Study On The Effect And Mechanism Of FTZ In Ameliorating Diabetic Kidney Disease Based On Th17/IL-17A Axis

Background: Diabetes has become a global epidemic with socio-economic development.Diabetic kidney disease(DKD)is one of the most common microvascular complications of diabetes,which greatly increases the risk of death in diabetic patients.There are no specific drugs for DKD treatment and the development of new drugs for DKD remains a priority and a challenge for the medical field.The renal inflammatory processes mediated by T helper 17(Th17)and its effector Interleukin(IL)-17 A have received significant attention in the pathogenesis of DKD,and therapeutic strategies to inhibit the Th17/IL-17 A axis could improve DKD.Professor Guo Jiao innovatively comes up with the therapy of “Tiao Gan Qi Shu Hua Zhuo” for the treatment of glucolipid metabolic diseases.Its representative formula Fufang Zhenzhu Tiaozhi capsule(FTZ)has a great effect on the treatment of glucolipid metabolic diseases in the clinic.However,its protective effect on the kidney and its mechanism have not yet been investigated.Therefore,this study investigates the role and mechanism of FTZ in the prevention and treatment of DKD based on the Th17/IL-17 A axis,and it provides an experimental basis and scientific evidence for the clinical prevention and treatment of DKD.Objective: The DKD mouse model was constructed by streptozotocin(STZ)combined with a high fat diet(HFD)and leptin receptor knockout to investigate the effects of FTZ on DKD,renal inflammation,and Th17 cells.Then the effects of FTZ on Th17 differentiation and effects were further investigated by high glucose culture of Jurkat T cells in vitro.Methods: 1.The construction of DKD model and treatment in groups:C57BL/6 mice induced by intraperitoneal injection of STZ combined with HFD were used to construct an animal model of type 2 DKD.Control group,model group,positive control(Losartan,30 mg/kg/d)group,the low dose of FTZ(FTZ-L,1 g/kg/d)group,and the high dose of FTZ(FTZ-H,2g/kg/d)group were set,respectively.db/db mice were used to construct an animal model of type 2 DKD.db/m group,db/db group,losartan group,FTZ-L group,and FTZ-H group were set,respectively.When the urine protein test was positive,it is considered the successful establishment of the DKD model.Mice were dissected and blood,urine,kidneys,and spleen were collected after 12 weeks treatment.2.Pharmacodynamic evaluation of FTZ: The levels of fasting blood glucose(FBG),triglycerides(TG),total cholesterol(TC),low-density lipoprotein-cholesterol(LDL-C),24 h proteinuria,serum creatinine(Scr),and urinary albumin creatinine ratio(UACR)were assayed by kits.Hematoxylin-eosin staining(H&E),Masson staining,periodic acid-Schiff staining(PAS),Sirian red staining,and electron microscopy were used to evaluate the pathological damage to the kidney.Finally,the accumulation of extracellular matrix(ECM)in the kidney was assessed by immunohistochemical staining or immunofluorescent staining.3.Investigation into the effects of FTZ on renal inflammation: ELISA was used to measure the levels of cytokines associated with renal inflammation and ECM deposition in renal,including tumor necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β),IL-6,IL-1β,and IL-17 A.The infiltration of neutrophils and macrophages in the kidney was detected using immunohistochemical staining.Finally,the expression of the nuclear factor kappa B(NF-κB)pathway in the kidney was measured by western blotting.4.Investigation into the effects of FTZ on T helper 17(Th17): The proportion of Th17 cells in the spleen of DKD mice was measured by flow cytometry.And the expression of retinoid orphan receptor γt(RORγt)and IL-17 A which are the transcription factor and the effector of Th17 in the kidney were measured by western blotting.5.Investigation into the effects of FTZ on Th17 differentiation and effect in vitro: Human leukemic T lymphocytes(Jurkat T cells)were stimulated with glucose(25 mmol/L).Normal glucose(NG)group,mannitol(MN)group,high glucose(HG)group,and FTZ(50/100/150μg/mL)group were set up respectively.After cultivation for 24 h,cells and medium were collected and investigated for the effect of FTZ on T cells.Results: 1.After the successful construction of the DKD model,the levels of 24 h proteinuria,Scr,and UACR were significantly elevated.FTZ significantly reduces the level of FBG,TG,TC,and LDL-C.24 h proteinuria,Scr,and UACR were significantly decreased with the administration of losartan and FTZ.The results of pathological staining showed that,compared with control mice,glomerular hypertrophy,mesangial expansion,accumulation of fibronectin,and collagen Ⅳ in the glomerular and tubular interstitium,and infiltration of the inflammatory cell were appeared in DKD mice,while the pathological phenomenons were improved after FTZ and losartan treatment.2.Compared to mice in the control group,the secretion of the cytokines(TNF-α,IL-6,IL-17 A,IL-1β,and TGF-β)and the expression of macrophages,neutrophils,and NF-κB pathway were significantly upregulated in the kidney of DKD mice.The administration of FTZ and losartan significantly reduced the secretion of TNF-α,IL-6,IL-17 A,IL-1β,IL-8,and TGF-β and downregulated the expression of macrophages and neutrophils and the NF-κB pathway.3.The proportion of Th17 cells in the spleen was significantly increased and the expression of RORγt and IL-17 A in renal was significantly upregulated in DKD mice.FTZ and losartan significantly reduced the proportion of Th17 cells and downregulated the protein expression of RORγt and IL-17 A.4.The protein expression of RORγt and IL-17 A was significantly upregulated in Jurkat T cells in the HG group compared to the NG group,and the secretion of IL-17 A into the medium was also increased.FTZ(50,100,and 150 μg/mL)significantly downregulated the expression of RORγt and IL-17 A and the secretion of IL-17 A in Jurkat T cells.Conclusion: 1.FTZ improves glucolipid metabolism,renal function,renal pathological damage,and ECM deposition in DKD mice.2.FTZ reduces the secretion of pro-inflammatory factors and the infiltration of neutrophils and macrophages and also downregulates the expression of the NF-κB pathway in the kidney of DKD mice.3.The protective effect of FTZ on the kidney and the inhibitory effect on inflammation may be closely related to the regulation of the differentiation and effects of Th17 cells.