and can enhance the immune function of mice and synergistically enhance the effect of chemotherapy. Its mechanism may be associated with regulating PI3 K/Akt/Fas/FasL signaling pathway related genes and protein expression.

Study on chemical constituents and pharmacodynamic changes of Bufonis Venenum with different drying methods / 中草药

Objective To study the changes of chemical constituents and pharmacodynamics with different drying methods (sun- drying, 50 ℃ vacuum-drying, 50 ℃ and 80 ℃ heat-drying, freeze drying method) in Bufonis Venenum. Methods HPLC method and TLC method was established for studying the changes of chemical constituents from B. Venenum before and after being dried, and determine the inhibitory effect of dried samples on five different tumor cell lines proliferation by MTT assay. Results The B. Venenum processed by five different methods were different in character, while no significant differences in the types and content of chemical constituents; The total content of resibufogenin and cinobufagin was more than 6%, which was consistent with the 2015 edition of Chinese Pharmacopeia; 50 ℃ and 80 ℃ heat-drying of B. Venenum showed more effective inhibitiory effect than other dry methods. Conclusion The appearance of B. Venenum met the criterion of pharmacoperia by sun-drying and 50 ℃ heat-drying method. Although the color of B. Venenum under the vacuum-drying and freeze drying method did not meet the requirements of Chinese Pharmacopeia, the main effective components have a few changes.

Effect of Huachansu capsule combined with hepatic arterial infusion chemotherapy on plasma fibrinogen and tumor marker boncentration in patients with Hepatocellular carcinoma / 中国生化药物杂志

Objective To study the effects of cinobufotalin capsule combined with transcatheter arterial chemoembolization in patients with hepatocellular carcinoma.Methods 68 patients with hepatocellular carcinoma from January,2013 to March,2016 were selected and divided into the combination group and chemotherapy group according to the random number table method,each with 34 cases.The combination group was treated with transcatheter arterial chemoembolization and hepatic artery infusion chemotherapy embolization.The levels of D-dimer,tumor markers,clinical efficacy and quality of life scores were observed before and after treatment in 68 patients.Results There was no significant difference in plasma fibrinogen(FIB)in combination group before and after treatment.Before treatment,the levels of FIB)was similar to chemotherapy group.After treatment,the FIB content in the chemotherapy group was significantly higher than before treatment and that in the combination group the differences were statistically significant(P<0.05).After treatment,the levels of cytokeratin 19,carcinoembryonic antigen and neuron-specific enolase in two group were significantly higher than those before treatment(P<0.05).But there was no significant difference between the two groups after treatment.The rate of disease control in the combination group was significantly higher than that in the chemotherapy group(77.5％vs.65.4％,P<0.05).Cases of diarrhea and insomnia in chemotherapy group were similar to the combination group,but the differences of cases of pain and fatigue between two group were statistically significant(P<0.05).Conclusion Cinobufotalin capsule combined with transcatheter arterial chemoembolization can improve the hypercoagulability of the patient's blood,and is beneficial to the treatment of cancer,improve the disease control rate and improve the life of patients.

Effect of Cinobufotalin Freeze-dry Powder on Heart Rate and Electrocardiograph of Anesthesia Rats / 医药导报

Objective To observe the effect of cinobufotalin freeze-dry powder on heart rate ( HR ) and electrocardiogram ( ECG) of SD rats and to provide experimental basis for monitoring its adverse effect on heart in clinical application. Methods The drug was administered into external jugular vein at constant speed throughout the whole experiment;standard-Ⅱ limb lead monitored the HR and ECG, and then the changes in HR and ECG before and after administration of cinobufotalin were compared. Results Thirty minutes after administration of cinobufotalin injection and cinobufotalin freeze-dry powder at middle dose and high dose, HR of the rats was significantly increased as compared with blank control group[(469±40) bpm, (466±29) bpm and (484±40) bpm vs. (411±17) bpm] (P0. 05). Conclusion Cinobufotalin freeze-dry powder has some side effects on rat heart and can increase HR, even lead to arrhythmia.

VEGF antisense oligodeoxynucleotides in enhancing the apoptosis-inducing effect of Cinobufotalin on K562 cells / 西安交通大学学报(医学版)

Objective To study the vascular endothelium growth factor(VEGF) antisense oligonucleotide(ASON) Cinobufotalin in enhancing apoptosis induced by human chronic myeloid leukemia K562 cell line.Methods The synthesis of VEGF ASON was transfected into the K562 cells;Cinobufotalin of four concentrations(1,3,5 and 7mg/L) acted on the K562 cell line for 24h,48h and 72h.Western blot was used to detect VEGF protein expression,while in situ apoptosis(TUNEL) and flow cytometry method(FCM method) were employed to detect the apoptosis.Results The different doses of Cinobufotalin all inhibited K562 cell line in time-and dose-dependent manners.K562 cell line transfected by VEGF ASON had a more pronounced induction of apoptosis.Conclusion The in vitro Cinobufotalin at a certain range of concentration can induce apoptosis in K562 cell line,and VEGF ASON enhances Cinobufotalin's effect in inducing apoptosis of K562 cells.

Clinical Study of Using Cinobufotalin and Chemotherapeutic Agents by Transcatheter Arterial with Oily Chemoembolization in the Treatment of Primary Liver Cancer / 实用放射学杂志

Objective To evaluate the effect of cinobufotalin and chemotherapeutic agents by transcatheter arterial with oilychemoembolization(TACE) in the treatment of primary liver cancer.Methods 144 patients with HCC proved histopathologically were divided into 2 groups.76 of them(group A) were treated by transcatheter arterial infusion(TAI) with cinobufotalin 100 ml,DDP and 5-FU,then embolism with iodized oil mixed ADM;while the other 68 patients(group B) were treated by TAI with DDP and 5-FU,then embolism with iodized oil mixed ADM.The serum T lymphocytes,HBV DNA,AFP and CT scan were acquired before and after treatment.Results The effective rate(PR+MR) of group A was 86.64%,the lymphocyte transformation rate(LTT),T lymphocytes CD_3~+,CD_4~+proportion and CD_4~+/CD_8~+ratio markedly increased;HBV DNA descended in 21 cases,unchanged in 46 cases,and elevated in 9 cases;1 and 2 year survival rate was 86.84%(66/76)and71.05%(54/76) respectively.The effective rate(PR+MR) of group B was 72.73%,LTT,T lymphocytesCD_3~+,CD_4~+proportion and CD_4~+/CD_8~+ratio markedly descended;HBV DNA descended in 2 cases,unchanged in 20 cases and elevated in 46 cases;1 and 2 year survival rate was 72.73%(48/68) and 54.41%(37/68) respectively.There were significant statistical differences between the two groups(P