| Objective: Diabetic nephropathy(DN)is a kind of microvascular complication caused by diabetes,which has seriously affected people’s health.Many studies have reported that inflammation induced by long-term hyperglycemia is involved in the development of DN.Among them,interleukin-6(IL-6),as a dual functional cytokine,is involved in a variety of autoimmune diseases,and anti-IL-6R antibody has been used to treat some autoimmune diseases.Therefore,IL-6 may be a potential target.This study explores the effect of anti-IL-6R single domain antibody fusion protein(VHH1-1)on changes in diabetic nephropathy rats and its related mechanisms,and provides a new way to improve the treatment of DN.Methods:(1)Preparation of anti-IL-6R single domain antibody fusion protein(VHH1-1): The recombinant plasmid of IL-6R single domain antibody fusion protein was transformed into BL21(DE3)cells,and the sequence was confirmed by gene sequencing.The engineered bacteria was induced by IPTG,followed by ultrasonic fragmentation,urea gradient washing and magnetic bead purification.(2)The activity analysis of VHH1-1: antigen antibody affinity analysis was performed by ELISA;MTT method and scratch test were used to test the proliferation and cell migration ability of RA-FLS cell line.(3)In vivo activity analysis of VHH1-1 in rats: Type 2 diabetic animals: Modeled 24-week-old SPF GK male rats(n = 24)were randomly divided into4 groups based on blood glucose levels: Model,Metformin 500mg/kg,VHH1-10.1mg/kg,and VHH1-1 0.5mg/kg group.Wistar rats(n = 6)were used as the age-matched;Type 1 diabetic animals: Contronl group(9-week-old SPF-grade SD male rats were divided into two groups,except for the Contronl group,the Model group was injected intraperitoneally with 90 mg/kg of STZ,and the Contronl group was injected with the same amount of sodium citrate buffer.Modeled rats were divided into 4 groups:Model,Benazepril 10mg/kg,VHH1-1 0.1mg/kg and VHH1-1 0.5mg/kg group).Rats were administered every three days,and the body weight and blood glucose levels were regularly checked.after the experiment,kidney tissues were stained(HE(PAS,Masson)to observe pathological changes.Blood was taken for detection of biochemical indicators.q RT-CR to detect changes in m RNA expression of inflammatory factors and related proteins in renal tissue;immunohistochemical observation of STAT3 and JNK1/2 protein expression.(4)D-glucose-induced rat mesangial cells(HBZY-1)were used to construct a high-glucose model in vitro.Cell proliferation activity was observed by MTT and flow cytometry experiments,followed by Western blot to determine the expression of proliferation-related signaling pathway proteins.Then,the expression of IL-6 in HBZY-1 cells was knocked down by small interfering RNA,and the expression levels of inflammatory factors(IL-6,TNF-α)and related protein m RNA were detected by q RT-PCR.Result:(1)VHH1-1 engineering bacteria were induced to express,then broken,washed,purified and other steps to obtain the target protein with high purity.ELISA analysis showed that VHH1-1 had specific affinity with IL-6R,that EC50 was 27.1nm.VHH1-1 antibody could inhibit the proliferation and migration of RA-FLS cells,and the mobility of VHH1-1 group(169,34,7pm)decreased by 22.78%,15.94%,7.82%respectively.(3)Compared with the control group,the biochemical indexes of blood glucose,Cr,bun,AST,ALT,the renal organ index and liver organ index in the model group were significantly higher,and the body weight was significantly lower(P < 0.05).Staining showed that glomerular hypertrophy,balloon adhesion,mesangial hyperplasia,basement membrane thickening,matrix accumulation increased.The m RNA expression of inflammatory factors(IL-6,TNF-α)and related proteins was significantly increased in renal tissue(p<0.05),and the expression of STAT3 and JNK1/2 was enhanced by immunohistochemistry.VHH1-1 treatment alleviated renal inflammation,morphologic damage and renal dysfunction in both GK T2 D rats and STZ-induced T1 D rats.However,VHH1-1 treatment could significantly reduce blood glucose levels in T2 D rats,but not in T1 D rats.(4)A high glucose model of HBZY-1 cells in vitro was successfully constructed.VHH1-1 significantly inhibited the proliferation of HBZY-1cells induced by high glucose with an IC50 of 5.48 p M.Flow cytometry showed that VHH1-1 increased the proportion of cells in G0-G1 phase and inhibited the transformation of cell cycle.Western blot showed that VHH1-1 could reduce the expression of cyclin D1 and cyclin E1.The results of q RT-PCR showed that it could up regulate the expression of p21 and p53,and further verify its effect on cell cycle;Western blot showed that VHH1-1 inhibited JAK2/STAT3 signaling pathway and the activity of downstream cyclin D1,resulting in cycle arrest.si RNA-IL-6 intervention can reduce the expression of inflammatory factors(IL-6,TNF-α)and related protein m RNA,and verify the pathway.Conclusion:(1)The constructed anti-IL-6R single domain antibody fusion protein(VHH1-1)displayed high-efficiency affinity and specificity,and can inhibit the migration and proliferation of RA-FLS cells.(2)VHH1-1 significantly improve the kidney of diabetic nephropathy rats.(3)Under the condition of high glucose,VHH1-1can inhibit the proliferation of HBZY-1 cells and reduce the inflammatory response.(4)The mechanism of renal protection of VHH1-1 may be related to anti-inflammatory and inhibition of STAT3 / JAK2 pathway. |
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