Futudan Improves Inflammatory Microenvironment By Regulating Macrophage Polarization In Diabetic Kidney Disease

ObjectiveFirstly,to analyze the polarization status of monocytes M1/M2 in peripheral blood of patients with type 2 diabetic nephropathy,and to investigate the relationship between polarization imbalance of monocyte macrophage system and the progression of type 2 diabetic kidney disease(DKD).Secondly,using the diabetic nephropathy mouse model(db/db),Futudan and Hyperoside(origin from Semen Cuscutae)were administered by gavage.To observe the status of body weight,glycolipid metabolism and the excretion rate of urine protein among db/db mice.To study the effect of drugs on the activation of M1 and M2 macrophage subtypes in kidney tissue,abdominal cavity,and spleen,providing experimental evidence for the treatment of DKD with Futudan and Hyperoside.Finally,bone marrow-derived macrophages(BMDMs)stimulated with high glucose were used to simulate the high glucose state in vitro,and two cell models were co-cultured with M2 macrophages and splenic T lymphocytes.From the cellular and molecular levels,observing the polarization effect of Hyperoside on macrophage M1/M2 phenotype and its corresponding immunological mechanism.Methods1.Clinical study on the polarization status of peripheral blood M1/M2 in patients with diabetic kidney disease.This study adopted a cross-sectional study method.Sixty-three patients with DKD inpatients or outpatients in Guangdong Provincial Hospital of Chinese Medicine were selected,and six healthy volunteers matched with their age were randomly selected at the physical examination center of our hospital as controls groups(NC group).All subjects were collected the general situation of the selected objects and the corresponding biochemical indicators.The elbow venous blood of the subject was collected on an empty stomach in the early morning,and the primary mononuclear cells in the peripheral blood were extracted using a mononuclear cell separation solution.The ratio of M1 and M2 mononuclear cells in the peripheral blood was detected by flow cytometry and enzyme-linked immunosorbent assay.Moreover,DKD patients were divided into three subgroups according to 24h urinary albumin excretion rate(UAER),to explore the relationship between mononuclear macrophage system polarization imbalance and the development of diabetic nephropathy.2.Study on the effect of Futu Dan and hyperoside on the polarization of db/db mouse macrophages.Selecting 35 8-week-old healthy male db/db mice and 6 normal healthy male db/m mice.Randomly divide 35 db/db mice into 5 groups according to the body weight,including model group(db/db),low-dose Futu Dan(db/db+FTD,40mg/kg/day),high-dose Futu Dan(db/db+FTD,80mg/kg/day),low-dose hyperoside group(db/db+LHPS,40mg/kg/day),high-dose hyperoside group(db/db+HHPS,80mg/kg/day),7 mice in each group.Real-time PCR was used to detect the effects of Futudan and hyperoside on the polarization of macrophages in the kidney tissues of each group of mice.Flow cytometry(FCM)analysis was used to detect the effect of Futudan and hyperoside on the polarization of macrophages derived from peritoneal cavity and spleen of mice in each group.In addition,the changes of podocyte number and morphology in the kidney tissue of mice in each group were observed under electron microscope.3.In vitro study of Hyperoside among macrophage polarization.BMDMs were inoculated into six-well plates to observe the effect of different concentrations of Hyperoside on the polarization of macrophages under the treatment with high glucose.The cells are grouped as follows:normal control group(Control),high glucose group(HG),Hyperoside high-dose group(HPS 50uM,HHPS),HG+Hyperoside middle-dose group(HPS 25uM,MHPS),HG+Hyperoside low-dose group(HPS 12.5uM,LHPS).qPCR was used to detect the expression of M1 type and M2 type markers of macrophages in each group.In addition,constructing M2-type macrophages and mouse spleens using the Transwell chamber with T lymphocyte co-culture model,and then intervene with different concentrations of Hyperoside.The experimental groups are as follows:single culture of T lymphocytes(T),co-culture of M2 macrophages and T lymphocytes(M2+T),M2+T+HPS 50uM(HHPS)and M2+T+HPS 12.5uM(LHPS).Flow cytometry was used to detect T lymphocyte subsets,mainly including Th2 cells(IL-4+CD4)and Tregs(Foxp3+CD4)immune cell subsets.Compare the presence of hyperoside on M2 and T cells Certain relationship.Results1.Clinical study.on the polarization status of peripheral blood M1/M2 in patients with diabetic kidney disease.Compared with the healthy control group,the levels of MO(P<0.001)and M2(P<0.0001)in the peripheral blood mononuclear cells of patients with incipient DN were significantly increased;Compared with the incipient DN,the level of M1 in Overt DN was significantly increased(P<0.05),but the M2 levels showed a downward trend in both the Overt DN and the stage of uremia,of which the CD206 level in the overt DN decreased by 63.85%(P<0.0001)compared with the incipient DN(P<0.0001).Compared with the incipent DN,CD206 decreased by 72.55%in uremia phase(P<0.001).Compared with the healthy control group,plasma TNF-α(M1 type)levels in patients with uremia were significantly increased(P<0.0001);compared with incipient DN patients,plasma TNF-α(M1 type)in patients with uremia significantly increased(P=0.0002,p<0.001);Compared with overt DN,plasma TNF-α(M1 type)level in uremic patients was significantly increased(P=0.0018,P<0.01).Compared with the healthy control group,the level of IL-4(M2 type)in the peripheral blood plasma of patients with incipient DN increased,but there was no statistical difference(P>0.05);With the progress of DN,compared with incipent DN,the level of IL-4 in patients with overt DN decreased by 47.1%(P=0.0035,P<0.01),and decreased by 56.25%in uremia stage(P=0.0037,P<0.01).The plasma IL-4 concentration in incipient DN was higher than that in the normal control group but there was,no statistical difference(P>0.05).It can be seen that compared with healthy people,there is an obvious inflammatory state in patients with DN.As the disease progresses,the concentration of TNF-α gradually increases,while the concentration of IL-4 gradually decreases.Spearman correlation analysis showed that serum TNF-α levels(M1 markers)in DN patients had no correlation with urine protein excretion rate(UAER)(r=0.5123,P=0.371>0.05),but was negatively correlated with glomerular filtration rate(EGFR)(r=0.7873,P=0.0005<0.01).IL-4 level(M2a marker)was positively correlated with glomerular filtration rate(eGFR(r=0.6608,P=0.0065<0.01),and There is no correlation between urinary protein excretion rate(UAER)(r=0.4683,P=0.0688>0.05).IL-10 level(M2c marker)have no correlation with urinary protein excretion rate(UAER)(r=0.3763,P=0.1362>0.05)and with glomerular filtration rate(eGFR)(r=0.2263,P=0.2993>0.05).2.Study on the effect of Futu Dan and hyperoside on the polarization of db/db mouse macrophages.Futu Dan and hyperoside can significantly reduce the levels of body weight,fasting blood glucose(FBG),cholesterol(TC),and low-density lipoprotein(LDL-C)and the Urine protein/creatinine(UACR)ratio.But it has little effect on ALT,AST,ALB,Urea,Cr,UA,TG,HDL-C,and there is no statistical difference.In addition,Hypericin can specifically excert a significant reduction effect of fasting blood glucose(FBG)in mouse serum.qPCR analysis of M1 and M2 polarization indicators.Compared with the control group,the indicators of M1 type macrophages(iNOS,TNF-α,MCP-1)in the db/db mouse model group increased significantly(P<0.05,P<0.01,P<0.0001).Compared with the model group,the expression of iNOS,TNF-α,MCP-1 was significantly reduced in the Futudan treatment group(P<0.05,P<0.05,P<0.0001)and in the hyperoside treatment group(P<0.05,P<0.05,P<0.01).Compared with the control group,the CD206 index of M2 macrophages in the db/db mouse model group decreased significantly(P<0.05).Although the levels of Arg-1 and CD163 had a downward trend,there was no statistical difference(P>0.05).Compared with the model group,the expression of Arg-1,CD206 and CD163 was significantly increased in the Futudan treatment group(P<0.0001,P<0.05,P<0.0001)and in the Hyperoside treatment group(P<0.001,P<0.05,P<0.0001).Flow cytometry analysis of spleen and peritoneal macrophage polarization subtype changes.Compared with the model group,Futudan and hyperoside can reduce the level of M0 and M1 macrophages in both the peritoneal cavity and spleen after under the treatment of Futudan and Hyperoside.In addition,the level of M2a macrophages in the spleen of mice could be significantly up-regulated,but there was no significant change in the peritoneal cavity.There was no significant difference in the levels of M2c cells in abdominal cavity and spleen with the intervention of Futudan and Hyperoside;Observed by the electron microscope,compared with the control group(db/m),the podocyte foot processes of model group(db/db)mice are extensively fused,podocytes are shed,and the number of cells is reduced.After intervention with Futu Dan,the above phenomenon has been improved,reflecting that Futu Dan can regulate podocyte damage,thereby reducing proteinuria in diabetic nephropathy and playing a role in kidney protection.3.In vitro study of Hyperoside among macrophage polarization.Compared with the normal control group,the expression levels of iNOS,MCP-1 and TNF-α onM1 macrophage surface markers in the high glucose group(HG)were significantly increased(P<0.01,P<0.01,P<0.01);Compared with the model group,the expression levels of iNOS,MCP-1 and TNF-α in the high-dose Hyperosie group were significantly reduced(P<0.05,P<0.05,P<0.05);Compared with the normal control group,the expression levels of CD163,CD206,and TGF-β on the surface markers of M2 macrophages in the high glucose group were significantly reduced(P<0.01),when different concentrations of Hyperoside were given.The expression level of M2 macrophage surface markers were increased to varying degrees(P<0.01),with statistical significance;By co-culture with M2 macrophages and T cells,it was found that compared with the control group(T),the expression of T cells in the model group(M2+T)was significantly reduced(P<0.0001),indicating that M2 has the effect of inhibiting T cell proliferation.Compared with the model group,the proliferation of T cells in the high dose hyperoside group was further reduced(P<0.0001),indicating that hyperoside has the effect of synergizing M2 to inhibit the proliferation of T cells.Compared with the control group(T),The model group(M2+T)can significantly increase the expression of IL4+CD4,indicating that M2 has the ability to induce T cells to differentiate into TH2.Compared to the model group,the high-dose hyperoside group can significantly increase the IL-4+CD4 Expression(P<0.05),indicating that hyperoside has the effect of synergizing M2 to promote the differentiation of T cells into TH2.Compared with the control group(T),the model group(M2+T)can significantly increase the expression of Foxp3+CD4(P<0.05);compared with the model group,the high-dose hyperoside group can significantly increase the expression of Foxp3+CD4(P<0.01),indicating that hyperoside has a certain effect on the expression of Foxp3+CD4+Treg cells.Conclusion1.In patients with type 2 diabetic nephropathy,there is a monnuclear macrophage M1/M2 type polarization imbalance,proinflammatory M1 type cells increase,and anti-inflammatory M2 type cells decrease.This polarization imbalance is related to the progresssion of diabetic nephropathy.2.Both Futu Dan and Hyperoside can reduce the body weight(BW),urinary protein/creatinine ratio(UACR)and blood cholesterol of db/db mice.In addition,Hyperoside can also significantly reduced the fasting blood glucose(FPG)levels in db/db mice,indicating that Hyperoside can specifically play a role in regulating the disorder of glucose and lipid metabolism in db/db mice to a certain extent.But There is no obvious effect in reducing blood creatinine and improving renal function under both Futudan and Hyperoside treatment group.Furthermore,Futu Dan and Hyperoside can down-regulate the expression level of M1 type macrophages in the kidney,spleen,and abdominal cavity of db/db mice,indicating that Futu Dan and Hyperoside have a role in reducing inflammation.In addition,Futu Dan and hyperoside can also up-regulate the expression level of M2 macrophages in kidney,M2a level in spleen and play an important role in tissue repair.Last but not least,Futudan can improve the phenomenon of extensive fusion of podocyte foot process in mice,podocyte shedding,cell number reduction,etc.,indicating that Futu Dan can reduce diabetic nephropathy proteinuria and play a role in kidney protection by regulating podocyte damage in db/db mice.3.Hyperoside can inhibit the expression of pro-inflammatory M1 markers induced by high glucose and up-regulate the expression of anti-inflammatory M2 markers,indicating that hyperoside has certain anti-inflammatory and tissue repairing effects.Through co-culture of M2 macrophages and T cells,it was found that Hyperoside and M2 macrophages synergistically inhibit T cell proliferation,promote T cell differentiation into TH2,and induce Foxp3+CD4+Treg cell expression.