The Regulation Effects Of Kaempferol On M1/M2 Phenotypic Transformation Induced By LPS/High Glucose And Their Impacts On Podocyte Injury In Vitro

Objective:1.The antioxidant effects of kaempferol were studied in vitro.2.The effects of kaempferol on lipopolysaccharide?LPS?and high glucose-induced RAW264.7macrophage polarized to classically activated macrophage?M1?and alternatively activated macrophage?M2?types were investigated.3.The effects of kaempferol on LPS-induced and high glucose-induced podocyte injury were tested directly,and indirectly via the macrophage polarization.Methods:1.The antioxidant activities of kaempferol were determined by1,1-diphenyl-2-trimitrophenylhydrazine?DPPH?method and oxygen free radical scavenging energy?ORAC?method.2.Griess assay was used to determine the effects of LPS and high glucose on the induction of nitric oxide?NO?in mouse RAW264.7 cells and to establish an in vitro model of macrophage transformation to M1 type.3.The effects of different concentrations of kaempferol on macrophage growth were determined by CCK8 method in LPS and high glucose environment.4.Western Blot was used to measure the protein expression of tumor necrosis factor-??TNF-??which represents M1polarization,and the protein of mannose receptor?CD206?and arginase 1?Arg-1?that indicates M2 polarization.The effects of kaempferol on those protein expressions were tested also.5.Griess assay was used again to measure the effect of kaempferol on the NO production in LPS and high glucose induced RAW264.7 cells.The effects of kaempferol on interleukin 1??IL-1??level in LPS and high glucose induced RAW264.7 cells were determined by ELISA.6.The effect of kaempferol on high glucose and LPS induced podocyte survival were determined by MTT assay.7.The collected macrophage supernatants?MS?,including LPS-treated,high glucose-treated and kaempferol-treated supernatants??LPS?MS,?HG?MS,?Kaem?MS?,were collected to determine the protective effects of kaempferol indirectly.Results:1.The half inhibitory concentration(IC₅₀)of kaempferol on scavenging DPPH was significantly lower than the positive control??5.3±1.1?g/ml vs?38.8±2.5?g/ml:P<0.05?.The ORAC value of kaempferol was significantly higher than that of positive control vitamin C??2678±272?molTE/g vs?2028±196?molTE/g:P<0.05?.2.The contents of NO production in 1.0?g/ml LPS and 33.3 mM glucose induced macrophage were significantly higher than that in the normal control group?P<0.05?.3.The survival rates of macrophage were higher than 90%after the cells were treated with kaempferol at4–8?M in 12–24 h separately.4.Both 33.3 mM high glucose and 1.0?g/ml LPS increased the TNF-?protein expression significantly compared with 11.1 mM control group?P<0.05?,while the expression of CD206 and Arg-1 decreased significantly?P<0.05?.Compared with the high glucose group,kaempferol at two tested concentrations decreased the expression of TNF-?significantly?P<0.05?,and increased the Arg-1expression significantly?P<0.05?.The inhibitory effect of 8?M kaempferol on TNF-?expresstion,and the increasing effect on Arg-1 and CD206 expression showed significant difference compared to the LPS group?P<0.05?.The effect of 4?M kaempferol had significant inhibitory effect on TNF-?expression compared to LPS group?P<0.05?.Two different doses of kaempferol showed significant difference in inhibiting effect on TNF-?expression and increasing effect on the expression of Arg-1?P<0.05?.5.When compared with control group,the production of NO and IL-1?level were significantly increased in high glucose and LPS induced macrophage?P<0.05?.Kaempferol showed significant effects at two tested concentrations on inhibiting NO production and decreasing IL-1?level.6.When treated with high glucose and LPS for 18h,the survival rate of podocyte was?82.0±2.6?%and?81.5±7.1?%,which was significantly lower than the control group?100±2.6?%separately?P<0.05?.Compared with the high glucose and LPS,the survival rate of kaempferol-treated podocyte did not show any difference?P>0.05?.7.The survival rate of podocyte in?high glucose?MS and?LPS?MS decreased to?53.5±5.7?%and?54.4±6.1?%,and was significantly lower than that of?control group?MS?100.0±3.1?%?P<0.05,respectively?,and even lower than that of high glucose and LPS-treated podocyte?P<0.05,respectively?.Compared with?high glucose?MS,the survival rate of kaempferol?4?M?and?8?M?MS increased significantly?P<0.05?.At the same time,the survival rate of kaempferol?4?M?and?8?M?MS was significantly increased compared with?LPS?MS?P<0.05?.Conclusion:1.Kaempferol showed antioxidant activity on scavenging DPPH,hydrogen peroxide?H₂O₂?and NO in vitro.2.The protein expression of TNF-?increased,while Arg-1 and CD206 expression decreased in high glucose and LPS induced RAW264.7cells.Kaempferol showed the increasing effects on Arg-1 and CD206 expression in LPS and high glucose induced macrophage.On the other hand,kaempferol decreased TNF-?expression and IL-1?.3.Kaempferol showed the indirect protective effect on LPS and high glucose-induced podocyte injury via macrophage differentiation.4.The protective effect of kaempferol in LPS and high glucose-induced podocyte injury was related with its antioxidant activity,the regulation of M1/M2 phenotype differentiation and the inhibition on IL-1?level.