The Therapeutic Effects Of Tanshinone-1 For Radiation-Induced Bladder Injury (RIBI) Via Action Of Nrf2/ARE Signal Pathway

Raditiaon induced baldder injury(RIBI)is one of common complications after radiotherapy to tumors located in pelvic region.Recently,specific treatment strategies for RIBI are very limited.Therefore,it’s important to explore the potential mechanism involved in the development of RIBI and specific treatment strategies.It has been demonstated that tanshinone-I(T-1)has pharmacological effect in relaxing blood vessel,scavenging free radicals and stabilize endothelial function,which is benifiecal for the treatment of cardiovascular diseases such as stroke,heart ischemia,and atherosclerosis.As one of the most popular focus on the researches of anti-oxidative stress,nuclear erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)signal pathway is reported to be related with the development of several diseases.The anti-oxidative stress effect of tanshinone-I has been demonstrated in the treatment of lung injury via action of Nrf2/ARE signal pathway.However,the effect of tanshinone-I and Nrf2/ARE signal pathway in the treatment of radiation induced bladder injury still remain unknown.In the present project,we intend to explore the therapeutic effect of tanshinone-I in the treatment of RIBI.We hypothesize that activation of Nrf2/ARE is crucial for the therapeutic effects of T-I for the treatment of RIBI.Our study includes three main parts:Part Ⅰ:the expression of Nrf2/ARE in radiation induced bladder injuryTo investigate the expression of Nrf2/ARE in RIBI through in vitro cell model amd in vivo animal model.In vitro cell model was established by irradiating human ureteral epithelial cells(sv-huc-1 cell line)with X-ray while in vivo animal model was established by irradiating mice bladder area with X-ray.Cell viability,oxidative stress markers and bladder histology were investigated.qPCR and WB were used to determine the expression of Nrf2 and its downstream target genes.siRNA was used to silence the gene expression of Nrf2 in cells.The results indicated the dose-dependent toxicity of X-ray to sv-huc-1 cells.The expression of Nrf2/ARE signal pathway was upregulated in RIBI.After silencing of Nrf2,the expression of Nrf2/ARE was downregulated.The toxicity of X-ray is greater in Nrf2 silenced cells.We conclude that Nrf2/ARE might paly an important role in the development of RIBI and could be a new target for the treatment of RIBI.Part Ⅱ:The therapeutic effects of tanshinone-I for RIBITo explore the protective effects of T-I for RIBI using the in vitro cell model and in vivo mice model established in Part I.The protective effects of T-I for sv-huc-1 cells was evaluated by testing cell viability and cell apoptosis.The protective effects of T-I for mice was evaluated by investigating bladder histology.The oxidative stress in cells and bladder tissue was determine by related oxidative stress markers.The results indicated that pretreatment of T-I could reduced X-ray induced cell injury and cell apoptosis.Also,the oxidatives stress was ameliorated in cells pretreated with T-I.Similarly,T-I could reduce the mucosal edema and inflammation in urothelium in the early phase of RIBI.The oxidatives stress was ameliorated in bladder tissue pretreated with T-I in the early phase of RIBI.Also,T-I could reduce the fibrosis and increase the vessel density in the late phase of RIBI.Our results showed the protective effect of T-I for RIBI,probably through anti-oxidative effects.Part Ⅲ:T-I reduces oxidative stress in RIBI via activation of Nrf2/ARE signal pathwayTo explore the role Nrf2/ARE signal pathway in the treatment of T-I for RIBI.qPCR and WB were used to determine the expression of Nrf2/ARE in sv-huc-I cells and mice bladder tissues in each experimental group.siRNA was used to silence the gene expression of Nrf2 in sv-huc-1 cells.The therapeutic effects of T-I was investigated in Nrf2 silenced sv-huc-1 cells.Gene knockout technology was used to silence the gene expression of Nrf2 in mice.The therapeutic effects of T-I was evaluated in Nrf2 silenced mice.Our results indicated that T-I could increase the expressions of Nrf2/ARE signal pathway in radiated cells and mice.T-I could protmote the transport of Nrf2 from the cytoplasm to the nuclei,and thereby activate the downstream pathway.The therapeutic effects of T-I was significantly reduced in Nrf2 silenced cells and mice.Our data suggests that the therapeutic effects of T-I for RIBI might be through activation of Nrf2/ARE signal pathway.