| Objective: Acute Kidney Injury(AKI)is a severe disease with limited treatment.This study established an acute kidney injury model in mice by intraperitoneal injection of cisplatin,and observed the improvement of renal function and inhibition of inflammation after Quercetin treatment.The potential mechanism of Quercetin on inhibition of inflammation of macrophage in AKI kidney provides scientific basis for prevention and treatment of acute kidney injury.Methods: In this study,cisplatin-induced AKI mouse models were established in vivo,and LPS-induced BMDM(Bone Marrow-derived Macrophages)inflammatory model was established in vitro.The effects of Quercetin on renal injury and inflammation of AKI were observed by adding different doses of Quercetin.A total of 60 SPF C57BL/6 male mice were divided into 6 groups,which were grouped after one week of adaptive feeding after purchase,with 10 mice in each group.Grouped into: saline control group,cisplatin-induced AKI group,cisplatin-induced AKI + Quercetin 50mg/kg/day group,cisplatin-induced AKI + Quercetin 100mg/kg/day group,Quercetin 100mg/kg/day Group,DMSO solvent control group.Quercetin was administered by intragastric administration one hour before intraperitoneal injection of cisplatin(20 mg/kg),and was administered daily until mice were sacrificed on day 3.The BMDM cells used in the in vitro experiments were induced from the bone marrow of male mice and induced in L929 cell supernatant for 7 days,then inflammatory BMDM cells were induced with LPS,and Quercetin was added for intervention.The cells were divided into: control group,LPS group,solvent DMSO control group,DMSO + LPS group,LPS + 4?M Quercetin group and LPS + 8?M Quercetin group.This study subsequently used a variety of methods to test samples from in vivo and in vitro experiments,including the use of Real-time PCR to detect inflammatory factor expression in BMDM cells and kidneys;Western blot to detect protein levels;and ELISA to analyze concentration of inflammatory factors in serum and cell supernatant;Serum creatinine and urea nitrogen kits were used to detect mouse kidney function indicators;H&E and PAS staining were used to analyze renal pathological changes;Immunofluorescence and immunohistochemistry were used to detect the expression and location of corresponding proteins in tissues and cells;Flow cytometry was used to detect changes of the expression of Mincle-related signaling proteins and macrophage polarization markers in macrophages in renal cell suspensions and BMDM cells.Results: 1.Quercetin significantly improved the inflammatory morphology of BMDM cells induced by LPS in vitro,inhibited the expression and secretion of inflammatory factors IL-1?,IL-6 and TNF-?(P<0.001),down-regulated the inflammation-related protein levels of iNOS and phosphorylated NF-?B(P<0.001);2.The results of in vivo experiments showed that compared with the normal group,creatinine and urea nitrogen were significantly increased in cisplatin-induced AKI mice(P<0.0001),and the renal tubular necrosis score also increased significantly(P<0.0001).After administration of Quercetin,renal function indexes of AKI mice were significantly reduced(P<0.001),and the renal tubular necrosis score was also decreased(P<0.01),as well as the expression and secretion of inflammatory factor IL-1?,IL-6 and TNF-? in the kidney and in the blood were significantly reduced(P<0.001),suggesting that Quercetin improves AKI kidney function and inhibits inflammation;3,Quercetin down-regulated the mRNA expression(P<0.01)and protein level(P<0.01)of Mincle and Syk(P<0.001)in vivo and in vitro.The in vivo results also showed that the infiltration of macrophage in the kidney of AKI mice was significantly increased.Immunofluorescence results showed that Minlce was abundantly expressed on the infiltrated macrophage in the kidney,and the flow cytometry results showed that Mincle and activated Syk were highly expressed macrophage of AKI kidney,which were significantly down-regulated after Quercetin was administered(P<0.001);4.Flow cytometry results showed that Quercetin can significantly inhibit polarization of macrophage in AKI kidney and LPS-induced BMDM,specially inhibit pro-inflammatory M1 macrophage activity(marker iNOS)(P<0.001),and increase anti-inflammatory M2 macrophage activity(marker CD206)(P<0.001);5.Mincle ligand was added to Quercetin-treated inflammatory BMDM cell.After overexpression of Mincle,the protein levels of iNOS,Syk,and NF-?B were increased(P<0.01).At the same time,the mRNA expression and secretion levels of inflammatory factors IL-1?,IL-6 and TNF-? were significantly increased(P<0.01),and the mRNA expression of iNOS,a marker of M1 macrophages,was also up-regulated(P<0.001)and the M2 macrophage marker CD206 was down-regulated(P<0.05).Conclusion: 1.Quercetin can significantly inhibit the inflammatory morphology of macrophages induced by LPS,down-regulate the expression and secretion of inflammatory factors,and shows a strong inflammatory inhibition effect;2.Quercetin improves the kidney function of AKI mice and reduces the pathological changes of the kidneys of AKI mice,and down-regulates the expression and secretion of renal inflammatory factors,indicating that Quercetin can improve AKI-induced kidney injury and inflammation;3.Quercetin can significantly inhibit expression and activation of Mincle and its downstream molecules Syk and NF-?B in macrophages in vivo and in vitro;4.Quercetin can inhibit the pro-inflammatory M1 macrophage phenotype and increase the anti-inflammatory M2 macrophage phenotype in vitro and in vivo;5.Cell recovery experiment results showed that overexpression of Mincle reversed the cellular inflammation inhibited by Quercetin,indicating that Quercetin reduces macrophage inflammation by down-regulating Mincle.The results of this study confirm that Quercetin inhibits the expression and activation of macrophages Mincle and its downstream signal molecules,thereby balancing the polarization of macrophage,down-regulating the inflammatory response of macrophage,improving AKI kidney function and reducing kidney damage. |
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