| PartⅠExpression of Fractalkine,Sirtuin3,S100A4 and macrophage polarization in lupus nephritisObjective: To construct chemokine Fractalkine(CX3CL1,FKN)knockdown lupus mice to explore the molecular mechanism of FKN,Sirtuin 3(SIRT3)deacetylase and S100A4,and the effect of FKN on renal function in lupus mice.Methods: Fourty 10-week-old C57BL/6J female mice and twenty MRL/lpr female mice were randomly divided into 4 groups.They were:(1)control group(C57BL/6J mice were injected negative control recombinant adeno-associated virus r AAV in situ),(2)FKN knockdown group(C57BL/6J mice were injected FKN recombinant adeno-associated virus r AAV in situ),(3)Lupus group(MRL/lpr mice kidney injection negative control recombinant adeno-associated virus r AAV)and(4)lupus FKN knockdown group(MRL/lpr mice kidney injection FKN recombinant adeno-associated virus r AAV).(1)The 24-hour urinary protein content and Serum Serum creatinine(Scr)and Blood Urea Nitrogen(Bun)levels of mice in each group were detected by urinary protein quantitative kit,creatinine detection kit and urea nitrogen detection kit,respectively.(2)Enzyme-linked immunosorbent assay(ELISA)was used to detect the anti-DSDNA and anti-Smith antibody levels in serum of mice of each group.(3)Hematoxylin-Eosin(HE)and Periodic Acid-schiff(PAS)staining were used to detect pathological changes of kidney tissue in all groups of mice.(4)The protein expression levels of FKN,SIRT3,S100A4,macrophage M1 polarization index(i NOS,IL-6)and macrophage M2 polarization index(ARG1)in renal tissue of each group were detected by Western blot and q RT-PCR.Results:(1)At the age of 20 weeks,MRL/lpr mice showed increased levels of urinary protein,blood creatinine and urea nitrogen,as well as increased levels of anti-SM antibody and anti-DSDNA antibody.Pathological findings showed a large number of inflammatory cell infiltration in the glomerulus and platinum ear changes,suggesting that lupus mice were in the state of LN.(2)Compared with the control group,the levels of 24-hour albuminuria,creatinine and urea nitrogen in lupus group were increased(P < 0.05),and the levels of these indexes were decreased in lupus FKN knockdown group compared with lupus group(P <0.05);(2)Compared with the control group,the serum anti-DSDNA and anti-Smith antibodies were increased in lupus group(P < 0.05),and the above antibody levels were decreased in FKN knockdown mice compared with lupus group(P < 0.05).(3)HE and PAS staining showed that compared with the control group,the renal pathological damage was more severe in the lupus group.Compared with lupus group,the pathological damage of kidney in lupus FKN knockdown mice group was reduced.(4)q RT-PCR and Western blot showed that compared with the control group,the expression of FKN and the expression of M1 macrophage polarization(i NOS,IL-6)were increased in lupus group(P < 0.05),suggesting that macrophages were transformed to M1 polarization in LN.Compared with lupus group,the polarization of FKN and M1 macrophages(i NOS,IL-6)was decreased in FKN knockout mice group(P < 0.05),and the polarization of SIRT3,S100A4 and M2 macrophages(ARG1)was increased in FKN knockout mice group(P < 0.05).It is suggested that FKN knockdown can promote the transformation of macrophages to M2 in MRL/lpr mice.Conclusion:Knockdown of FKN gene can improve renal function in lupus mice by promoting the transformation of macrophages into M2.PartⅡFractalkine regulates S100A4 to participate in IFN-γ induced macrophage polarization by SIRT3Objective: Based on animal experiments,to investigate whether FKN can regulate S100A4 through SIRT3 and then affect the polarization of Raw264.7 macrophages.Methods: Raw264.7 macrophages were cultured in vitro,transfected with FKN knockdown lectin virus,and interfered with IFN-γ and SIRT3 agonists.It can be divided into8 groups:(1)Blank control group,(2)IFN-γ group,(3)SIRT3 group(SIRT3-specific agonist group),(4)IFN-γ+SIRT3 group,(5)FKN-Si group(FKN knockdown group),(6)FKN-Si+IFN-γ group,(7)FKN-Si +SIRT3 group,(8)FKN-Si +IFN-γ+SIRT3 group.The protein expressions level of FKN,SIRT3,S100A4,M1 macrophage polarization(i NOS,IL-6)and M2 macrophage polarization(ARG1)were detected by Western blot.Results: Compared with blank control group,IFN-γ induced polarization of Raw264.7macrophages towards M1,the expressions of M1 macrophages polarization indices(i NOS,IL-6)were increased(P < 0.05),and the expressions of SIRT3,S100A4 and M2 polarization indices(ARG1)were decreased(P < 0.05).Compared with IFN-γ group,the expressions of SIRT3,S100A4 and M2 polarization indexes in FKN-si+IFN-γ group were increased(P <0.05).Compared with FKN-si+IFN-γ group,S100A4 and M2 polarization index ARG1 in FKN-si+IFN-γ+SIRT3 group were further increased(P < 0.05).Conclusion: Knockdown of FKN gene can increase the expression of S100A4 protein by activating SIRT3 and promote the M2 polarization of Raw264.7 macrophages. |
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