| Objective: Diabetic nephropathy(DN)is a common complication of diabetic patients and one of the important causes of end-stage renal disease(ESRD).With the research and understanding of diabetic nephropathy,it is now widely believed that diabetic nephropathy is a chronic inflammatory disease.The long-term microinflammation state causes changes in the renal glomeruli and tubules,which in turn triggers microalbuminuria and even large amounts of proteinuria.Many scholars now believe that macrophages play an important role in the development of diabetic nephropathy.Bruton's tyrosine kinase(Btk)is a non-receptor tyrosine kinase belonging to the Tec kinase family.Recent studies have shown that Btk is an important molecule of Toll-like receptor 4(TLR4)and Toll-like receptor 2(TLR2)signaling pathways,and participates in and enhances TLR signaling.It also leads to the activation of NF-?B and MAPKs signaling pathways,which in turn initiates the inflammatory response.At the same time,Btk is expressed in myeloid cells other than T lymphocytes and histoplasm cells,which is superior to TLR signaling pathway.This study explored the role and molecular mechanism of Btk in diabetic macrophage activation and renal inflammation.The treatment of kidney disease provides new ideas and measures.Methods:Part 1:Macrophages derived from mouse bone marrow were extracted and identified by flow cytometry after maturation.The effects of different concentrations of Btk inhibitor PCI-32765 on macrophage activity were detected by CCK-8 assay.Then the expression of p-Btk at different time and different inhibitor concentrations was detected by Western blot to optimize the experimental conditions.Macrophages were divided into low glucose group(LG,5mmol/L),inhibitor control group(LG +PCI-32765),high glucoce(HG,30 mmol/L),high glucoce +PCI-32765 group(HG+PCI-32765).The chemotaxis of macrophages was observed by m RNA and total protein Transwell.The concentrations of IL-1?,TNF-? and MCP-1 in supernatant were detected by ELISA,IL-1?,TNF-? and MCP-1m RNA were detected by real-time quantitative PCR.The expression of i NOS,Btk,p-Btk,ERK,p-ERK,JNK,p-JNK,p38,pp38,NF-?B p65,p-NF-?B p65,I ?B and p-I ?B were detected by western blot,and the activation of macrophages and the nuclear metastasis of NF-?B p65 were detected by confocal laser.Part 2:Fourteen Btk knockout mice and 14 wild-type mice of the same background were selected as the study objects.The diabetic group was injected with 50mg/kg.d STZ for 5 days,and the blood glucose at the tail tip of each mouse was detected after one week.Blood glucose ? 16.7mmol/L was regarded as successful modeling.The experiment was divided into four groups: 7 wild-type mice as normal control group(WT,n=7),7 diabetic mice(Diabetic,n=7),Btk knockout mice group(Btk-/-,n=7)and Btk knockout diabetic mice group(Btk-/-diabetic,n=7).After 12 weeks,the body weight,blood glucose,kidney weight,blood samples,24 hours urine and kidney were collected.Enzyme-linked immunosorbent assay(ELISA)was used to detect urinary protein excretion rate of 24 hours in mice,pathological changes of kidney were observed by light microscope,IL-1? and MCP-1 were detected by immunohistochemistry and CD68 was detected by confocal laser.The expression of IL-1?,MCP-1 and TNF-? was detected by real-time PCR.The expression of IL-1?,i NOS,TNF-?,Btk,p-Btk,ERK,p-ERK,JNK,p-JNK,p38,pp38,NF-?B p65,p-NF-?B p65 were detected by western blot.Results:Part 1:F4/80 and CD11 b double positive cells accounted for 94.5% of the total number of cells.The purity and maturity of the cells met the requirements of the experiment.After optimization of the experimental conditions,the concentration of high glucose was 25 mmol/L and the intervention concentration of PCI-32765 was 10-6 mmol/L;The results of Transwell chemotaxis test showed that the number of macrophage chemotaxis was significantly higher in high glucose group than that of low glucose group(P< 0.05),and the number of macrophages induced by PCI-32765 was significantly lower than that of high glucose group(P<0.05).The results of real-time quantitative PCR showed that the levels of IL-1 ?,TNF-? and MCP-1m RNA in the high glucose group were significantly higher than those in the low glucose group(P< 0.05),and the m RNA levels in the inhibitor group were significantly lower than those in the high glucose group(P<0.05).ELISA results showed that the concentrations of IL-1?,TNF-? and MCP-1 in the supernatant of high glucose group were significantly higher than those in low glucose group(P<0.05).The concentrations of IL-1?,TNF-? and MCP-1 in inhibitor group were significantly lower than those in high glucose group(P< 0.05).The results of western blot showed that the expression of i NOS,p-Btk,p-ERK,p-JNK,pp38,p-NF-?B p65,p-I?B in high glucose group was significantly higher than that in low glucose group(P<0.05).The ratio of i NOS,p-Btk,p-ERK in inhibitor group was significantly lower than that in high glucose group(P<0.05).The expression of p-NF-?B p65 and p-I?B inhibited significantly(P< 0.05)by PCI-32765,but the expression of p-ERK and p-JNK did not change(P> 0.05).The results of laser confocal showed that the expression of i NOS,macrophage activation marker,and the nuclear metastasis of NF-?B p65 in high glucose group was significantly higher than that in low glucose group,but the expression of these in HG+PCI-32765 group was lower than that in high glucose group.Part 2:Compared with WT group,diabetic mice showed significant changes in blood glucose,glycosylated hemoglobin,kidney weight/100 g body weight and 24 hour urinary protein excretion rate(P<0.05),and compared with Diabetic group,urinary protein excretion rate decreased significantly(P<0.05)in Btk-/-diabetic group,but blood glucose,glycosylated hemoglobin and kidney weight/100 g body weight did not change significantly(P<0.05).Under the light microscope,the glomerular volume,basement membrane thickening and mesangial cell proliferation of Diabetic group were larger than those of normal group,and that of Btk-/-diabetic group was better than that of Diabetic group.The results of real-time quantitative PCR showed that the expression of renal inflammatory factors in diabetic mice was significantly higher than that in normal controls(P<0.05).The level of renal inflammation in Btk-/-diabetic mice was significantly lower than that in diabetic mice(P<0.05).Immunohistochemistry and laser confocal focus showed that the infiltration of macrophages in diabetic mice was significantly increased,and the infiltration of macrophages decreased with the knockout of Btk.The results of western blot showed that the expression of p-Btk,p-ERK,p-JNK,p-NF-?B p65,p-I?B in kidney of diabetic group were significantly higher than that of normal mice(P<0.05),and the expression of these proteins decreased significantly in the kidneys of Btk-/-diabetic mice(P<0.05).Conclusions:1.Stimulated by high glucose,macrophages derived from bone marrow were activated,the expression of p-Btk,p-ERK,p-JNK,pp38,p-NF-?B p65,p-I?B was up-regulated,and the expression of inflammatory factors IL-1? and MCP-1,TNF-? were increased.Btk knockout inhibited the activation of MAPK and NF-?B signaling pathway and down-regulated the expression of inflammatory factors in macrophages.2.In animal experiment,the expression of p-Btk in kidney of diabetic mice was obviously increased,the excretion rate of urinary protein was increased.Glomerular hypertrophy,thickening of basement membrane and deposition of extracellular matrix were observed.In addition,renal inflammation and macrophage infiltration increased,which were decreased after Btk knockout,suggesting that Btk may be involved in the development of diabetic nephropathy.3.In diabetic mice,the expression of p-Btk,p-ERK,p-JNK,pp38,p-NF-?B p65 and p-I?B were up-regulated,the synthesis of inflammatory factors IL-1?,TNF-? and MCP-1 was increased.The NF-?B and MAPK signaling pathway and the expression of inflammatory factors were down-regulated after Btk knockout,which suggest that the mechanism of renal injury induced by Btk may be related to the activation of MAPK and NF-?B signaling pathway. |
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