Tangshenping Study The Protective Effect And TGF-β ₁ Pathway -Smad2 / 3-ILK Signal Transduction DKD Rat Kidney Right

Diabetic kidney disease is one of the serious and common microvascular complications of diabetes mellitus, its incidence is increasing year by year, seriously affecting the quality of life of patients. The basic pathological changes of DKD include glomerular hypertrophy, extracellular matrix deposition, glomerulo sclerosis and renal interstitial fibrosis, but its etiology is very complex, pathogenesis has not yet entirely clear. In recent years, epithelial-mesenchymal transformation (EMT) is considered to be one of the important mechanisms leading to the occurrence of renal fibrosis, transforming growth factor-β1(TGF-β1) is a key factor induced fibrosis, can participate transdifferentiation through multiple signal transduction pathways. Studies have shown that high blood sugar, oxidative stress, advanced glycation end products, angiotensin II and other factors can lead TGF-β/Smad signal transduction pathway activation when DKD, mediated the imbalance between extracellular matrix generation and degradation, mesangial cell proliferation, renal tubular epithelial-to-mesenchymal transdifferentation, podocyte injury and other pathological changes, to accelerate the process of renal fibrosis. Integrin-linked kinase (ILK) is an important downstream factor of TGF-β/Smad signal transduction pathway, play an important role in TGF-β-mediated EMT. TGF-β1increased expression of ILK through Smad2/3, The latter increases the expression of fibronectin, matrix metalloproteinase-2and-9, to promote extracellular matrix deposition, damage the integrity of the basement membrane, and lead to the occurrence of EMT. Recent studies show that, podocytes that as an important component of the glomerular filtration barrier can undergo phenotypic change and EMT in the stimulation of high glucose, oxidative stress and other factors, thereby damage the integrity of the glomerular filtration barrier and lead to proteinuria, accelerate the process of renal fibrosis. Podocyte EMT provides a new explanation for the relationship between podocytes and proteinuria, become an important starting point of the the mechanism of the proteinuria and renal fibrosis. My tutor Professor Zhao Zongjiang proposed DKD "consumptive kidney disease" hypothesis combining with the traditional and modern medicine and composed Tangshenping capsule (hereinafter referred to as Tangshenping) on the basis of the "consumptive kidney disease" hypothesis and clinical experience, it achieved good clinical efficacy for the treatment of DKD. Our research group previous studies have found that Tangshenping can reduce the expression of MCP-1, CTGF protein and mRNA in the DKD rats kidney, reduce podocyte injury and apoptosis, and relieve the pathological changes of DKD rats kidney by inhibiting TGF-β1/Smad7, NF-κB and TGF-β1/p38MAPK signal transduction pathway, protect the renal function of DKD rats. The study takes podocyte EMT as the starting point, to explore the functional mechanisms of Tangshen Ping on the intervention of DKD TGF-β1-Smad2/3-ILK signal transduction pathway by immunohistochemistry, in situ hybridization, western blot and RT-PCR method. The study provides abundant experimental data for the research on DKD "consumptive kidney disease" hypothesis.Objective:Animal experiments:we use intraperitoneal injection of streptozotocin one-time to establish the DKD animal models, while irbesartan as the controlled drugs, and observe the influence of Tangshenping to the pharmacodynamics effects, oxidative stress level of DKD rats, and we also observe its influence to the expression of TGF-β1, Smad2/3, ILK, CD2AP, α-SMA protein and mRNA in the DKD rats kidney by immunohistochemistry, in situ hybridization and RT-PCR method.Cell cultured experiments in vitro:we observe the effects of high glucose and LPS on podocyte phenotype and the expression of TGF-β1, Smad2/3, ILK, CD2AP, α-SMA protein and mRNA by western blot and RT-PCR method.Methods:1. Pharmacodynamics effects of Tangshenping:74clean level Wistar rats were randomly divided into normal group (10rats) and other group (64rats) according to mass. Rats in the other group were intraperitoneally injected with STZ (45mg/kg) to establish DM model. Then the successful of the64rats are randomly divided into model group, irbesartan group, low dose group and high dose group of tangshenping according to mass and gavage treatment. We observe the general condition and detect body mass,24hours urinary protein quantity of rats. At14th week, the blood samples of femoral artery were detected to obtain the level of BUN, Scr, TG; removal of the kidney and weight kidney mass; ordinary HE, mallory, Masson, PASM-HE stain paraffin sections of the renal tissues for observing the pathological changes and TEM for observing the ultrastructural changes of the glomerular basement membrane of blood vessels and foot processes.2. ELISA method:The level of serum NO, MDA, SOD were detected with this method.3. Immunohistochemistry:The level of TGF-β1, Smad2/3, ILK, CD2AP, α-SMA protein in kidney of DKD rats were detected with this method.4. In situ hybridization:The level of TGF-β1, Smad2/3, CD2APmRNA in kidney of DKD rats were detected with this method.5. RT-PCR method:The level of TGF-β1, Smad2/3, ILK, CD2AP, α-SMAmRNA in kidney of DKD rats were detected with this method.6. Cell experiments:Using cultured rat glomerular podocytes as the object of study, making model with high glucose (25mmol/L), LPS (1μg/mL) stimulating podocytes, and use Tangshenping serum intervening in podocyte36h to observe the podocyte phenotype and detect the expression level of TGF-β1, Smad2/3, ILK, CD2AP, α-SMA protein and mRNA.7. The experimental data were statistically processed with SPSS17.0.Results:1. The pharmacodynamics effects of Tangshenping on DKD rats induced by STZ.After the treatment with Tangshenping, the general state of rats had improvement; compared with the model group, the mass of the rats had different degrees of growth (P<0.01), the ratio of kidney mass to body mass decreased definitely (P<0.01),24hours urinary protein quantity decreased definitely (P<0.01), the content of serum BUN, TG decreased definitely (P<0.01), Scr decreased (P<0.05), glomerulosclerosis index and renal interstitial injury index decreased significantly (P<0.05) in the high and low dose group of Tangshenping, the glomerular basement membrane thickened slightly and foot processes fused slightly in the low dose group of Tangshenping, the glomerular basement membrane foot processes were normal basically in the high dose group of Tangshenping, renal pathology changes reduced definitely.2. The effects of Tangshenping on oxidatie stress of DKD rats induced by STZ.Compared with the model group, the content of NO, SOD increased definitely (P<0.01), the content of MDA decreased definitely (P<0.01) in the high and low dose group of Tangshenping.3. The effect of Tangshenping on TGF-β1, Smad2/3, ILK, CD2AP, α-SMA protein expression of DKD rats kidney tissue.Results of immunohistochemistry:A small amount of TGF-β1, Smad2/3, ILK and a-SMA protein were expressed in kidney inherent cells in normal group, significantly lower than the model group (P<0.01); Compared with the model group, the expression of TGF-β1and ILK protein in kidney inherent cells in treatment groups decreased (P<0.01), the expression of Smad2/3and a-SMA protein in treatment groups decreased significantly (p<0.01); and the effect of high dose group of Tangshenping is best. A great amount of CD2AP protein were expressed in podocytes in normal group, significantly higher than the model group (P<0.01); Compared with the model group, the expression of CD2AP protein in podocytes in irbesartan group and the high dose group of Tangshenping increased significantly (P<0.01). the expression of CD2AP protein in podocytes in the low dose group of Tangshenping is higher than the model group.4. The effect of Tangshenping on TGF-β1, Smad2/3, ILK, CD2AP, α-SMAmRNA expression of DKD rats kidney tissue.4.1Results of in situ hybridization:A small amount of TGF-β1, Smad2/3mRNA were expressed in kidney inherent cells in normal group, significantly lower than the model group (P<0.01); Compared with the model group, the expression of TGF-β1, Smad2/3mRNA in kidney inherent cells in the high dose group of Tangshenping decreased significantly (P<0.01), the expression of TGF-β1, Smad2/3mRNA in kidney inherent cells in the low dose group of Tangshenping decreased. A great amount of CD2APmRNA were expressed in podocytes in normal group, significantly higher than the model group (P<0.01); Compared with the model group, the expression of CD2APmRNA in podocytes in treatment groups increased significantly (p<0.01), and the effect of high dose group of Tangshenping is best.4.2Results of RT-PCR:Compared with the normal group, the expression of TGF-β1, Smad2/3, ILK and a-SMAmRNA in kidney tissue in model group significantly increased (P<0.01); Compared with the model group, the expression of TGF-β1, Smad2/3, ILK and a-SMAmRNA in kidney tissue in the high dose group of Tangshenping decreased significantly (p<0.01), the expression of Smad2/3, ILK and α-SMAmRNA in kidney tissue in the low dose group of Tangshenping decreased significantly (P<0.01), and the effect of the high dose group of Tangshenping is better than the low dose group of Tangshenping. Compared with the normal group, the expression of CD2APmRNA in kidney tissue in model group significantly decreased (p<0.01); Compared with the model group, the expression of CD2APmRNA in kidney tissue in treatment groups significantly increased (P<0.01).5. Podocytes phenotypeNormal podocytes are forked, have irregular shape, have a longer, thirmer, more cell’s foot processes, some foot process peripheral will separate the dendritic bifurcation. After high glucose and high glucose plus LPS stimulating, adherent cells lose their foot processes, have larger cell body. Podocyte morphology of Tangshenping groups have improved, foot processes increase, have smaller cell body, the high dose group of Tangshenping is the most obviously.6. The effect of Tangshenping on podocytes TGF-β1, Smad2/3, ILK, CD2AP, α-SMA protein and mRNA expression.6.1Results of western blot:A great amount of CD2AP protein and a small amount of TGF-β1, P-Smad2/3, ILK, α-SMA protein were expressed in podocytes in normal group; Compared with the normal group, the expression of CD2AP protein in podocytes decreased significantly (P<0.01) and the expression of TGF-β1, P-Smad2/3, ILK and α-SMA protein in podocytes increased significantly in the high glucose group and high glucose plus LPS group (P<0.01). Compared with the high glucose plus LPS group, the expression of TGF-β1, P-Smad2/3and α-SMA protein in podocytes decreased significantly (P<0.01) and the expression of CD2AP protein in podocytes increased significantly (P<0.01) in the low, moderate and high dose group of Tangshenping.6.2Results of RT-PCR:Compared with the normal group, the expression of CD2AP mRNA in podocytes decreased significantly (P<0.01) and the expression of TGF-β1, Smad2/3, ILK and α-SMA mRNA in podocytes increased significantly in the high glucose group and high glucose plus LPS group (P<0.01). Compared with the high glucose plus LPS group, the expression of TGF-β1, Smad2/3, ILK and α-SMA mRNA in podocytes decreased significantly (P<0.01) and the expression of CD2AP mRNA in podocytes increased significantly (P<0.01) in the low, moderate and high dose group of Tangshenping.Conclusion:1. Tangshenping can improve general state of rats, increase rats body mass, reduce the ratio of kidney mass to body mass,24hours urinary protein quantity, significantly relieve the pathological changes of DKD rats kidney, have a protective effect on DKD rat kidney. The effect of the high dose group of Tangshenping is better than the low dose group of Tangshenping.2. Tangshenping can decrease the content of serum MDA, increase the content of serum NO and SOD, improve DKD rats oxidative stress, and play a role in protecting renal function.3. Tangshenping can reduce the expression of TGF-β1, Smad2/3, ILK, α-SMA protein and mRNA of the renal tissues and potocytes, and increase the expression of CD2AP protein and mRNA of the renal tissues and potocytes, thus reverse podocytes EMT. Tangshenping can relieve podocytes EMT by suppressing TGF-β1-Smad2/3-ILK signal transduction pathway, and protect podocytes.Above all, Tangshenping can intervene podocytes EMT by TGF-β1-Smad2/3-ILK signal transduction pathway, maintain the stability of podocyte morphology and function, ensure the glomerular filtration barrier function, reduce glomerular sclerosis and renal interstitial fibrosis of consumptive kidney disease, protect DKD renal function, and delay its progression.