Study On Shelong Capsule On Decreasing Proteinuria In Rats And The Mechanism Of Podocyte Protection

Objective: To explore the mechanism of Shelong capsule on decreasing proteinuria in rats and the role of podocytes.by using pharmacological,pathological,molecular biology and electron microscopy.Method:To 72 SPF healthy male rats were studied,12 were randomly selected as normal control group,the remaining 60 using two times of intravenous injection of adriamycin(ADR)method to establish the animal model of renal proteinuria,the detection of urine protein positive were randomly divided into model group,positive control group(Tripterygium Group),compound capsule,"low and high dose group.Groups were given intragastric gavage treatment volume was 2 ml/100 g·d.Tripterygium wilfordii group with5 times of adult clinical dosage,namely 5 mg/kg·d Rats as the dosage;low dose group with3.75 times of adult clinical dosage,which is equivalent to 0.375 mg/kg ·d.Rats as the dosage in the dose group,adult clinical dosage 7.5 times,equivalent to 0.75 mg/kg·d..Rats as dose,high dose group of adult clinical dose equivalent to 15 times the drug 1.5 mg/kg·d.Rats as dose,normal group and model group were given equal volume of students Saline.Respectively administered for 0 weeks,2 weeks,4 weeks,6 weeks,24 hours urinary protein excretion in 8 weeks;4 weeks of continuous administration,each group of 6 rats were randomly selected,determination of total protein,serum albumin,total cholesterol,triglycerides and other biochemical indicators,and morphological observation of renal cortex each group;the remaining rats continued after 4 weeks of dosing,detection and observation of these indicators.Generation of conditionally immortal mice podocyte MPC5 as the research object,modeling and dose of compound capsule treatment dose ADR screening approach by using the CCK-8 kit.Respectively in the intervention after the completion of each related indexes:the use of RT-PCR and Western blot were detected from the gene and protein level of podocyte specific protein nerphrin,podocin,synaptopodin and apoptosis factor cyt-C,caspase9,Caspase3;the protein level of detection of cAMP response element binding protein(CREB)expression level detection by cAMP;double antibody sandwich method;detection using apoptosis of Annexin V-FITC/PI cells detection kit Cell apoptosis;mitochondrial membrane using Jc-1 kit to detect potential changes;use than ATP level detection method;the morphological changes were observed by transmission electron microscopy.Result:1.Shelong capsule can effectively reduce the excretion of 24 h urinary protein in rats,increase the total protein and albumin,lower total cholesterol and triglycerides,thereby maintaining lipid homeostasis and protect renal function.At the same time,the morphology observation shows that: under the light microscope,the normal group of GBM rats,mesangial cells and matrix to no hyperplasia.The medicine group compared with the model group,podocytes slight swelling,matrix and mesangial cells showed mild hyperplasia,renal tubular lumen and a small amount of protein casts,interstitial mild inflammatory cell infiltration;under electron microscope,the blank group rats GBM complete smooth,no mesangial cells and matrix hyperplasia,podocyte arrangement neat,no fusion That gives the medicine group than in model group,the matrix and mesangial cell proliferation is light,but only a small amount of fusion of foot process,renal tubular cell edema,mitochondria swelling degree less lysosome reduced.2.The ADR dose was 0.25 g/ml,the intervention time was 24 h;the establishment of Shelong capsule high low dose group,respectively 100 ng/ml,75 ng/ml,50 ng/ml,the intervention time was 24 h.3.Shelong capsule can upregulated in podocytes associated specific factors nerphrin,podocin,synaptopodin gene and protein levels,apoptosis inhibition factor cyt-C,caspase9,caspase3 gene and protein levels,inhibition of apoptosis,increase the level of cAMP,reduce the down-regulation of mitochondrial membrane potential,improve the level of ATP.Morphological observations show that: under the transmission electron microscope.The drug group compared with model group,podocyte swelling of mitochondria to a lesser extent,mitochondrial crista disorder to a lesser extent,lysosomal rupture to a lesser extent,endoplasmic reticulum degranulation process The degree of reduction is mild and the degree of pyknosis is mild.Conclusion:1.Shelong capsule can effectively reduce the excretion of urine protein in rats.2.The possible mechanism of Shelong capsule reduce proteinuria and effect onpodocyte protection through regulating the cAMP level regulating downstream protein CREB phosphorylation,while maintaining the podocyte specific protein nerphrin,podocin,synaptopodin,on the other hand maintain mitochondrial structure and function,inhibit apoptosis,decrease of mitochondrial membrane potential,improve the content of ATP and to protect podocytes,and maintain the integrity of the filtration barrier,reduce protein leakage,so as to reduce the proteinuria.