Protective Effect And Mechanism Of Myricetin Nanomedicine On Drug-induced Kidney Injury

ObjectiveCisplatin is a highly effective platinum-based anticancer drug.It is one of the earliest and most widely used metal-based chemotherapy drugs.One of the common side effects of cisplatin therapy is acute kidney injury(AKI).However,the pathogenesis of AKI has not been clarified and effective intervention measures are lacking.Recent studies have found that the c GAS-STING signaling pathway plays a key role in the regulation of inflammatory diseases and may be a potential target for the regulation of AKI.Myricetin(Myr)has been shown to have remarkable biological activities,including anti-inflammatory and anti-oxidative activities,suggesting its potential in the prevention and treatment of AKI.However,the low solubility of myricetin in aqueous solution and poor oral bioavailability limit its further research.It is promising to improve the oral application of myricetin through drug delivery systems.Therefore,in this study,by constructing myricetin loaded nanomedicine,we investigated its role in the treatment of cisplatin-induced AKI,and explored its mechanism of action in the treatment of AKI by regulating c GAS-STING signaling pathway.The development of this study is expected to provide a new treatment plan for AKI,which has important research value and potential clinical significance.Methods1.HS15-myricetin nanomicelles(HS15-Myr)were constructed by thin film hydration method,characterized and analyzed,and the optimal formulation ratio was screened;Its storage stability was investigated to determine its antioxidant activity and drug release in vitro,and its intracellular uptake and localization were studied.Human renal proximal tubular epithelial cells(HK-2 cells)were used to establish acute kidney injury(AKI)model in vitro,and the renoprotective effect of HS15-Myr was evaluated;The effects of HS15-Myr on reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were detected;Immunofluorescence and Western Blot were used to detect the effect of HS15-Myr on cisplatin-induced DNA damage and c GAS-STING signaling pathway.Cisplatin-induced AKI mouse model was established and HS15-Myr was orally administered.The renal protective effect of HS15-Myr was evaluated by body weight,renal tissue H&E staining,serum urea nitrogen(BUN)and creatinine(SCr);The effects of HS15-Myr on renal related antioxidant enzymes such as SOD and GSH were detected;ELISA was used to detect the effect of HS15-Myr on IL-1β and TNF-α in kidney tissue;Immunohistochemistry was used to detect the effect of HS15-Myr on DNA damages-c GAS-STING signaling pathway in mice.2.Myricetin loaded micelles(RA-Myr)were constructed using rebaudioside A(RA)as the carrier,characterized and analyzed,and the optimal formulation ratio of RA-Myr was determined;Its storage stability was investigated,its antioxidant activity and drug release were measured in vitro,and its intracellular uptake and localization were studied.Acute kidney injury(AKI)was induced in human renal proximal tubular epithelial cells(HK-2 cells)using cisplatin(RA-Myr);The effects of RA-Myr on reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were detected.Immunofluorescence assay was used to detect the effect of RA-Myr on cisplatin-induced DNA damage and c GAS-STING signaling pathway.The effects of RA-Myr combined with RU.521 on the proliferation,ROS accumulation and MMP of HK-2 cells were evaluated by adding the c GAS inhibitor RU.521,and the effects of RA-Myr combined with RU.521 on DNA damage-c GAS-STING signaling pathway were detected.The mechanism of the renoprotective effect of RA-Myr was elucidated by regulating the c GAS-STING signaling pathway in vitro.Cisplatin induced AKI mouse model was established and RA-Myr was orally administered.The in vivo renal protective effect of RA-Myr was evaluated by body weight,renal tissue H&E staining,serum urea nitrogen(BUN)and creatinine(SCr);RT-PCR was used to detect the effects of RA-Myr on renal related antioxidant enzymes SOD,NQO1,HO-1 and inflammatory factors such as TNF-α,IL-1β and IL-6;RT-PCR and immunohistochemistry were used to detect the effect of RA-Myr on DNA damage-c GAS-STING signaling pathway in mice.Results1.HS15-Myr was prepared in this study.Its encapsulation efficiency was high,its particle size was 12.76±5.8 nm,its polydispersity index(PDI)was 0.062±0.005,and its Zeta potential was-7.31 ± 9.0 m V;It has excellent storage stability;In vitro antioxidant assay showed that HS15-Myr enhanced the antioxidant activity of Myr;HS15-Myr significantly increased the solubility and cellular uptake of Myr.HS15-Myr mitigated cisplatin-induced HK-2 cell death,ROS accumulation and MMP reduction in vitro;Further mechanistic results showed that HS15-Myr inhibited cisplatin-induced DNA damage and c GAS-STING signaling pathway activation.In vivo results showed that HS15-Myr ameliorated cisplatin-induced AKI,including body weight loss,renal histopathological damage,and elevated BUN and SCr levels,indicating renoprotective potential of HS15-Myr(P<0.05);Further mechanistic studies showed that HS15-Myr inhibited cisplatin-induced changes in the levels of antioxidant enzymes and inflammatory cytokines,and inhibited cisplatin-induced activation of DNA damage-c GAS-STING signaling pathway(P<0.05).2.RA-Myr was prepared with high encapsulation efficiency.The mean size of the micelles was 112.55±0.25 nm,the polydispersity index(PDI)was 0.290±0.004,and the Zeta potential was 13.92±1.06 m V;And has a very considerable storage stability;Results from the in vitro antioxidant assays showed that RA-Myr enhanced the antioxidant activity of Myr;RA-Myr significantly enhanced cellular uptake of Myr.RA-Myr mitigated cisplatin induced HK-2 cell death,ROS accumulation and MMP reduction in vitro;Mechanistic results showed that RA-Myr inhibited cisplatin-induced DNA damage and c GAS-STING signaling pathway activation.RA-Myr combined with RU.521 further alleviated cisplatin induced HK-2 cell death,ROS accumulation and MMP decline,and inhibited cisplatin-induced DNA damage and c GAS-STING signaling pathway activation.RA-Myr ameliorated the AKI symptoms in cisplatin-induced mice,including weight loss,increased renal index,renal histopathological damage,and elevated serum BUN and SCr levels(P<0.05);Mechanistic studies showed that RA-Myr enhanced the activities of renal antioxidant enzymes,alleviated renal inflammation,and inhibited cisplatin-induced activation of DNA damage-c GAS-STING signaling pathway in AKI mice(P<0.05).ConclusionMyricetin nanomicelles improve the therapeutic effect of myricetin on cisplatin-induced AKI in vitro and in vivo,and the mechanism may be related to the inhibition of DNA damage-c GAS-STING signaling pathway.