| BackgroundDKD is one of the most important microvascular complications of diabetes and the leading cause of ESRD.Its pathogenesis is complex,involving genetic factors and environmental factors,and is related to inflammation,oxidative stress,activation of RAAS system,and fibrosis.The current comprehensive therapy based on RAAS blockers can improve the condition of DKD patients,but it can't completely prevent DKD progressing to ESRD.Therefore,it is important to develop safe and effective new drugs to prevent or delay the initiation and progress of DKD.Symptoms of DKD belong to the categories of "thirst quenching","edema","guange",and"depletion" in the traditional Chinese medicine.At present,the relatively well-known name of DKD in Chinese medicine is "thirsty kidney disease".The etiology is related to insufficient congenital endowment,inadequate diet,the loss of internal organs,and injury to the kidney.The pathogenesis is deficiency of spleen and kidney,deficiency of qi and yin,and long-term stagnation of wet phlegm,turbidity,and blood stasis.It can be summarized as:deficiency,stasis,and turbidity.Deficiency is the basic condition,stasis is the core pathogenesis,and turbidity is the final outcome.The traditional Chinese medicine Polygonum cuspidatum is a traditional Chinese herbal medicine for the treatment of diabetes.As stated on "the Nature of Medicine" by Zhen Quan of Tang Dynasty,Polygonum cuspidatum can cure hot fever,irritability,thirst,urinate,and suppress all heat poison.Polygonum cuspidatum,slightly bitter and slightly cold,has the functions of clearing heat and detoxifying,relieving biliary and yellowing,relieving cough and resolving phlegm,dispersing stasis and relieving pain,and is suitable for the core pathogenesis of DKD "stasis".At the same time,the theory of traditional Chinese medicine believes that traditional Chinese medicine of clearing away heat and detoxification often has relatively obvious anti-inflammatory effects.Many studies have confirmed that DKD is related to oxidative stress and inflammation caused by hyperglycemia and renal hypoxia.In the development of DKD,the inflammatory pathway is the core and is an important factor leading to the progress of glomerulosclerosis.The levels of chemokines(IL-1? and MCP-1),C-reactive protein(CRP),and tumor necrosis factor-?(TNF-?)are related to the occurrence and progression of DKD.Polydatin is the main active ingredient of Polygonum cuspidatum,and it is a glycoside form of resveratrol.They have similar pharmacological effects,but Polydatin has stronger stability and antioxidant effect.At the same time,polydatin is a structural analog of phlorizin,and it is speculated that it may also have SGLT inhibitory effect.Therefore,Polydatin may have a certain renal protective effect,may have a novel mechanism of action,and both benefit from hypoglycemic and protecting kidney,and it is expected to become a potential drug for preventing and treating DKD.Objective1.Using the methods of network pharmacological,To explore the signal pathway and mechanism of Polygonum cuspidatum multi-component synergistic therapy for DKD.2.From two aspects of cell experiments and animal experiments.,to investigate the inhibitory effects of polydatin on the activities of SGLT1/2 and GLUT1/4,and the effects on glucose transporters,glucose homeostasis,and kidney damage in STZ-induced diabetic rats.Methods1.Network pharmacologyUsing the TCMSP to search and screen for Polygonum cuspidatum activity and superior chemical components and potential targets,search for DKD related targets from multiple disease databases.The target network diagram of "Polygonum cuspidatum active ingredient-DKD" was constructed by Cytoscape.Using ClueGO analysis tools,GO and KEGG genes were enriched and analyzed for biological processes and pathways of action targets of Polygonum cuspidatum treating diabetic nephropathy.2.Cell experiment To observe the effect of polydatin on HEK293 cells and 3T3-L1 adipocytes and the detection of related indicators.HEK293 cells stably expressing SGLT1 or SGLT2 were used as vectors,14C-AMG as substrate to evaluate the activity of inhibiting SGLTs in vitro,calculate the radioactivity and corresponding IC50 value of each well using a top counting microplate scintillation counter,evaluate the effect of polydatin at different concentrations.Using 3T3-L1 adipocytes,with or without insulin,effects of different concentrations of polydatin on 2-DG intake,measuring radioactive signals with a liquid scintillation counter,calculating the percentage of 2-DG inhibition,the inhibitory activity of polydatin GLUT1/4 was evaluated.3.Establishment of rat model of type 2 DKD,intervention of polydatin in DKD model rats and detection of related indicatorsHigh-sugar and high-fat rats were fed for 8 weeks to induce insulin resistance,then a single intraperitoneal injection of STZ(35mg/kg bwt)was used to establish a DKD rat model,two consecutive fasting blood glucose(FBG)>16.7 mmol/L,24-hour urine protein>20 mg is the success criterion for modeling.The rats were randomly divided into metabolic cages and divided into 4 groups:the normal control group(Normal Control),the DKD model group(STZ),the positive control group(phlorizin),and the treatment group(polydatin).Rats of each group were orally administered different doses of polydatin(20-120 mg/kg bwt),phlorizin(120 mg/kg bwt),and a control solvent(0.25%sodium carboxymethyl cellulose solution)for 6 weeks.Regularly monitor rat feed and water intake,detect blood glucose.HbAlc,urea nitrogen,creatinine,24h urine glucose,and 24h urine protein of rats in each group,ELISA method to determine the levels of serum IL-1?,TNF-?,MCP-1 and CRP,and explore the relationship between time and quantity.One-half left kidney tissue was taken,and the tissue sections were stained with H&E,and the morphological changes of the pathological structure were observed under a light microscopeResults1.Network pharmacology Through OB and DL screening and literature search,15 superior active ingredients of Polygonum cuspidatum were obtained,and 290 potential targets were predicted.Through Venn analysis,37 targets for diabetic nephropathy were obtained,and 17 key targets were further screened.Using ClueGO enrichment analysis,47 significant pathways such as AGE-RAGE signaling pathway,adhesion junction,HIF-1 signaling pathway,insulin resistance,and VEGF signaling pathway and reactive oxygen metabolism processes were enriched in diabetic complications 87 significant biological processes including positive regulation of small molecule metabolic processes,positive regulation of vascular system development,and reactive oxygen biosynthesis processes.2.In vitro cell experiments Polydatin was used to evaluate the activity of SGLT1 and SGLT2 in vitro,and Phlorizin was selected as a positive control.The results of the study showed that Polydatin significantly inhibited SGLT1 and SGLT2,IC50 is 1968nM and 878.5nM respectively.Compared with the phlorizin group,Polydatin has moderate SGLT2 inhibitory activity,indicating that polydatin may be a potential dual inhibitor of SGLT1/2.The inhibitory effect of polydatin on GLUT1 and GLUT4 was evaluated with cytochalasin B as a control group The results showed that in the presence of insulin,1 ?M Polydatin only slightly inhibited the uptake of 2-DG,while 10 ?M and 100 ?M Polydatin produced 32%and 78%inhibition of 2-DG uptake,respectively,indicating that polydatin inhibit the activity of GLUT4 in a dose-dependent manner.On the other hand,in the presence of insulin,cytochalasin B significantly inhibited the activity of GLUT4 by 96%.However,in the absence of insulin,the inhibitory effect of polydatin on GLUT1 was also dose-dependent,but 10 ?M polydatin produced only 27%inhibition of 2-DG uptake,and 10 ?M cytochalasin B increased GLUT1 activity to 90%.It indicated that Polydatin could be a potential dual inhibitor of SGLT1/SGLT2,with high selectivity for GLUT1 and GLUT4.3.In vivo animal experiments In this study,the relationship between time-effect and dose-effect relationship showed that Polydatin significantly reduced the blood glucose and promoted the excretion of urine glucose in diabetic rats in a dose-dependent manner.Polydatin produced the best hypoglycemic effect at a dose of 120 mg/kg bwt.Long-term pharmacological studies have shown that Polydatin exhibits a continuous hypoglycemic effect throughout the treatment process,which can significantly reduce HbAlc in DKD model rats.Polydatin treatment can reduce renal function indexes such as urinary protein,serum creatinine and urea nitrogen in DKD rats,and improve symptoms such as polyuria,polydipsia and polydipsia related to DKD.The levels of serum inflammatory factors IL-1?,TNF-?,MCP-1 and CRP were significantly increased in the DKD model group rats,and after treatment with polydatin,the levels of the above serum inflammatory factors in DKD rats were significantly reduced.At the same time,the results of histopathological examination of kidneys in this experiment showed that compared with the normal control group,the renal tubules and glomeruli of the DKD model group significantly deteriorated,accompanied by a large number of inflammatory cell infiltration.However,after treatment with Polydatin,the cells of the kidney tissue were regularly arranged,the structure of the glomeruli and tubules was relatively complete,and the inflammatory cells were significantly reduced.Conclusion1.Polygonum cuspidatum main active ingredients can regulate multiple targets in important pathways of DKD.2.Polydatin has significant inhibitory effects on SGLT1/2 and GLUT1/4 in vitro,with moderate SGLT2 inhibitory activity,but its selectivity to SGLT1 is five times higher,indicating that polydatin may be a potential dual inhibitor of SGLT1/2.3.Polydatin significantly reduce blood glucose,promoted urinary glucose excretion in a dose-dependent manner,and reduce the level of HbAlc,urine protein,serum creatinine,urea nitrogen,and inflammatory factors IL-1?,TNF-?,MCP-1,and CRP.It indicated that Polydatin could be a novel dual-target inhibitor of SGLT1/SGLT2,which has certain renal protection,and its renal protection mechanism may be through a variety of pathways such as hypoglycemic,anti-inflammatory and antioxidant pathways. |
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