Effect And The Potential Mechanism Of Triptolide-Polypeptide-Liposome In Diabetic Nephropathy

Objective:Diabetic nephropathy(DN)is the leading cause of end stage renal disease around the world,the pathological changes of which include glomerular basement membrane thickening,mesangial expansion and focal or diffuse sclerosis.Triptolide is one of the most active and the most promising components extracted from the traditional Chinese herb Tripterygium Wilfordii Hook F.The potent inhibitory effects of triptolide on inflammation and fibrosis endow it with promising therapeutic potential for kidney disease.However,triptolide also has some disadvantages,such as poor aqueous solubility,narrow therapeutic window,and multiple organ toxicity,which limit its application in kidney diseases.Polypeptide-liposome named CREKA-Lip is a new drug delivery system which overcomes the disadvantage of poor water solubility and targets fibronectin(FN).This study aimed to evaluate if triptolide delivered by CREKA-Lip could enhance therapeutic effects of free triptolide on diabetic nephropathy and reduce its multiorgan toxicity,furthermore,to explore the mechanism of triptolide based on network pharmacology and in vivo and in vitro research validation.Materials and Methods:This study is composed of four parts.Part 1,db/db mice were used to construct animal models of type 2 diabetes and diabetic nephropathy,and db/m mice were used as normal control mice.Eight-week old db/m or db/db mice were randomly divided into db/m group,db/db group,db/db + triptolide(free TP)group and db/db + CREKALip/triptolide(CREKA-Lip/TP)group.The latter two groups were given free triptolide and CREKA-Lip/TP respectively,50μg/kg body weight,intravenous injection,twice a week.After 10 weeks of continuous administration,the mice were sacrificed to collect blood,urine,kidney,heart,liver and testis.Blood glucose,serum creatinine,blood urea nitrogen,serum albumin and urinary albumin were tested and renal pathology was evaluated.And multiorgan toxicity was measured based on HE staining of heart,liver,and testis.q PCR and Western Blot were used to detect renal inflammation,fibrosis,apoptosis,fatty acid synthesis and β – oxidation.And oil red O staining was used to detect renal lipid deposition.Part 2,network pharmacology was used to find the potential target of triptolide on DN.Firstly,TCMSP 、 Zinc 、 Ch EMBL 、 Binding DB 、 Stitch 、 Super Pred 、Swiss Target Prediction and TTD databases were used to search triptolide related targets,and Mala Cards database was used to search DN related targets.Secondly,Cytoscape and the plug-in Bisogenet were used to establish the protein-protein networks.Thirdly,Cytoscape and plug-in Cyto NAC were used to determine the core targets.Finally,the core targets were imported into online tool DAVID for signal pathway enrichment analysis.The molecular docking model of triptolide with predicted target was constructed by Auto Dock.Part 3,to verify the mechanism of triptolide on DN in vivo,based on the animal models in the first part.Transcriptomics and Western blot were used to detect the expression of the predicted target in each group.And immunohistochemistry and immunofluorescence were used to observe the localization of the predicted target in the kidney.Part 4,to verify the effects and mechanism of triptolide on high glucose(30m M)stimulation in vitro in mouse mesangial cells,mouse podocytes and human podocytes.Firstly,CCK8 experiment was carried out to explore the relationship between triptolide concentration and toxicity.According to the results,low,medium and high doses were selected for follow-up experiments.Western blot was used to detect the inhibitory effect of triptolide on the predicted target,collagen production of mesangial cells,and apoptosis and lipid metabolism of podocytes.Podocyte apoptosis was also detected by flow cytometry.The data were analyzed by Graph Pad Prism 8,and Western Blot and immunohistochemistry were analyzed by Image J.Continuous variables were expressed as mean ± SD.T test was used to compare the two groups,one-way ANOVA was used to compare the three groups or more,Tukey post hoc test was used to compare the three groups or more.P<0.05 was regarded statistically significant.Results:Part 1,the fasting blood glucose and urinary albumin-to-creatinine ratio of 18-week old db/db mice were significantly higher than those of db/m mice,suggesting successful DN modeling.The positive areas of immuno-histochemical staining for FN in kidney sections of db/db mice were significantly increased compared to db/m mice.q PCR and Western Blot also showed increased FN expression in the kidneys of db/db mice,indicating that there were targets of CREKA-Lip in the kidneys of db/db mice.Immuno-fluorescence double staining also revealed that FN colocalized with nephrin,a podocyte marker,suggesting that CREKA-Lip/TP protects the kidney by acting on podocytes.Compared to db/m mice,db/db mice had significantly higher blood glucose and urine albumin,lower serum albumin,and PAS staining of kidney tissue sections demonstrated typical DN pathological changes: mesangial proliferation,along with a significant increase in Masson’s and Sirius red staining positive areas.Both biochemical parameters and pathology improved after triptolide treatment,and the CREKA-Lip/TP group improved to a greater extent than the free TP group.Next,we verified that CREKA-Li/TP significantly reduced renal inflammation,fibrosis,inhibit apoptosis and improve lipid metabolism disorders using q PCR,Western Blot.Also oil red O staining demonstrated that CREKA-Lip/TP reduced renal lipid deposition.The above curative effect of CREKA-Lip/TP was also better than that of the free TP.HE staining of heart,liver and testis sections did not reveal any obvious organ toxicity of CREKA-Lip/TP.Part 2,121 and 87 proteins were dug out as the targets of triptolide and DN,respectively.The protein-protein network was made by topology analysis,and a total of 5164 proteins were included in the triptolide predicted target protein-protein network and 2277 proteins were included in the DN predicted target protein-protein network.After merging,we got 1442 proteins shared by triptolide and DN.And 327 core targets were finally screened based on the strength of association.Core target enrichment analysis revealed that triptolide might improve DN by binding to STAT1 and thereby inhibiting the JAK2-STAT1 pathway.The docking mode of triptolide with STAT1 was predicted by Auto Dock.The binding site of triptolide to STAT1 was consistent with that of STAT1 to DNA.Part 3,transcriptomics and Western Blot were performed to verify that renal JAK2-STAT1 pathway was activated in db/db mice and inhibited after triptolide treatment.The immunohistochemical findings suggested that pSTAT1 Tyr701 was localized primarily in renal tubules,whereas pSTAT1 Ser727 was localized in both tubules and glomeruli.Immunofluorescence double staining revealed that FN colocalized with pSTAT1 Ser727 in glomeruli,suggesting a uniformity of the CREKA-Lip/TP targeting area to the lesion area.Part 4,the suitable therapeutic dosages of triptolide for CCK8 experiment were selected as 1.25ng/m L(low concentration group),2.5ng/m L(middle concentration group),5ng/m L(high concentration group).In mice mesangial cells,the results of Western Blot showed that after triptolide treatment the JAK2-STAT1 pathway was inhibited,and collagen production was reduced,in a dose-dependent manner.In both mice and human podocytes,the results of Western Blot showed that after triptolide treatment the JAK2-STAT1 pathway was inhibited,BAX and caspase-3 were decreased while Bcl2 was increased,and the expression of fatty acid synthesis related proteins was increased but fatty acid β oxidation related proteins was decreased,in a dose-dependent manner.The results of flow cytometry showed that the sum ratios of early and late apoptotic cells were 7.54%,26.21%,12.87%,10.87% in normal human cells,high glucose group,middle triptolide concentration group,and high triptolide concentration group,respectively,in mice podocytes experiment.And the sum ratios of early and late apoptotic cells were 8.21%,26.22%,20.48%,8.92% in normal human cells,high glucose group,middle triptolide concentration group,and high triptolide concentration group,respectively,in human podocytes experiment.The results indicated that triptolide can inhibit podocyte apoptosis induced by high glucose stimulation.Conclusion:Targeted delivery of triptolide by polypeptide-liposomes(CREKA-Lip)enhanced its therapeutic effect against diabetic nephropathy,and reduced its multiorgan toxicity.Triptolide attenuated inflammation,fibrosis and improved apoptosis and lipid homeostasis in diabetic nephropathy through inhibiting JAK2-STAT1 pathway.