| ObjectiveBleomycin(BLM),a glycopeptide antibiotic antineoplastic agent extracted from Streptomyces,is one of the broad anti-tumor drugs in clinic.In clinical practice,it combined with chemotherapy drugs for the treatment of malignant lymphoma,germ cell tumors,squamous cell carcinoma,melanoma and malignant pleural effusion has a good effect,because of it has no immunesuppression and myelosuppression,in the treatment of acute myeloid leukemia and parkinson’s disease also have great prospects.However,there are side effects of pneumonia-like symptoms and symptoms of pulmonary fibrosis,which greatly limits its clinical usage.It has been reported that 20%of patients treated with BLM would develop into pulmonary fibrosis,which manifests itself in a variety of forms,the most serious of which is pulmonary fibrosis.Therefore,how to improve the clinical value of BLM,to find adjuvant drugs that can enhance the antitumor effect of BLM and reduce the side effects has become a hot topic.Wild chrysanthemum is a dried flower head of Chrysanthemum indicum Linn.,Which belongs to the edible flower.It is widely used in the tea,beverage and food additives.It can be used for the treatment of various infectious diseases,such as headache,eye diseases and a variety of immune-related diseases,with high efficiency and low toxicity.Pharmacological studies have shown that wild chrysanthemum extracted has anti-bacterial,anti-inflammatory,anti-cancer and other pharmacological effects.The supercritical extract of Wild Chrysanthemum(CISCFE)has been widely used in many classic prescriptions and daily life,including functional foods,cosmetics and beverages.There has a report that CISCFE can act as a chemotherapy drugs,such as cisplatin-induced nephrotoxicity has a protective effect.Our previous study confirmed that CISCFE has a significant protective effect on LPS-induced acute lung injury in mice,it can significantly attenuate the symptoms of lung injury in mice;at the same time for the UV-induced photoaging mice has significant protective effect.These studies shown that CISCFE has good anti-inflammatory,anti-oxidant and lung protective bioactivity.However,there have no studies about CISCFE and BLM combined treatment of tumor-bearing mice,suggesting that it may have a potential synergistic effect with BLM.Therefore,in this study,the classical H22 tumor-bearing mice model used to explore the potential synergistic effect of CISCFE combined with BLM and its possible mechanism,and provide a theoretical basis for improving the clinical efficacy of BLM.Methods1.Composition analysis of CISCFEThe supercritical extract of wild chrysanthemum(CISCFE)extracted three times with n-hexane and 75%methanol,and the n-hexane layers were concentrated and analyzed by GC-MS,the 75%methanol layers were analyzed by HPLC-PAD.2.Drug efficacy study of CISCFE combined with BLMIn this study,KM mice used as experimental animals to establish H22 tumor-bearing mice model.H22 hepatoma cells were injected intraperitoneally into the abdominal cavity of mice at a dose of 1×10~7 cells/mL,each 0.2 mL.Once succeed,BLM(7.5 mg/kg)was intraperitoneally injected.Meanwhile,the CISCFE drug vehicle(240,360,480 mg/kg)were intragastrically administered to the tumor-bearing mice.By measuring the abdominal diameter,body weight,energy activity and survival curve to observe whether CISCFE and BLM have synergistic effect.According to survival analysis results,the follow-up experiment was carried out with the medium dose of CISCFE.After the model was successfully established,the mice were treated by intraperitoneal injection of BLM 7.5 mg/kg and CISCFE drug solute 360 mg/kg.The weight and abdominal diameter of mice with ascites were measured every day,and the volume of ascites was measured on the last day.Flow cytometry was used to detect the apoptosis of H22 ascites cells and to observe whether CISCFE could enhance the anti-tumor effect of BLM.At the same time,the lung tissue was stripped and stained with HE and Masson to observe the pathological changes of lung tissue in mice,and to study whether CISCFE could alleviate the pulmonary fibrosis induced by BLM.3.The mechanism study of synergistic effect of CISCFE with BLMBased on the above studies,the medium dose of CISCFE was selected to continue the follow-up mechanism study.The activities of Caspase 3 and Caspase 8 in ascitic cells were detected by caspase activity assay kit,and the expression of p53 protein in ascites cells were analyzed by western blot to further study the mechanism of CISCFE enhancing the anti-tumor effect of BLM.In addition,the activity of MPO and MDA in lung tissue was measured by biochemical quantitative method,and the level of inflammatory factor TNF-αand IL-6 was measured by ELISA kit,and the expression of TGF-β1 protein was detected by western blot to investigate the mechanism of CISCFE in improving pulmonary fibrosis of BLM.Finally,QRT-PCR used to evaluate the expression of miR-29b gene in ascitic cells and lung tissues.Results1.Compositions analysis of CISCFEGC-MS analysis showed that the layer of n-hexane fraction contain 30 compounds,mainly terpenoids and alkenes.The most abundant of them are E,E,Z-1,3,12-Nonadecatriene-5,14-diol(13.491%),Octacosane(8.885%),P21entacosane(8.62%),Tetratriacontan(6.341%),Tricosane(5.271%),Heptacosane(2.885%),Pentacosane(1.950%),Octadecane,3-ethyl-5-(2-ethylbutyl)(1.769%).HPLC results showed that the layer of 75%methanol fraction mainly consist of five components,including luteolin,apigenin,quercetin,linarin,acacetin.2.Efficacy study on synergistic effect of CISCFE with BLMIn the H22 tumor-bearing mouse model,compared with the normal group,the body weight and abdominal diameter of the model group increased sharply,and the survival time was greatly shortened.CISCFE alone(240,360 and 480 mg/kg)groups had no obvious effect on the survival time of H22 tumor-bearing mice,compared with model group.BLM alone dose group could significantly prolong the survival time of H22 tumor-bearing mice;Combined treatment group(especially 360 and 480 mg/kg)could significantly improve the survival time of tumor-bearing mice,according to the results of survival analysis,CISCFE dose continued to follow-up mechanism of experimental study.In addition,the combination of BLM and CISCFE could significantly increase the apoptosis rate of ascites cells,compared with BLM alone group,thus enhancing the anti-tumor effect of BLM.At the same time,combined treatment could significantly improve the pulmonary fibrosis and inflammatory infiltration induced by BLM,compared with BLM alone group.The preliminary results showed that CISCFE could alleviate the pulmonary fibrosis induced by BLM.3.The mechanism study of synergistic effect of CISCFE with BLMThe results of caspase activity test kit showed that the combined administration of Caspase 3 and Caspase 8 significantly increased the activities of in ascitic cells,and upregulated the expression of p53 protein,thus enhancing the anti-tumor effect of BLM,compared with BLM alone.Combined therapy could significantly reduce the levels of oxidized factor MPO,MDA and TNF-α,IL-6 in lung tissue,and down-regulate the expression of TGF-β1 protein expression,thus alleviating the pulmonary fibrosis induced by BLM,compared with BLM alone group.QRT-PCR results showed that compared with the single group,the expression of miR-29b in ascitic cells and lung tissues were significantly increased after combined administration.ConclusionIn this study,we first found that CISCFE has better synergistic and attenuated effects on BLM by applying H22 ascites tumor mice model.Its synergistic and attenuated mechanism may be related to regulate the balance of p53 and TGF-β1 signaling pathways.The anti-tumor effect of BLM was enhanced by promoting the apoptosis and up-regulating the expression of p53 protein in ascitic cells.At the same time,the expression of TGF-β1 was down-regulated by reducing pulmonary inflammation,oxidative reaction and lung injury. |
