| Diabetic nephropathy(DN)is a microvascular complication,which is caused by hyperglycemia in vivo.It is the most common complication of diabetes.At present,it leads to end-stage kidney disease and chronic kidney disease,Pathophysiological tissue sections usually show the characteristics of glomerular basement membrane thickening,glomerular mesangial expansion,nodular sclerosis,glomerular sclerosis,interstitial sclerosis,inflammation,and renal tubular atrophy.According to statistics in 2019,our country grown-up diabetic’s quantity already surpassed 100 million,about could develop diabetes nephrosis 25%to 40%diabetic.Not only diabetes nephrosis affects patient’s quality of life,also gives the patient and the family member brings the serious economic burden.At present,there are few drugs directly used to treat diabetic nephropathy on the market,mainly relying on the combined application of blood sugar,blood pressure and blood lipid lowering drugs,kidney dialysis and kidney transplantation.Therefore,there is an urgent need to develop a drug directly for the treatment of diabetic nephropathy.Garlic,as a traditional Chinese herbal medicine with the same food and medicine,has been proven to have a variety of pharmacologically active ingredients and has its special medicinal value.The effective active ingredients such as organic sulfur extracted from garlic have become a hot spot in the research of traditional Chinese medicine and western medicine.Among them,S-allylmercaptocysteine(SAMC)is a water-soluble organic sulfur compound extracted from garlic.A large number of literature studies have shown that SAMC has the effects of anti-oxidative stress,anti-inflammatory,anti-tumor,inhibiting mucus secretion,protecting liver,treating kidney injury caused by cisplatin,and treating acute lung injury.Although SAMC has a relatively good medicinal value,because the solubility of SAMC in water and organic solvents is low,the effective dose of SAMC cannot be achieved in vivo,and the application of SAMC has not been effectively and widely developed.Based on this,this experimental project intends to improve the solubility of SAMC by preparing the dosage form of SAMC to reach the required level of administration in vivo and in vitro,and then explore the efficacy and related molecules of SAMC in the treatment of renal fibrosis caused by diabetic nephropathy Biological mechanism of action.The research content of this experimental subject mainly includes the following three parts:(1)Preparation and characterization of SAMC cyclodextrin inclusion compound(SAMC-β-CD);(2)In vitro anti-kidney fibrosis effect of SAMC and its related molecular biology(3)Research on the therapeutic effect and mechanism of SAMC-β-CD on renal fibrosis caused by diabetic nephropathy in vivo.1.Preparation and characterization of SAMC-β-CD inclusion compound:This research part includes the establishment of the method for determination of SAMC API content,the screening of stabilizers,the preparation process of SAMC-β-CD inclusion compound,and the inclusion rate is used as an index to screen the best prescription,solubility experiment to verify the influence of inclusion effect on the solubility of SAMC and the characterization of SAMC-β-CD inclusion compound;firstly,the standard curve of SAMC was established by HPLC and used for content determination and later Screening of prescriptions;through the screening of stabilizers,the experimental results show that PVP-K30 is a more suitable stabilizer.After the inclusion of β-cyclodextrin as the inclusion material and PVP-K30 as the stabilizer,SAMC was included in the single-factor screening The best prescription was screened by the method of HPLC to determine the inclusion rate.The experimental results showed that the best ratio of β-cyclodextrin to SAMC is 10:1,and the best ratio of SAMC-β-CD to PVP-K30 is 3:1.The formation of SAMC-β-CD inclusion compound was verified by infrared spectroscopy(FT-IR)and differential scanning calorimetry(DSC);the solubility experiment proved that the SAMC bulk drug was prepared into SAMC-β-CD inclusion compound can improve its solubility,2.In vitro anti-kidney fibrosis effect of SAMC and exploration of related molecular biological pathways:this research part mainly consists of three modules establishment of in vitro renal fibrosis model,determination of cell proliferation protein expression,and molecular signal pathway research.In the experiment,high-concentration glucose was used to stimulate rat renal mesangial cells(RMC)to establish an in vitro renal fibrosis model.The cell morphology and cell viability were determined by MTT method.The optimal glucose modeling concentration was 30 mM,and the most suitable SAMC was selected.The administration concentration range is between 0-100 μM,and 10,25,and 50 μM are selected as low,medium and high dosing concentrations;Western blot method is used to determine the related protein pathways,as well as the expression of fibronectin Fibronectin and PCNA.Experimental results show that after SAMC treatment,the expression of fibronectin Fibronectin and proliferating cell nuclear antigen PCNA is significantly reduced,and the signal pathway studies have shown that SAMC can inhibit the proliferation of RMC cells by affecting the NF-κB p65 signaling pathway,indicating that SAMC is in vitro It has anti-proliferation and treatment of renal fibrosis.3.Research on the therapeutic effect and mechanism of SAMC-β-CD on renal fibrosis caused by diabetic nephropathy:This research part mainly includes the establishment of diabetic nephropathy mouse models,and the effect of SAMC-(3-CD on renal fiber in diabetic nephropathy mice.Therapeutic effects of chemistry,biopsy of kidney pathological tissues,and the study of related molecular biological mechanisms.In this experiment,a one-time injection of 130 mg/kg streptozotocin(STZ)into the mouse’s intraperitoneal cavity was used to establish a diabetic nephropathy mouse model.Mice with fasting blood glucose≥11.0 mmol/L were selected for grouping;the body weight was measured every other week and fasting blood sugar.At the same time,SAMC-β-CD(50,100 mg/kg)was administered intragastrically for 8 weeks,and enalapril(1.5 mg/kg)was used as the positive drug.At the 7th week,the mice were collected 24 h urine for determination of urinary microalbumin;8 weeks later,the mice were sacrificed,and the left kidney was collected after cardiac perfusion for PAS and Masson staining to observe the morphological changes,mesangial expansion,and extracellular matrix of the mice.Deposition and glycogen accumulation;collect mouse serum for determination of renal function parameters;explore its related molecular biological mechanism by Western blot.The results of the study show that the administration of SAMC-β-CD can significantly improve renal fibrosis.Masson staining results show that the renal mesangial expansion is alleviated.The PAS staining results show that SAMC-β-CD reduces glycogen deposition;The level of urinary microalbumin was reduced,which effectively alleviated the phenomenon of proteinuria;the renal function parameters of serum urea nitrogen(SCr)and serum urea nitrogen(BUN)were reduced;total triglycerides(TG),total cholesterol(T-CHO)indicates that the lipid metabolism disorder caused by hyperglycemia has been improved;the ELISA kit measures the levels of IL-1β and IL-6 and the RT-PCR method measures the expression of inflammatory factors at the gene level,and it is found that SAMC-β-CD can play an anti-inflammatory effect in the body;Western blot assay results show that SAMC reduces the expression of extracellular matrix marker proteins fibronectin,collagen-I,E-cadherin,and vimentin,thereby showing the efficacy of SAMC against renal fibrosis and affects epithelial-mesenchymal transdifferentiation(EMT)related.From the perspective of signaling pathways,SAMC-β-CD can affect the Wnt 3a/β-catenin signaling pathway by affecting the expression of LRP5/6 on the one hand,and on the other hand by inhibiting the NF-κB p65/HIF-la pathway.The pathways work together to affect the EMT process by regulating the downstream transcription factor Snail.In summary,in order to increase the solubility of SAMC in water to reach the required dosage in vivo,this experimental subject designed and prepared SAMC-β-CD,screened out the best process prescription and characterized the preparation.Using glucose stimulation,an in vitro renal fibrosis model was established,and the effect of SAMC on the anti-cell proliferation treatment of renal fibrosis and its mechanism were investigated and verified.The use of STZ to establish an in vivo diabetic nephropathy model proved that SAMC-β-CD can effectively alleviate renal fibrosis and inflammation caused by diabetic nephropathy.Its mechanism of action is mainly to inhibit the Wnt 3a/β-catenin pathway by reducing the expression of LRP5/6 activation,while inhibiting the activation of the NF-κB p65/HIF-1α signaling pathway,the two pathways jointly regulate the EMT process to reduce ECM accumulation and inhibit the fibrosis of diabetic nephropathy. |
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