| ObjectiveBleomycin(BLM),is a kind of antibiotic broader range of chemotherapy drugs with a history of more than 50 years of clinical use which can be used for a variety of cancers and incurable diseases.The unique mechanism of BLM develops advantages of no immunosuppression and bone marrow inhibition of the adverse event making BLM stand out from other chemotherapeutic drugs.And it also has good application prospect in the treatment of acute myeloid leukemia and malignant tumors.However,due to the large amount of BLM distributed in the lungs after entering the human body,and the lack of BLM hydrolase in the lungs,BLM induces relatively serious pulmonary toxicity,mainly for pulmonary fibrosis.The adverse reaction of pulmonary toxicity severely limits the clinical use value of BLM.Therefore,in order to efficiently and safely use BLM in clinic,it is of great significance to find an adjuvant to reduce lung toxicity of BLM.Scutellarin(SCU)is a kind of flavonoids isolated from natural medicines S.baicalensis Georgi and S.Barbara D.Don,and is abundant in these two medicines.S.baicalensis Georgi and S.Barbara D.Don have the effect of detoxification,and S.baicalensis Georgi is a traditional medicine commonly used in the treatment of lung diseases.Studies have shown that SCU could be used against cancers,fibrosis,inflammatory diseases and so on.Moreover,it possesses efficacy enhancing and toxicity reducing effects on cisplatin.Our previous research found that SCU had anti-bacterial activity and could be used in treatment of gastritis,gastric cancer and other diseases.However,whether SCU could reduce the pulmonary toxicity of BLM without affecting the anti-tumor efficacy of BLM remained poorly understood.On the basis of the above studies,this study used BLM-induced pulmonary fibrosis animal model and TGF-?1-induced cell model to explore the attenuation of SCU on BLM and its related mechanism.Because the pulmonary toxicity of BLM is mainly reflected in its clinical application,the research group will continue to investigate the effects of SCU on the pulmonary toxicity and antitumor activity of BLM in H22 ascites tumor models in vitro and in vivo.Methods1.Study on the effect of SCU on BLM-induced pulmonary fibrosisIn order to investigate whether SCU can reduce lung toxicity caused by BLM,this study adopted the trachea drip BLM(5 mg/kg)to construct the pulmonary fibrosis model in SD rats.After treatment of different doses of SCU(30,60,90 mg/kg),related indicators were detected to evaluate attenuated effect of SCU on BLM.Firstly,lung coefficient value and HYP levels were measured.Besides,the lung tissues of rats were stained by HE and Masson staining methods,and the protective effect of SCU on BLM-induced pulmonary fibrosis was evaluated according to the pathological conditions of staining reactions.ELISA kits were used to detect the contents of TNF-? and IL-6 in lung tissues.RT-PCR was used to detect the mRNA levels of MMP-3,MMP-9,TIMP-1,VEGFA and VEGFR2 in lung tissues.Western blot method was also used to detect the expression of collagen-I,?-SMA,E-cadherin,vimentin and TGF-?1 in lung tissues.Then the protective effect of SCU on BLM-induced pulmonary fibrosis was studied from the perspectives of angiogenesis and epithelial-mesenchymal transformation.2.Study on the mechanism of SCU on BLM-induced pulmonary fibrosisIn order to further explore the mechanism of toxicity attenuating ability of SCU on BLM,TGF-?1-induced A549 and MRC-5 cell models were used in vitro and the pulmonary fibrosis model of SD rats was established by tracheal infusion of BLM(5 mg/kg)in vivo.After treatment with SCU(60 mg/kg),the related indicators in the lung tissues of rats were detected at the 14th and 28th days.Immunofluorescence assay was used to measure the expression of E-cadherin,collagen-I in A549 cells and MRC-5 cells.RT-PCR was used to detect the mRNA levels of MMP-3,MMP-9 and TIMP-1 genes in lung tissues at the 14th and 28th days.Western blot was used to detect the expression of collagen-?,?-SMA,E-cadherin,vimentin,TGF-?1,Smad2/3,p-Smad2/3 in A549 cells and MRC-5 cells as well as lung tissues at the 14th,28th days.The expression of p-Smad2/3 protein in lung tissue was also detected by immunohistochemistry at day 14th and 28th.Combined with the detection results of related indicators,it can be clarified whether SCU affects the epithelial mesenchymal transformation in lung tissues mediated by TGF-?1/Smad2/3 signaling pathway through inhibiting the expression of TGF-?1 protein,and thus plays the attenuated effect of BLM.In addition,to clarify whether SCU can also affect the activation of the VEGFA/PI3K/Akt signaling pathway mediated by inhibiting the expression of TGF-?1 protein,and inhibit angiogenesis in lung tissues,so as to achieve the effect of BLM attenuation,RT-PCR was used to measure the mRNA levels of VEGFA and VEGFR2 genes in lung tissues at the 14th and 28th days.Moreover,the expression of PI3K,p-PI3K,Akt and p-Akt in lung tissues at the 14th and 28th days were detected by western blot.The protein expressions of vWF,p-PI3K and p-Akt in lung tissues at the 14th and 28th days were detected by immunohistochemistry.3.Study on anti-H22 ascites tumor effect of SCU combined with BLM in vitro and in vivoThe pulmonary toxicity is a significant limitation of BLM in its clinical use.Therefore,this study attempts to explore whether SCU can also reduce lung toxicity in the anti-tumor process of BLM.Firstly,H22 cells and MRC-5 cell models were used in vitro,and the cytotoxicity of SCU and BLM on the two cells were detected by MTT assay.The fluorescence intensity of ?-SMA protein in MRC-5 cells was detected by immunofluorescence assay.To evaluate the pulmonary toxicity and antitumor efficacy of SCU on BLM in vitro,flow cytometry was used to detect the apoptosis rate of H22 cells,and western blot method was used to detect the expression of fibrosis associated protein ?-SMA,collagen-I,TGF-?1 in MRC-5 cells and the expression of p53 in H22 cells.Then H22 ascites tumor model of mice was used in vivo.After oral administration of SCU(30,60,90 mg/kg)and intraperitoneal injection of BLM(7.5 mg/kg),the survival cycle of mice was observed in a certain period of time.Then the H22 ascites tumor model was established and the mice were treated with SCU(60mg/kg)and BLM(7.5 mg/kg).To evaluate the attenuation effect of SCU on BLM according to the pathological conditions of the slices,the lung tissues of mice were stained with HE and Masson.At the same time,the contents of oxidative stress factors MPO and MDA,inflammatory factors TNF-? and IL-6 in lung tissues were detected by biochemical kit and ELISA kit.In addition,to illustrate the effect of SCU combined with BLM on the anti-tumor efficacy of BLM,the life cycle,body weight,abdominal circumference and the volume of the ascites were observed.The apoptosis rate of mouse ascites cells was detected by flow cytometry.The biochemical kit was used to detect the content of activated caspase 3 and caspase 8 in mouse ascites.The expression of p53 protein in ascites cells were detected by western blot.The expression of miR-29b gene in lung tissues and ascites cells was detected by RT-PCR.Results1.Study on the effect of SCU on BLM-induced pulmonary fibrosisIn the pulmonary fibrosis model,different doses of SCU could reduce the value of lung coefficient and the content of hydroxy proline in lung tissues,and had an inhibitory effect on BLM-induced pulmonary fibrosis.Compared with model group,60 and 90 mg/kg SCU reduced the inflammatory infiltration and collagen deposition in lung tissues,and alleviated the pathological injury in lung tissues.It could reduce the content of TNF-? and IL-6 in lung tissues and alleviated lung inflammation caused by BLM.Compared with the model group,it also reduced the mRNA levels of MMP-3,MMP-9,TIMP-1,VEGFA and VEGFR2 genes in lung tissues.In addition,60,90 mg/kg of SCU could reduce the expression of collagen-I,?-SMA,vimentin,TGF-?1 in lung tissues and increased the expression of E-cadherin.These results suggested that SCU could inhibit BLM-induced epithelial mesenchymal transformation and abnormal angiogenesis in lung tissue,and alleviated BLM-induced pulmonary fibrosis.2.Study on the mechanism of SCU on BLM induced pulmonary fibrosisIn the A549,MRC-5 cell model induced by TGF-?1,the expression of collagen-I,?-SMA,vimentin and the mRNA levels of MMP-3,MMP-9 and TIMP-1 could be reduced by SCU.Meanwhile,the expression level of E-cadherin protein could be up-regulated suggesting SCU could inhibit the occurrence of epithelial mesenchymal transformation to a certain extent.Further studies on in vitro cell models and in vivo animal models of pulmonary fibrosis showed that SCU could reduce the p-Smad2/3 protein expression in TGF-?1 induced A549,MRC-5 cells and the lung tissues of 14th,28th day,and could reduce the expression of TGF-?1 in the lung tissues of 14th,28th days.In addition,in the BLM-induced pulmonary fibrosis model,the immunohistochemical results of lung tissues on days 14 and 28 showed that SCU could reduce the number of new blood vessels in the lung tissues.Immunohistochemistry,RT-PCR and western blot results of lung tissues at the 14th and 28th days showed that SCU could reduce the gene levels of VEGFA and VEGFR2 in lung tissues,and also decrease the expression levels of p-PI3K and p-Akt in lung tissues.In other words,SCU could reduce abnormal angiogenesis in lung tissues and reduce the pulmonary toxicity of BLM by inhibiting the expression of TGF-?1 protein and affecting the activation of VEGFA/PI3K/Akt pathway.3.Study on anti-H22 ascites tumor effect of SCU combined with BLM in vitro and in vivoMTT results showed that BLM significantly inhibited the proliferation of MRC-5 cells,while SCU had no significant inhibitory effect on MRC-5 cells.Immunofluorescence showed that SCU combined with BLM could decrease the expression of ?-SMA protein in MRC-5 cells.Western blot results showed that SCU combined with BLM could reduce the fibrosis related indicators of ?-SMA,collagen-1,TGF-?1 in MRC-5 cells.At the same time,in the model of H22 ascites tumor,SCU combined with BLM could reduce the pathological injury and collagen deposition of lung tissues.In addition,compared with BLM alone group,SCU combined with BLM could reduce the contents of MPO,MDA,TNF-?,IL-6 and the expression of TGF-?1 protein in lung tissues.These results indicated that SCU combined with BLM could also reduce the pulmonary toxicity of BLM on H22 ascites tumor animal model.MTT results also showed that BLM significantly inhibited H22 cell proliferation.SCU developed inhibitory effect on H22 cell proliferation when the concentration was lower than 40 ?M,and SCU(20,30,40 ?M)combined with BLM(20,30,40 ?M)showed synergistic effect on H22 cell proliferation.In H22 ascites tumor model,compared with BLM alone group,SCU combined with BLM could significantly prolong the life cycle of mice,and reduce the body weight,abdominal circumference and the volume of ascites.Flow cytometry showed that SCU combined with BLM could enhance the apoptosis rate of H22 cells and mouse ascites cells.At the same time,SCU could increase the levels of cleaved caspase 3,8 in ascites cells,and increase the expression of p53 protein in H22 cells and ascites cells.Moreover,the expression level of miR-29b gene in ascites cells and lung tissues were up-regulated.These results indicated that SCU combined with BLM did not affect the anti-tumor effect of BLM.In conclusion,SCU combined with BLM could reduce the pulmonary toxicity of BLM and improve the anti-tumor efficacy of BLM.Conclusion1.In the BLM-induced pulmonary fibrosis model,it was found that SCU inhibited BLM-induced pulmonary fibrosis,and the related mechanism may be related to the epithelial-mesenchymal transformation and angiogenesis mediated by TGF-?1 protein.2.In vivo and in vitro studies on the mechanism of action of SCU on BLM-induced pulmonary fibrosis,it was found that SCU inhibited the expression of TGF-?1 protein,affected the activation of TGF-?1/Smad2/3 and VEGFA/PI3K/Akt signaling pathway,alleviated the epithelial mesenchymal transformation and abnormal angiogenesis in lung tissue,and alleviated the pulmonary toxicity of BLM.3.Through in vivo and in vitro studies on the combination of SCU and BLM against H22 ascites tumor,it was found that SCU combined with BLM could reduce the pulmonary toxicity of BLM without affecting its antitumor efficacy.In conclusion,SCU could inhibit the epithelial mesenchymal transformation and abnormal angiogenesis in lung tissues to reduce the pulmonary fibrosis toxicity caused by BLM,and it could be further developed as an adjuvant of BLM for tumor treatment in the future. |
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